A significant sexual dichotomy was discovered, coupled to muscle and age-specific gene regulation. As such, Adnp was shown to regulate myosin light chain (Myl) in the gastrocnemius (GC) muscle, the language acquisition gene forkhead box protein P2 (Foxp2) in the tongue and the pituitary-adenylate cyclase activating polypeptide (PACAP) receptor PAC1 mRNA (Adcyap1r1) in the bladder, with PACAP linked to bladder function. A tight age regulation was observed, coupled to an extensive correlation to muscle function , placing ADNP as a muscle-regulating gene/protein.The activity-dependent neuroprotective protein (ADNP), a double-edged sword, sex-dependently regulates multiple genes and was previously associated with the control of early muscle development and aging. Here we aimed to decipher the involvement of ADNP in versatile muscle gene expression patterns in correlation with motor function throughout life. Using quantitative RT-PCR we showed that  Adnp mRNA is 90% identical to human ADNP mRNA [Adnp-deficient heterozygous (haploinsufficient) mouse model [The activity-dependent neuroprotective protein (ADNP) ,2, partlse model  predictese model .ADNP syndrome children, suffering from de novo mutations in ADNP [ADNP syndrome motor deficiencies manifest as hypotonia as well as opposing acute muscle tightness, signs of atrophy and abnormal gait [Adnp haploinsufficiency [Delayed motor development characterizes a large majority (96%) of  in ADNP ,17,18. Aficiency , exhibitficiency .ADNP and its paralogue protein ADNP2 in the vastus lateralis and bicep brachii muscles are significantly upregulated in the human elderly population, compared to young subjects, in a sex dependent manner. Thus, in the vastus lateralis ADNP is increased with aging in males and females, while ADNP2 is increased with aging in females only. In the bicep brachii muscles, ADNP is increased with aging only in males and not in females, while ADNP2 is increased with aging exclusively in females [ADNP expression was highly correlated with 24 genes, with nicotinamide nucleotide adenylyl (NAD) transferase 1 (NMNAT1) being the leading gene/protein [+ salvage capacity in human skeletal muscle declines with aging, suggesting a causative or a compensatory role for ADNP content [We have also shown that the expression levels of  females . ADNP ex/protein . As such content .ADNP syndrome. For example, the bladder is slow in development with 81% of children suffering from the syndrome, exhibiting bladder training delay, and many are still not toilet trained when approaching puberty [ADNP syndrome is speech delay, which presented in 98.6% of individuals and 19% had no language development at all [Importantly, not only the skeletal limb muscles are affected in the  puberty . Anothert at all . Apraxiat at all .Mechanistically, ADNP is found in the nucleus as well as in the cytoplasm, and cytoplasmic representation increases in mature neurons, where ADNP is essential for neurite maintenance . As suchAdnp deficiency is also associated with reduced autophagy that is dependent on microtubule integrity [ntegrity , with ADntegrity . Interesntegrity ,29,30,31ntegrity ,33, exhintegrity . Furtherntegrity .The cytoplasmic interactions of ADNP with LC3 and EB1/EB3 are enhanced in the presence of the ADNP snippet, NAP  containing an EB1/EB3 and self-interacting SxIP motif ,24,26,27Given the significant effect of ADNP mutations and ADNP deficiency on motor functions , and the+/–Adnp and +/+Adnp mice, previously extensively studied for brain function and behavior (methods) [ADNP syndrome patients suffer from delays in motor development, language acquisition, and bladder training [Adnp and the reference gene Hprt [Adnp, Adnp2, Akap6, Bmp4, Foxp2, Myl2 and Myl9.To evaluate a potential mechanistic basis for the effects of ADNP on muscle development/maintenance, we examined the water-based DD- formulated/NAP-treated methods) . We chostraining . Given ttraining , males aene Hprt  in the yAdnp heterozygosity (haploinsufficiency), which were corrected by the ADNP snippet, drug candidate NAP, further attesting for specificity.We chose to concentrate on gene transcripts changed as a consequence of Adnp reduction as a consequence of Adnp gene copy deficiency (+/−Adnp) was significant in all tested tissues at the youngest tested age in both males and females, with significantly higher expression in +/+Adnp males compared with females , Myl2 , Myl9, and Nmnat1 showed Adnp/NAP as well as age/sex-dependent regulation in the GC muscle  and two  at the oldest age tested .Tongue sex/age/Adnp genotype/NAP-dependent regulation was shown in 5 out of the 16 tested genes at the youngest age tested , which were corrected by the ADNP snippet, drug candidate NAP , while Myl9 correlated with Adnp in both sexes, and Myl2 correlated with Adnp only in females  detected in males. Similarly, all transcripts tested showed significant correlations with Adnp in the female GC muscle, while only Adnp2, Akap6, and Myl9 were significant in the males. Akap6, Foxp2 and Myl2 correlated with Adnp in the old female tongue, with Akap6 also correlating with Adnp in males, and Myl9 correlating with Adnp only in the male and not in the female tongue.At 8 months of age, all gene transcripts tested showed significant correlations with Foxp1 in the youngest GC muscle and for