Epimedium brevicornu, has provided new impetus for the development of prenylated flavonoids in the field of oncology. Flemingia macrophylla is reported to contain characteristic prenylated flavonoids which can regulate the p53 protein. We aimed to isolate these constituents and conduct activity evaluation, structure–activity relationship, and mechanism studies to provide candidate compounds for antitumor drug development.The successful launch of icaritin, a therapeutic drug for liver cancer derived from Flemingia macrophylla methanol extract. The cytotoxic activity of the constituents was evaluated using an MTT assay with A549 and H1975 cells as the model. The binding mechanism between the compounds and the p53 protein was investigated with molecular docking and validated with cellular thermal shift assay (CETSA). Western blotting (WB) was employed to detect the expression of p53 protein and apoptosis-related proteins in cells.In this study, chromatographic techniques combined with spectroscopic methods were used to separate, purify, and identify the constituents of 1 and 7 provided two pairs of undescribed enantiomers (1a/1b and 7a/7b), along with eight known compounds (2 − 9) isolated from Flemingia macrophylla roots. Their structures were elucidated by spectroscopic analysis, and the absolute configurations of the enantiomers were determined from experimental and calculated electronic circular dichroism data. Compounds 1 − 7, and the non-prenyl analogues 10 − 13, were evaluated for cytotoxic activity against the human lung cancer A549 and H1975 cell line. Compounds 5 − 7 displayed better cytotoxicity than the positive control icaritin in A549 and H1975, with IC50 values ranging from 4.50 to 19.83 μmol·L-1 and < 5 μmol·L-1, respectively. The structure–activity relationships of the chromone or flavonoid analogues against A549 cells were discussed. Molecular docking results demonstrated that compound 7a has strong interaction with p53 and WB indicated that 7a induced apoptosis by increasing the p53 protein, decreasing the anti-apoptotic protein Bcl-2, and activating the caspase family in A549 cells. These results suggest that prenylated flavonoids are potential p53 protein activators.Chiral HPLC separation of racemates Flemingia macrophylla is rich in prenylated flavonoid constituents, among which compounds 5 and 7 exhibited significant cytotoxic activity against A549 cells and served as reference candidates for the design and development of prenylated compounds as antitumor therapeutic drugs.This study demonstrates that The online version contains supplementary material available at 10.1186/s13020-023-00860-3. 1a/1b and 7a/7b) were isolated from Flemingia macrophylla.Two pairs of undescribed enantiomers (5 − 7 exhibited significant cytotoxicity against A549 cells.Prenylated flavonoids Structure-activity relationship study revealed that the presence of B-ring and 2ʹ-OH substitution and the cyclization of 6/8-pentenyl group with 7-OH were beneficial for cytotoxicity.Molecular docking results showed that Cys124 and Cys277 were the key amino acid residues for binding.7a induced apoptosis of A549 cells by activating the p53 protein.Compound The online version contains supplementary material available at 10.1186/s13020-023-00860-3. According to the Global Cancer Observatory (GLOBOCAN) in 2020, lung cancer is the second most diagnosed cancer and is the most prevalent cause of cancer deaths . Non-smaFlemingia macrophylla (Willd.) Merr., a shrub belonging to the Fabaceae family, is widely distributed in southern China, India and Indonesia  25 D − 0.09 ; UV (MeOH) λmax (log ε) 265 (4.11), 273 (4.14), 312 (3.63), 358 (3.01) nm; IR (KBr) νmax 2971, 2914, 1647, 1629, 1584, 1516, 1443, 1397, 1298, 1245, 1127, 979, 925 cm−1; 1H (500 MHz) and 13C (125 MHz) NMR data in CDCl3, see Table m/z 345.1621 [M + H]+ . 1a: [α] 25 D + 24.39 ; ECD (MeOH) λmax (Δε) 214 (+ 5.41), 241 (+ 0.09), 269 (+ 7.51), 297 (− 2.28), 316 (+ 1.71) nm. 1b: [α] 25 D − 25.16 ; ECD (MeOH) λmax (Δε) 203 (− 2.94), 239 (− 0.76), 260 (− 2.75), 295 (+ 2.44), 318 (− 1.20) nm.2-ematin E  yellow a7) yellow amorphous powder; [α]25 D − 1.09 ; 1H (500 MHz) and 13C (125 MHz) NMR data in CDCl3, see Table 7a: [α]25 D + 82.18 ; ECD (MeOH) λmax (Δε) 218 (6.06), 249 (− 2.42), 298 (+ 7.86), 320 (− 1.09) nm. 7b: [α]25 D − 87.15 ; ECD (MeOH) λmax (Δε) 219 (− 0.17), 258 (+ 2.94), 298 (− 7.66), 322, (+ 1.29) nm.Flemiphilippinin F (1 − 7) isolated from F. macrophylla and the commercial non-prenyl analogues (10 − 13) were tested. Compounds were dissolved in DMSO and diluted to 5, 10, 20, and 40 μM with RPMI 1640 containing 10% FBS. Fresh culture medium was used as a blank, and icaritin as a positive control. To measure the viability of A549 and H1975 cells, after treatment with the trial compounds, MTT solution (5 mg/mL) was added to test the cytotoxicity of the compounds in vitro. Cells were seeded in a 96-well plate at a density of 5 × 103 cells/well for growth overnight, and then incubated with the trial compounds at different concentrations for 24 h. The original medium in each well was replaced with the MTT reagent diluted 1/10 with RPMI 1640 containing 10% FBS, and the cells were cultured for 4 h under light-protected conditions. The culture medium and cell supernatant were discarded and the cell precipitate was carefully washed to remove impurities from the culture medium. DMSO (100 μL) was added to each well to dissolve the crystalline material completely by shaking on a shaker. Absorbance was measured at 490 nm with a multimode plate reader 25 D − 0.09 . Its molecular formula C20H24O5, with nine indices of hydrogen deficiency, was assigned from HR-ESI–MS m/z 345.1621 [M + H]+ . The IR spectrum showed absorption bands characteristic of olefinic bond (1647 cm−1), conjugated carbonyl (1629 cm−1), and aromatic  functionalities. The UV spectrum showed absorption maxima at 265, 273, 312, and 358 nm, indicating that 1 was a pyranochromanone derivative 25 D + 82.18) and 7b ([α]25 D − 87.15). By comparing the experimental and calculated ECD spectra  and (2ʺS), respectively. Therefore, compounds 7a and 7b were established as ( +)-(2ʺR)-flemiphilippinin F and ( −)-(2ʺS)-flemiphilippinin F, respectively.Compound  reports –29, the 2 [3 [4 [5 [6 [8 [S)-stigmasterol-4-en-3-one 9 [The other known compounds, eriosemaone A 2 , khonklo2 [3 , 6,8-dip2 [3 [4 , khonklo[3 [4 [5 , derrone[4 [5 [6 , juniper[5 [6 [8 , and  were stronger than those of non-prenyl chromones and flavonoids (10 − 13) (Table 1 − 7), compound 4 was the weakest  significantly enhanced inhibition of A549 cells. This indicates that the number of prenyl groups had little effect on the activity, but that their structural form was significant, especially in the γ,γ-dimethylpyran ring form. Moreover, compared to compound 6 that had a γ,γ-dimethylpyran ring formed by the prenyl at C-8 and the hydroxy at C-7, the 2-methyl-3,3-dimethyl tetrahydrofuran ring formed by the prenyl at C-8 and the hydroxy at C-7 (7a and 7b) increased cytotoxicity, indicating higher activity of the 5-membered than the 6-membered ring. The SAR described above indicated that the prenyl group was essential for chromones and flavonoids to exert toxicity on A549 cells, and that prenyl in cyclized form could enhance activity, especially in the 2-methyl-3,3-dimethyl tetrahydrofuran form. It is worth noting that compounds 1a, 1b, and 2, which lack the B-ring, had lower activity against A549 cells than compounds 5 − 7 which bear a B-ring substituted at C-2 or C-3. Comparing the activities of non-prenyl chromones and flavonoids 10 − 13 , indicating that the B-ring of the flavonoids was positively associated with cytotoxic activity against A549 cells. Notably, compared to compound 3, introduction of a 2ʹ-OH substituent (5) increased the cytotoxic activity against A549 cells. Compared to (R)-1a, (S)-1b exhibited higher cytotoxic activity, indicating that the chirality at C-2 significantly affected cytotoxic activity against A549 cells. Meanwhile, comparing 7a to 7b, chirality at C-2ʺ appears to have little impact on the toxicity against A549 cells.It has been reported that prenyl (3-methyl-2-butenyl or isopentenyl) substitution on a flavonoid’s aromatic ring can enhance affinity to P-glycoprotein and permeability to cell membranes, and significantly improve their bioactivity . Conside3) Table , it coulIn conclusion, the increase in activity due to prenyl substitution was not related to the number of prenyl groups, but rather to the position, form, B-ring substitution, and parent structure. The cyclization of the 6/8-prenyl group with 7-OH was crucial for the activity, and the B-ring and 2ʹ-OH substitution increased cytotoxicity. Based on these results of the inhibitory effects of chromones and flavonoids on A549, the SAR of these compounds is summarized in Fig. 5 > 7a = 7b > 6 = 3 > 13 > 2 > 11 > icaritin > 4 > 1b > 1a > 12 > 10. To further investigate the interaction between P53 and the flavonoids, the molecular docking results are summarized in Additional file 1, compared to their parent structure, the prenyl chromones or flavonoids showed significantly increased binding energy and activity with p53 without an increase in the number of hydrogen bonds. This would indicate that alkyl and π-alkyl interactions were key driving forces for the binding of prenyl flavonoids to p53.The inhibition mechanism was confirmed from the docking data summarized in Table 1a (2-OMe binding with Cys124) and 1b   or DMSO and underwent CETSA thermal pulse treatment. As shown in Fig. 7a-treated group. To further clarify the mechanism of prenyl flavonoid-induced A549 cell toxicity, we detected p53 protein and apoptosis-related proteins, as shown in Fig. 7a, the expression of p53 protein increased, activating Bax expression and increasing the expression of Bax protein, thus competitively reducing the expression of Bcl-2. Bcl-2 protects cells from apoptosis by inhibiting the activation of caspase enzymes. Therefore, reduced Bcl-2 expression led to a decrease in the inhibitory effect on caspase enzyme activity. Herein, we observed a significant decrease in the expression of Pro-Casp8, Pro-Casp9, and Pro-Casp3 in cells relative to the blank group, with a corresponding significant increase in the expression of cleaved-Casp8, cleaved-Casp9, and cleaved-Casp3 relative to the blank group. These data indicate that compound 7a directly acts on the p53 protein, activating the Bax protein, increasing the expression of cleaved-Casp8, cleaved-Casp9, and cleaved-Casp3, and promoting the cytotoxic apoptosis of A549 cells -1a-aat B3LYP/6-31G(d) level in gas phase. Table S2. Standard orientation of (R)-1a-bat B3LYP/6-31G(d) level in gas phase. Table S3. Standard orientation of (R)-7a-aat B3LYP/6-31G(d) level in gas phase. Table S4. Standard orientation of (R)-7a-bat B3LYP/6-31G(d) level in gas phase. Table S5. Standard orientation of (R)-7a-cat B3LYP/6-31G(d) level in gas phase. Figure S1. ( +) HR-ESI–MS sepectrum of compound 1. Figure S2. IR spectrum of compound 1. Figure S3. UV spectrum of compound 1 in MeOH. Figure S4.1H NMR sepectrum of compound 1 in CDCl3. Figure S5.13C NMR sepectrum of compound 1 in CDCl3. Figure S6. DEPT-135 spectrum of compound 1 in CDCl3. Figure S7.1H-1H COSY spectrum of compound 1 in CDCl3. Figure S8. HSQC spectrum of compound 1 in CDCl3. Figure S9. HMBC spectrum of compound 1 in CDCl3. Figure S10. Chiral HPLC analysis of compound 1. Figure S11. ECD spectrum of compound 1a in MeOH. Figure S12. ECD spectrum of compound 1b in MeOH. Figure S13. 1H NMR sepectrum of compound 7 in CDCl3. Figure S14. 13C NMR sepectrum of compound 7 in CDCl3. Figure S15. Chiral HPLC analysis of compound 7. Figure S17 ECD spectrum of compound 7b in MeOH. Figure S18. The molecule docking results of chromones and chromanones with p53. Figure S19. The molecule docking results of flavanones with p53. Figure S20. The molecule docking results of isoflavones and icaritin with p53.


