This appears to be related to reduced phosphorylation of serine residues in both N-terminal and C-terminal regions of the p53 molecule. Furthermore, the S100P expression results in lower levels of pro-apoptotic proteins, in reduced cell death response to cytotoxic treatments, followed by stimulation of therapy-induced senescence and increased clonogenic survival. Conversely, the S100P silencing suppresses the ability of cancer cells to survive the DNA damage and form colonies. Thus, we propose that the oncogenic role of S100P involves binding and inactivation of p53, which leads to aberrant DNA damage responses linked with senescence and escape to proliferation. Thereby, the S100P protein may contribute to the outgrowth of aggressive tumor cells resistant to cytotoxic therapy and promote cancer progression.S100P belongs to the S100 family of calcium-binding proteins regulating diverse cellular processes. Certain S100 family members (S100A4 and S100B) are associated with cancer and used as biomarkers of metastatic phenotype. Also S100P is abnormally expressed in tumors and implicated in migration-invasion, survival, and response to therapy. Here we show that S100P binds the tumor suppressor protein p53 as well as its negative regulator HDM2, and that this interaction perturbs the p53-HDM2 binding and increases the p53 level. Paradoxically, the S100P-induced p53 is unable to activate its transcriptional targets  S100P belongs to the S100 family of calcium-binding proteins linked with various diseases. S100P is one of the cancer-related family members implicated in cell migration-invasion and tumor metastasis . SeveralThese discordant effects may be caused by S100P crosstalk with diverse pathways in different tumor types, including activation of the MAPK/ERK, PI3K/AKT and NFkB signaling mediated by the binding of the extracellular S100P to RAGE receptor [The wild-type p53 tumor suppressor protein plays a key role in cellular responses to DNA damage and other forms of stress, including oncogenic activation, hypoxia, and red-ox disturbance. Its expression and activation usually leads either to cell cycle arrest or to apoptosis, depending on the stress type, extent and duration, and is often associated with reduced tumor growth and good therapy response, although recent data suggest that the wild-type p53 may also contribute to treatment failure through therapy-induced senescence , 12. StaUnder basal/unstressed conditions, the wild-type p53 is a short-lived protein undergoing proteasome degradation induced by its binding with the HDM2 ubiquitin ligase. Upon the DNA damage, p53 accumulates due to its phosphorylation by stress-induced kinases  followed by the disruption of the p53-HDM2 interaction. Additional kinases then activate the ability of p53 to act as a transcription factor. This leads to cell cycle arrest and/or apoptosis –16. TumoInterestingly, the cancer-related members of the S100 family  can interact with p53 and/or HDM2, but the modes of their interactions differ and consequences range from cancer-suppressing to cancer-promoting –21. HereTo study the S100P-p53 interaction, we performed pull-down experiments using the GST-wtS100P protein and the GST-mutS100P protein with the dimerization-disabling F15A mutation. The proteins were immobilized on affinity matrix and incubated with extracts of T47D cells expressing the mutated p53 and of RKO cells expressing the wild-type p53, with or without calcium ions. We found that S100P could bind both wild-type and mutant p53 in a calcium-dependent manner Figure . Moreove2) to induce p53. Expectedly, the PLA signal was rare in MCF-7 cells under basal conditions and abundant mainly in the nuclei of the treated MCF-7 cells , , and dems Figure .Because the wild-type p53 protein is kept under negative control by HDM2, we wanted to learn, whether S100P interferes with the p53-HDM2 interaction. We performed the PLA with the p53- and HDM2-specific antibodies in RKO cells and in their transient S100P-transfectants. Both mock- and S100P-cells were either untreated or UV irradiated to elevate the p53 expression Figure . This reNext we asked whether the S100P-p53 interaction could affect the p53 expression and/or function. Thus, we analyzed the p53 protein levels in A549 and RKO cells, which normally express low levels of the wild-type p53, and display either moderate expression (A549) or absence of S100P (RKO), . We examhdm2, WAFp21, and bax  and thus the observed changes could be attributed to initial cell responses to the DNA damage.We found clear differences between the mock-transfected and transiently S100P-transfected RKO cells both under basal and drug-treated conditions, as exemplified on the profile of the camptothecin-treated cells Figure . The mosIn order to better understand biological effects of S100P, we evaluated cell proliferation and cytotoxic responses in the real-time setting using the xCELLigence system, which measures the electrical impedance across the gold microelectrodes integrated in the bottom of microplates. There, the attachment, spreading and growth of cells resulting in an increased coverage of the bottom area increase the impedance, whereas detachment and cell death cause its reduction. We evaluated the RKO-mock cells versus transiently transfected RKO-S100P cells either in control conditions or after the treatment with 5 nM or 25 nM PTX Figure .Both S100P-positive and negative non-treated RKO cells displayed continuous proliferation, which appeared to be faster for the S100P transfectants especially in the first phase (at 27-52 h post-plating). Subsequently, growth of the RKO-S100P cells was slightly slower (at 57-72 h post-plating) until reaching the plateau (at 76-86 h), Figure . The mocTo understand this finding, we inspected the appearance of the RKO cells treated with CPT for 72 h and then allowed to restore in fresh medium for additional 72 h, fixed and stained. Interestingly, the S100P-transfectants that survived the drug treatment contained rare cells with a senescence-like morphology characterized by spread, flattened shape with an increased cytoplasmic granularity Figure . These cWe then wanted to know, whether these flattened cells express S100P and/or p53. Thus, we triple-stained the cells surviving the drug treatment with antibodies against S100P and p53 and with DAPI to visualize the nuclei. Confocal microscopic analysis showed the nuclear p53 staining in both mock- and S100P-transfected cells, but the p53-positive nuclei of the subset of S100P-expressing cells were much larger and had an aneuploid-like appearance, which is another feature of senescent cells Figure . These dTo verify the assumption that S100P can support the onset of therapy-induced senescence, we performed the SA-β-gal assay. The blue color resulting from the increased lysosomal activity of the senescent cells was virtually not present in the non-treated cells. However, treatment with the PTX and ETP induced a strong SA-β-gal staining in the S100P-transfectants, whereas the mock-transfected cells showed only a faint signal suggesting the S100P involvement in the therapy-induced senescence Figure .Cellular senescence induced by therapy is currently perceived as one of the mechanisms protecting tumor cells from death and allowing them to temporarily resist cytotoxic drugs –30. ThisMoreover, we accomplished knockdown experiments leading either to transient or stable S100P silencing in MCF-7 breast carcinoma cells that display endogenous S100P expression. Despite the fact that the level of the endogenous S100P protein is lower compared to the ectopic S100P level in the transfected cells, the effects of silencing versus scrambled control could be seen with respect to an increased p53 transcription and p21 transactivation Figure , reducedThis study aimed at better understanding of the role of S100P protein in the response of tumor cells to cytotoxic therapy. This issue has remained controversial, since certain studies claim the S100P involvement in therapy resistance, whereas the others suggest its role in chemosensitivity . The sitWe decided to look closer at this phenomenon in conjunction with the p53-related responses. We were inspired by the fact that cancer-related S100 family members interact with p53 and modulate its DNA binding, oligomerization and/or transactivation activity –34.WAF and upregulation of Bax. This seems to result in elimination of cells expressing the wild-type p53 and selection of cells with aggressive phenotype [Interestingly, the modes of the p53 binding by the S100 proteins and impacts on the p53 activity are not identical, albeit all seem to be calcium-dependent. Binding of S100 proteins to the tetramerization domain (TET) of p53 seems to be a general property of the family, whereas binding to the C-terminal negative regulatory domain (NRD) is not common . Some S1henotype , 38.Here we showed that also S100P protein binds p53 in a calcium- and dimerization-dependent manner and that the binding reduces the p53 phosphorylation at Ser15 and Ser46 in the TAD and Ser392 in the NRD. This suggests that the S100P hinders the access of relevant kinases to their substrate serine residues. In addition, the N-terminal region of p53 mediates interaction with an oncogenic ubiquitin-ligase HDM2, which maintains the p53 level low by the proteasome