Myl2 in the oldest tested tongue tissue  negative correlation was observed in the females for sue red, A. Molecusue red, B.Adnp and Myl2 with age in the male muscle (blue), contrasting a decrease of Adnp in the female (pink), while a decrease in Adnp between one  and three months of age was apparent in both sexes in the tongue  exhibited significant positive correlations with ed genes  in the Ged genes  addressiBmp4 and Chl1 correlated with a number of different CatWalk behaviors in females . Previous findings linked dysregulation of Adnp/Adnp2 correlations with aberrant synaptic function in neuropsychiatric diseases [ADNP, ADNP2 expression in Alzheimer’s disease (lymphocytes) [ADNP2 is a less studied gene compared to ADNP [ADNP2 deletion (together with other genes) to autism [Additionally, here, in the GC muscle, diseases . Intereshocytes)  and in shocytes) ,42. Whil to ADNP ,45, receo autism . Furthero autism , suggestAdcyap1r1 encoding the PACAP-specific, PAC1 receptor, did not correlate with Adnp in the bladder, despite the significant increase seen in the +/−Adnp females compared to +/+Adnp females and the amelioration with NAP treatment. These findings are of significant interest, as intra-bladder administration of the PAC1 receptor antagonist, PACAP(6–38), reduces urinary bladder frequency and pelvic sensitivity in mice exposed to repeated variate stress [Adnp-deficiency exacerbated stress response [e stress , with PA, PACAP6–8, reduceresponse ,17,49 anresponse , indirecresponse . We alsoresponse .Myl2 and Myl9 in correlation with Adnp expression. In this respect, MYL2 is linked to cardiac development and function [ADNP syndrome children suffering cardiac as well as gastrointestinal problems [Interestingly, here, we also found sexually/developmentally differential expression of function , while Mfunction , with ADproblems . Furtherproblems  as well problems ,54. Anatproblems . Myl9 maADNP syndrome. Indeed, Foxp2 showed distinct sexual dichotomy in the tongue. Further sexual differences were observed in Foxp2 also in correlation with Catwalk performance. Previous studies have indicated a close correlation of the Foxp1/Foxp2 transcript expression and regulation of ultrasonic vocalization in mice. Foxp1 and the androgen receptor are co-expressed in striatal medium spiny neurons and brain-specific androgen receptor KO (ArNesCre) mice exhibit reduced Foxp1 expression in the striatum at E17.5 and P7.5 and an increased Foxp2 level in the cortex at P7.5 [Similarly, Foxp2 is implicated as critical for central control for normal bladder voiding behavior  and may  at P7.5 . Our pre at P7.5  and our  at P7.5 , with AD at P7.5 ,60. Futu+/−Adnp associated deficiencies, it did not correct all  A DD-group consisting of mouse pups that were subcutaneously administrated NAP  for 21 consecutive days at the following doses: 40, 80, 120, and 160 μL on P9–P10, P11–P14, P15–P18, and P19–P21, respectively. At 21 days of age, these mice were treated daily with intranasal NAP for another 6 days  (tested (2) 1-month-old mice were treated daily with intranasal NAP for 2 months (0.5 μg NAP/5 μL DD per mouse) (tested at 3 months of age) .(3) 1-month-old mice (from group 1), were continuously treated daily with intranasal NAP for additional 7 months (0.5 μg NAP/5 μL DD per mouse) (tested at 8 months of age) .CatWalk XT  was used ,69,70. Ig, 10 min, 4 °C) to pellet cell debris, and the supernatants were transferred to new tubes. All samples were processed as per the manufacturer’s protocol supplied with the TRI Reagent. The quantity and quality of RNA were analyzed by Nanodrop  [Mice described above were sacrificed and the following tissues were immediately frozen with liquid nitrogen after excision: GC muscle, tongue, and bladder. Total RNA was isolated using TRI Reagent  kit according to the manufacturer’s instructions, with slight modifications as described before . BrieflyMA, USA) . Total RHprt [CT−Δ method [CT = CT (a target gene)−CT  [Real-time PCR was performed using PerfeCTa SYBR Green FastMix, Low ROX  and QuantStudio 12K Flex Real-Time PCR System , employing the default thermocycler program for all genes. RNA expression levels of each gene in each tissue were normalized to Hprt . Primer Hprt  and syntT method ,71, with., Hprt) .Biological and technical replicates were used for gene expression analysis as detailed in the Figure legends.Adnp and muscle regulation, during development and aging, including a potential involvement with the acto-myosin muscle system (Myl2 and Myl9), energy metabolism (Nmnat1), and language acquisition Foxp1/Foxp2 tongue expression. Our discovery of the extensive sexual dichotomy paves the path to better experimental design, looking separately at males and females both in basic research and most importantly in clinical studies. Our studies further suggest biomarkers for Adnp’s crucial activities regarding muscle function such as gait regulation and suggest a feedback mechanism with the ADNP regulator PACAP, with Adnp regulating bladder PAC1.Despite the obvious study limitation of reliance upon subtle, albeit significant, gene expression changes, our results suggest a connection between