Small RNAs (sRNAs) within 15-30 nt such as miRNA, tsRNA, srRNA with 3’-OH have been identified. However, whether these sRNAs are the major 15-30 nt sRNAs is still unknown. Here we show about 90% mammalian sRNAs within 15-30 nt end with 2’,3’-cyclic phosphate (3’-cP). TANT-seq was developed to simultaneously profile sRNAs with 3’-cP (sRNA-cPs) and sRNA-OHs, and huge amount of sRNA-cPs were detected. Surprisingly, sRNA-cPs and sRNA-OHs usually have distinct sequences. The data from TANT-seq were validated by a novel method termed TE-qPCR, and Northern blot. Furthermore, we found that Angiogenin and RNase 4 contribute to the biogenesis of sRNA-cPs. Moreover, much more sRNA-cPs than sRNA-OHs bind to Ago2, and can regulate gene expression. Particularly, snR-2-cP regulates Bcl2 by targeting to its 3’UTR dependent on Ago2, and subsequently regulates apoptosis. In addition, sRNA-cPs can guide the cleavage of target RNAs in Ago2 complex as miRNAs without the requirement of 3’-cP. Our discovery greatly expands the repertoire of mammalian sRNAs, and provides strategies and powerful tools towards further investigation of sRNA-cPs. The authors uncover the major 15-30 nt mouse and human small RNAs (sRNAs), which mainly end with 2’,3’-cyclic phosphate, and show that many of these sRNAs can be generated by Angiogenin or RNase 4 and function in Ago2 complex as miRNAs. However, construction of the cDNA libraries of 15–30 nt sRNAs including miRNA for sequencing is usually based on ligation of adapter to 3′-OH of sRNAs6. Modifications of RNA have been identified in many different types of small RNAs9, and whether there is any modification at 3′-end severely hampering the ligation is still largely unknown. Furthermore, whether sRNAs with 3′-OH (sRNA-OHs) are the major 15–30 nt sRNAs is yet to be elucidated.High-throughput sequencing has substantially facilitated the discovery of mammalian sRNAs such as miRNA, piRNA, tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA)11. The mammalian genome encodes four AGO proteins (Ago1–4), and Ago2 is the most highly expressed and the only AGO protein able to cleave a target that is fully complementary to the guide strand of the miRNA12. Interestingly, srRNA, tsRNA, snoRNA, snRNA, small intron RNA (sinRNAs), small mature mRNA (smRNA), piRNA, small repeat-derived RNA (srpRNA) and small miscRNA (smcRNA) have also been found in Ago2 complex16. For example, tsRNA CU1276 associated with AGO proteins modulates proliferation and DNA damage response by targeting 3′UTR of RPA117. Small RNAs from snoRNA ACA45 existed in Ago2 complex have miRNA-like functions by targeting 3′UTR of CDC2L616. However, whether mammalian sRNAs in Ago2 complex have 3′-end modification is still unclear, and whether sRNA-OHs are the major 15–30 nt sRNAs in Ago2 complex remains unknown.miRNAs play important gene-regulatory roles in animals by guiding Argonaute (AGO) proteins to target sites in the 3′UTR of mRNAsTo investigate the 3′-end of sRNAs, 15–30 nt sRNAs from mouse liver were analyzed by ligation assay for sRNA-OHs. Only about 10% of the sRNAs were ligated, whereas the synthetic sRNA-OHs can be almost completely ligated under the same condition Fig. . To furt18, a powerful method to simultaneously quantify more nucleosides and nucleotides was developed to analyze the 15–30 nt sRNAs. Numerous RNA modifications were detected, such as ψ, 2′,3′-cCMP, 2′,3′-cUMP, m1A, Um, m6A, m3C, m22G, m5C, I, 2′,3′-cAMP and 2′,3′-cGMP  to remove 3′-phosphate (3′-P) and 3′-cP or alkaline phosphatase (AP) to remove 3′-P, and then analyzed by ligation assay or polyadenylation assay. Treatment with AP only had a slight effect, but treatment with T4 Pnk led to almost completely ligation or polyadenylation of the sRNAs Fig. . These rAll these data demonstrate that both mouse and human 15–30 nt sRNAs mainly end with 3′-cP, and about 90% of 15–30 nt mammalian sRNAs can be categorized as either sRNA-cPs or sRNA-OHs.T4 Rnl2/AP/NaIO4/T4 Pnk (3′ phosphatase minus)/RtcB-based sRNA-seq (TANT-seq) was used for library construction and high-throughput sequencing to simultaneously profile sRNA-OHs and sRNA-cPs  method was developed to simultaneously detect sRNAs with 3′-OH, 3′-P (sRNA-Ps) and 3′-cP. Briefly, the total RNA was treated with vehicle, AP or T4 Pnk, then polyadenylated, reverse transcribed and detected by qPCR  Hepa 1–6 cell line, and validated by sequencing and immunoblot , RNase 4 KO (RKO) and Ang/RNase 4 double KO (DKO) Hepa 1–6 cell lines, and validated by sequencing and immunoblot  in Arabidopsis and a 3′UTR-derived small RNA  in bacteria can function as small interfering RNAs56. Interestingly, we further demonstrated that several sRNA-cPs can guide the cleavage of target RNAs in Ago2 complex as miRNAs, which indicates that much more sRNA-cPs can function like miRNAs to exert versatile biological functions, although it still needs to be confirmed in the future.Interestingly, we found that a lot of sRNAs including sRNA-cPs are in Ago2 complex. Consistently, various types of sRNAs besides miRNA have been found in Ago2 complexOur studies greatly expand the repertoire of mammalian sRNAs, and provide an exciting avenue and powerful tools towards further recognization of sRNAs, especially sRNA-cPs, for their discovery, biogenesis and important roles in regulating gene expression and related biological functions.DNA and RNA oligos, RNA mimics and siRNAs used in this study were listed in Supplementary Data All animals were maintained and used in accordance with the guidelines of the Institutional Animal Care and Use Committee .ad libitum in a room with an ambient temperature of 20–24 °C and humidity of 40–60% on a 12 h light/dark cycle. To study the effect of high-fat diet, male C57BL/6 mice at the age of 8 weeks were randomly assigned to feed chow or high-fat diet with 60 kcal% fat (Research Diets) for 16 weeks. Male db/db mice at the age of 16 weeks were used.To perform the fasting experiment, male C57BL/6 mice at the age of 8 weeks were randomly grouped and maintained in standard cages with or without access to food for 24 h, and water was available Tissues of interest were collected and snap-frozen in liquid nitrogen immediately after resection and stored at −80 °C.Hepa 1–6, AML12, NIH/3T3, Hep G2 and 293 T cells, obtained from National Collection of Authenticated Cell Cultures, CAS, China, were maintained in DMEM with 10% fetal bovine serum. The cells with 80–90% confluence were used for total RNA extraction and isolation of sRNAs.https://imagej.nih.gov/ij/index.html).For denatured PAGE analysis, RNA or DNA oligo samples were mixed with equal volume of 2 × RNA loading buffer . After incubation at 70 °C for 10 min, the samples were immediately placed on ice for 15% denaturing PAGE containing 7 M urea, and the gel was stained by SYBR Gold nucleic acid gel stain (Invitrogen). Gels were visualized and quantified with ImageJ . Finally, the sRNAs or DNA oligos were reconstituted in RNase-free water.DNA oligos were adenylated using a 5′ DNA Adenylation Kit (New England Biolabs). After the adenylation reaction, the samples were separated by 15% denatured PAGE, and stained with SYBR Gold nucleic acid gel stain. The adenylated oligos were excised from the gel and extracted.57. Briefly, 100 pmol synthetic sRNA with 5′-OH and 3′-P were incubated in 1 × Adenylation reaction buffer, 50 μM ATP and 5 μM Mth RNA ligase in a total volume of 20 μl at 65 °C for 60 min, followed by heat inactivation at 85 °C for 5 min. Then, 1 μl 10 × CutSmart buffer, 1 U shrimp alkaline phosphatase (New England Biolabs) and RNase-free water were added to a final volume of 30 μl and incubated at 37 °C for 30 min to convert the remaining 3′-P to 3′-OH. After heat inactivation at 65 °C for 20 min, 1 µl 10 × Poly(A) polymerase reaction buffer, 4 μl 10 mM ATP, 10 U recombinant ribonuclease inhibitor (Takara), 10 U Poly(A) polymerase (New England Biolabs) and RNase-free water were added to a final volume of 40 μl, and incubated at 37 °C for 30 min. Then the samples were separated by 15% denatured PAGE, and the sRNAs with 5′-OH and 3′-cP were excised from the gel and extracted.Preparation of sRNAs with 3-cP was performed using Mth RNA ligase in 5′ DNA Adenylation Kit as previously describedTo obtain sRNAs with 5′-P and 3′-cP, 300 pmol sRNA with 5′-OH and 3′-cP, 5 μl 10 × T4 Pnk reaction buffer, 5 μl 10 mM ATP, 40 U recombinant ribonuclease inhibitor, 10 U T4 Pnk (3′ phosphatase minus) (New England Biolabs) and RNase-free water were mixed in a total volume of 50 μl, and incubated at 37 °C for 60 min. After heat inactivation at 65 °C for 20 min, the samples were recovered with QIAquick Nucleotide Removal Kit (Qiagen) and dissolved in RNase-free water to obtain sRNAs with 5′-P and 3′-cP.58. sRNAs ( < 200 nt) in the supernatants were precipitated with ethanol-NaAc overnight at −80 °C, and dissolved in RNase-free water and stored at −80 °C. sRNAs ( < 200 nt) were separated by 15% denatured PAGE, and the gel was stained by SYBR Gold nucleic acid gel stain. The 15–30 nt sRNAs were excised from the gel and extracted.Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. High molecular weight RNAs ( > 200 nt) were removed from total RNA by precipitation with 5% polyethylene glycol 8000 and 0.5 M NaCl on ice for 30 min as described previouslyLigation assay for sRNA-OHs was carried out in 20 μl final volume containing 50 ng sRNAs, 20 pmol adenylated DNA oligo, 2 μl 10 × T4 RNA ligase reaction buffer, 4 μl 50% PEG8000, 20 U recombinant ribonuclease inhibitor, 200 U T4 RNA ligase 2, truncated KQ (T4 Rnl2) (New England Biolabs) and RNase-free water. The mixtures were incubated at 16 °C for 16 h, and the ligation products were analyzed by denatured PAGE.RNA polyadenylation assay was performed in a volume of 20 μl containing 50 ng RNA samples, 2 µl 10 × Poly(A) polymerase reaction buffer, 2 μl 10 mM ATP, 20 U recombinant ribonuclease inhibitor, 5 U Poly(A) polymerase and RNase-free water, and incubated at 37 °C for 30 min. The polyadenylated products were analyzed by 15% denatured PAGE.O-methyladenosine (Am), 1-methyladenosine (m1A), N6-methyladenosine (m6A), N6-isopentenyladenosine (i6A), 2′-O-methyluridine (Um), 5-methyl-2-thiouridine (m5s2U), 5-methoxyuridine (mo5U), 5-methyluridine (m5U), 4-thiouridine (s4U), 3′-O-methylurindine (3′-OMeU), 2′-O-methylcytidine (Cm), 5-methylcytidine (m5C), N4-acetylcytidine (ac4C), 2′-O-methylguanosine (Gm), N2-methylguanosine (m2G), 7-methylguanosine (m7G), inosine (I), adenosine 2′,3′-cyclic monophosphate , uridine 2′,3′-cyclic monophosphate , cytidine 2′,3′-cyclic monophosphate  and guanosine 2′,3′-cyclic monophosphate  were purchased from Sigma-Aldrich. 