degradation. Interestingly, S100P also binds HDM2 and compromises the p53-HDM2 interaction as evidenced by our PLA and co-immunoprecipitation data. Therefore it is not surprising that the presence of S100P is linked with the elevated p53 protein that presumably escapes recognition by HDM2 and degradation in the proteasome. This is in agreement with the data obtained with other S100 proteins that can also bind HDM2 but do not form ternary complex with HDM2 and p53 [Although the S100P interaction with p53 results in its elevated expression, it is linked with the decreased activation of the p53 transcriptional targets in response to DNA damage. Based on these data we believe that S100P reduces the wild-type p53 transactivation activity through the mechanisms that may involve the S100P-p53 binding and either the steric inhibition of the p53 phosphorylation or, based on the analogy with the related S100 proteins, inhibition of the p53 oligomerization. Both phosphorylation and oligomerization were shown to be required for the p53-mediated responses to the DNA damaging treatments, although the extent of their involvement and the threshold required for the full p53 activity appear to be cell type- and cell context-dependent .The p53-mediated transactivation is known to have a profound impact on molecular and cellular responses of cancer cells to cytotoxic drugs, generally inducing cell cycle arrest or cell death, and suppressing senescence, with the outcome depending on the level/extent of p53 activation, and on the severity/duration of stress. Actually, DNA damaging drugs used at concentrations that do not induce p53 to levels and activities sufficient for death, can permit the therapy-induced senescence . In addiBecause the S100P protein reduces the p53 transactivation activity, we expected that it could interfere with these cellular processes. Interestingly, the S100P-expressing, drug-treated RKO cells differed from the mock-transfected cells by the reduced expression of several important pro-apoptotic proteins, including the p53 target Bax, thus indicating a down-regulation of the death-related signaling. This down-regulation was observed shortly after the drug addition (coincidently with reduced p53 phosphorylation) and was also reflected by the increased viability of the S100P-expressing cells during the first two-to-three post-treatment days. During that period, cell numbers declined as indicated by the lowered impedance values, FACS data, values, FACS and appearance of cell monolayers  S100P binds p53 protein and increases its level, (b) this binding leads to reduced p53 phosphorylation and transactivation activity in response to DNA damaging treatments, (c) through the inactivation of p53, S100P permits the onset of therapy-induced senescence and supports survival of the drug-treated tumor cells , paclitaxel (12.5 or 25 nM), UV irradiation (12 J/m2), and camptothecin (2 μM) for different time periods depending on experimental settings.Human lung carcinoma cells A549, colon carcinoma RKO, and breast carcinoma T47D and MCF-7 cells  were cultured in DMEM with 10% FCS (Biochrome), at 37°C in humidified air containing 5% COThe full-length S100P cDNA was subcloned into the pcDNA 3.1 plasmid (Invitrogen) from the pSG5C-S100P vector . ConstruIn order to transiently suppress the S100P expression, MCF-7 cells were transfected with 10 nM S100P siRNA (h): sc- 61488 (Santa Cruz Biotechnology) using the Gene Silencer siRNA Transfection Reagent (Genlantis) according to the manufacturer's instructions. Ten nanomolar Silencer Negative Control siRNA (Applied Biosystems) was used as control. 48 h after transfection, the cells were treated with PTX and UV and incubated for additional 24 hours. The RNA was isolated and analyzed by real-time quantitative PCR as described above.For the stable S100P suppression, the MCF-7 cell line was transfected by pRNATin-1.2/Hygro/shRNA scr (negative control) and pRNATin-1.2/Hygro/sh-S100P, respectively, and selected in Hygromycin B.Following pairs of oligonucleotides were cloned into the BamHI/HindIII-digested and dephosphorylated pRNATin-1.2/Hygro: siS100P top strand 5′-GATCCGTG CCGTGGATAAATTGCTCAATTCGTGGATAAATTGCTCAATTGATATCCGTTGAGCAATTTATCCACGGCATT TTTTGGAAA-3′ and bottom strand 5′-AGCTT TTCCAAAAAATGCCGTGGATAAATTGCTCAACGGATATCAATTGAGCAATTTATCCACGGCACG-3′); control scr top strand 5′-GATCCGAGATCCGTATAGTGTACCTTATTGATATCCGTAAGGTACACTATACGGA TCTTTTTTT GGAAA-3′ and bottom strand 5′-AGCTTTTCCAAAAAAAGATCCGTATAGTGTACCTTACGGATATCAAAAGGTACACTATACGGATCTCG-3′. The selected cells were expanded and analyzed for senescence and colony formation as described below.Primary antibodies, including the mouse monoclonal antibody (MAb) DO-1 specific for p53 , the mou2 or 0.7 mM EGTA. After repeated washing, the samples were resolved in SDS-PAGE and analyzed by immunoblotting using DO-1 and 2A9 antibodies [The recombinant proteins GST-wtS100P, GST-mutS100P (containing F15A mutation), and GST in supernatants from bacterial lysates were immobilized on Glutathione-S-Sepharose 4B beads. Washed beads were incubated overnight with pre-cleared lysates of RKO and T47D cells containing either 0.7 mM CaCltibodies , 51.Cells grown in confluent monolayers were rinsed twice with cold PBS, re-suspended in lysis buffer  containing the cocktail of protease inhibitors (Roche) and cleared by centrifugation. Proteins were quantified using the BCA protein assay (Pierce). Protein extracts (100 μg/lane) were resolved in 10% SDS-PAGE and transferred to a PVDF membrane (Macherey-Nagel). The proteins were detected with appropriate antibodies and visualized using an enhanced chemiluminescence kit . Far Western blotting was performed as described in the legend to the 3 cells/well . The impedance was recorded in 15 min intervals for 24 h. After administration of 25 nM paclitaxel, the impedance was recorded in 5 min intervals for additional 36 h in quadruplicates. Recorded values were presented as Cell Index (CI) calculated as a relative change in the electrical impedance.RTCA was used for monitoring of cell proliferation and viability in real-time. Experiments were set up in E-Plates 16 (Roche). Background impedance was measured in 100 μl of cell culture medium/well. RKO-empty pcDNA 3.1 and RKO-S100P cells were plated at 7×10PLA was used for in situ detection of the protein-protein interactions . Cells w6 cells/sample), washed in PBS, labeled and analyzed by flow cytometer FACSCanto™ II (Beckton Dickinson) equipped with 488 nm laser used for dye excitation. Labeling of viable cells was performed in 300 μl of PBS with 10 nM fluorescein diacetate (FDA) for 25 minutes at room temperature in the dark followed by propidium iodide (PI) at final concentration of 5 μg/ml. Emitted fluorescence was collected using the 530/30 filter for FDA and 585/42 filter for PI. Viable populations showed positive staining for FDA and negative staining for PI. Samples for subG1 analysis were treated with 0.05% Triton X100 in 300 μl of PBS for 25 minutes at 37°C and stained with 50 μg/ml PI. Emitted PI fluorescence was collected with the 585/42 filter. Data for the assessment of cell viability and SubG1% were analyzed with the FCS Express version 4.0 (De Novo Software).Treated cells were harvested . The membranes with immobilized antibodies were incubated with the lysates of RKO cells transiently transfected with empty pcDNA 3.1 or pcDNA 3.1-FL-S100P, untreated or treated with paclitaxel (25 nM for 4 h), etoposide (25 μM for 6 h), or camptothecin (2 μM for 4h). Washed membranes were developed by ECL and exposed to X-ray films. The films were scanned and pixel density was evaluated by quantifying the mean duplicate spots densities from two separate experiments. For the quantification, the spot volume was determined, corrected for background and values were expressed as % difference between S100P-expressing versus control cells.Total RNA was isolated using Instapure solution (Eurogentech). Reverse transcription of RNA was performed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Amplification was performed in the Stratagene Mx 3005P thermal cycling block (Agilent Technologies). PCR was carried out in 20-μl volumes using Maxima Syber Green PCR Master Mix (Fermentas) for 10 min at 95°C for initial denaturation followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, using primers listed in the 2. The cells were viewed using the phase contrast microscope. Senescent cells were recognized according to blue staining.SA-β-Gal activity was detected by the Senescence β-Galactosidase Staining Kit . Transfected RKO cells were seeded in 30-mm Petri dishes. Following the drug treatment for 72 h, the cells were washed, fixed and stained for 24 h at 37°C in absence of COThe cells were plated at 10% density in 6 cm dishes and exposed to camptothecin or paclitaxel (at 100 nM and 25 nM concentrations) in triplicates for three days. On day four, the cells were recovered in fresh media for additional 14 days with the growth medium replacement every 4 days. After extensive washing with PBS and fixation with 4% paraformaldehyde, the remaining colonies were stained with 0.5% Coomassie blue or Giemsa for 5 min at room temperature, extensively washed with PBS, dried and photographed.Results were analyzed by two-tailed unpaired t test (Student's test), and p<0.05 was considered significant.


Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure–activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR.Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR–tyrosine kinase inhibitors (EGFR–TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR–TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells  Resistance to EGFR-tyrosine kinase inhibitors in non-small-cell lung cancer is mediated by the C797S/T790M/activating mutation. Here, the authors identify brigatinib as a potential drug that in combination with anti-EGFR can overcome resistance in mutated cells. Non-small-cell lung cancer (NSCLC) harbouring an epidermal growth factor receptor (EGFR)-activating mutation accounts for ∼30–40% of NSCLC in the Japanese population and ∼15% in Caucasians2345678et al.Before clinical application of EGFR–TKIs, the overall survival of NSCLC patients was only approximately a year, as shown in the trials verifying the efficacy of platinum doublets chemotherapy in metastatic NSCLC1213et al.Although the benefits of molecularly targeted drugs are substantial, most patients experience a recurrence of disease within ∼1–2 years due to acquired resistance. The acquired resistance against the first-generation EGFR–TKIs gefitinib and erlotinib has been revealed to be mainly caused by a gatekeeper mutation16182627et al.N-ethyl-N-nitrosourea mutagenesis method, are totally refractory to the third-generation EGFR–TKIs WZ-4002, osimertinib and CO-1686. In cases without the C797S mutation, although loss of T790M was reported as specific resistant mechanism to third-generation EGFR–TKIs, bypass pathway activation, such as c-MET activation or small-cell lung cancer (SCLC) transformation, in resistant tumours is thought to be the mechanism of resistance similar to that known in first-generation EGFR–TKIs333435363738HER2 amplification, Met amplification, BRAF mutation and SCLC transformation have been observed in osimertinib-resistant cases40Approval of osimertinib will influence the treatment tactics for EGFR-mutated lung cancer, but, again, resistance to osimertinib will be a major obstacle. In 2015, various mechanisms of the acquired resistance against osimertinib were independently published by several groups. An EGFR mutation involving substitution of cysteine at position 797 with serine (C797S) was detected in cell-free plasma DNA from osimertinib-refractory patients and was shown to induce osimertinib resistanceet al.et al.in trans of the T790M allele, a combination of first- and third-generation EGFR–TKIs may be effective enough for clinical use; however, when the C797S and T790M mutations developed in cis, all sensitivity to any of the existing EGFR–TKIs, including the third-generation ones, was lost. However, we currently have no information on whether the in vitro efficacy of the combination of first- and third-generation EGFR–TKIs for trans C797S is clinically reproducible. The C797S mutations found in the samples obtained from participants in the osimertinib trial mentioned above were all in cis alleles except for one case of in transThe osimertinib resistance due to the loss of T790M or bypass pathway activation is expected to be overcome using existing methods, for example, exchange to or addition of a first-generation EGFR–TKI or concurrent combination therapy of an inhibiting alternative pathway, respectively. However, we now have no clinically available strategy to conquer the C797S/T790M/activating-mutation (triple-mutation). Recently, Jia in vitro and in vivo assays. Structure–activity relationship analysis and computational simulation reveal the key component determining the affinity and the binding mode to triple-mutant EGFR that are expected to attribute to the future development. Finally, the combination with anti-EGFR antibody strikingly reduces the IC50 of brigatinib and prolongs the survival of the triple-mutant EGFR xenograft-bearing mice. These findings in this study may help overcome acquired resistance to third-generation EGFR–TKIs.To investigate the therapeutic strategies for treating patients with triple-mutant EGFR, we performed drug screening and found brigatinib to be the promising candidate against triple-mutant EGFR with less potency against wild-type EGFR according to the Currently, there are four EGFR–TKIs available in the clinical setting—gefitinib, erlotinib, afatinib and osimertinib. Gefitinib and erlotinib are so-called first-generation EGFR–TKIs that were proven to be efficacious for NSCLC harbouring an EGFR mutation . Afatinib is a second-generation EGFR–TKI irreversibly targeting the pan-HER signal pathway. Osimertinib and EGF-816 are third-generation EGFR–TKIs that covalently bind to EGFR and are effective against the T790M-mutated EGFR, the most common mechanism of acquired resistance to first-generation EGFR–TKIs. EGF-816 is not yet accessible except for clinical trials. All classes of EGFR–TKIs are active against the EGFR-activating mutation alone. Therefore, we evaluated the sensitivity of the EGFR–TKI-resistant mutations introduced into Ba/F3 cells (T790M/activating mutation or C797S/T790M/activating mutation (triple-mutation)) to the clinically relevant EGFR–TKIs gefitinib, afatinib, osimertinib and EGF-816.in vitro –TKI45activity . The difactivity . These eactivity . Compariactivity .in silico docking simulation and molecular dynamic simulation to assess the binding compatibility between brigatinib and the triple-L858R mutant EGFR and to clarify whether the efficacy of brigatinib against the triple-mutant EGFR depends on targeting of the ATP-binding site  and double-mutant (T790M/del19) or triple-mutant (C797S/T790M/del19) cells was inhibited by gefitinib, osimertinib and brigatinib with a pattern similar to that observed in Ba/F3 cells expressing the corresponding mutation type . In westin vivo experiments by administering brigatinib or osimertinib to nude mice into which EGFR-triple-del19 expressing PC9 lung cancer (PC9-triple mutant) cells had been subcutaneously injected. As a result, the mice treated with brigatinib showed significant inhibition of the growth of PC9-triple mutant cells compared with vehicle controls and osimertinib-treated group without explicit toxicity  and brigatinib (75 mg kg−1) both successfully suppressed the growth of T790M/del19-expressing PC9 cells , MGH121-resistant-2 (EGFR-C797S/T790M/del19), PC9 parent (amplified EGFR-del19), PC9 T790M (amplified EGFR-T790M/del19) and PC9 triple-mutant (amplified EGFR-C797S/T790M/del19) cells were cultured in RPMI with 10% serum. These cells were kindly provided by Drs. Niederst and Engelman. Ba/F3 cells harbouring EGFR mutations were cultured in low-glucose Dulbecco's minimal essential medium (DMEM) with 10% fetal bovine serum (FBS). The MGH121 resistant-2 cells were established from MGH121 by treating with 1 μM WZ-4002, and the PC9 T790M cells were obtained from PC9 parental cells by treating with 1 μM gefitinib as The full-length wild-type EGFR was synthesized from cDNA obtained from A549 cells. The EGFR-activating mutation, del19 or L858R, was obtained from the cDNA of EGFR-exon 19 deletion (del19)-harbouring HCC827 cells or L858R-harbouring lung cancer specimens, respectively. Each EGFR was amplified using polymerase chain reaction and then cloned into a pENTR vector. The T790M and/or C797S mutants were generated by QuikChange site-directed mutagenesis using the following primers: T790M F- 5′-CCGTGCAGCTCATCATCCAGCTCATGCCCTTC-3′, and T790M R- 5′-GAAGGGCATGAGCTGCATGATGAGCTGCACGG-3′, C797S F- 5′-CATGCCCTTCGGCTCCCTCCTGGAGCTA-3′, and C797S R – 5′-TAGTCCAGGAGGGAGCCGAAGGGCATG-3′.The resulting pENTR-EGFR mutation constructs were sequenced and used as a template to make the pLenti6.3 lentiviral vector using LR clonase II. The lentivirus was made by transfecting the pLenti6.3 constructs along with helper plasmids (ViraPower) in 293FT cells. Virus production, collection and infection were completed following the manufacturer's protocol. The Ba/F3 cells were selected by culturing for 1 week with 7 μM blasticidin in DMEM with interleukin-3 (IL-3)-supplemented 10% FBS and then in DMEM without IL-3 to obtain the EGFR signalling-addicted cells.−1 to 10 μmol l−1. After 72 h of drug treatment, cell viability was measured using the CellTiter-Glo assay (Promega).Three-day cell viability assays were carried out by plating 2,000, 1,500 and 2,000 cells per well of Ba/F3, PC9 or MGH121, respectively, into black transparent-bottom 96-well plates. On the same day for Ba/F3 cells and the following day for PC9 and MGH121 cells, the cells were treated with each TKI across a 10-dose range from 0.3 nmol lBa/F3 cells of EGFR-del19, -T790M/del19, -C797S/del19 and -C797S/T790M/del19 were plated with 2,000 cells per well into black 96-well plates and treated for 3 days with a panel of 30 inhibitors including dimethylsulfoxide (DMSO) controls prepared in-house. After the incubation, cell viability was measured using CellTiter-Glo assay. The relative cell viability was calculated as a ratio of each value to that of the DMSO control. Experiments were repeated three times independently, and the average relative cell viability was calculated and shown as a heat map . Lysates were transferred to microtubes, boiled for 5 min at 100 °C and then vortexed. Protein quantification was performed using the BCA Protein Assay Reagent (Pierce) according to the manufacturer's protocol. Western blot analyses were conducted after separation by SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking in 5% BSA with Tris-buffered saline/Tween 20 (TBS-T) or 5% skim milk/TBS-T, membranes were incubated with phospho-EGFR antibody , total EGFR , phospho-Akt , total Akt , phospho-ERK , total ERK1/2 , phospoh-S6 , total S6  or β-actin .−1 substrate and added to a kinase protein–inhibitor mixture, and then incubated for 60 min at room temperature. After the kinase reaction, an equal volume of ADP-Glo Reagent was added to terminate the kinase reaction, and the remaining ATP was depleted. The Kinase Detection Reagent was added both to convert ADP to ATP and to allow the newly synthesized ATP to be measured using the luciferase/luciferin reaction. The light generated was measured using a luminometer.The recombinant proteins of the kinase domain of wild-type EGFR and EGFR-C797S/T790M/L858R were purchased from Signal Chem. The inhibitors were purchased as described earlier. The appropriate amount of target proteins calculated on the basis of the ADP-Glo assay manufacturer's protocol was incubated in 96-well half area white plates with serially diluted inhibitor over a 10-dose range from 0.0002, nM to 10 μM for 10 min at room temperature. ATP at concentration of 1, 10, 100 and 1,000 nM was mixed with 100 μg ml6 cells) on Cytomics FC500 (BECKMAN COULTER). The assessed cells were incubated with cetuximab 5 ng for 5 min before the addition of the PE antibody to cancel the cross-activity with cetuximab.To evaluate the level of EGFR, fluorescence-activated cell sorting (FACS) analysis was performed with PE Mouse Anti-Human EGF receptor  or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 106) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River). Tumour growth was monitored twice weekly by bilateral caliper measurement, and tumour volume was calculated as 0.5 × length × width × width (mm3). When the average tumour volume reached ∼200 mm3, the mice were randomized into vehicle and treatment groups using the restricted randomization such that the mean tumour size of each group was equivalent . The mice whose implanted tumour size was ranked in the top 5% and bottom 5% were excluded from randomization to minimize the variation of tumour sizes. The mice were treated once daily by oral gavage for the indicated period. Relative tumour volume was calculated by dividing by the tumour volume on day 0. The body weights of the mice were measured twice weekly. Implanted tumours were resected from the mice on day 15 of drug treatment and were fixed with formalin. The mice were euthanized when the tumour size exceeded 700 mm3 within several days. The investigators performing tumour measurements were not blinded to treatment groups. The sample size (minimum n=6 per treatment group) was selected to ensure satisfactory inter-animal reproducibility. The Mann–Whitney U test was used for the statistical analysis of the mice experiments.All mouse studies were conducted through Institutional Animal Care and Use Committee-approved animal protocols according to the institutional guidelines. PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells , which shares the largest common basic structure with brigatinib. The structure of a disordered loop (residues Leu989 – Asp1003) and the side chains of Ser797 and Arg858 were modelled using the Structure Preparation module in Molecular Operating Environment  version 2013.08 ref. . A compo3 periodic box, including roughly 13,000 water molecules. Charge-neutralizing ions were added to neutralize the system. All MD simulations were carried out using the GROMACS 4 programmeTen kinds of representative binding poses of brigatinib into the C797S/T790M/L858R triple-mutant EGFR were extracted by the docking simulation and used as initial structures of the molecular dynamics (MD) simulation. Brigatinib conformation was optimized, and the electrostatic potential was calculated at the HF/6-31G* level using the GAMESS programmet-tests as appropriate. Significant probability (P)-values are indicated as ***P<0.001, **P<0.01 and *P<0.05.Data were analysed using GraphPad Prism software (GraphPad Software). In cell growth inhibition experiments analysis, the curves were fitted using a nonlinear regression model with a sigmoidal dose response. Unless otherwise specified, data displayed are mean±s.d. Pairwise comparisons between groups  were made using paired or unpaired Student's The authors declare that all the other data supporting the findings of this study are available within the article and its How to cite this article: Uchibori, K et al. Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer. Nat. Commun.8, 14768 doi: 10.1038/ncomms14768 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary TableOriginal dataset of Figure 1a.