Background: Colorectal cancer (CRC) is the third most lethal malignancy in the world, wherein colon adenocarcinoma (COAD) is the most prevalent type of CRC. Exploring biomarkers is important for the diagnosis, treatment, and prevention of COAD. Methods: We used GEO2R and Venn online software for differential gene screening analysis. Hub genes were screened via Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape, following Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Finally, survival analysis and RNA expression validation were performed via UALCAN online software and real-time PCR. Immunohistochemistry (IHC) was performed to verify the protein expression level of hub genes from tissues of COAD patients. Results: In the present study, we screened 323 common differentially expressed genes (DEGs) from four GSE datasets. Furthermore, four hub genes were selected for survival correlation analysis and expression level verification, three of which were shown to be statistically significant. Conclusion: Our study suggests that Serpin Family E Member 1 (SERPINE1), secreted phosphoprotein 1 (SPP1) and tissue inhibitor of metalloproteinase 1 (TIMP1) may be biomarkers closely related to the prognosis of CRC patients. Colorectal cancer (CRC) originates in the colon or rectum , and typGene microarray technology has been extensively applied in several fields of medicine and biology. This high-throughput technology can effortlessly acquire gene expression profile information, which is very useful for exploring various molecular mechanisms . RecentlIn the present study, we screened the common differentially expressed genes (DEGs) in four GSE datasets from the Gene Expression Omnibus (GEO) repository and constructed a protein–protein interaction (PPI) network. Via integrated bioinformatics analysis, we identified the four hub genes associated with survival of COAD patients and verified the mRNA expression levels in CRC cells. Finally, our results showed that secreted phosphoprotein 1 (SPP1), Serpin Family E Member 1 (SERPINE1) and tissue inhibitor of metalloproteinase 1 (TIMP1) could be potential biomarkers for prognostics of COAD in patients.The high-throughput gene expression profiles of normal colorectal tissues and COAD tissues were extracted from the GEO database. The independent datasets of GSE37364, GSE41328, GSE81558, and GSE110225 were selected which included 14 COAD tissues and 37 normal tissues, 10 COAD tissues and 10 normal tissues, 23 COAD tissues and 9 normal tissues, and 17 COAD tissues and 17 normal tissues, respectively.The DEGs between normal colorectal tissues and COAD tissues were screened via the GEO2R tool. We limited the cut-off criterion of |logFC| ≥ 1 to be statistically significant. Then, the DEGs in the four datasets were screened out using online Venn software. The DEGs with logFC ≥ 1 were considered as up-regulated genes, while DEGs with logFC ≤ −1 were considered as down-regulated genes.In order to characterize the functional roles of the DEGs, we used Database for Annotation, Visualization and Integrated Discovery  databasThe PPI network was built via the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING)  databaseP-value <0.05 was considered statistically significant.UALCAN  was used2 humidified incubator in Dulbecco’s modified Eagle’s medium (HyClone) containing 10% fetal bovine serum  at 37°C.The normal colon epithelial cell line, NCM460, and COAD cell lines, HCT116, HCA-7, HT-29, and SW480 were maintained by our laboratory. And all cells were cultured in a 5% COTotal RNA from cell cultures was collected by using Total RNA Extraction Kit . The specific primers were designed by using Primer Express 3.0 software. Each sample was used in triplicate for the qPCR using PowerUp™ SYBR™ Green Master Mix  on Applied Biosystems™ StepOnePlus instruments, and analyzed with the StepOne software, using GAPDH as a control. The primers used in the present study are designed as follows: TIMP1: Forward: 5′-GGGACACCAGAAGTCAACCAG-3′; Reverse: 5′-CAATGAGAAACTCCTCGCTGC-3′; C–X–C motif chemokine ligand 2 (CXCL2): Forward: 5′-CCAAACCGAAGTCATAGCCAC-3′; Reverse: 5′-TGCCATTTTTCAGCATCTTTTC-3′; SPP1: Forward: 5′-TGAGCATTCCGATGTGATTGAT-3′; Reverse: 5′-TTATCTTCTTCCTTACTTTTGGGGT-3′; SERPINE1: Forward: 5′-CTGGGTGAAGACACACACAAAAG-3′; Reverse: 5′-CACAGAGACAGTGCTGCCGT-3′; GAPDH: Forward: 5′-TCAAGGGCATCCTGGGCTACAC-3′; Reverse: 5′-TCAAAGGTGGAGGAGTGGGTGTC-3′.Sixty paired primary cancer tissues and adjacent tissues from colon cancer patients who underwent surgery between September 2011 and April 2020, were collected at the Tianjin Xiqing Hospital. No chemotherapy and radiotherapy was given to patients before surgery. All the pathology materials were reviewed independently by two pathologists. Informed consent was obtained from each patient, and the collection of tissue specimens was approved by the Institutional Review and Ethics Boards of the Tianjin Xiqing Hospital. The two-step EnVision method was used to perform immune histochemical experiments, along with different primary antibodies against CXCL2 (1:50), SERPINE1 (1:50), TIMP1 (1:50) and SPP1 (1:50). The slides were analyzed using the Image-Pro Plus software program .P-values were calculated via Student’s t test, wherein *, **, and *** represented P<0.05, P<0.01 and P<0.001, respectively.Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 8.0 software. Results were presented as the mean ± standard deviation. The statistically significant changes were indicated with asterisks and the In the present study, we selected and downloaded four GEO datasets and extracted DEGs on the basis of the cut-off criteria. The present study covered 64 COAD and 73 normal colorectal tissues in total. The results showed that 2729, 957, 1864, and 617 genes were down-regulated while 1917, 770, 1282, and 469 genes were up-regulated in GSE37364, 41328, 81558, and 110250, respectively. By using the Venn diagram software, we detected a total of 323 common DEGs in the COAD samples, composed of 111 up-regulated genes and 212 down-regulated genes .We analyzed the DEGs via the DAVID software and identified 125 significant enrichment terms, including BP (71), MF (24), and CC (26). With regard to BP, DEGs were seen to be significantly enriched during cell adhesion, negative regulation of cell proliferation, angiogenesis, cytoskeleton organization, steroid hormone-mediated signaling pathway, inflammatory response, negative regulation of protein kinase activity, and so on. Whereas, for MF, DEGs were significantly enriched during chemokine activity, hormone activity, CXCR chemokine receptor binding, receptor binding, calcium ion binding, structural constituent of cytoskeleton, protein kinase inhibitor activity, oxidoreductase activity etc. With regard to CC, DEGs were significantly enriched in extracellular space, apical plasma membrane, extracellular exosome, extracellular region, cell surface, plasma membrane, basal plasma membrane, and so forth. The top 20 GO terms are depicted in Via the STRING and Cytoscape analysis, we drew the PPI network complex constructed with 268 nodes and 709 edges, consisting of 97 down-regulated and 171 up-regulated genes. Then we conducted further analyses by applying Cytoscape MCODE plus, and found 32 central nodes that included 20 up-regulated genes and 12 down-regulated genes .CXCL2, SERPINE1, SPP1, and TIMP1, were remarkably related to the survival of COAD patients (We used UALCAN to identify the survival data of 32 central genes and found that the expression levels of four hub genes, namely patients . Furtherpatients . Furtherpatients . Moreovepatients .CRC is the third main cause of deaths from malignant cancers worldwide, and the fourth most common cancer in China ,16. One In the present study, we screened DEGs via GEO2R, and selected 111 up-regulated genes and 212 down-regulated genes found in COAD tissues. Then we analyzed their enrichment in GO and signaling pathways and found that the DEGs were enriched in 11 pathways, including the PI3K-Akt signaling pathway. Previous studies showed that several signal pathways could be involved in the progress and development of CRC . PI3K/AKCXCL2, SERPINE1, SPP1, and TIMP1, were selected. Furthermore, we tested the expression of these four genes at the mRNA as well as protein levels and found that SPP1, TIMP1, and SERPINE1 had higher expression levels in COAD cell lines than the colon epithelial cell line as well as in CRC tissue compared with para-cancerous tissue.Furthermore, we constructed a PPI network and performed survival analysis along with verification of the RNA expression levels via the GEO database, DAVID and STRING online tools, Cytoscape software, and UALCAN online analysis. After the above series of operations, four hub genes, SPP1, named as osteopontin, is a phosphoprotein secreted by malignant cells. Cheng et al.  found thin vitro, whereas there is weakened tumorigenesis and metastasis in vivo via the FAK-PI3K/Akt and MAPK pathways. The elevated TIMP1 levels in serum were related to lower tumor grade and shorter overall survival times of CRC patients [TIMP1 is one of the members of the TIMP family which can bind to MMP substrates and mediate MMP degradation that is necessary for matrix renewal . TIMP1 hpatients .SERPINE1, also called PAI-1, can regulate plasmin formation from plasminogen . The expCXCL2 belongs to the chemokine superfamily, and is responsible for the attraction of leukocytes to inflammatory sites apart from the regulation of angiogenesis, tumorigenesis . DietricSERPINE1, SPP1, and TIMP1 as key genes which may be closely related to the prognosis of COAD in patients. Further investigations need to be performed to explore the mechanisms of these genes. These results would consequently provide candidate targets for the diagnosis and prognosis of CRC in patients, as well as new ideas for treatment.In summary, in the present study, we screened 323 DEGs from four COAD GEO datasets and identified


Hepatocellular carcinoma (HCC) metabolism is redirected to glycolysis to enhance the production of metabolic compounds employed by cancer cells to produce proteins, lipids, and nucleotides in order to maintain a high proliferative rate. This mechanism drives towards uncontrolled growth and causes a further increase in reactive oxygen species (ROS), which could lead to cell death. HCC overcomes the problem generated by ROS increase by increasing the antioxidant machinery, in which key mechanisms involve glutathione, nuclear factor erythroid 2-related factor 2 (Nrf2), and hypoxia-inducible transcription factor (HIF-1α). These mechanisms could represent optimal targets for innovative therapies. The tumor microenvironment (TME) exerts a key role in HCC pathogenesis and progression. Various metabolic machineries modulate the activity of immune cells in the TME. The