5,2′-O-dimethyluridine (m5Um), 3-methyluridine (m3U), 2-thiocytidine (s2C), 5-hydroxymethylcytidine (hm5C), N2-dimethylguanosine (m22G), pseudouridine (ψ), 1-methylpseudouridine (m1ψ) and 2′-O-methylinosine (Im) were obtained from Berry and Associates Inc. 3′-O-methyladenosine (3′-OMeA), 2-thiouridine (s2U), 3-methylcytidine (m3C), 3′-O-methylcytidine (3′-OMeC), 5,2′-O-dimethylcytidine (m5Cm), N4-acetyl-2′-O-methylcytidine (ac4Cm), 1-methylguanosine (m1G), 3′-O-methylguanosine (3′-OMeG), N2,N2,7-trimethylguanosine , 5′-O-methylthymidine (5′-OMeT) and 3′-O-methylinosine (3′-OMeI) were purchased from Carbosynth. Guanosine 5′-monophosphate  was obtained from Cambridge Isotopes Laboratories. Guanosine 5’-monophosphate  at 140 μg/ml was hydrolyzed by calf intestine alkaline phosphatase (Takara) at a final concentration of 1 U/μl at 37 °C for 24 h to obtain guanosine . Enzymatic digestion of sRNAs and LC–MS/MS analysis were performed as previously described18. Briefly, 100 ng sRNAs were digested with 0.2 U nuclease P1 (Sigma-Aldrich) in 60 μl of 50 mM NH4OAc, pH 5.3 at 50 °C for 3 h and then treated with 0.04 U phosphodiesterase I (USB) for 2 h at 37 °C. Subsequently, the RNA samples were treated with 2 U alkaline phosphatase (Sigma-Aldrich) for 2 h at 37 °C. Proteins were removed by centrifugation through Nanosep 3 K device with Omega membrane . Then, the samples were lyophilized and stored at −80 °C and were resuspended with 17.5 ng/ml G  in 100 μl of 2 mM ammonium acetate for LC–MS/MS analysis. Analysis of nucleoside mixtures was performed on an API 4000 Q-TRAP mass spectrometer (Applied Biosystems) with an Agilent 1200 HPLC system and a diode array UV detector (190–400 nm) and equipped with an electrospray ionization source. The calibration curves for LC–MS/MS analysis are shown in Supplementary Data Adenosine (A), uridine (U), cytidine (C), guanosine (G), 2′-50 ng indicated synthetic or isolated sRNAs, 1 μl 10 × CutSmart buffer, 20 U recombinant ribonuclease inhibitor, 1 U shrimp alkaline phosphatase and RNase-free water were mixed in a total volume of 10 μl, and incubated at 37 °C for 30 min. After heat inactivation at 65 °C for 20 min, the samples were used for ligation assay or RNA polyadenylation assay.50 ng indicated synthetic or isolated sRNAs, 1 μl 10 × T4 Pnk reaction buffer, 20 U recombinant ribonuclease inhibitor, 10 U T4 Pnk (New England Biolabs) and RNase-free water were mixed in a total volume of 10 μl, and incubated at 37 °C for 30 min. After heat inactivation at 65 °C for 20 min, the samples were used for ligation assay or RNA polyadenylation assay.2, 1 μl 10 mM GTP, 15 pmol RtcB ligase , 20 U recombinant ribonuclease inhibitor and RNase-free water. The mixtures were incubated at 37 °C for 2 h, and the ligation products were analyzed by 15% denatured PAGE.15–30 nt sRNAs used for RtcB ligation assay were treated with T4 Pnk (3′ phosphatase minus) to phosphorylate the 5′-OH of sRNAs according to the manufacturer’s instructions. After heat inactivation, the samples were extracted with phenol-chloroform, precipitated with ethanol-NaAc, and dissolved in RNase-free water. RtcB ligation assay was carried out in 20 μl final volume containing 50 ng sRNAs, 20 pmol adapter (OH-RNA8-OH), 2 μl 10 × RtcB reaction buffer, 2 μl 10 mM MnCl2) on ice in Stratalinker 1800 (Stratagene). Subsequently, 500 μl lysis buffer ) was added and incubated on ice for 10 min. The lysates were centrifuged at 12000 g for 10 min at 4 °C to collect supernatant.Cells cultured in 10 cm dishes at a confluence of about 80% were washed twice with ice-cold PBS. After aspiration of the PBS, the cells were treated with UV irradiation at 254 nm  was washed with lysis buffer and incubated with 2 μg anti-Ago2 antibody (Abcam) or normal rabbit IgG antibody (CST) in 200 μl lysis buffer for 4 h at 4 °C. The antibody-bound beads were washed with lysis buffer for three times, and incubated with the supernatant of cell lysates for 4 h at 4 °C. Then the beads were washed with lysis buffer for three times, and subsequently used for immunoblot or RNA extraction.For RNA extraction, the beads were resuspended in 400 μl 100 mM Tris–HCl pH 7.5, 50 mM NaCl, 10 mM EDTA containing 10 mg/ml protease K (Millipore) and incubated for 30 min at 37 °C. Finally, the Ago2-binding RNA was obtained by phenol-chloroform extraction, precipitation with ethanol-NaAc, and reconstitution in RNase-free water.4, 40 U recombinant ribonuclease inhibitor and RNase-free water were added to a final volume of 200 μl and incubated at 0 °C for 40 min in the dark to convert the 2′ and 3′-OH at 3′-end into dialdehydes59. After precipitation with ethanol-NaAc, the sRNAs at the size of 15-46 nt were purified with 15% denatured PAGE, and treated with T4 Pnk (3′ phosphatase minus) to phosphorylate the 5′-OH of sRNAs, followed by phenol-chloroform extraction and precipitation with ethanol-NaAc. After dissolved in 10 μl RNase-free water, 10 pmol OH-RNA13-ddC containing the barcode for 3′-cP sublibrary, 2 μl 10 × RtcB reaction buffer, 2 μl 10 mM MnCl2, 1 μl 10 mM GTP, 15 pmol RtcB ligase, 20 U recombinant ribonuclease inhibitor and RNase-free water were added to a final volume of 20 μl, and incubated at 37 °C for 2 h, followed by heat inactivation at 70 °C for 5 min. The 3′-adapter-ligated sRNAs at the size of 31–46 nt were purified with 15% denatured PAGE. The purified sRNAs were mixed with 5 pmol OH-RNA14-OH, 3 μl 10 × T4 RNA ligase reaction buffer, 6 μl 50% PEG8000, 1 μl 10 mM ATP, 30 U T4 RNA ligase 1 (New England Biolabs), and 20 U recombinant ribonuclease inhibitor in a final volume of 30 μl, and incubated at 25 °C for 2 h. Then, 3 pmol OH-DNA15-OH, 200 U SuperScript IV Reverse Transcriptase (Invitrogen), 4 μl 5 × SSIV buffer, 2.5 μl 100 mM DTT, 2.5 μl 10 mM each dNTP, and 20 U recombinant ribonuclease inhibitor and RNase-free water were added to a final volume of 50 μl, and incubated at 50 °C for 1 h, followed by heat inactivation at 80 °C for 10 min. The cDNA was amplified using Phusion High-Fidelity DNA Polymerase (New England Biolabs) with RNA PCR primer and RNA PCR index primers (Supplementary Data T4 Rnl2/AP/NaIO4/T4 Pnk (3′ phosphatase minus)/RtcB-based sRNA-seq is termed TANT-seq. Negative control library, 3′-OH library and 3′-OH & 3′-P & 3′-cP library were constructed as the above description, except T4 Rnl2 and RtcB ligase, RtcB ligase, shrimp alkaline phosphatase were replaced with nuclease free water respectively. For AlkB treatment, 2 μg sRNAs ( < 200 nt) were incubated in 20 μl reaction mixtures containing 2 μl 10 × AlkB Buffer I, 2 μl 10 × AlkB Buffer II, 20 U AlkB (Beyotime), 40 U recombinant ribonuclease inhibitor at 37 °C for 60 min. Then, the mixtures were used for denatured PAGE analysis, or extracted with 500 μl TRIzol reagent to obtained purified AlkB-treated sRNAs. The AlkB-treated sRNAs were subsequently used for TANT-seq. The method using AlkB-treated sRNAs for TANT-seq is termed ATANT-seq.To construct the library for high-throughput sequencing, 1 μg sRNAs ( < 200 nt) in a volume of 9 μl mixed with 1 μl spike-in mixture , or 100 ng RNA obtained from crosslinking immunoprecipitation in a volume of 10 μl, were incubated at 70 °C for 5 min and immediately placed on ice. Then, the samples were mixed with 20 pmol App-DNA12-ddC containing the barcode for 3′-OH sublibrary, 4 μl 10 × T4 RNA ligase reaction buffer, 8 μl 50% PEG8000, 400 U T4 RNA ligase 2, truncated KQ, 40 U recombinant ribonuclease inhibitor and RNase-free water in a 40 μl reaction volume, and incubated at 16 °C for 16 h. After heat inactivation at 70 °C for 5 min, 1 μl 10 × CutSmart buffer, 20 U recombinant ribonuclease inhibitor, 2.5 U shrimp alkaline phosphatase and RNase-free water were added to a final volume of 50 μl and incubated at 37 °C for 30 min. Subsequently, 20 μl 100 mM NaIO60 to remove adapter sequences except the barcode to distinguish 3′-OH and 3′-cP sublibraries. Then low-quality (Phred score <30) reads, and reads shorter than 21 nt or longer than 46 nt were filtered out. The sequences with the barcode GACGTA in App-DNA12-ddC or CTATCG in OH-RNA13-ddC were assigned to 3′-OH sublibrary and 3′-cP sublibrary respectively, and then the barcodes were removed.Raw reads were first trimmed with fastp (v0.20.0)61 and mitotRNAdb62 before mapping. The reads were first mapped to spike-ins (Supplementary Data NR_030686.1), mmu-5.8 S rRNA (NR_003280.2), mmu-12S rRNA (NC_005089.1), mmu-16S rRNA (NC_005089.1), mmu-18S rRNA (NR_003278.3), mmu-28S rRNA (NR_003279.1), mmu-45S rRNA (NR_046233.2) and mouse rRNA from Ensembl63, or human rRNAs including has-5S rRNA (NR_023363.1), has-5.8 S rRNA (NR_145821.1), has-12S rRNA (NC_012920.1), has-16S rRNA (NC_012920.1), has-18S rRNA , has-28S rRNA , has-45S rRNA  and human rRNA from Ensembl, then sequentially mapped to mouse or human tRNA modified from GtRNAdb with additional 3′-CCA, mitochondrial tRNA modified from mitotRNAdb with additional 3′-CCA, miRNA from miRbase64 and Ensembl, piRNA from piRNAdb (https://www.pirnadb.org), snRNA, snoRNA, lncRNA, miscRNA and other ncRNAs from Ensembl, intron obtained from the information of exon sequence from Ensembl, mature mRNA, IG gene, TR gene, Pseudogene and TEC from Ensembl, repeat sequence  from UCSC Genome Browser (http://genome.ucsc.edu), genome (GRCm38/mm10 or GRCh38/hg38) and mitochondrial genome (NC_005089.1 or NC_012920.1), and the perfectly matched reads were assigned to the mapped biotype classes. Then the unmapped reads were sequentially mapped to the above database again allowing mismatches + gaps ≤2, and assigned to the mapped biotype classes. Unmapped sequences were discarded.Subsequently, the reads were sequentially mapped to the corresponding reference database using ncbi-BLAST + 2.11.0. The CCA sequence was added to the 3’-ends of all tRNAs from GtRNAdb65, were manually picked and assigned to pseudogene. Some high abundant sRNAs for each biotype class were manually checked with NCBI nucleotide-nucleotide BLAST. In mouse samples, the sequences mapped to repeat sequence matched to small nucleolar RNA, C/D box 118 (Snord118) (NR_028566.3) or Gm25313 (XR_004935972.1) were manually picked and assigned to snoRNA or snRNA respectively. The sequences mapped to mRNA sequence perfectly matched to Snord14c (NR_028276.2) were manually picked and assigned to snoRNA. In human samples, the sequences mapped to repeat sequence perfectly matched to U5A small nuclear 1 (RNU5A-1) (NR_002756.2), and U5B small nuclear 1 (RNU5B-1) (NR_002757.3) were manually picked and assigned to snRNA. The sequences mapped to repeat sequence matched to small nucleolar RNA C/D box 118 (SNORD118) (NR_033294.1) were manually picked and assigned to snoRNA.The sequences mapped to repeat sequence assigned to pseudogene in DfamThe reads mapped to rRNAs and tRNAs were classified into srRNA and tsRNA respectively. The reads mapped to intron, lncRNA, genome, mature mRNA, repeat sequence and miscRNA were termed as small intron RNA (sinRNA), small lncRNA (slncRNA), small genome-derived RNA (sgmRNA), small mature mRNA (smRNA), small repeat-derived RNA (srpRNA), small miscRNA (smcRNA) respectively. A small RNA nomenclature was established for sRNAs with average reads more than 2 according to its biotype class, description of matched sequence, start position, length and 3′-modification, meanwhile a simplified small RNA nomenclature was also established according to its biotype class, rank of abundance and 3′-modification. tsRNAs were further classified into 5′-tsRNA , 3′-CCA-tsRNA , 3′-CC-tsRNA  and internal-tsRNA.For replicate analysis, independent biological replicates of TANT-seq experiments were compared by computing the Pearson correlation coefficient by GraphPad Prism 7.0 between any two replicate experiments.The relative abundance of 15–30 nt sRNAs or each biotype class of sRNA with 3′-OH and 3′-cP was calculated as the following. The ligation efficiency of 15–30 nt sRNAs with 3′-OH to 3′-adapter by T4 Rnl2 was calculated with the data shown in Fig. 1e,66.Venn diagrams were generated by VennDiagram package67.To obtain the nucleotide enrichment, the nucleotide abundance was calculated from the terminal five nucleotides in 3′-OH or 3′-cP sublibrary. The sequence logos were constructed with the R package ggseqlogoP values were calculated by edgeR68, and adjusted P value was calculated by Benjamini-Yekutieli method as described previously69. sRNAs with adjusted P value < 0.01 and log2(fold change) ≥2 were considered as differentially expressed. The sequences with average read ≥2 were shown in the volcano plot.For differential expression analysis of sRNAs, the fold changes and https://ggplot2.tidyverse.org).The