Plesiomonas shigelloides 78/89 lipopolysaccharide directly on bacteria revealed the characteristic structural features of the O-acetylated polysaccharide in the NMR spectra. The O-antigen profiles were unique, yet the pattern of signals in the, spectra along with their 1H,13C chemical shift values, resembled these of d-galactan I of Klebsiella pneumoniae. The isolated O-specific polysaccharide (O-PS) of P. shigelloides strain CNCTC 78/89 was investigated by 1H and 13C NMR spectroscopy, mass spectrometry and chemical methods. The analyses demonstrated that the P. shigelloides 78/89 O-PS is composed of →3)-α-d-Galp-(1→3)-β-d-Galf2OAc- analysis of  Plesiomonas shigelloides is a facultative anaerobic Gram-negative flagellated, rod-shaped bacterium belonging to the Enterobacteriaceae family [P. shigelloides are generally related to visiting countries with low sanitary standards [P. shigelloides is not entirely understood, lipopolysaccharide (LPS) is considered the main virulence factor. LPS is a major component of the outer leaflet of the external membrane of Gram-negative bacteria. These are amphiphilic molecules isolated from smooth bacterial strains (S-LPS). Structurally, they can be divided into three distinct regions: lipid A, core oligosaccharide, and O-specific polysaccharide. These segments are important for the biological activity [O-specific polysaccharide  determines bacterial O-serotype and constitutes a fingerprint of bacteria [O-specific polysaccharides provides the serological distinction between bacterial strains. The variability of O-antigen structures is one of the strategies used by bacteria to avoid recognition by host organisms and to hamper the host’s defenses. Despite this variability, serological cross-reactivities between various species occur, indicating the presence of common structural epitopes within O-antigens. To date only several LPS structures out of 102 O-serotypes of P. shigelloides [P. shigelloides LPSs, i.e., the lack of phosphate groups, the presence of uronic acid residues in the core oligosaccharides, and the unusual hydrophobicity of the O-specific polysaccharides [Plesiomonas species and distinguish them from other members of the Enterobacteriaceae family. However, some O-antigens of P. shigelloides have shown cross-reactivity with antisera directed against LPS of Shigella spp. The structure of P. shigelloides serotype O17 was found to be identical to the Shigella sonnei phase I O-chain [P. shigelloides share a type-specific antigen with S. flexneri and S. dysenteriae [l-Rhap(1→2)-α-l-Rhap described in S. flexneri serotype 6 is shared with P. shigelloides and Klebsiella pneumoniae O-antigens [O-antigens is desirable as it can provide broad protection against heterologous bacteria.e family . It is we family . These btandards ,4, drinktandards ,6. Thesetandards , gastroetandards , and diatandards . Althougactivity  and takeactivity . The hydactivity . The corbacteria . The higelloides  have beeelloides ,23,24,25charides . Most of O-chain ,26,27. Tenteriae ,28. The antigens . In someO-antigen of P. shigelloides, structurally similar to this of K. pneumoniae strain Kp20. Herein, we present the chemotyping of the O-antigen of P. shigelloides strain CNCTC 78/89 by high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy in situ alongside the structural analysis of the isolated O-specific polysaccharide, which supports this preliminary observations. The shared epitopes of Klebsiella and Plesiomonas O-antigens, responsible for their serological cross-reactivities, have also been determined.We have now identified a new O-antigens of P. shigelloides 78/89 were initially investigated by HR-MAS NMR spectroscopy as the technique allows for the direct identification of the flexible O-antigen molecules on the bacterial cells in situ. The screening of the whole bacteria of P. shigelloides 78/89 using HR-MAS NMR technique provided data on the O-PS spectral pattern of this strain. The HR-MAS NMR spectra of P. shigelloides 78/89 bacteria were complex and contained signals for anomeric and ring protons, as well as resonances of other surface molecules and metabolites . The observed pattern of resonances was compared with the O-antigen structural data available in our laboratory, including LPSs of various Klebsiella strains, and the data published previously. This O-antigen profile was unique, however, in that a subset of signals in the 1D and HSQC-DEPT spectra as well as the 1H, 13C chemical shift values were similar to these of K. pneumoniae strain Kp20 LPS  mass spectrometry and NMR spectroscopy, and identified as the O-specific polysaccharide fraction (PSI), fraction composed of short O-specific chains substituted by core oligosaccharide (OSII) and the core oligosaccharide (OSIII and OSIV) (The LPS of nd OSIV) .P. shigelloides strain 78/89 has been deduced from the analysis of the partially hydrolyzed PSI fraction. The PSI fraction was subjected to a partial acid hydrolysis with 0.5 M TFA. The MALDI-TOF mass spectrum  of the O-acetyled variant of the oligosaccharide. The observed mass differences indicated the disaccharide (Δ 324 Da) and the O-acetylated disaccharide (Δ 366 Da) O-repeats.As the attempts to obtain the MALDI-TOF spectra of the intact PSI failed, the mass of the repeating unit of spectrum  showed tP. shigelloides described to date. The acquired NMR data of the OSIV appeared very much like the previously published core oligosaccharide structure of P. shigelloides O33:H3 (strain CNCTC 34/89) [pNAc-(1-residue in the core of P. shigelloides 78/89 is not O-acetylated. The disaccharide element in the outer core is built of -4)-α-GalpNAc-(1→6)-α-GlcpN- [P. shigelloides O51 reacted vividly with the fast migrating bands of the SDS-PAGE-separated LPS fractions composed of the core oligosaccharide linked to lipid A. Reactions were also observed for the LPS fractions composed of the lipid A-core oligosaccharides substituted by the increasing number of the O-repeats (The combined NMR and MS data of the more abundant core oligosaccharide fraction (OSIV) indicated the presence of 11 sugar residues, together having a monoisotopic mass of 2015.65 Da. The chemical shift values of the spin systems of the OSIV were compared with these for other core types of C 34/89) , with tws O33:H3 . The OSI 110/92) . The str-repeats .P. shigelloides 78/89 bacteria and LPS using HR-MAS NMR spectroscopy provided a structural fingerprint of the O-antigen chemotype, including the 1H and 13C chemical shift values for the O-specific polysaccharide in situ. Composition analysis of the PSI polysaccharide fraction together with determination of the absolute configuration confirmed the presence of d-galactose residues in galacto-furanose and galacto-pyranose ring forms. The NMR spectra of the isolated PSI contained main signals for three anomeric protons and a distinct signal (δH 2.11 ppm) in the region of acetyl groups. The spin systems for each residue  were assigned using COSY, TOCSY, HSQC-DEPT, HSQC-TOCSY, HMBC, and NOESY NMR experiments  was identified as a 3-substituted 2-O-acetyl-β-d-galactofuranose  on the basis of the high chemical shift values of anomeric carbon (δ 107.1), C-2 (δ 81.8), C-3 (δ 82.1) and C-4 (δ 82.9) as well as the similarities of the chemical shift values of H-2, H-3, and H-4 to the published data [H 5.25) and its multiple bond correlation with a carbonyl carbon resonance at δC 173.1 ppm in the HMBC spectrum indicated an ester substitution at this position by an O-acetyl group (δH/δC 2.11/20.5). Similarly, residue B  was recognized as the 3-substituted-β-d-galactofuranose devoid of the O-acetyl. The chemical shift values except the H-2 were similar to these of residue A. The relatively high chemical shift of the C-3 signal (δ 84.5) indicated a substitution by another residue. Residue C with the H1/C1 signal at δH/δC 5.07/99.5 ppm, 1JC1,H1 ~175 Hz was assigned as a 3-substituted α-d-galactopyranose  based on the large vicinal couplings between H-2 and H-3, the small vicinal coupling constants between H-3, H-4 and H-5, as well as relatively high chemical shift value of the C-3 signal (δ 76.9). The heteronuclear multiple bond correlations between H-1 and both C-3 and C-5 observed in the HMBC spectrum confirmed the pyranose ring size. The presence of the C* variant (δH/δC 5.08/99.4 ppm) of residue C reflected the changes of chemical shifts induced by the O-acetylation of the disaccharide repeating unit and indicated that two forms of the disaccharide repeating units exist in the O-specific polysaccharide: the O-acetylated A-C* and non-O-acetylated B-C.Residue H/δC 5.11/95.4 ppm, residue C’) and the corresponding spin system observed at a lower contour level in the 2D spectra of the PSI showed chemical shift values similar to the unsubstitited α-d-galactopyranose [C, defining the biological repeating unit of the O-specific polysaccharide.The minor anomeric signal  in the 1H NMR spectrum of PSI relative to the resolved resonance of an anomeric proton at 5.20 ppm . The ratios of the integral values have shown that ~32% of the repeating units were O-acetylated in the PSI, generating the O-acetylation-related heterogeneity. This level of substitution was sufficient to disguise the chemotype of pure d-galactan I. Interestingly, the NMR spectra also indicated that there was no O-acetylation of the repeating units in the OSII fraction -β-d-Galf-(1→3)-α-d-Galp--α-d-Galp-(1→3)-β-d-Galf2OAc--d-galactopyranose known as d-galactan I [Klebsiella. This disaccharide is considered a valuable model for studies of biochemical pathways for formation of the Galf-contaning glycans present in the bacterial cell wall complex  [P. shigelloides 78/89 expresses LPS with the modified d-galactan I structure of the O-antigen.The β-lactan I  is presecocalyx) . These gcocalyx) . P. shigKlebsiella and Plesiomonas are Gram-negative organisms that are frequently co-isolated from intestinal infections. They are also isolated from different clinical specimens including respiratory tract. Their common residence and a niche-specific selection may have contributed to the fact that the strains share epitopes. The cross-reactivities of P. shigelloides O-antigens with antisera raised against some strains of the genus Shigella have been previously reported [Plesiomonas and Shigella either had some common epitopes or structures of their O-antigens were identical. The structural determinant →3)-α-d-Galp-(1→3)-β-d-Galf- serogroups cause over 50% of all Klebsiella infections worldwide [reported ,28. Thes antigen , which iorldwide .O-serotypes of Klebsiella and Plesiomonas were classified using cross-absorbed sera, indicating the differences among the epitopes recognized by polyclonal antibodies with no immediate correlation to the chemotypes of their O-antigens.Typically, O-antigens are appealing targets for active and passive immunizations