deregulated metabolic activity of tumor cells could impair antitumor response. Lactic acid–lactate, derived from the anaerobic glycolytic rate of tumor cells, as well as adenosine, derived from the catabolism of ATP, have an immunosuppressive activity. Metabolic reprogramming of the TME via targeted therapies could enhance the treatment efficacy of anti-cancer immunotherapy. This review describes the metabolic pathways mainly involved in the HCC pathogenesis and progression. The potential targets for HCC treatment involved in these pathways are also discussed. Hepatocellular carcinoma (HCC) is the sixth most common cancer and the fourth leading cause of cancer-related death ,2,3. HCCCancers have an altered metabolism with an increase in glucose uptake and lactate production in the presence of oxygen. This increased energy supplied by aerobic glycolysis is called “the Warburg effect” ,7. OxidaAlterations in metabolic pathways could generate advantages for tumor proliferation and growth by ensuring energy and substrates. These alterations could create advantageous reducing conditions in the tumor microenvironment (TME) that are optimal for cancer survival ,12.In the current review, we discuss the role of glucose metabolism and oxidative stress in HCC pathogenesis and progression and the potential therapeutic targets for new HCC treatments.GLUT1 expression is significantly associated with HCC tumorigenicity, tumor invasiveness, and growth. GLUT1 protein expression is increased in HCC cancer tissues and is associated with an increase in 18F-FDG PET-CT  uptake .,82.81,82The MAPK pathway regulates cell growth, differentiation, and apoptosis through a phosphorylation cascade that can start from tyrosine-kinase receptors (TKRs), and activates downstream protein effectors Ras, Raf, MAP/ERK kinases (MEK1/2), and extracellular regulated kinases (ERK1/2) . AnotherERK1/2 are required for G1/S phase transition after mitogen action. ERK1/2 are involved in the induction of proliferation, in the suppression of apoptosis, and in the promotion of cell survival . MEK/ERKJNK has a role in cell cycle progression, cell proliferation, and migration . ImmunohGankyrin is an oncoprotein highly expressed in HCC, with an active role in tumor initiation and progression . It enhap38α protein regulates the production of pro-inflammatory cytokines, controls differentiation and apoptosis, and negatively regulates proliferation . Mice prThe PI3K/AKT/mTOR pathway can be activated by TKRs, cytokines, and hormones or by Ras ,95. OncePIK3CA, which encodes for the catalytic subunit of the PI3K protein. The PIK3CA mutation frequency is about 7%. PIK3CA mutations can be found in the helical domain (E545K) or in the kinase domain (H1047R) [SGK3 gene deletion was shown [PIK3CA may predict sensitivity to inhibitors of the PI3K/AKT mTOR pathway [RPS6KA3, and half of these mutations cause a premature stop codon or an altered splicing. To a lesser extent, missense mutations were found in close proximity to codons encoding for Ser227 or Thr557 phosphorylation sites required for RSK2 activation and thus inactivating its function [The PI3K/AKT pathway is affected by mutations in HCC. One mutated gene is (H1047R) ,99,100. (H1047R) ,99,100. function ,102.NFE2L2 gene) pathway and GSH metabolism [Cancer cells, in order to reduce the overproduction of ROS and the related toxic effects while maintaining cell proliferation and survival conditions, can activate two major response mechanisms: the Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor erythroid 2-related factor 2 (Nrf2)  stress . Specifiduce ROS .NFE2L2 [NFE2L2 harbored these mutations that cause the inhibition of the binding of KEAP1 [NFE2L2 mutations in 3% and KEAP1 mutations in 5% of the cases, respectively [NFE2L2 mutations are very recurrent and they occur in the early stages of tumorigenesis, suggesting an active role in HCC activation and development [NFE2L2 knockout mice were able to withstand diethylnitrosamine-induced hepatocarcinogenesis [Somatic mutations that interfere with Nrf2–KEAP1 interaction were identified in HCC cases ,109. TheNFE2L2 . The DLGof KEAP1 . Anotherectively . A chemiogenesis . This reogenesis .GSH is a natural non-enzymatic and endogenous antioxidan ,112. It 2O2 and GSH into H2O, O2 and GSSG, and glutathione reductase (GRd) which reduces GSSG to GSH again [GSH is produced through two steps requiring ATP. One step is the reaction of glutamate and cysteine catalyzed by γ-glutamylcysteine synthetase (GCS) to produce γ-glutamylcysteine; the other step is the reaction of γ-glutamylcysteine with glycine catalyzed by GSH synthetase (GS) to produce GSH . GCS is SH again ,117.Huang et al. detected a 2-fold increase in the GSH protein level, and an increase in GCS-HS mRNA and protein level and in GS mRNA level in HCC tumor samples when compared to the normal liver counterparts . This inIn HCC tissues, higher GSH and GPx activity were detected when compared to the adjacent normal liver counterpart. GSH and GSSG levels detected in pre-operative plasma were low, while after resection, their concentration increased, suggesting that GSH may be used to decrease the oxidative stress during carcinogenesis and that tumor tissue may uptake GSH from plasma . Aldehyde dehydrogenases are a group of enzymes that catalyze the oxidation of endogenous