distribution of abundance and unique reads for sRNAs was visualized in weighted scatter plots using the function geom_count from the package ggplot2 in R . Universal qPCR reverse primer 1 and the indicated synthetic RNA qPCR forward primers listed in Supplementary Data triplex-3′-end qPCR (TE-qPCR) to detect sRNAs with 3′-OH, 3′-P or 3′-cP was shown in Fig. Schematic representation of In a total 500 μl volume, 1 mg total RNA were hybridized with 5 μl 10 μM biotin-labeled DNA oligos complementary to an indicated tRNA , 100–200 ng 15–30 nt sRNAs, or 100–200 ng RNA immunoprecipitated by anti-Ago2 antibody were separated by 15% denatured PAGE and transferred to positively charged Nylon Membranes (Roche). Subsequently, the membranes were air-dried and UV crosslinked, and pre-hybridized with DIG Easy Hyb buffer (Roche). Then, the membranes were incubated overnight with 10 nM 3’-end digoxigenin-labeled oligonucleotides probes as shown in Supplementary Data Escherichia coli BL21 Rosetta (DE3) cells and purification with His-Tag Purification Resin (Roche) and ion-exchange column . Purified Ang and RNase 4 were analyzed by SDS-PAGE and Coomassie blue staining, and then dialyzed to storage buffer  and stored at −80 °C.The Angiogenin (Ang) and RNase 4 cDNA were obtained by RT-PCR from mouse liver total RNA with primers listed in Supplementary Data 4 μg mouse liver total RNA was treated with 0.5 ng RNase A (Thermo Fisher Scientific), 1 U RNase T1 (Thermo Fisher Scientific), 0.32 μg Ang, 0.08 μg RNase 4 or the indicated amount of Ang or RNase 4 in a final volume of 20 μl at 37 °C for 30 min. Then, the samples were extracted with phenol-chloroform, precipitated with ethanol-NaAc, and dissolved in RNase-free water for TE-qPCR or Northern blot.GSE164819)70. The expression levels were presented using Fragments Per Kilobase of exon model per Million mapped fragments (FPKM).RNA-seq data of mouse liver were obtained from the Gene Expression Omnibus (GEO) database  or RNase 4 knockout (RKO) Hepa 1–6 or Hep G2 cells, and RNH1 knockout (RNH1 KO) Hepa 1–6 cells.The Hepa 1–6 or Hep G2 cells were transfected with lentiCRISPR v2 vector or the constructed plasmids by Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The transfected cells were screened with 2 ng/μl puromycin (Thermo Fisher Scientific) for 48 h. Thereafter, the cells were digested and diluted to obtain single-cell clones. Knockout of the indicated genes was confirmed by immunoblot and/or sequencing to obtain wild-type (WT), Ang and RNase 4 double knockout (DKO) Hepa 1–6 and Hep G2 cells.RKO Hepa 1–6 cells and RKO Hep G2 cells were transfected with px330-mp-mAng-sgRNA2 and px330-mp-hAng-sgRNA2 respectively using Lipofectamine 3000 transfection reagent. After transfection for 48 h, the cells with red fluorescence were sorted with flow cytometry into a 96-well plate containing 1 cell per well. Subsequently, the single-cell clones were collected, and knockout of the indicated genes was confirmed by immunoblot and/or sequencing to obtain RNH1 KO Hepa 1–6 cells were transfected with 2 μg Ang or RNase 4 using Xfect protein transfection reagent (Takara) in 6-well plates according to the manufacturer’s protocol. After transfection for 6 h, the cells were washed twice with PBS for subsequent total RNA extraction with Trizol reagent.Wild-type or Recombinant ribonuclease inhibitor (RNH1) was added into the cell culture media of WT, AKO, RKO or DKO Hepa 1–6 or Hep G2 cells at a final concentration of 2000 U/ml. After incubation for 48 h, the cells were used to extract total RNA with Trizol reagent for TE-qPCR, Northern blot or TANT-seq.The cells were lysed in RIPA lysis buffer containing 50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS. Immunoblot was performed with antibodies against RNH1 , Angiogenin , RNase 4 , Ago2 , Bcl2 , Nr3c2 , Twf1 , Me1 , Caspase-3 , Actin  or Tubulin .4 μg total RNA from WT, AKO, RKO or DKO Hepa 1–6 cells pretreated with recombinant ribonuclease inhibitor for 48 h was incubated with or without T4 Pnk to quantitate 15–30 nt sRNAs with 3′-(OH + P+cP) or 3′-OH in 10 μl final volume. Then, 20 pmol 16 nt App-DNA12-ddC, 1 μl of 10 × T4 RNA ligation buffer, 2 μl 50% PEG8000, 20 U recombinant ribonuclease inhibitor, 200 U T4 Rnl2, truncated KQ and RNase-free water were added to a final volume of 20 μl and incubated at 16 °C for 16 h. The ligation products were analyzed by 15% denatured PAGE. Northern blotting was performed with DIG-labeled oligonucleotides probe which was reverse complementary to App-DNA12-ddC as shown in Supplementary Data 72. Tandem 3′UTR luciferase reporter plasmids, including pGL3-20-3′UTRs, pGL3-19-3′UTRs, pGL3-22-3′UTRs-a, pGL3-22-3′UTRs-b and pGL3-24-3′UTRs, were constructed by cloning the predicted target 3′UTRs into pGL3-Promoter (Promega) near XbaI site by homologous recombination as shown in Supplementary Data Target 3′UTRs of sRNAs were predicted by miRandapGL3-3′UTR plasmid was obtained by insertion of CATATGCCGCGGGATATCCTGCAGGACTAGTCGAATTCCCTAGA into XbaI site of pGL3-Promoter. Luciferase reporter plasmids containing a single 3′UTR were constructed by insertion of a synthetic target 3’UTR sequence at XbaI and EcoRI sites of pGL3-3′UTR as shown in Supplementary Data For luciferase reporter assay, Hepa 1–6 cells cultured in a 24-well plate were transfected with the indicated luciferase reporter plasmids, pRL-TK (Promega) and the indicated antisense sRNAs or sRNA mimics using Lipofectamine 3000. After transfection for 48 h, cells were harvested for luciferase reporter assay using Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalized to Renilla luciferase activity.72.The alignment of sRNA sequence and the corresponding biotin-labeled target RNA fragment sequence was predicted by miRandaFull-length Ago2 cDNA was obtained by RT-PCR using mouse liver total RNA with primers listed in Supplementary Data 73, containing 5 μl Myc-Ago2 complexes purified from cell lysates co-transfected with the indicated sRNA mimics, 1 nM 5’ end biotin-labeled target RNA .Ago2 cleavage assay for in vivo loaded sRNAs was essentially performed in 20 μl final volume as described previouslyAgo2 cleavage assay for in vitro loaded sRNAs, 5 μl Myc-Ago2 complexes purified from cell lysates without co-transfection with sRNA mimics were mixed with 5 nM indicated sRNA mimics or sRNA in the presence of 1 × cleavage buffer in 10 μl final volume, and incubated at 37 °C for 30 min. Then 1 nM 5′ end biotin-labeled target RNA  with RNA PCR primer and RNA PCR index primer  and cycloheximide (1 µg/ml) were added and incubated for 4 h. The cells were harvested for immunoblot or flow cytometry analysis with Annexin V-FITC/PI apoptosis detection kit (Vazyme).P < 0.05. * or a indicates P < 0.05, ** or b indicates P < 0.01, *** or c indicates P < 0.001.All measurements were taken from distinct samples. Except indicated, data are expressed as mean ± SD of at least three independent experiments, and statistical significance was assessed by Student’s t-test or edgeR. Except indicated, differences were considered statistically significant at Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Reporting SummarySource Data


Introduction: Chronic non-healing wound is a considerable clinical challenge and research into the discovery of novel pro-healing agents is underway as existing therapeutic approaches cannot sufficiently meet current needs.Method: We studied the effects of corylin in cell line fibroblasts and macrophages by Western blots, PCR, Flow cytometry assay, Immunofluorescence.Results: We showed that corylin, a main flavonoid extracted from Psoralea corylifolia L, reduced inflammatory responses, promoted collagen deposition, and accelerated the healing of full-thickness skin wounds in mice. Exploration of the underlying mechanisms showed that corylin activated the PI3K/AKT signaling, leading to fibroblasts’ migration, proliferation, and scratch healing. Corylin also activated sirtuin 1 (SIRT1) signaling, enhanced the deacetylation and cytoplasmic translocation of NF-κB p65, and therefore reduced lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. Furthermore, inhibition of PI3K/AKT and sirtuin 1 pathway with LY294002 and EX527 prevent the therapeutic potency of corylin against chronic wounds.Conclusion: In summary, our results suggested that corylin may be a candidate for the development of novel pro-healing agents. As the natural physical barrier to the external environment, the skin plays a crucial role in temperature regulation, as well as protection against microbial infection, and other environmental factors . HoweverPsoralea corylifolia L. is a herb that has been widely used for the treatment of leucoderma and skin inflammatory-related diseases , seeking the highest grade commercially available unless otherwise indicated. To study the role of Sirt1, PI3K, and AKT in corylin-induced pharmacological actions, cells were treated with Sirt1 inhibitor EX527, ER antagonist Fulvestrant, PI3K inhibitor LY294002 and AKT inhibitor Miltefosine, respectively. EX527 (Cat. E129892), Fulvestrant (Cat. F125644), LY294002 (Cat. L408397), and Miltefosine (Cat. N130417) were purchased from Aladdin . Corylin was purchased from MedChemExpress (Cat. HY-N0236).n = 6) received corylin , EX-527 (0.5 mg), LY294002 (0.5 mg) or acetone vehicle control once daily for 8 days. The doses of corylin, LY294002 and EX527 were based on the preliminary experiments. The administration and route of corylin at the animal level were determined in the pharmacokinetic studies. The wounds were photographed, on day 0, day 2, 4, 6, and 8, using a digital camera. Wound areas (S) were measured by counting the total amount of grayscale pixels in the cell-free area with ImageJ software at 0 and 24 h. The migration rate (%) was calculated using the following formula: migration rate (%) = 1 - S (24 h)/S (0 h). FGF2 was used as the positive control. Where SW0 is the size of the initial wound area and SWn is the size of the wound area on day n post-surgery  were anesthetized with pentobarbital sodium and back cutaneous hair was removed by electrical shaving. One 10 mm diameter full-thickness skin wound was made on the dorsal surface with a round skin biopsy punch. For corylin treatment, mice  for 15 min. Slides were then incubated with primary antibodies: rabbit anti-α-SMA  or rabbit anti-COL1A1  at 4°C for 10 h. After incubation, sections were washed with 0.1 M PBS and incubated with goat anti-rabbit IgG-Alexa Fluor 488  or goat anti-rabbit IgG-Alexa Fluor 555  at room temperature for 1 h. After an additional rinse, sections were mounted by VECTASHIELD mounting medium with DAPI  and observed under confocal microscopy  . The fluTotal RNA was extracted from skins and cell samples with Trizol  and measured by spectrophotometer  using a previously described method . cDNA waα-SMA: 5’-ACTGGGACGACATGGAAAAG-3’ (forward); 5’-CATCTCCAGAGTCCAGCACA-3’ (reverse).TGF-β: 5’-TGATACGCCTGAGTGGCTGTCT-3’ (forward); 5’-CACAAGAGCAGTGAGCGCTGAA) -3’ (reverse).COL1A1: 5’-GAGCGGAGAGTACTGGATCG-3’ (forward); 5’-TACTCGAACGGGAATCCATC-3’ (reverse).COL3A1: 5’-ATGCCCACAGCCTTCTACAC-3’ (forward); 5’-ACCAGTTGGACATGATTCACAG -3’ (reverse).Fibronectin: 5’-GATGTCCGAACAGCTATTTACCA-3’ (forward); 5’-CCTTGCGACTTCAGCCACT-3’ (reverse).IL-1β: 5’-TCGCTCAGGGTCACAAGAAA-3’ (forward), 5’-CATCAGAGGCAAGGAGGAAAAC-3’ (reverse);IL-6: 5’-ACAAGTCGGAGGCTTAATTACACAT-3’ (forward), 5’-TTGCCATTGCACAACTCTTTTC-3’ (reverse);iNOS: 5’-CCCGTCCACAGTATGTGAGGAT-3’ (forward), 5’-CATTACCTAGAGCCGCCAGTGA-3’ (reverse);CCL-20: 5’-AAGACAGATGGCCGATGAAG-3’ (forward), 5’-TCTTGACTCTTAGGCTGAGGA-3’ (reverse);TNF-α: 5’-TGAACTTCGGGGTGATCGGTC-3’ (forward), 5’-AGCCTTGTCCCTTGAAGAGGAC-3’ (reverse);GAPDH: 5’-AGTGGCAAAGTGGAGATT-3’ (forward); 5’-GTGGAGTCATACTGGAACA-3’ (reverse).Single-cell suspensions of skin samples were prepared using a previously reported method . Single-2 atmosphere at 37°C, overnight until 50% confluence. Cells were then incubated with vehicle (0.1% DMSO), corylin (20 μM), EX-527 (1 μM), Fulvestrant (0.1 μM), LY294002 (10 μM) and Miltefosine (10 μM) for 24 h. Cells were then collected for Western blot and PCR studies  for 24 h  at 37°C for 4 h. The supernatant was then removed and DMSO (0.15 mL) was added. The reaction mixture was maintained at 37°C for 10 min, and continuously scanned at a wavelength of 490 nm for 3 min. Percent cell proliferation was defined as the relative percentage (%) of treated cells relative to the vehicle-treated control group . At leasProteins were extracted from NIH/3T3 cells and Raw264.7 cells using RIPA lysis buffer and quantified by the BCA protein assay kit. Western blots were performed using the standard sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis method and enhanced chemiluminescence detection reagents  . Primary2 atmosphere at 37°C, overnight until 80% confluence. The medium was then replaced with serum-free DMEM containing mitomycin C  and NIH/3T3 cells were cultured for 90 min to prevent cell proliferation. Scratch lines were made using a sterile pipette tip. Cells were then incubated with vehicle (0.1% DMSO), corylin (20 μM), EX-527 (1 μM), Fulvestrant (0.1 μM), LY294002 (10 μM) and Miltefosine (10 μM) for 36 h. Scratches lines were photographed 0–36 h after scratching. At least three replicates were analyzed for each treatment.Wound scratch assays were performed in NIH/3T3 cells using a previously reported method . NIH/3T3post-hoc multiple comparison tests. p < 0.05 was considered statistically significant.Data are presented as means ± SEM. Analyses were performed with either GraphPad Prism 9.0.5. Three or more different groups were analyzed by one-way ANOVA with Dunnett’s First, we examined whether corylin could promote the repair of full-thickness skin wounds in mice. Round wounds, penetrating the full-skin thickness of mice, were generated by a sharp punching pipe tool, and then treated once daily, either by a topical application of vehicle or by corylin for 8 days. As shown in + leukocytes into wounded skin tissues (Next, we investigated the effects of corylin on inflammatory responses, an important factor that could delay wound healing , in full tissues . Further tissues . These dThe proliferation of fibroblasts is a critical process during wound healing . We thenMacrophages play an important role during wound healing . They taWe examined the protein expressions of p-IκBα in Raw264.7 cells after treatment with LPS and corylin for 24 h. As shown in in vitro mechanism studies, we further confirmed the signal pathways underlying the effects of corylin in vivo. PI3K inhibitor LY294002 and SIRT1 inhibitor EX527 were topically applicated to wound tissues in corylin-treated mice. Corylin-induced collagen deposition and α-SMA expression during wounds healing were significantly suppressed by LY294002 (Encouraged by the LY294002 . In addiLY294002 . AlthougLY294002 .We also examined whether PI3K and SIRT1 signaling participated in the anti-inflammation actions of corylin in mice. Inhibition of SIRT1 activity by EX527 reversed the protective effect of corylin against leukocyte infiltration in skin-wounded tissues, as demonstrated by H&E staining and flow cytometric analysis . SimilarChronic non-healing wound is a serious clinical problem and research into finding effective wound healing strategies is underway there is no ideal treatment due to the complexity of the healing process . In the + immune cells’ recruitment into wounded tissues (Healing of wounds is a dynamic and complex process that develops through 4 typical stages: hemostasis, inflammation, proliferation, and tissue remodeling with scar formation . Inflamm tissues . Moreove tissues . These a tissues . Macroph tissues . They al tissues . Our dat tissues .Moreover, we found that corylin could promote the production and deposition of collagen and expressions of α-SMA in wounded tissues . Both acPsoralea coryliforlia, and has been demonstrated to exhibit various biological properties through many signaling pathways, including SIRT1, NF-κB, PI3K/AKT and ERα signaling (Corylin is a flavonoid that is purified from ignaling . Chen etignaling . In addiignaling . SIRT1/Nignaling . Previouignaling . Consistignaling , 6A–C, iignaling . Our datignaling . In addiIt must be pointed out that one limitation of our study is that we have only used male mice in experiments. Further studies are necessary to fully characterize the potential effects of corylin in female animal. The other limitation is that we only studied the effects of corylin in cell line fibroblasts and macrophages. Future studies are necessary to purify primary fibroblasts and test the effects of corylin in these primary cells.In summary, our results revealed that corylin promoted wounds healing through the PI3K/Akt pathway and SIRT1/NF-κB pathway. Our results also identify corylin as a promising therapy for chronic wounds healing.


Diacylglycerol kinase (DGK) α, which is a key enzyme in the progression of cancer and, in contrast, in T-cell activity attenuation, preferentially produces saturated fatty acid (SFA)– and/or monounsaturated fatty acid (MUFA)–containing phosphatidic acids (PAs), such as 16:0/16:0-, 16:0/18:0-, and 16:1/16:1-PA, in melanoma cells. In the present study, we searched for the target proteins of 16:0/16:0-PA in melanoma cells and identified heat shock protein (HSP) 27, which acts as a molecular chaperone and contributes to cancer progression. HSP27 more strongly interacted with PA than other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate. Moreover, HSP27 is more preferentially bound to SFA- and/or MUFA-containing PAs, including 16:0/16:0- and 16:0/18:1-PAs, than PUFA-containing PAs, including 18:0/20:4- and 18:0/22:6-PA. Furthermore, HSP27 and constitutively active DGKα expressed in COS-7 cells colocalized in a DGK activity–dependent manner. Notably, 16:0/16:0-PA, but not phosphatidylcholine or 16:0/16:0-phosphatidylserine, induced oligomer dissociation of HSP27, which enhances its chaperone activity. Intriguingly, HSP27 protein was barely detectable in Jurkat T cells, while the protein band was intensely detected in AKI melanoma cells. Taken together, these results strongly suggest that SFA- and/or MUFA-containing PAs produced by DGKα selectively target HSP27 and regulate its cancer-progressive function in melanoma cells but not in T cells. Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to convert to phosphatidic acid (PA) , 2, 3, 42+-binding EF-hand motifs and is activated by Ca2+ - and/or monounsaturated fatty acid (MUFA)-containing PAs, such as 16:0/16:0-, 16:0/18:0-, and 16:0/16:1-PA in serum-starved AKI melanoma cells . MoreoveIn the present study, to explore how DGKα plays reverse roles in cancer cells and T lymphocytes, we searched for the target proteins of 16:0/16:0-PA in human melanoma cells. We identified heat shock protein (HSP) 27, which acts as a molecular chaperone and is a biomarker of cancer , as a noA). By in-gel digestion and liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, the proteins of this band were identified, and we focused on HSP27 , which was reproducibly detected in the precipitates of 16:0/16:0-PA-liposomes as a candidate 16:0/16:0-PA-binding protein from the ∼28 kDa band .We previously reported that DGKα preferentially generated SFA/MUFA-PA species, such as 16:0/16:0-, 16:0/18:0-, and 16:0/16:1-PA, in AKI human melanoma cells . In the Fig. 1HSP27 is a molecular chaperone and is known to be a biomarker of cancer, renal injury and fibrosis, and neurodegenerative and cardiovascular disease . In partAB020027) from the mRNAs of AKI cells and ligated it with the pET-28a vector. 6× His-tagged HSP27 protein was produced in Escherichia coli cells and purified by affinity chromatography using nickel-nitrilotriacetic acid agarose. We confirmed that 6× His-HSP27 was detected as a single band with a molecular mass of approximately 30 kDa, which was recognized by an anti-6× His antibody and was thus successfully purified (C).Next, we cloned human HSP27 cDNA (accession number: purified C.A and B). However, approximately 90% of HSP27 was sedimented with 16:0/16:0-PA-containing liposomes (A and B), indicating that HSP27 more intensely binds to 16:0/16:0-PA liposomes than PC liposomes (background control) and 16:0/16:0-PS liposomes (acidic phospholipid control). Moreover, we analyzed 16:0/16:0-PC-binding activity of HSP27 and confirmed that it was almost the same with 16:0/16:0-PS (C and D). Endogenous HSP27 expressed in AKI melanoma cells was mainly recovered in 200,000g sup  (E and F). The endogenous HSP27 recovered in soluble fractions also showed stronger interaction activity with 16:0/16:0-PA liposomes than 16:0/16:0-PS and PC liposomes (G and H).We next verified the interaction activity of 6 ×His-HSP27 with 16:0/16:0-PA using a liposome precipitation assay as described previously . Only apiposomes , A and B/16:0-PS , C and Ds, ∼80%) , E and Fiposomes , G and HA). However, HSP27 did not show marked binding activity to liposomes at 25 μM PC (A). The dissociation constant (Kd) of HSP27 for 16:0/16:0-PA was determined to be 13.3 μM (B). The extrapolated Kd for 16:0/16:0-PS was calculated to be approximately 100 μM (B). These results indicate that HSP27 has a markedly higher affinity for PA than PS and PC.We also determined the affinity of HSP27 for 16:0/16:0-PA by measuring binding activity at 0 to 25 μM PA. Liposome-binding HSP27 was augmented in a 16:0/16:0-PA and 16:0/16:0-PS concentration-dependent manner A. Howeve25 μM PC A. The di 13.3 μM B. The exy 100 μM B. These A and B) and 1000 nm (C and D)) of liposomes containing 16:0/16:0-PA), and the binding activity was stronger than those of 16:0/16:0-PS- and PC-containing liposomes (A–D). No substantial differences between 100 nm and 1000 nm liposomes were observed (A–D). Therefore, it is likely that different membrane curvatures and shapes formed by various liposome diameters fail to substantially affect the interaction of HSP27 with PA.The PA-binding activity of clathrin coat assembly protein AP180 was affected by liposome diameters . Therefo 1000 nm , C and Diposomes , A–D. Noobserved , A–D. ThA). However, other acidic lipids, such as phosphatidylinositol (PI), PS, phosphatidylglycerol, and 3-sulfogalactosylceramide, and neutral lipids, such as triglyceride, DG, PC, phosphatidylethanolamine, cholesterol (Chol), and sphingomyelin, failed to interact with HSP27 (A). PI-4-monophosphate (PI(4)P), PI-4,5-bisphosphate P2), and cardiolipin (CL), which are acidic lipids, are also associated with HSP27 (A). However, their binding intensities were lower than that of PA (B). Therefore, these results indicate that HSP27 selectively and most intensely binds to PA.To measure the lipid binding selectivity of HSP27 in more detail, we carried out a lipid overlay assay using a nitrocellulose membrane spotted with diverse lipids that included 16:0 as fatty acid chains. HSP27 exhibited an intense interaction with 16:0/16:0-PA A. Howeveth HSP27 A. PI-4-mth HSP27 A. Howeveat of PA B. TherefA). The binding activities of these PA species were almost the same as that of 16:0/16:0-PA (B). However, PUFA-containing PAs, 18:0/20:4- and 18:0/22:6-PA, showed substantially lower binding activities to HSP27 (B). These results indicate that HSP27 preferably binds to SFA- and/or MUFA-containing PAs.To assess the PA