with a potential for therapeutic applications. However, it is important to uncover subtle structural attributes of LPS molecules that may have impact on the desired specificity of antibodies [O-antigen structures in situ, providing robust information on the chemotype of the O-antigens.The tibodies . It is wP. shigelloides strain CNCTC 78/89, classified as serovar O12:H12 according to Aldova’s antigenic scheme [K. pneumoniae strain 20  is a bloodstream clinical isolate and was kindly provided by Szilvia Melegh  [c scheme , was obtHungary) . SamplesO-specific polysaccharide was isolated by mild acid hydrolysis (1.5% acetic acid containing 2% SDS at 100 °C for 15 min) of LPS (50 mg). SDS was removed by extraction with 96% ethanol and the pellet was suspended in water and centrifuged. The supernatant was fractionated by gel permeation chromatography, on Bio-Gel P-10 (1.6 × 100 cm) equilibrated with 0.05 M pyridine/acetic acid buffer, pH 5.6. The chromatography yielded a main fraction containing O-specific polysaccharide , separated from shorter O-specific polysaccharide chains linked to the core  and two fractions containing unsubstituted core oligosaccharides . The eluates were monitored with a Knauer differential refractometer and all fractions were checked by NMR spectroscopy MALDI-TOF mass spectrometry (MS).LPS was isolated from bacterial cells by the hot phenol/water extraction  and puri−1. The absolute configurations of the sugars were determined as described by Gerwig et al. [Monosaccharides were analyzed as their alditol acetates by GC-MS. Methylation was performed on the isolated PS according to the method described by Hakomori . Alditolg et al.  using (−3 (1 mL) at room temperature for 16 h, after which the solution was diluted with water and freeze-dried. The product was analyzed by NMR spectroscopy.Polysaccharide (5 mg) was treated with aqueous 12.5% NHThe polysaccharide (0.5 mg) was used for hydrolysis with 0.5 M trifluoroacetic acid (1 mL) at 80 °C. A sample (20 µL) was taken every 30 min for 2 h and the progress of hydrolysis was checked by MALDI-TOF MS.O-antigen of P. shigelloides O12 and for the OS of P. shigelloides O33 in separate experiments. Immunoblotting was done as previously described [405nm value in the range (0.8–2.2) was mixed with 100 µL of inhibitor solution (inhibitor concentration range from 250 µg/mL down to 0.49 µg/mL) and incubated for 1 h at 37 °C. The mixture (100 µL) was then transferred to a microtiter plate with LPS, incubated for 15 min with shaking and followed by the subsequent steps of standard ELISA protocol.The LPS was analyzed by SDS-PAGE according to the method of Laemmli with modifications . The LPSescribed . An ELISescribed  of the mescribed . The detescribed . BrieflyMALDI-TOF MS spectra of the PSI, the partially hydrolyzed PSI and the OS fractions were acquired using Bruker Autoflex III  time-of-flight instrument. Spectra were recorded in positive and negative modes. 2,5-Dihydroxybenzoic acid was used as matrix.2H2O  were obtained using the HR-MAS technique on a Bruker Avance III 600 MHz spectrometer . HR-MAS NMR experiments were carried out at a spin rate of 4 kHz at 27 °C  using a Bruker 4 mm HR-MAS probe and a ZrO2 rotor, as previously described [2H2O solutions at 30 °C on Bruker Avance III 600 MHz spectrometer using a 5 mm inverse detection QCI cryoprobe and 1.7 mm TXI microprobe. The polysaccharide and oligosaccharide fractions were repeatedly exchanged with 2H2O, with intermediate lyophilization. The data were acquired and processed using Topspin 3.1 . The processed spectra were assigned with the help of the SPARKY program [NMR spectra of bacteria (~4 mg dry mass) and LPS (~3 mg) suspensions in escribed . NMR spe program . The sig program .O-acetyl groups in the O-specific polysaccharides was determined by integration of the relevant signals from their 1D 1H NMR spectra acquired with a 30° pulse and a relaxation delay of 1 s. Volumetric integration of the selected resonances in the HSQC-DEPT spectra was performed by the Gaussian fit method using built-in functions of the SPARKY program.The relative content of the


In farm animals, mitochondrial DNA (mtDNA) effect on economic performance remains hot-topic for breeding and genetic selection. Here, 53 maternal lineages of Small-tailed Han sheep were used to investigate the association of mitochondrial DNA variations and the lambing litter size.tRNA-Lys gene was associated with litter size (P < 0.05), manifesting 0.29 lambs per litter between the G and T carriers. Furthermore, using the mixed linear model, we assayed the potential association of the ovine litter size and haplogroups and multiple-level mtDNA haplotypes, including general haplotypes, assembled haplotypes of electron transport chain contained sequences (H-ETC), mitochondrial respiratory complex contained sequences (H-MRC) and mitochondrial genes . The strategy for assembled mitochondrial haplotypes was proposed for the first time in mtDNA association analyses on economic traits, although none of the significant relations could be concluded (P > 0.05). In addition, the nuclear major gene BMPR1B was significantly correlated with litter size in the flock (P < 0.05), however, did not interact with mtDNA T7719G mutation (P > 0.05).Sequence sweeping of the mitochondrial coding regions discovered 31 non-synonymous mutations, and the association study revealed that T7719G in mtDNA tRNA-Lys gene be a potentially useful marker for selection of sheep litter size.Our results highlight mutations of ovine mitochondrial coding genes, suggesting T7719G in The online version of this article (doi:10.1186/s40104-017-0160-x) contains supplementary material, which is available to authorized users. BMPR1B was identified as one of causative genes for sheep prolificacy [BMPR1B effect [BMPR1B effect and interaction with mtDNA mutations were analyzed.Mitochondria are responsible for ATP production in the electron transport chain (ETC) in cells. The ETC consists of five mitochondrial respiratory complexes (MRCs), of which complex I, complex III, complex IV and complex V are constructed by both mitochondrial and nuclear encoded proteins, and complex II is entirely encoded by nuclear genes. Mitochondrial genome codes 13 polypeptides, 2 rRNAs and 22 tRNAs , dairy cB effect . HoweverB effect –17, whicBMPR1B at >2-month age.In total, 117 lambing Small-tailed Han sheep from 53 maternal lineages  of the same flock were performed blood sample collection, and recorded one or more times of litter size .ys), parity number (parity), service ram (ram), BMPR1B genotype (BMPR1B), mtDNA mutations , the interaction between BMPR1B and mtDNA mutations (BMPR1B × mutations), the polygenic effect (ID), the permanent environmental effect (EP), and the random residual (e) were included. The response variable was the ewe litter size (ls). Each cell of these effects contained observations. The polygenic effect corrected the genetic background by the additive genetic relationship matrix, i.e. the pedigree information. The permanent environmental effect dealed with the repeated measurement data.In the model, the effects of lambing year-season  in the R project (R version 3.2.5) .In total, 95 mutations in mitochondrial coding genes were discovered , and the interaction between BMPR1B and haplotype or haplogroup also did not significantly affect litter size (P > 0.05), while the BMPR1B was positively associated with litter size (P < 0.05).The 31 mitochondrial non-synonymous mutations assigned the Small-tailed Han sheep flock to 44 haplotypes  (Table P > 0.05) (Table BMPR1B was strongly associated with litter size (P < 0.05) (Table BMPR1B and mtDNA mutations was not remarkable (P > 0.05).Totally 35 H-ETCs were constituted by 19 missense mutations in protein coding sequences, while 15 missense mutations in MRCI assembled 33 H-MRCIs, and both 2 mutations in MRCIV and MRCV constituted 3 H-MRCIVs and 3 H-MRCVs respectively. Intensively, H-12SrRNA sorted the sheep flock into 4 haplotypes (H-12SrRNA), and the 5 mutations in 16SrRNA constituted 15 haplotypes (H-16SrRNA) (Table P > 0.05) (Tables BMPR1B genotype was remarkably associated with litter size (P < 0.05) (Table P > 0.05).The 3 mutations in tRNA-Lys affected litter size by 0.29 lambs per litter between the G (1.79) and T (1.50) carriers (P < 0.05). Even though the BMPR1B was in prominent correlation with litter size (P < 0.05) (Table P > 0.05)  Table , there wen the G .79 and TIn human diseases, mutations in mitochondrial coding genes led to changes of oxidative phosphorylation enzyme complexes . It is rIn order to investigate the overall mtDNA effects, a novel strategy of assembling mitochondrial haplotypes based on biology functions is proposed. Extensively, there are four levels of mitochondrial haplotypes for mitochondrial biological functions. The general haplotype is assembled by all non-synonymous mutations to reflect the integrated characteristics of mtDNA coding regions. Subsequently, the ETC based haplotype (H-ETC) represents the general feature of all ETC-contained mtDNA sequences. The MRC based haplotype infers the integrated signal of particular MRC-contained mtDNA sequences. H-MRC includes four types, i.e. MRC-I, MRC-III, MRC-IV and MRC-V. The last is the gene level, which indicates the information of a particular gene-contained mtDNA sequence.BMPR1B genotype, mtDNA mutations and their interaction. We started a full model (Model I) with all the environmental and genetic factors to test if environmental factors were noises. Results revealed that the effects of parity of dam and service ram were not significant on litter size. Subsequently, these two factors were excluded in the aims to construct an optimized model (Model II), which was used to test the significance of genetic factors, especially the effects of mtDNA mutations on litter size.In the mixed linear model, unnecessary factors interfere with the estimation of interested factors, as the degree of freedom is wasted. Therefore, based on the thoughts of multiple models  and varitRNA-Asp A7551G mutation [tRNA-Ile A4263G mutation [tRNA-Lys was reported to affect litter size in an Afec-Assaf flock [tRNA-Lys was significantly associated with ewe litter size in Small-tailed Han sheep, with the means of 1.79 and 1.50 for the G and T carriers, manifesting a difference of 0.29 lambs per litter. Furthermore, the mutation was predicted to produce a transversion of U to G at DHU loop on 2D cloverleaf of the tRNA-Lys structure  and interested traits. The present study discovered the mtDNA T7719G was linked with ewe litter size. For traits of low heritability, the marker assisted selection could increase the accuracy of breeding and selection. For the further study, the mtDNA T7719G should be put into post-association validation, and may become a genetic marker in the sheep breeding programs.