and exogenous aldehydes, and are also necessary for the production of retinoic acid . ALDH, bIn the HCC TME, immune system cells can be affected by metabolites released from cancer cells, in particular lactic acid–lactate ,122,123.Another type of immune cells that can be affected by lactate are the myeloid-derived suppressor cells (MDSCs) ,129. In Tumors can escape from dendritic cells (DCs) immunosurveillance by responding to several signals ,130, incCytotoxic T lymphocytes (CTL) can be affected by lactic acid, which is capable of inhibiting their cell growth and cytokine secretion ,132.Adenosine (ADO) is derived from ATP catabolism . During High ADO levels in the TME induce suppression of antitumor activity through the activation of ADO receptors expressed on immune cells . Four suADO also interferes with the differentiation of monocytes in DCs and with their activation. By the activity of A2AR and A2BR, it impairs the capability of DC to induce the Th1 immune response, whereas Th2 and Th17 responses are stimulated. ADO enhances the production of interleukins (ILs) IL-5, IL-6, IL-10, transforming growth factor- β (TGF-β), indoleamine-pyrrole 2,3-dioxygenase-1 (IDO-1), arginase 2, programmed cell death ligand 2 (PD-L2), and VEGF whereas it reduces the expression of tumor necrosis factor-α (TNF-α) and IL-12 ,144,145.The interaction between ADO and its receptors compromises the differentiation of monocytes into macrophages ,146. By The activation of A2AR by ADO impairs NK cells’ maturation, proliferation, and the secretion of interferon-γ (IFN-γ) and TNF-α. The stimulation of A2AR decreases the NK cell cytotoxic capability .A2BR stimulation boosts IMC differentiation toward MDSCs . A2AR-memicroRNA (miRNAs) are small non-coding RNAs that play key roles in many cellular functions, including metabolism . SeveralThe liver-specific miR-122 exerts a key role in the sustenance of the physiological liver metabolism . AlteratThe miR-520 family is significantly downregulated by Tat-activating regulatory DNA-binding protein (TARDBP). High TARDBP levels have been found in HCC, where it regulates the expression of PFKP .miR-7 acts as a tumor suppressor in HCC. Specifically, miR-7 reduces tumor cell proliferation and migration through PI3K/AKT/mTOR signaling .Another regulator of the PI3K/AKT/mTOR pathway is miR-125a, which impairs cell proliferation and migration .MCT4, responsible for lactate efflux, is targeted by miR-145 . The regABCB1 gene, and breast cancer resistance protein (BCRP), encoded by ABCG2 gene, need to be briefly mentioned. MDR1 is an ATP-binding cassette able to recognize a broad number of hydrophobic molecules, such as chemotherapeutic compounds, and extrudes them by ATP hydrolysis across the extracellular compartment [BCRP mRNA expression was found to be upregulated in liver diseases and in HCC compared to normal tissues, particularly in less differentiated HCC [Molecular pumps that move the pharmacological compounds across the plasma membranes of tumor cells are the major mechanisms that induce drug resistance . Most ofpartment . Upregulpartment ,158. MDRpartment . The BCRpartment . BCRP mRated HCC . The expated HCC .Disruption of KEAP1 renders HCC cells resistant to regorafenib and lenvatinib, with an increase in Nrf2 activity . Nrf2 upAnother drawback of current HCC therapies is the formation of a hypoxic environment ,168,169.MDR1 gene and MDR1 protein [During hypoxia, HIF-1α can also regulate the expression of the  protein . A posit protein . HIF-1α  protein ,173. In  protein . A simil protein .Three main pathways are upregulated in HCC due to a metabolic switch and elevated ROS production: glycolysis, MAPK pathway, and PI3K/AKT/mTOR pathway, which can interact with one another to increase their effects in tumor proliferation and survival  82,94,1,194,175.clinicaltrials.gov, there are some completed trials regarding the efficacy of treatment combinations using PI3K and MAPK inhibitors as anti-cancer therapies, although without straightforward results [On  results ,182,183.Long-term sorafenib exposure causes chemoresistance with an upregulation in the PI3K/AKT/mTOR pathway ,185. BasPI3K and MAPK pathways are regulators of anaerobic glycolysis . The firAn alternative strategy to treat HCC is to increase ROS levels in order to augment oxidative stress . The sorAnother possible approach is to act on the hypoxic TME. LS081 was shown to inhibit cell growth through prolyl hydroxylation of HIF-1α and its degradation . GefitinClinicalTrials.gov, there are several ongoing clinical trials based on small-molecule tyrosine-kinase inhibitors capable of altering HCC metabolism, and thus inhibiting tumor growth or recurrence. These trials are listed in To date, on Despite recent advances in therapeutic options, curative therapies for HCC are still not available ,209,210.To extend the impact of immunotherapy to more patients, targeting additional mechanisms of tumor immune evasion will be critical . The bio