molecular species selectivity of HSP27, we performed a liposome precipitation assay using various PA species, including 16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/18:1-, 18:0/18:0-, 18:0/20:4- and 18:0/22:6-PA. SFA- and/or MUFA-containing PAs, 16:0/18:1-, 18:1/18:1-, 18:0/18:1- and 18:0/18:0-PA, intensely interacted with HSP27 A. The bi/16:0-PA B. Howeveto HSP27 B. These A), and expressed in E. coli and highly purified them (B). HSP27-NT interacted moderately more strongly with 16:0/16:0-PA than HSP27-CT (C and D), indicating that while the N-terminal region more strongly contributes to the 16:0/16:0-PA binding, both the N- and C-terminal regions of HSP27 are important for 16:0/16:0-PA binding.To determine the 16:0/16:0-PA-binding region in HSP27, we divided HSP27 into two parts, the N-terminal region  and the C-terminal region  of HSP27 including an α-crystallin domain A, and exied them B. HSP27-HSP27-CT , C and Dhttps://www.biogem.org/tool/chou-fasman/) and found a sole Arg-containing α-helix (aa 26–32) in HSP27-NT (aa 1–80). Therefore, Arg27 was replaced with Glu (HSP27-R27E) (A), expressed in E. coli, and highly purified (B). However, the 16:0/16:0-PA-binding activity of HSP27-R27E was not considerably reduced (G and H). These results indicate that Arg27 is not a critical residue for the 16:0/16:0-PA binding of HSP27.We next attempted to determine residues in HSP27 critical for 16:0/16:0-PA binding. Some reports have indicated that α-synuclein has α-helices containing several basic residues, which bind to acidic phospholipids , 41, 42.27-R27E) A, exprespurified B. Howeve reduced , G and HA–C). Moreover, mCherry-HSP27 coexpressed with EGFP-DGKα-CA showed a higher plasma membrane/cytosol ratio of HSP27 at the membrane region where DGKα-CA is colocalized with HSP27 (∼3.1) than mCherry-HSP27 coexpressed with EGFP alone (∼1.6) (A–C). Notably, the plasma membrane/cytosol ratio (∼2.4) of HSP27 was significantly attenuated by CU-3 (10 μM), a DGKα-selective inhibitor (A and C), although DGKα-CA was located at the plasma membrane even in the presence of CU-3 (A and B). These results indicate that the colocalization of EGFP-DGKα-CA with HSP27 was markedly reduced by CU-3. In addition, although EGFP-DGKα-CA-KD, a kinase-dead inactive mutant of DGKα-Δ1–196 in which Gly-435 is substituted with Asp (D and E), plasma membrane/cytosol ratio (∼3.0) of mCherry-HSP27 coexpressed with the inactive mutant was lower than that (∼4.0) with EGFP-DGKα-CA (D and F). These results indicate that the inactive mutant was less strongly colocalized with mCherry-HSP27 than EGFP-DGKα-CA. Therefore, these results indicate that the colocalization between HSP27 and DGKα occurs in a DGK activity (PA production)-dependent manner, suggesting that HSP27 can interact with PA produced by DGKα in cells.We next investigated whether HSP27 can associate with PA in cells. When EGFP-DGKα-CA (a constitutively active mutant lacking EF-hand motifs (Δ1–196)), which produces SFA- and/or MUFA-containing PAs  , was expe (∼1.6) , A–C. Nonhibitor , Fig. 8A–C. MoreA). In the presence of PC and 16:0/16:0-PS, HSP27 was electrophoresed to the same position as that in the absence of phospholipids (It was reported that HSP27 forms a large oligomer (12-mer–35-mer)  and thatFig. 9A and B), although the protein band was strongly detected in AKI melanoma cells (A and B), suggesting that SFA/MUFA-PAs function via HSP27 only in melanoma cells but not in T cells.To analyze whether HSP27 is substantially expressed in T cells, where DGKα plays roles different from cancer cells , 34, we ma cells , A and BIn the present study, we demonstrated for the first time that a chaperone, HSP27, is a novel PABP that prefers SFA- and/or MUFA-containing PA species. Several PABPs have been reported to recognize different PA species , 14, 15.2 with lower affinity than PA    comparedolipids) . TherefoEssentially the same results  were obtained by the liposome sedimentation  and 4 anG and H). However, the sedimentation activity is almost the same as PC-binding activity (G and H). Moreover, the PS-binding activity was not detected in the lipid overlay assay  also affect HSP27 function. Indeed, lysoPA acyltransferase isoform expression has been shown to enhance the proliferation of cancer cells and correlates with an increased risk of tumor development and aggressiveness of tumors . MoreoveAlthough HSP27 exists as a large oligomer (12-mer–35-mer) , Ser/Thrvia direct binding to HSP27 and activation of HSP27 phosphorylation pathways.PA binding may have effects similar to Ser/Thr phosphorylation because both PA binding and phosphorylation introduce negative charge(s) of a phosphate group to the protein. It was reported that HSP27 is phosphorylated by mitogen-activated protein kinase (MAPK)-activated protein kinase (MAPKAPK) 2 and 3, MAPKAPK5, PKC, cGMP-dependent kinase, Akt/protein kinase B, and protein kinase D . The acthttps://www.proteinatlas.org/ENSG00000106211-HSPB1/tissue). Therefore, this expression pattern may explain at least in part how DGKα plays reverse roles in T cells (attenuator) and cancer cells (enhancer) (Overexpressed DGKα recruited HSP27 to the plasma membrane in COS7 cells in a DGK activity (PA)-dependent manner , suggestnhancer) . HoweverRecently, it has been revealed that DGK isozymes utilize different DG species and thus produce distinct PA species in different cells , 70. DGKvia activation of the Ras–Raf–MAPK/ERK kinase–ERK pathway , 1,2-dipalmitoyl-sn-glycero-3-phosphate (16:0/16:0-PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0/18:0-PA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (16:0/18:1-PA), 1-stealoyl-2-oleoyl-sn-glycero-3-phosphate (18:0/18:1-PA), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1/18:1-PA), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate (18:0/20:4-PA), and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphate (18:0/22:6-PA) were purchased from Avanti Polar Lipids . Cholesterol (Chol) was purchased from Wako Pure Chemical Industries . Membrane Lipid Strips were purchased from Echelon Biosciences .2.AKI cells (a human melanoma-derived cell line) and COS-7 cells (fibroblast-like cell lines derived from monkey kidney tissue) were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 μg/ml streptomycin . The cells were maintained at 37 °C in an environment containing 5% COThe following lipid mixtures were used to identify 16:0/16:0-PA-specific binding proteins from AKI melanoma cells: 16:0/16:0-PS liposomes [Chol (30 mol%) and PC Mix (from egg yolk) (60 mol%) and 16:0/16:0-PS (10 mol%)] and 16:0/16:0-PA liposomes [Chol (30 mol%) and PC Mix (from egg yolk) (60 mol%) and 16:0/16:0-PA (10 mol%)]. The combined dried lipid mixture was hydrated at 95 °C in HEPES buffer  for 45 min and vortexed for 1 min once every 15 min during hydration. The liposomes were then subjected to five freeze–thaw cycles  . LiposomThe following lipid mixtures were used to determine the properties of the HSP27 protein: the control liposome [Chol (30 mol%) and PC Mix (from egg yolk) (70 mol%)], PS liposome , and the PA liposome . For the lipid-binding assay, the combined dried lipid mixture was hydrated at 95 °C in HEPES buffer for 45 min and vortexed for 1 min once every 15 min during hydration. The liposomes were then subjected to five freeze–thaw cycles . Liposomes were formed by sonication at 90 °C using a Branson Sonifier 450, or the liposomes were further extruded 11 times through a 100 nm or 1000 nm polycarbonate membrane using a Mini Extruder (Avanti Polar Lipids) . The extg for 30 min at 4 °C). AKI cell lysates were incubated with the PC liposomes at 4 °C for 30 min, and nonspecific protein bound to the vesicles were removed by centrifugation at 200,000g for 1 h at 4 °C. The resultant supernatant was incubated with the 16:0/16:0-PS or 16:0/16:0-PA liposomes at 4 °C for 30 min and then centrifuged at 200,000g at 4 °C for 1 h. The precipitates were dissolved in HEPES buffer containing 25 mM HEPES, pH 7.4, 100 mM NaCl, and 1 mM dithiothreitol. The 16:0/16:0-PA-binding proteins were separated by SDS-PAGE and visualized by silver staining , 100 mM NaCl, and 1 mM dithiothreitol. After sonication, insoluble materials were removed by ultracentrifugation , and total RNA was isolated as previously described  and collected by centrifugation (500escribed . cDNA waAB020027) was amplified using the primers 5′-GGTGGTGGATCCATGACCGAGCGCCGC-3′ (forward) and 5′-GGTGGTCTCGAGTTACTTGGCGGCAGTCTC-3′ (reverse) from prepared cDNA (human AKI cells) by PCR, ligated with pET-28a vector , which carries an N-terminal 6 ×His tag, and transfected into Rosetta 2 (DE3) E. coli cells (Novagen). The expression and purification of the 6× His-tagged HSP27 protein using nickel-nitrilotriacetic acid agarose  were performed as previously described . The membranes were blocked with 2% skim milk in phosphate-buffered saline (pH 7.4) for 1 h at 4 °C. After blocking, 10 ml of 3% fatty acid–free bovine serum albumin and 0.1% Tween 20 in phosphate-buffered saline (pH 7.4) containing 6× His-tagged HSP27  was added to the membranes. The membrane was incubated for 20 min at 4 °C and was then incubated with an anti-6× His antibody for 1 h at 4 °C, followed by incubation with anti-mouse IgG conjugated with horseradish peroxidase  antibody. Finally, lipid-bound 6× His-HSP27 was visualized using an enhanced chemiluminescence Western blotting detection system .g at 4 °C for 1 h. The precipitate was dissolved in HEPES buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, and 1 mM dithiothreitol. SDS-PAGE (15% acrylamide) was conducted, and separated proteins were stained with Coomassie Brilliant Blue or detected by Western blotting using an anti-6× His antibody.The purified 6× His-tagged HSP27 protein  was dissolved in HEPES buffer and incubated with the PA-containing or control liposomes at 4 °C for 30 min. Samples were ultracentrifuged at 200,000g for 30 min at 4 °C). The cell lysates were incubated with the PA-containing or control liposomes at 4 °C for 30 min. Samples were ultracentrifuged at 200,000g for 1 h at 4 °C. The precipitate was dissolved in HEPES buffer. SDS-PAGE (15% acrylamide) was conducted, and separated proteins were detected by Western blotting using an anti-HSP27 antibody.To measure the binding activity between PA-containing liposomes and endogenous HSP27, AKI cells were washed two times with phosphate-buffered saline and lysed in HEPES buffer containing 25 mM HEPES (pH 7.4), 100 mM NaCl, and 1 mM dithiothreitol by sonication. After sonication, insoluble materials were removed by ultracentrifugation  , and pEGCOS-7 cells seeded on coverslips were transiently transfected with plasmids using PolyFect reagent (Qiagen) as described by the manufacturer. After 20 h of transfection, the cells were incubated with the DGKα selective inhibitor CU-3   was dissolved in HEPES buffer and incubated with the PA-containing or control liposomes at 4 °C for 30 min. BN-PAGE (4% stacking gel and 6% separation gel) was carried out as previously described . The gelhttps://imagej.nih.gov/ij/index.html) as described Band intensities were quantified by densitometry using ImageJ software  to determine any significant differences. p < 0.05 was considered significant.Data are represented as the means ± SD and were analyzed using one-way analysis of variance followed by Tukey's or Dunnett's post hoc test for multiple comparisons or two-tailed sakane@faculty.chiba-u.jp] upon reasonable request.The data that support the findings of this study are available from the corresponding author [The authors declare that they have no conflicts of interest with the contents of this article.