Aims. The study was designed to explore whether hydrogen sulphide (H2S) and nitric oxide (NO) generation changed in D-galactose-  induced ageing, the possible effects of exogenous H2S supplementation, and related mechanisms. Results. In D-gal-induced senescent mice, both H2S and NO levels in the heart, liver, and kidney tissues were decreased significantly. A similar trend was observed in D-gal-challenged human umbilical vein endothelial cells (HUVECs). Sustained H2S donor (NaHS) treatment for 2 months elevated H2S and NO levels in these mice, and during this period, the D-gal-induced senescent phenotype was reversed. The protective effect of NaHS is associated with a decrease in reactive oxygen species levels and an increase in antioxidants, such as glutathione, and superoxide dismutase and glutathione peroxidase activities. Increased expression of the H2S-producing enzymes cystathionine γ-lyase (CSE) and cystathionine-β-synthase (CBS) in the heart, liver, and kidney tissues was observed in the NaHS-treated groups. NaHS supplementation also significantly postponed D-gal-induced HUVEC senescence. Conclusions. Endogenous hydrogen sulphide production in both ageing mice and endothelial cells is insufficient. Exogenous H2S can partially rescue ageing-related dysfunction by inducing endogenous H2S and NO production and reducing oxidative stress. Restoring endogenous H2S production may contribute to healthy ageing, and H2S may have antiageing effects. With advancements in medical care, the population of people over 65 years old is growing rapidly. However, age-related health deterioration has not received enough attention, which may pose new problems for the healthcare system . Ageing Low-dose administration of D-galactose  in vitro  and in vivo  is widely accepted as an experimental model for the study of accelerated ageing .2S regulates several key proteins involved in cellular oxidative stress, it could have a protective effect against ageing. For example, H2S induces the S-sulphydration of Keap1, which leads to Nrf2 activation and nuclear translocation and results in the synthesis of antioxidative proteins [2S also inhibits mitochondrial ROS production and prevents activation of the adaptor protein p66Shc [Cellular senescence can be postponed by scavenging intracellular reactive oxygen species (ROS), and cell survival can thus be improved , 11. Givproteins . H2S alsn p66Shc . Higher n p66Shc .2S or manipulating its endogenous production. The regulatory role of exogenous H2S in C. elegans ageing was first reported by Miller and Roth [2S in senescence. Our previous study reported decreased heart H2S levels in long-term fructose-fed ageing mice, which may contribute to the pathogenesis of diabetic cardiomyopathy [2S protected the ageing kidney by alleviating oxidative stress, inducing endogenous H2S production and Nrf2 nuclear translocation [2S and NO, during the ageing process is not clear. Moreover, whether H2S supplementation manipulates NO production during ageing and whether it has a protective effect against ageing-related tissue damage is far from well understood. The present study aims to study the effect and possible mechanism of the action of H2S on cellular senescence during accelerated ageing both in vitro and in vivo.Recently, studies have concentrated on the possibility of lifespan extension by administrating exogenous Hand Roth  and lateand Roth . In mousand Roth , indicatmyopathy . We alsolocation . Howeverμmol/kg/day), D-gal with medium-dose NaHS (50 μmol/kg/day), and D-gal with high-dose NaHS (100 μmol/kg/day). All animal studies were approved by the Ethics Committee of Experimental Research, Fudan University Shanghai Medical College. For in vitro studies, subacute senescence was induced in human umbilical vein endothelial cells (HUVECs) by incubating cells with 50 mmol/L D-gal for 48 h [D-Galactose was purchased from Sigma-Aldrich (Germany). Eight-week-old male C57BL/6 mice were obtained from the Department of Laboratory Animal Science of Fudan University and raised under controlled conditions . A total of 60 mice were randomly assigned to the control group with normal saline and four D-gal model groups with hypodermic injections of 50 mg/kg D-gal daily for 2 months. The mice in each D-gal model group received different intraperitoneal injections of NaHS  or normal saline once a day at the same time: D-gal with normal saline, D-gal with low-dose NaHS  for 40 min on a shaker at room temperature. The reaction was terminated by adding 20% formic acid, and H2S levels were measured by a gas chromatograph-mass spectrometer .Hescribed . BrieflyAn NO assay kit  was used to measure nitrite/nitrate (NOx) via the Griess reaction. Nitrate was converted into nitrite by using nitrate reductase, and then, nitrite was measured.μm) were obtained by using a frozen tissue slicer. Heart tissue sections or HUVECs were incubated with 10 nmol/L DHE for 30 min at 37°C and observed under a laser confocal microscope (Zeiss LSM710) at the excitation/emission wavelengths of 488/610 nm. Fluorescence values were normalized to the control groups.ROS levels in the heart tissue and HUVECs were measured by using dihydroethidium (DHE) staining . Mouse heart tissue sections (7 μm) were stained with haematoxylin and eosin (HE). eNOS immunohistochemical staining was performed according to the manufacturer's instructions. Renal pathological changes were observed under an optical microscope .The kidney tissues were excised, fixed in 10% formalin, and embedded in paraffin. Kidney sections . Briefly, HUVECs cultured in 12-well plates were fixed and washed with phosphate-buffered saline (PBS) thrice. Cell-staining working solution (0.5 mL) was added into each well and incubated at 37°C for 12 h. After washing with PBS, senescent cells were identified as blue-stained cells under an inverted microscope . For each well, a minimum of 500 cells was counted to determine the percentage of SA-β-gal positive cells.The percentage of SA-μL CCK-8 in 100 μL PBS, Beyotime Biotechnology, China) and incubated in a CO2 incubator for 2 h. Afterward, cells were washed thrice with PBS, and the optical density (OD) in each well was determined by using a microplate reader at 450 nm.Cell proliferation was measured by the Cell Counting Kit-8 (CCK-8)  assay. Briefly, HUVECs were cultured in 96-well plates, exposed to D-gal for 46 h and various dosages of NaHS for 22 h. Subsequently, the cell culture medium was replaced with CCK-8 buffer . Protein samples were loaded on a sodium dodecyl sulphate polyacrylamide gel (10%), separated through electrophoresis, transferred onto a polyvinylidene fluoride membrane , and incubated with primary antibodies (1 : 1000 dilution) against CSE, CBS, 3-MST , p-eNOS, eNOS , p16, p21, or p53  at 4°C overnight. The blots were washed thrice with Tris-buffered saline containing Tween 20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The blots were then washed thrice and visualized by using chemiluminescent substrate (ECL). The densities of the immunostained bands were analysed by using a scanning densitometer  and Bio-Rad's image analysis software.μg total RNA using a reverse transcription kit . Real-time PCR was tested using the SteponePlus Real-Time PCR System  in a total volume of 20 μL reaction mixture containing 2 μL cDNA, 10 μL 2× SYBR Green PCR Master Mix , 0.8 μL of each primer (10 μM), and 6.4 μL ddH2O. Three-step real-time PCR of denaturing, annealing, and extension reactions was performed for 40 cycles of 10 s at 95°C, 30 s at 60°C, and 20 s at 72°C. For the eNOS gene, the forward primer was 5′-AGGTCTGTGGGTCTGGTTTG-3′ and the reverse primer was 5′-GCTCATTCTCCAGGTGCTTC-3′. For the GAPDH gene, the forward primer was 5′-CCACCCATGGCAAATTCC-3′ and the reverse primer was 5′-GATGGGATTTCCATTGATGACA-3′.Total RNA from HUVECs was prepared using Trizol reagent  according to the manufacturer's instructions. cDNA was generated from 0.5–2 t-test. Differences were considered statistically significant when P < 0.05.Results are expressed as the means ± SEM. Statistical analysis was performed by using SPSS software, version 21.0 . Comparisons among groups were performed by one-way ANOVA. Paired data were evaluated by two-tailed Student's 2S levels decreased significantly in the D-gal model group. NaHS supplementation for 8 weeks mitigated this decrease. However, the optimal NaHS concentration was not identical in different tissue types. In the heart, 100 μmol/kg/day of NaHS was the most effective dose, while in the kidney, 50 μmol/kg/day of NaHS was optimum , the expressions of both CSE and CBS, but not the 3-MST, increased significantly .Since eNOS is the major NO-producing enzyme in endothelial cells, we checked eNOS expression as well as its phosphorylation in D-gal-challenged HUVECs. The levels of both p-eNOS (s1177) and eNOS were decreased in the D-gal group. NaHS treatment at 50 and 100 is trend . We alsoμmol/L could induce the phosphorylation of AKT at Ser473, without altering the expression of total AKT  exogenous administration of the H2S donor NaHS mitigates age-related dysfunction, and the protective effects of NaHS may at least partially be due to improved endogenous H2S and NO production and the antioxidative ability of H2S.In this study, we adopted an accelerated ageing model to investigate the effects of