Colorectal cancer (CRC), the third most common type of cancer, is the second leading cause of cancer-related mortality rates worldwide. Although modern research was able to shed light on the pathogenesis of CRC and provide enhanced screening strategies, the prevalence of CRC is still on the rise. Studies showed several cellular signaling pathways dysregulated in CRC, leading to the onset of malignant phenotypes. Therefore, analyzing signaling pathways involved in CRC metastasis is necessary to elucidate the underlying mechanism of CRC progression and pharmacotherapy. This review focused on target genes as well as various cellular signaling pathways including Wnt/β-catenin, p53, TGF-β/SMAD, NF-κB, Notch, VEGF, and JAKs/STAT3, which are associated with CRC progression and metastasis. Additionally, alternations in methylation patterns in relation with signaling pathways involved in regulating various cellular mechanisms such as cell cycle, transcription, apoptosis, and angiogenesis as well as invasion and metastasis were also reviewed. To date, understanding the genomic and epigenomic instability has identified candidate biomarkers that are validated for routine clinical use in CRC management. Nevertheless, better understanding of the onset and progression of CRC can aid in the development of early detection molecular markers and risk stratification methods to improve the clinical care of CRC patients. Colorectal cancers (CRCs) encompass two types of highly aggressive and common types of cancers, namely, colon and rectal. Globally, while colon cancer is the fourth most common malignancy, rectum cancer is the eight most common one . CollectAdenomatous polyposis coli (APC), Kirsten ras (KRAS), Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA), Small mothers against decapentaplegic 4 (SMAD4), and Tumor protein 53 (TP53) [Two molecular pathological classifications for CRC are described ,4. The COn the other hand, Guinney et al. (2016)  aggregatTo determine the extent of CRC spread, according to the American Joint Committee on Cancer, CRC staging is done according to the tumor-nodes-metastasis (TNM) system .CRC pathogenesis is highly complex and diverse and is induced by several risk factors including sporadic, familial, and inherited . SporadiOn the other hand, genetic and epigenetic alterations are involved in events that lead to the initiation of neoplastic transformation of healthy epithelium that progresses toward the malignant stages; in 1990, Fearon and Vogelstein defined the genetic paradigm of CRC formation . In theiToday, CRC is one of the leading causes of morbidity and represents a formidable health burden. Therefore, understanding the molecular and genetic features of its onset and progression is essential. This review focused on several novel cellular signaling pathways associated with CRC metastasis, including Wnt/β-catenin, p53, cyclooxygenase (COX), TGF-β/SMAD, nuclear factor kappa beta (NF-κB), Notch, vascular epidermal growth factor (VEGF), and Janus kinase/signal transducer and activator of transcription 3 (JAKs/STAT3) pathways. Additionally, targeted therapeutic agents developed based on these pathways are also briefly discussed.As mentioned above, both genetic and epigenetic alterations are involved in regulating tumorigenesis of CRC . The adeKRAS and BRAF), inactivation of TSGs (APC and TP53), and a loss of heterozygosity for the long arm of chromosome 18 (18q LOH), thus, promoting CRC tumorigenesis [Chromosomal instability refers to a significant increase in the gain or loss of either the entire or the large portions of chromosomes and is the most commonly occurring genetic instability in CRC. CIN is found in around 85% of adenocarcinoma transitions ,17 and iigenesis ,16.APC, followed by oncogenic KRAS mutations in the adenomatous stage, and, finally, deletion of chromosome 18q and inactivation of TP53 occurring during the transition to malignancy  syndrome (OMIM#: 608456), attenuated FAP, Gardner syndrome (OMIM#: 175100), Turcot syndrome (OMIM#: 276300), and other major hereditary predisposition events leading to CRC development [APC gene [APC mutations (frameshift or nonsense mutations) are present in more than 70–80% of sporadic CRCs and 5q LOH is reported in approximately 40% of CRC cases [ cancers ,34,35,36elopment . FAP, anelopment ,39. CIN elopment ,41,42,43APC gene . More imAPC gene . On the RC cases ,47,48.APC gene inhibits transition from the G0/G1 to the S phase of the cell cycle. The Wnt signaling pathway maintains the undifferentiated stem cells in the base of the colonic crypts, allowing survival of both normal and cancer stem cells [APC mutations are also present in sporadic CIN tumors. Activating mutations in the gene for β-catenin (CTNNB1) block APC-regulated breakdown and are present in colorectal neoplasia. Although CTNNB1 mutations are more common in adenomas (12.5%) than invasive cancer (1.4%), they are found in the preliminary stages of CRC pathogenesis and plausibly substitute APC mutations in cancer onset and progression [APC, the mitotic checkpoint protein BubR1 plays a vital role [In both familial and sporadic CRCs, the APC/β-catenin/Wnt-Tcf pathway plays a major role in the onset and progression of CRC carcinogenesis . The APCem cells . In thisem cells . The wilem cells ,52. Disrem cells . Mutatiogression ,55,56. Ital role . BubR1 ital role . Bindingtal role ,59, thustal role .cyclin D1 (CCND1) was implicated in APC signaling: Mutated APC cells activate downstream targets, such as cyclin D1 and Myc [CCND1, along with other cyclin-dependent kinases (CDKs) that block cyclins, such as p27 (CDKN1B) and p21 (CDKN1A), are vital for cell growth and apoptosis as well as cell