Epithelial barrier is important for asthma development by shaping immune responses. Airway expressing-IL-1 receptor-associated kinase (IRAK)-M of Toll-like receptor pathway was involved in immunoregulation of airway inflammation through influencing activities of macrophages and dendritic cells or T cell differentiation. Whether IRAK-M has effect on cellular immunity in airway epithelial cells upon stimulation remains unclear.We modeled cellular inflammation induced by IL-1β, TNF-α, IL-33, and house dust mite (HDM) in BEAS-2B and A549 cells. Cytokine production and pathway activation were used to reflect the effects of IRAK-M siRNA knockdown on epithelial immunity. Genotyping an asthma-susceptible IRAK-M SNP rs1624395 and measurement of serum CXCL10 levels were performed in asthma patients.IRAK-M expression was significantly induced in BEAS-2B and A549 cells after inflammatory stimulation. IRAK-M knockdown increased the lung epithelial production of cytokines and chemokines, including IL-6, IL-8, CXCL10, and CXCL11, at both mRNA and protein levels. Upon stimulation, IRAK-M silencing led to overactivation of JNK and p38 MAPK in lung epithelial cells. While antagonizing JNK or p38 MAPK inhibited increased secretion of CXCL10 in IRAK-M silenced-lung epithelium. Asthma patients carrying G/G genotypes had significantly higher levels of serum CXCL10 than those carrying homozygote A/A.Our findings suggested that IRAK-M has effect on lung epithelial inflammation with an influence on epithelial secretion of CXCL10 partly mediated through JNK and p38 MAPK pathways. IRAK-M modulation might indicate a new insight into asthma pathogenesis from disease origin.The online version contains supplementary material available at 10.1186/s12931-023-02406-5. Our understanding of asthma immunopathogenesis has been advanced over the last decade . Much evGenetic studies have linked IRAK-M SNP variants to both early- and late-onset asthma, suggesting that airway-expressing IRAK-M may be involved in inappropriate activation of innate immunity in the bronchial cells , 7. AberGiven the multiple lines of evidence indicating that IRAK-M is involved in modulation of innate immunity, whether IRAK-M regulates airway epithelial inflammation and downstream signaling pathways has not been well understood. Here we performed in vitro investigations to test this possibility by examining the effect of IRAK-M knockdown on cellular immunity in immortalized lung epithelial cell lines under inflammatory stimulations. We found that IRAK-M showed effects on inflammatory events of lung epithelial cells, with an impact on Th1 cytokine CXCL10 production possibly through JNK and p38 MAPK pathways.2.Human airway epithelial cell line BEAS-2B cells and human alveolar epithelial cell line A549 cells were purchased from Shanghai Institute of Life Sciences, Chinese Academy of Sciences . BEAS-2B cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) media  and A549 cells were cultured in F-12K media . All media were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (HyClone). All cells were cultured at 37 °C in a humidified incubator of 5% COIRAK-M siRNA  were obtained from GenePharma . Nontargeting pool-negative siRNA  were used as controls. siRNAs transfections of BEAS-2B cells and A549 cells were performed using GP-transfect-Mate transfection reagents. siRNAs and transfection reagents were mixed following manufacturer’s protocol and incubated with the cells for 48 h. Cells were harvested with TRIzol reagent  for quantitative real-time polymerase chain reaction (qRT-PCR) or harvested with protein lysis buffer , 3 ml Tris–HCl , 5 ml 100% glycerol, 32 ml H2O) freshly added 1 mM phosphatase and protease inhibitor  for western blot.IL-1β , or TNF-α , or IL-33 , or HDM  was separately added to BEAS-2B or A549 cells. Cells were harvested at 0, 6, 12, 24 h for Western blot. BEAS-2B and A549 cells were starved in serum-free media for 24 h after IRAK-M siRNA transfection. The cell-free supernatant and cells were collected at 24 h after stimulation with IL-1β, or TNF-α, or IL-33, or HDM for analysis of cytokines. To reflect the effect of IRAK-M on the JNK and p38 MAPK signaling pathways in airway epithelial cells, BEAS-2B and A549 cells were harvested at 0, 10, 30, 60 min for Western blot after IRAK-M siRNA transfection. Cells were starved and stimulated. BEAS-2B and A549 cells were incubated with JNK inhibitor SP600125  or p38 MAPK inhibitor SB203580  for 2 h. After 24 h of stimulation, cell-free supernatant and cells were harvested for cytokines analysis.Whole-cell protein extracts were separated on 10% SDS–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose filter membranes. The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 1 h. Then the membrane was cut and incubated with the respective primary Abs at 4 °C overnight. The Abs were as follows: anti-mouse IRAK-M, anti-mouse JNK, anti-mouse Phospho-JNK, anti-mouse p38 MAPK, anti-mouse Phospho-p38 MAPK and anti-mouse GAPDH . Primary Ab application was followed by incubation with HRP-conjugated secondary Abs (rabbit anti-mouse IgG)  at room temperature for 1 h. After washing for three times with TBS-T, membranes were scanned using Odyssey CLx imaging system . Semi-quantitation was analyzed with Image J software . Protein expression levels were normalized to GAPDH.Commercial ELISA kits were used to measure the concentrations of the cytokines, including IL-6 (R&D system), IL-8 (R&D system), CXCL10 , CXCL11 (R&D system), and IFN-γ (R&D system), in supernatants collected from cells.qRT-PCR was used to reflect mRNA expression of IRAK-M and cytokine and chemokines, including IL-6, IL-8, CXCL10, CXCL11 and IFN-γ. Total RNA prepared from the BEAS-2B or A549cells using TRIzol reagent was reverse transcribed into cDNA using the ALL-IN-ONE RT MasterMix . qRT-PCR was performed on the CFX Connect Real-Time PCR Detection System  in a 20 μl of final reaction volume containing 1 μl of cDNA and qPCR MasterMix . The relative quantities of mRNAs were calculated with the comparative cycle threshold method and normalized using human GAPDH as an internal control. The primer sequences are shown in Additional file A total of 137 individual patients with asthma was recruited from Asthma Clinic of Beijing Aviation General Hospital between September 2021 and March 2022. Asthma was diagnosed by the respiratory specialists. All patients were fulfilled the diagnostic criteria of the Global Strategy for Asthma Management and Prevention . The stuGenomic DNA was extracted from peripheral blood leukocytes by using TIANamp Genomic DNA Kit  according to manufacture protocol. Two SNPs of IRAK-M  were genotyped by SNaPshot. The PCR products were sequenced on an ABI 3730xl DNA Analyzer (Applied Biosystems) and analyzed using the GeneMapper 4 software. The genotyping efficiency for IRAK-M SNP was > 95% and minor allele frequency was > 5%.Fasting blood samples were harvested from patients with asthma in the morning and centrifuged at 12,000 rpm. Serum was stored at − 80 °C until measurement. Serum CXCL10 concentrations were measured using a commercial ELISA kit (R&D system) following the manufacturer’s instructions.t-test . P-value < 0.05 was considered significant.Data were expressed as mean ± SEM. Multiple comparisons were performed by one-way ANOVA with Tukey post-hoc test. Comparisons between two groups were made by unpaired Student To investigate IRAK-M expression by lung epithelial cells in response to the exogenous stimuli, we performed a time course study to examine the dynamic change of IRAK-M protein in BEAS-2B and A549 cells after stimulation with IL-1β, or TNF-α, or IL-33, or HDM. IRAK-M protein was significantly induced by stimulation in BEAS-2B cells at indicated time points, except for IL-33 stimulation  and SB203580  for 2 h and then exposed to IL-1β for 24 h. The JNK inhibitor significantly reduced the activation of JNK pathway , the mainstay for asthma therapy, target the established airway inflammation but fail to modulate the underlying mechanisms that drive asthma initiation. Our current observations might suggest a previously unrecognized important role for IRAK-M in regulating cellular inflammation in lung epithelium and indicate that appropriate manipulation of airway-expressing IRAK-M could be a novel target for asthma treatment from disease origin.Additional file 1: Table S1. qRT-PCR primers used in this study.Additional file 2: Figure S1. IRAK-M expression is inducible by multiple stimuli in A549 cells. IRAK-M protein expression at 0, 6, 12, 24 h after (a) IL-1β (1 ng/ml), (b) TNF-α (10 ng/ml), (c) IL-33 (100 ng/ml) and (d) HDM (10 μg/ml) exposure. IRAK-M protein expression was normalized to GAPDH. Values are expressed as mean ± SEM (n = 3). *P < 0.05.Additional file 3: Figure S2. IRAK-M expression was attenuated by IRAK-M siRNA in BEAS-2B and A549 cells. (a) Expression of IRAK-M at both mRNA level and protein level in BEAS-2B cells after IRAK-M knockdown by siRNAs. (b) Expression of IRAK-M at both mRNA level and protein level in A549 cells after IRAK-M knockdown by siRNAs. Both mRNA and protein expression of IRAK-M were normalized to GAPDH. Values are expressed as mean ± SEM (n = 3). *P < 0.05.Additional file 4: Figure S3. Impact of IRAK-M knockdown on cytokines production in A549 cells. Expression of IL-6, IL-8, CXCL10, CXCL11 and IFN-γ at both mRNA level and protein level after (a) IL-1β (1 ng/ml), (b) TNF-α (10 ng/ml), (c) IL-33 (100 ng/ml) and (d) HDM (10 μg/ml) stimulation for 24 h. mRNA expression of IL-6, IL-8, CXCL10, CXCL11 and IFN-γ were normalized to GAPDH. Values are expressed as mean ± SEM (n = 3). *P < 0.05. CON, si-control. KD, si-IRAK-M.Additional file 5: Figure S4. Effect of IRAK-M on activation of JNK and p38 MAPK pathways in A549 cells. Protein expression of p-JNK, JNK, p-p38, p38 after (a) IL-1β (1 ng/ml), (b) TNF-α (10 ng/ml), (c) IL-33 (100 ng/ml) and (d) HDM (10 μg/ml) stimulation for 10, 30 and 60 min. P-JNK was normalized to total JNK, while p-p38 was normalized to total p38. Values are expressed as mean ± SEM (n = 3). *P < 0.05.Additional file 6: Figure S5. The activation of JNK and p38 MAPK pathway was blocked by inhibitor in A549 cells. (a) Protein expression of p-JNK and JNK after SP600125  pretreated for 2 h and IL-1β stimulated for 24 h. (b)Protein expression of p-p38 and p38 after SB203580  pretreated for 2 h and IL-1β stimulated for 24 h. P-JNK was normalized to total JNK, while p-p38 was normalized to total p38. Values are expressed as mean ± SEM (n = 3). *P < 0.05.Additional file 7: Figure S6. Treatment with JNK or p38 MAPK inhibitor attenuated IRAK-M knockdown-mediated CXCL10 secretion upon stimulation in A549 cells. Expression of CXCL10 at both mRNA and protein level after SP600125  and SB203580  incubation for 2 h and (a) IL-1β (1 ng/ml), (b) TNF-α (10 ng/ml), (c) IL-33 (100 ng/ml) and (d) HDM (10 μg/ml) stimulation for 24 h. Values are expressed as mean ± SEM (n = 3). *P < 0.05. CON, si-control. KD, si-IRAK-M.


To investigate the impact of treatment time and patterns in inoperable stage III non-small cell lung cancer (NSCLC) following concurrent chemoradiotherapy (cCRT) ± immune checkpoint inhibitors (ICIs).Patients were stratified by treatment year: A (2011–2014), B (2015–2017) and C (2018–2020). Tumor- and treatment-related characteristics regarding locoregional recurrence-free survival (LRRFS), progression-free survival (PFS) and overall survival (OS) were investigated.p = 0.007). Median OS was 19.9 months, 23.4 months and not reached (NR), respectively. In group C, median LRRFS and PFS were 27.2 vs. NR; and 14.2 vs. 26.3 months in patients treated with and without ICI. On multivariate analysis planning target volume (PTV) ≥ 700 cc was a negative prognosticator of LRRFS , PFS  and OS ; ICI was a predictor of LRRFS , PFS  and OS . In the non-ICI cohort, multivariate analyses revealed PTV ≥ 700 cc (p = 0.047) and a maximum standardized uptake value (SUVmax) ≥ 13.75 (p = 0.012) were predictors of PFS; PTV ≥ 700 cc (p = 0.017), SUVmax ≥ 13.75 (p = 0.002) and a total lung V20 ≥ 30% (V20 ≥ 30) (p < 0.05) were predictors of OS.One hundred and thirty-six consecutive patients were analyzed. All patients completed thoracic radiotherapy (TRT) to a total dose ≥ 60.0 Gy; 36 (26%) patients received ICI. Median PFS in subgroups A, B and C was 8.0, 8.2 and 26.3 months (Patients treated after 2018 had improved survival regardless of ICI use. Implementation of ICI resulted in further significant increase of all tested survival endpoints. PTV ≥ 700 cc and ICI were only prognosticators for LRRFS, PFS and OS in the analyzed cohort. Lung cancer is the most common cause of cancer-related deaths worldwide  in multimodal treatment strategies led to significant improvement in patient outcomes. The PD-L1 inhibitor durvalumab led to a paradigm shift following the unprecedented results of the PACIFIC trial with a median progression-free survival (PFS) of 16.9 months and a median OS of 47.5 months  from 189 consecutive patients treated between February 2011 and November 2020 were enrolled in the study. Eligible patients were diagnosed with inoperable stage IIIA-C (UICC 8th edition) NSCLC and received cCRT ± ICI as part of a multimodal approach. ICI consisted of either consolidation administration of durvalumab according to the PACIFIC trial or concurrent and sequential administration of nivolumab as part of the