sustained NaHS treatment in the ageing process both in vivo and in vitro. Our work reveals two key findings: (1) lowered heart, liver, and kidney H2S has an essential role in internal environment homeostasis. Endogenous H2S production is insufficient in several diseases, such as hypertension and myocardial infarction [2S production in the heart tissue is believed to play a role in protecting the myocardium [2S levels might change during D-gal-induced ageing. The measurement of endogenous H2S levels confirmed decreased H2S levels in the heart, liver, and kidney tissues in D-gal mice. We also observed decreased CSE expression in the heart and kidney. Interestingly, sustained NaHS treatment for 2 months significantly elevated the expressions of CSE and CBS in the heart, liver, and kidney tissues. A major difference between the expression changes in CSE and CBS lies in the effective dosages of NaHS. NaHS supplementation at 50 and 100 μmol/kg/day raised CBS expression in all three tissue types. With regard to CSE expression, NaHS treatment at both 50 and 100 μmol/kg/day was effective in the heart tissues. Additionally, the NaHS dosage regimes of 50 and 100 μmol/kg/day enhanced CSE expression in the liver and kidney, respectively. A certain degree of tissue specificity may result from the diverse distribution and abundant expression of H2S-producing enzymes in the given tissue types. For D-gal-challenged HUVECs, NaHS treatment elevated endogenous H2S production by increasing the expression of all three H2S-producing enzymes. Similar to previous reports, our findings indicate that age-related damage was inversely correlated with endogenous H2S production.Endogenous Hfarction , 21. Enhocardium , 21. We 2S reverses D-gal-induced ageing through decreased ROS generation and lipid peroxidation, but the detailed mechanism is not clearly understood [In the present study, all animals were freely fed with normal food. Additionally, D-gal-treated mice received hypodermic injections of 50 mg/kg D-gal daily for 2 months. Long-term administration of D-gal increases the concentration of intracellular D-gal. As a reducing sugar, D-gal reacts with the amino groups in various intracellular proteins and lipids . The oxiderstood . In D-gaderstood , 30. Ourderstood  and usinderstood  indicatederstood . Auditorderstood . Anotherderstood . ConservOxidative stress upregulates the tumour suppressor p53 and its target genes, such as p21 . The mec2S are indispensable for diverse cellular and systemic functions [2S interact under physiological and disease conditions remains unclear. Our previous study first reported that the novel proangiogenic effect of H2S was dependent on Akt phosphorylation [2S induced p38 MAPK/Akt and eNOS phosphorylation, which was followed by increased NO production [2S could induce Akt phosphorylation in D-gal-challenged HUVECs. Additionally, exogenous H2S increased both the expression of eNOS and its phosphorylation at S1177. As expected, the cellular NO content was elevated. When endogenous H2S production was inhibited pharmacologically, the cellular NO content decreased. We found that eNOS mRNA expression in HUVECs did not change after D-gal treatment, but it was increased with 50 μmol/L NaHS supplementation. We speculate that eNOS protein stability may play a role in D-gal-caused NO level reduction, and NaHS supplementation may mitigate this reduction by influencing eNOS mRNA expression and/or eNOS protein stability. These data indicate crosstalk between the H2S and NO signalling pathways in D-gal-treated senescent cells. More studies are needed to reveal the mechanism of crosstalk between the NO and H2S signalling pathways. The elucidation of their relationship may improve our understanding of the pathogenic mechanisms underlying age-related diseases.Interestingly, endogenous NO levels are also decreased in D-gal-accelerated ageing. Since NO is an important endothelial-derived vasodilator and cardioprotector, low NO levels may contribute to D-gal-induced tissue damages. The two gaseous signalling molecules NO and Hunctions , 35, 36.unctions . Howeverrylation . Altaanyoduction . In this2S were insufficient in D-gal-accelerated ageing, which was caused by the impaired expression of H2S-producing enzymes. Sustained exogenous H2S treatment could protect D-gal-accelerated ageing both in vitro and in vivo by reducing oxidative stress and increasing eNOS expression and NO contents as well as increasing endogenous H2S production.We demonstrated that endogenous levels of HFIGURE S1. Influence of NaHS treatment on eNOS mRNA expression in ageing HUVECs. N=5. Values are the means ± SE. ∗P < 0.05.


Interleukin-7 receptor (IL-7R) is involved in the abnormal function of solid tumors, but the role and regulatory mechanisms of IL-7R in HBV-related hepatocellular carcinoma (HCC) are still unclear.Gene and protein expression levels of IL-7R were examined in hepatoma cells transfected with hepatitis B virus (HBV) plasmids and in hepatoma cells transfected with the multifunctional nonstructural protein X (HBX). The expression of HBX and IL-7R was measured by immunohistochemical analysis in HBV-related HCC tissues. The role of NF-κB and Notch1 pathways in HBX-mediated expression of IL-7R in hepatoma cells was examined. Activation of IL-7R downstream of intracellular signaling proteins AKT, JNK, STAT5, and the associated molecules CyclinD1 and matrix metalloproteinase-9 (MMP)-9, was assessed in HBX-positive cells with or without treatment with IL-7R short hairpin RNA (shRNA). Additionally, the role of IL-7R in HBX-mediated proliferation and migration of hepatoma cells was investigated.The expression of IL-7R was increased in hepatoma cells transfected with HBV plasmids; HBX was responsible for the HBV-mediated upregulation of IL-7R. Compared to adjacent tissues, the expression of HBX and IL-7R was increased in HBV-related HCC tissues. Additionally, the relative expression levels of HBX were associated with IL-7R in HBV-related HCC tissues. The activation of NF-κB pathways and expression of Notch1 were increased in hepatoma cells transfected with HBX, and inhibition of NF-κB and Notch1 pathways significantly decreased HBX-mediated expression of IL-7R. The activation of AKT and JNK and the expression of CyclinD1 and MMP-9 were increased in HBX-positive cells. When cells were treated with IL-7R shRNA, the activation of AKT and JNK, as well as the expression of CyclinD1 and MMP-9, were significantly inhibited. Additionally, IL-7R was responsible for HBX-induced proliferation and migration ability of hepatoma cells.Our data demonstrate that HBX can upregulate IL-7R via NF-κB and Notch1 pathways to facilitate the activation of intracellular pathways and expression of associated molecules, and contribute to proliferation and migration of hepatoma cells. Hepatocellular carcinoma (HCC) is a common malignant solid tumor with poor prognosis. According to epidemiological data, most cases of HCC are closely associated with chronic infection with the hepatitis B virus (HBV). In China, over 50 % of HCC patients are HBV carriers , 2. HBV Interleukin-7 receptor (IL-7R) is a heterodimeric receptor containing a specific α chain (IL-7Rα) and a common cytokine receptor γ chain; IL-7R plays a key role in regulating lymphocyte development as well as maintaining survival during differentiation and activation of T cells . Upon enIn this study, we examined the expression of IL-7R in HBV-infected hepatoma cells and HBV-related HCC tissues. Further, we investigated the role of HBX in the upregulation of IL-7R, the activation of downstream intracellular pathways of IL-7R, and the expression of associated molecules in inducing cell proliferation and migration. Our data provides novel evidence on the mechanisms involved in HBX-mediated progression of HCC and may aid in the development of therapeutic targets for HBV-associated HCC.The HBV plasmid pUC18-HBV1.2 (a vector containing HBV genome) and pcDNA3.1-X  were obtained as previously described . The pUCThe genes of HBS, preS2, preS1, HBC, HBe, and HBP proteins, encoded by the three overlapping open reading frames  of the HBV genome, were amplified by polymerase chain reaction (PCR) using the pUC18-HBV1.2 plasmid and cloned into pcDNA3.1 vectors using Nhel and EcoRI or HindIII and EcoRI sites. The primer sequences encoding viral proteins were as follows: HBS (681 bp): CCCAAGCTTATGGAGAACATCGCATCAGGAC and CCGGAATTCTTAAATGT ATACCCAAAGACAAAAGAAAAT. preS1 (1203 bp): CAAGCTTGATGGGAGG TTGGT and CGGAATTCTTAAATGTATACCCAAA. preS2 (846 bp): CCCAAGC TTATGCAGTGGAACTCCACCACTTTCCACCAAAC and CCGGAATTCTTAA ATGTATACCCAAAGACAAAAGAAAAT. HBC (564 bp): CTATAGGGAGAC CCAAGCTGGCTAGCATGGACATTGACCCGTATAAAGAATTTGGAGC and CA CTGTGCTGGATATCTGCAGAATTCCTAACATTGAGATTCCCGAGATTGAGAT CTTC. HBe (651 bp): CTATAGGGAGACCCAAGCTGGCTAGCATGCAACTTTT TCACCTCTGCCTAATCATC and CACTGTGCTGGATATCTGCAGAATTCCTAA CATTGAGATTCCCGAGATTGAGATCTTC. HBP (2544 bp): CTATAGGGAGACC CAAGCTGGCTAGCATGCCCCTATCTTATCAACACTTCCGGAAAC and CACT GTGCTGGATATCTGCAGAATTCTCACGGTGGTCTCCATGCGACGTGCAGAG. The gene sequences encoding viral proteins in the pcDNA3.1 vectors were verified by sequencing.The anti-HBX mouse polyclonal antibody was purchased from Millipore . E-cadherin rabbit anti-human polyclonal antibody, vimentin mouse anti-human polyclonal antibody, and β-catenin mouse anti-human polyclonal antibody were purchased from Ruiying Biological . The p-P65 rabbit anti-human polyclonal antibody, rabbit anti-human P65 polyclonal antibody, p-JNK rabbit anti-human polyclonal antibody, JNK rabbit anti-human polyclonal antibody, p-STAT5 rabbit anti-human polyclonal antibody, STAT5 rabbit anti-human monoclonal antibody, p-AKT rabbit anti-human polyclonal antibody, AKT rabbit anti-human monoclonal antibody, CyclinD1 rabbit anti-human polyclonal antibody, and MMP-9 rabbit anti-human polyclonal antibody were obtained from UCallM Biotech Co, Ltd. . GAPDH mouse anti-human polyclonal antibody, β-actin goat anti-human polyclonal antibody, IL-7R rabbit anti-human polyclonal antibody, and Notch1 goat anti-human polyclonal antibody were purchased from Santa Cruz Biotechnology, Inc. . Anti-HBsAg mouse polyclonal antibody was obtained from Bioss Biotechnology . IgG-HRP goat anti-mouse, IgG-HRP rabbit anti-goat, and IgG-HRP goat anti-rabbit secondary antibodies were purchased from ZSJQ-Bio . NF-κB pathway inhibitor BAY11-7082 and Notch1 inhibitor GSI-953 were purchased from Sigma-Aldrich . Recombinant human IL-7 was from PeproTech . Trizol reagent and Lipofectamine 2000 were purchased from Invitrogen . The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories . Clarity™ Western ECL substrate was purchased from BIO-RAD . Transwell cluster plates with 8.0-μm pore were obtained from Corning Costar . BCA protein kit and crystal violet staining solution were obtained from Beyotime Institute of Biotechnology . TIANScript RT Kit was purchased from TIANGEN Biotech . LO2, Huh-7, and HepG2 cells were obtained from Cell Bank of Chinese Academy of Sciences. BEL7402 and SMMC7721 were obtained from XiangYa central experiment laboratory, Hubei, China. HepG2.2.15 was a gift from the Peking University Hepatology Institute, Beijing, China.https://www.gcbi.com.cn) and significant differentially expressed genes (DEGs) with fold change ≥ 1.5 were identified. Additionally, the microarray data presented in this study have been deposited in the Gene Expression Omnibus  under the accession number GSE83489.Huh-7 cells were transfected with the HBV plasmid (Huh-7-HBV) and control plasmid (Huh-7-Mock) and incubated for 48 h. Total cellular RNA was extracted using Trizol reagent following manufacturer’s instructions. The purity of prepared RNA was confirmed using agarose gel electrophoresis. cDNA labeling and microarray hybridization using Affymetrix GeneChip HuGene-1.0ST array platform were performed by the Genminix Informatics Company, Shanghai, China. The microarray data were interpreted, normalized, and log2 scaled using the online analysis tool GCBI ; double-distilled water was used to terminate the DAB reaction, and then sections were counterstained with hematoxylin.Forty HBV-related HCC tissues and 10 adjacent tissues were collected at the Department of Pathology, Affiliated Hospital of Xuzhou Medical University, from 2012 to 2015. The study was approved by the ethics committee of Xuzhou Medical University, and informed consent was obtained from all participants. Immunohistochemical analyses were performed using 4-μm formalin-fixed paraffin-embedded (FFPE) tissue sections. First, tissue sections were deparaffinized, rehydrated, and incubated in 0.01 M sodium citrate 5 times for 2 min each time to retrieve the antigens. After incubating with 3 % HThe detection of relative expression levels of target proteins was based on a semiquantitative system that relied on two parameters: staining intensity and proportion of positive tumor cells as described by Soslow RA et al. , 14. Sta4 cells/mL or 2 × 104 cells/mL was placed into 96-well plates with five wells per group. After the plates were incubated for 24, 48, 72, and 96 h, the number of viable cells was tested using the CCK-8. The relative rate of proliferation was determined by optical density (OD) values at 450 nm detected with the ClinBio-128 plate reader .The protocol for cell viability assay has been described previously . BrieflyThe plate clone formation assay has been described previously . Five hu4 or 4 × 104 cells were resuspended in serum-free medium and placed in the upper part of a transwell plate; the bottom chamber contained 10 % fetal bovine serum (FBS) used as chemoattractant. The cells were then incubated for 24 h. The cells that did not pass through the polycarbonate membrane were scraped off with a cotton swab. The cells that passed through the polycarbonate membrane were designated as cells that had migrated, fixed with 4 % paraformaldehyde, and stained with crystal violet. Finally, the cells were counted as described previously [The transwell array was performed as described in our previous study . Brieflyeviously .The cells were plated in 6-well dishes and allowed to reach 90 % confluence in 2 mL of culture medium. Then, a wound was created in the cells using a micropipette tip. Images were taken using a microscope (×400 magnification), and migration distance was calculated as previously described .p value less than 0.05 was considered significant.All data are presented as mean ± SD. Statistical analysis was carried out by Student’s t test, one-way ANOVA, and Mann-Whitney tests where appropriate. A chi-square test was used to analyze the relative expression levels of target proteins in HBV-related HCC tissues and adjacent non-tumor tissues. Additionally, correlations between HBX and IL-7R in HCC tissues were analyzed by Pearson correlation coefficients. A To investigate the role of HBV in genetic alteration of hepatoma cells, we first transfected the pUC18-HBV1.2 plasmid and control plasmid into Huh-7 cells. After 48 h, total RNA was extracted and an Affymetrix GeneChip Human Gene 1.0 ST array was used to assess gene expression in both HBV-transfected and control cells. As shown in Fig. To confirm the HBV proteins responsible for HBV-mediated upregulation of IL-7R, pcDNA 3.1 plasmids containing the genes of seven viral proteins  encoded by the four overlapping ORFs  of the HBV genome were transfected into Huh-7 and HepG2 cells for 48 h. The role of different viral genes in the expression of IL-7R was then detected by RT-PCR and western blot. The results showed that only HBX upregulated the expression of IL-7R at the gene and protein levels, while other viral genes had no significant effect on the expression of IL-7R Fig. . HBX is 2 = 27.2, p < 0.01). Additionally, our data indicates that the relative expression level of HBX in HBV-related HCC tissues was significantly higher than that in pericarcinous tissues . Furthermore, positive correlation between the intensity of IL-7R and HBX expression was found in HCC tissues using the Pearson correlation analysis  in lung cancer cells . BecauseActivation of IL-7R is mainly based on its interaction with IL-7. We detected the expression of the IL-7 gene in HBX-positive cells; the results showed that expression of IL-7 gene was increased in HBV-transfected cells compared with that in control cells. Additionally, the increased expression of IL-7 gene mediated by HBV was also mainly relied on HBX Fig. . Next, wOur previous reports indicate that HBX promotes the proliferation and migration of hepatoma cells . In thisWe next used the transwell array and wound healing assay to explore the function of IL-7R in HBX-mediated cell migration. The results indicated that HBX could enhance the migration of hepatoma cells. When HBX-positive cells were treated with IL-7R shRNA, the migration of hepatoma cells mediated by HBX was inhibited Fig. . In addiChronic HBV infection is responsible for the development of HCC. The multifunctional regulatory protein HBX plays an important role in hepatocarcinogenesis mediated by HBV infection. HBX can interfere with a variety of cellular processes and uses various molecular mechanisms to impact the progression of HCC , 5. FurtPrevious studies have shown that the expression of IL-7R was increased in various tumors such as those of the lung, bladder, and breast , 18, 23.Current studies have identified the different signaling pathways responsible for IL-7R expression. Miller ML et al. indicated that NF-κB pathways mediate the transcription of IL-7 receptor, thereby controlling the responsiveness of quiescent naïve T cells to IL-7 . Tian B Activation of IL-7R increased the phosphorylation levels of downstream signaling proteins, including STAT5, AKT, and JNK , 18. Cur2 cells transfected with HBX (L-O2-X cells) and showed that the expression of IL-7 gene was increased in L-O2-X cells [Based on whole-genome sequencing, Kan et al. reported the amplification of IL-7 gene in 88 HCC tumor tissues, compared to matched adjacent tissues, and even 81 of these tumor tissues were HBV-related HCC . Zhang W-X cells . Consist-X cells ; therefo-X cells . This suSeveral studies have demonstrated that IL-7R is involved in proliferation and migration of tumor cells , 42. We In conclusion, we found that HBV could upregulate the expression of IL-7R via HBX to increase the activation of intracellular pathways and expression of associated molecules, and contribute to the proliferation and migration of hepatoma cells. Our data broadens the current understanding of the role and associated mechanisms of IL-7R in HBV-related HCC and may aid in the exploration of treatment targets for HCC with HBV infection.