ETOP 6–14 NICOLAS phase II study  ≥ 13.75 , total lung V20 ≥ 30% (V20 ≥ 30) and smoking history ≥ 20 pack years in regard to locoregional recurrence-free survival (LRRFS), PFS and overall survival (OS). All survival parameters were calculated from the last day of TRT.All patients received cCRT consisting of two cycles of platinum-based chemotherapy (cisplatin/carboplatin combined with vinorelbine/pemetrexed) and conventionally fractionated thoracic radiotherapy (TRT) to a total dose of ≥ 60 Gy. Analysis of all parameters was performed for the entire cohort and additionally for patients that had not received ICI (non-ICI cohort). Patients were stratified by year of initial TRT and divided into three subgroups: subgroup A (2011–2014), subgroup B (2015–2017) and subgroup C (2018–2020). We analyzed treatment- and patient-related characteristics: age ≥ 65, application of VMAT, use of ICI, tumor histology, planning target volume (PTV) ≥ 700 cc, maximum standardized uptake value (SUVIn 133 (97.8%) patients, baseline staging was performed using positron emission tomography (PET)-computed tomography (CT). Cranial contrast-enhanced MRI was performed in 88 (64.7%) patients before starting multimodal treatment, all other patients received cranial contrast-enhanced CT. Pulmonary function testing and routine blood work were performed in all patients. All patients were discussed at the multidisciplinary tumor board and deemed eligible for cCRT. Eligible patients were required to have an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0–1 and adequate lung function: diffusing capacity of lung for carbon monoxide corrected for hemoglobin (DLCOc) ≥ 40%, forced expiratory volume in 1 s (FEV1) ≥ 1 L. Treatment plans for TRT were based on conventional planning-CT and PET-CT. These scans were acquired with patients in the corresponding treatment position. Patients were treated in the supine position with their arms positioned overhead in a dedicated positioning and immobilization device-WingSTEP™ . Target volumes were defined according to an in-house protocol in close accordance with the later published guidelines of the European Society for Radiotherapy and Oncology-Advisory Committee on Radiation Oncology Practice (ESTRO-ACROP)  for lung treatments, TRT consisted of 50 Gy in 2 Gy single-dose fractions, followed by a sequential 16 Gy boost. After the full implementation of IMRT, TRT-regimens consisted of 30 fractions with simultaneous integrated boost (SIB) of 2.0/2.12 Gy to the gross tumor volume (GTV) lymph node (LN)/GTV primary tumor (PT) to a total dose of 60.0/63.6 Gy. TRT was delivered on a linear accelerator (LINAC) with megavoltage capability, using either VMAT, 3D-CRT or step-and-shoot IMRT. Image guidance was performed at least twice a week on a cone-beam CT.ICI maintenance treatment was administered for a duration of up to 12 months and/or until disease progression or evidence of unacceptable toxicity. Durvalumab was given at a dose of 10 mg/kg of body weight every two weeks; nivolumab was administered concurrently at a dose of 360 mg, every third week and after CRT at a dose of 480 mg, monthly up to 1 year.Diagnostic measures like routine blood work, lung function testing, clinical examination and imaging with CT or PET-CT scans were performed every 3 months in the first 2 follow-up years after treatment, every 6 months in the following 2 years and annually thereafter. Additional imaging, e.g. contrast-enhanced magnetic resonance imaging (MRI) and bone-scintigraphy, was performed if deemed necessary.p < 0.05) in univariate analysis. All analyses including univariate and multivariate analysis were performed using SPSS version 26 .Tumor progression was assessed according to RECIST 1.1. CT, PET-CT or MRI scans were used to document local relapse (LR), while pathological confirmation was not obligatory. LR was defined as recurrence in the ipsilateral lung or mediastinum. Median follow-up was defined as the time from the end of TRT to loss or end of follow-up in patients that had not died up to that point. LRRFS was defined as the time from the end of TRT until either LR or death. Similarly, PFS was defined as the time from the end of TRT to locoregional/systemic progression or death, while OS was defined as the time to death from any cause or last follow-up. Volumetric parameters were derived from the radiation treatment plans. The impact of each parameter on LRRFS, PFS and OS was analyzed by means of Kaplan–Meier analysis using the log-rank test. Multivariable Cox-regression analysis was performed with all parameters, which had shown to be significant  NSCLC were analyzed. A summary of patient- and tumor characteristics is shown in Table max was available for 118 (87%) patients and was ≥ 13.75 in 59 (43%) cases. All patients finished cCRT, with 60 (44%) receiving induction chemotherapy. Thirty-six (26%) patients received either durvalumab or nivolumab. In the entire cohort, median follow-up was 35.7 (range 0.9–111.9) months, median LRRFS, 12- and 18-month LRRFS rates were 23.2 (95% CI 14.9–31.4), 58% and 45%, respectively. Median PFS, 12- and 18-month PFS rates were 10.1 months (95% CI 7.4–12.8), 38% and 27%, respectively. Median OS, 12- and 18-month OS rates were 27.4 months (95% CI 16.0–38.7), 70% and 54%, respectively (Table Subgroup A consisted of 37 27.2%) patients, who received TRT between 2011 and 2014, subgroup B consisted of 49 (36.0%) patients, that were treated between 2015 and 2017 and subgroup C consisted of 50 (36.8%) patients with treatment between 2018 and 2020. The median age was 66.9 (range 33.6–82.5) years with 78 (57%) patients older than 65 years. Forty-three (32%) were female and 93 (68%) were male. Fifty-six (41%) patients had squamous cell carcinoma (SCC), 69 (51%) had adenocarcinoma (AC) and in 11 (8%) patients the tumor was classified as not otherwise specified (NOS); 92 (68%) patients were current or former smokers with more than 20 pack-years, 21 (15%) had less than 20 pack-years and for 23 (17%) patients the smoking status could not be discerned. All patients completed conventionally fractionated radiotherapy to a total dose ≥ 60.0 Gy. Of these, 82 (60%) were treated with VMAT, while 35 (26%) received 3D-CRT and 19 (14%) patients were treated with step-and-shoot IMRT. The median PTV was 700.5 cc (range 172.5–2293.2). Sixty-eight (50%) patients had a PTV ≥ 700 cc. SUV7.2% patiFor detailed results of the uni- and multivariate analyses, refer to Tables p = 0.007), PFS (p = 0.007) and OS (p = 0.001). Median LRRFS was 14.1 (95% CI 7.7–20.5) months in subgroup A, 16.9 (range 7.9–25.9) months in subgroup B and not reached in subgroup C. Median PFS was 8.0 (95% CI 6.6–9.4), 8.2 (95% CI 2.8–13.6) and 26.3 (95% CI 7.5–45.1) months for groups A, B and C, respectively. Median OS for subgroups A and B was 19.9 (95% CI 10.7–29.1) and 23.4 (95% CI 15.1–31.6) months, whereas it was not reached for C.In the univariate analysis for the entire cohort, stratification into treatment groups A, B and C was significantly associated with established endpoints. Namely, group C had significantly longer LRRFS (p = 0.004). Median PFS was 8.1 (95% CI 5.9–10.2) months vs. 22.8 (95% CI 7.8–37.7), respectively (p = 0.002). Median OS was 21.1 (95% CI 15.4–26.7) months vs. not reached (p = 0.001).In patients that received no ICI median LRRFS was 17.0 (95% CI 9.4–24.5) months vs. not reached for patients that received ICI (p = 0.151). Median PFS was 9.8 (95% CI 7.1–12.5) vs. 11.8 (95% CI 5.4–18.2) months for patients over and under 65 years (p = 0.609). However, median OS was 23.4 (95% CI 16.3–30.5) vs. 73.4 (95% CI 0.3–146.5) months (p = 0.016) for patients ≥ 65 and < 65, respectively.Median LRRFS of patients over 65 was 23.2 (95% CI 14.4–31.9) months vs. 21.2 (95% CI 5.3–37.0) months in patients under the age of 65 (p = 0.012). Median PFS was 14.0 (95% CI 3.4–24.6) vs. 7.4 (95% CI 5.1–9.7) months (p = 0.001), and median OS was 52.2 (95% CI 9.9–94.5) vs. 19.9 (95% CI 14.2–25.6) months, respectively (p = 0.001).We analyzed the use of VMAT in the entire cohort and observed significantly longer survival in patients that received VMAT vs. patients with other forms of TRT. Median LRRFS was 30.3 (95% CI 15.2–45.4) vs. 12.1 (95% CI 4.7–19.4) months (p = 0.057).Univariate analysis of tumor histology revealed no significant association between histology and any of the endpoints; however, there was a trend toward longer LRRFS for patients with AC (p < 0.05). Median PFS was 6.9 (95% CI 4.8–9.1) vs. 15.4 (95% CI 9.0–21.7) months (p = 0.011), and median OS was 16.3 (95% CI 10.8–21.8) vs. 52.2 (95% CI 27.9–76.6) months (p = 0.001).PTV ≥ 700 cc was significantly associated with all endpoints. Median LRRFS of patients with PTV ≥ 700 cc vs. patients with PTV < 700 cc was 11.4 (95% CI 8.9–13.9) vs. 43.1 (95% CI 22.8–63.5) months . Similarly, median PFS was 5.8 (95% CI 0.0–12.2) vs. 11.0 (95% CI 8.6–13.4) months (p = 0.030), and median OS was 12.2 (95% CI 7.5–16.8) months vs. 34.9 (95% CI 16.0–53.8) months (p = 0.002).Median LRRFS in the entire cohort was 8.9 (95% CI 2.9–15.0) months for patients with total lung V20 ≥ 30% vs. 27.2 (95% CI 18.1–36.3) months for patients with V20 < 30% (p = 0.042).Median OS was 27.4 (95% CI 13.6–41.1) vs. 77.9 (95% CI 0.4–155.4) months for patients with more vs. patients with less than 20 pack-years (p = 0.323); however, we observed a trend for longer PFS (p = 0.095) and OS (p = 0.083) in subgroup C.In patients without ICI, stratification by treatment year did not show a significant association with LRRFS (p = 0.057).We observed no significant association between age ≥ 65 and LRRFS or PFS amongst patients in the non-ICI cohort, but a trend toward longer OS for patients under 65 (p = 0.051).There was no significant association of VMAT with LRRFS and OS amongst patients in the non-ICI cohort but a trend toward longer PFS (We observed no significant association between tumor histology and established endpoints in patients without ICI.p = 0.001). Median PFS was 6.2 (95% CI 3.5–8.9) and 11.2 (95% CI 6.9–15.6) months, respectively (p = 0.042). Median OS was 14.7 (95% CI 10.0–19.5) months and 43.1 (95% CI 15.9–70.3) months, respectively (p = 0.007).In subgroup analysis of patients without ICI, median LRRFS was 9.5 (95% CI 7.2–11.9) months for patients with PTV ≥ 700 cc and 37.8 (95% CI 16.0–59.5) months for patients with PTV < 700 cc  months for patients with SUVmax ≥ 13.75 vs. 25.1 (95% CI 14.1–36.0) months, for patients with SUVmax < 13.75 (p = 0.015). Median PFS was 5.4 (95% CI 3.5–7.2) vs. 14.1 (95% CI 5.6–22.8) months (p = 0.019), and median OS was 15.4 (95% CI 9.9–20.9) months and 31.2 (95% CI 9.4–53.1) months, respectively (p = 0.027).SUVp = 0.016). Median OS amounted to 12.1 (95% CI 7.6–16.7) months and 25.6 (95% CI 14.6–36.5) months (p = 0.002). Furthermore, we observed a trend toward shorter median PFS for patients with V20 ≥ 30: 5.6 (95% CI 1.1–10.0) vs. 9.2 (95% CI 6.6–11.7) months (p = 0.060).  vs. 20.5 (95% CI 13.2–27.8) months for patients with V20 ≥ 30 and V20 < 30, respectively ( 95% CI 1.6–36.5 mp = 0.226). Median PFS for patients with no ICI was 14.2 (95% CI 0.0–35.4) months and 26.3 (95% CI 6.7–45.9) months for patients with ICI (p = 0.629). Median OS was not reached for patients of subgroup C, regardless of ICI use (p = 0.583).In subgroup C, median LRRFS was 27.2 (95% CI 18.4–35.9) months for patients without ICI, while it was not reached for patients that had received ICI ] and PFS [HR 1.522 ]. For OS, age ≥ 65 years [HR 2.228 ], PTV ≥ 700 cc [HR 2.883 ] and 20 + PY [HR 2.546 ] remained significant prognosticators. Furthermore, there was a clear trend toward longer LRRFS [HR 0.497 ], PFS [HR 0.571 ] and OS [HR 0.447 ] in patients treated with ICI.On multivariate analysis, PTV ≥ 700 cc remained significantly associated with LRRFS [HR 2.194 ], PFS [HR 1.630 ] and OS [HR 1.958 ]. Similarly, SUVmax ≥ 13.75 remained a prognosticator of LRRFS [HR 2.252 ], PFS [HR 1.859 ] and OS [HR 2.405 ]. V20 ≥ 30 was significantly associated with LRRFS [HR 2.248 ] and OS [HR 3.357 ].In patients without ICI, PTV ≥ 700 cc was also associated with LRRFS [HR 2.065 .Consecutive patients that completed cCRT ± ICI at a single tertiary cancer center from 2011 to 2020 were evaluated. Initial staging and treatment planning were performed according to an in-house protocol in close accordance with the published ESTRO-ACROP guidelines   were associated with longer median survival time and superior PFS compared to patients with larger tumors  especially when large PTV was combined with UICC stage IIIC disease , as well as OS (p = 0.005) . Also, SUVmax ≥ 12 was a prognosticator for OS (p = 0.087)  and invasive  diagnostics, analysis/reduction of tumor motion and strict image guidance during radiation treatment. Intensified induction regimes including chemoimmunotherapy could therefore further improve this new tri-modal approach by ensuring a reduction in the GTV and consequently PTV.The present study found that patients with inoperable stage III NSCLC treated after 2018 demonstrated improved survival regardless of ICI use. However, implementation of ICI resulted in further improvement of patient outcome. PTV ≥ 700 cc and ICI were the only prognosticators of LRRFS, PFS and OS in the entire cohort. Further optimization strategies should consider both parameters and their interaction as most relevant for long-term patient outcome.