cycle control, majorly during the transition from G1 to S phase [CCND1 by APC mutation leads to the onset of colonic neoplasia by allowing the cell to divert from apoptosis [CCND1 in normal colonic mucosa, adenoma, and adenocarcinoma and confirmed its high expression in colonic tumors, thus indicating CCND1 expression as an early event during multistage process of CRC tumorigenesis that may deregulate cell-cycle control in benign adenomas and stimulate tumor progression [Recently, activation of leucine-rich repeat-containing G-protein-coupled receptors (LGR-4 and LGR-5) triggered signaling by binding with proteins in the R-responding family ,62,63,64 and Myc ,66. CCND S phase . Prolongpoptosis . Arber agression .Notch1 stimulates the assembly of active β-catenin protein in the absence of ligand-receptor activation [CDK8 (cyclin-dependent kinase-8) gene amplification and occurs in over 60% of CRC cases. Increased CDK8 enhances both β-catenin [Notch1, thus stimulating transcription and cell differentiation [On the other hand, β-catenin activity can be indirectly triggered by aberrations in oncogenes controlling its activity at different levels. β-catenin mutually interacts with various members of the Notch pathway that are vital regulators of cell differentiation and play a role in CRC carcinogenesis ,71. Kwontivation . Further-catenin  and Notcntiation ,74.TP53 gene, located on the short arm of chromosome 17, is known as the “guardian of the genome” and encodes proteins regulating cell cycle, DNA repair, senescence, and apoptosis [TP53 mutations or loss of function are reported in 50–75% of CRC cases [p53-mediated pathways of apoptosis is an important determinant of progression from adenoma to malignant tumor [p53 loss of function enhances high cell proliferation activities and uncontrolled cell cycle, a key step in colorectal carcinogenesis [TP53 mutations in CRC [The poptosis . TP53 muRC cases ; loss ofnt tumor . The p53ogenesis . Researcs in CRC ,79, follp53 is considered a controller for BubR1 transcription and expression; loss of p53 expression downregulates BubR1, thus comprising checkpoint function to mitotic aberrations leading to progression of CRC [WAD-1, a gene highly induced to suppress tumor cell growth in the p53 pathway [Commonly, n of CRC . Additio pathway .CDK are likely to occur during CRC development and progression [p53 and induces AMPK-dependent cell-cycle arrest that upregulates the cyclin-dependent kinases inhibitor 1A (CDKN1A or p21). This eventually controls cell cycle regulation, cellular senescence, and stem cell aging [p21 occurs in a p53-dependent pathway; p21 also inhibits the activity of cyclin D1 [p21 is significantly associated with poor prognosis in CRC [TP53 loss of function is CDK inhibitor 1B (CDKN1B or p27) [TP53-negative cancers [Moreover, mutations in gression . Adenosill aging . Furtheryclin D1 . Loss ofs in CRC . Another or p27) . Specifi or p27) . It has  cancers .cyclooxygenase-2 (COX-2) and is involved in enhancing inflammation and CRC cell proliferation [COX-2-positive tumors are significantly linked with cancer-specific mortality regardless of p53 status, thus suggesting COX-2 as an independent CRC prognostic factor [The p53 also interacts with feration . Interesc factor ,89.Deleted in Colorectal Carcinoma (DCC), SMAD2, and SMAD4; loss of expression of 18q LOH plays a significant role in CRC pathogenesis [Loss of heterozygosity (LOH) refers to the absence of one of the two copies or alleles of a gene, with the remaining allele frequently being affected by mutation . LOH in ogenesis .DCC, located on chromosome 18q21.2, encodes netrin-1 and is indicated as a plausible TSG [DCC gene region is present in approximately 70% of CRCs. Moreover, a few somatic mutations in DCC are found in CRC [DCC gene inhibits binding of netrin-1 to DCC transmembrane protein, leading to abnormal cell survival [ible TSG ; LOH in d in CRC . Netrin-d in CRC . Furthersurvival .SMAD2 and SMAD4 are present on 18q21.1, the prevalent region lost during CRC progression, and correlate with adenoma development and adenocarcinoma progression in mice models, thus suggesting a plausible tumor suppressor role for SMAD genes [SMAD2 and SMAD4 is comparatively low in CRC [SMAD genes encode for TGF-β [TGF-βR1, TGF-βR2, and TGF-β superfamily members Activin Receptor type 2 (ACVR2) are reported in CRC [TGF-βR2 are present in ~30% of all CRC cases and correlate with malignant transformation of late adenomas. TGF-βR2 mutations are most frequent in MSI tumors; however, they are also present in around 15% of MSS tumors [On the other hand, AD genes ,96. FurtAD genes . Since tw in CRC , other Tor TGF-β . Dysreguor TGF-β . Additiod in CRC . FunctioS tumors ,20,101.Another type of genomic instability in CRC is microsatellite instability (MSI), a distinctive characteristic of cancerous cells . MSI is TGF-βR2, Bax, MSH3, ActRIIB, SEC63, AIM2, NADH-ubiquinone oxidoreductase, COBLL1, and EBP1) in >20% of tumors. TGF-βR2 was the most commonly mutated loci and instability in the poly-adenine tract of TGF-βR2 is present in approximately 85% of MSI-H CRCs [A subset of tumors with unstable loci in ≥30% markers are defined as “Microsatellite high” (MSI-H), a subset of tumors with 10–29% unstable loci are classified “Microsatellite low” (MSI-L), and “microsatellite stable” (MSS) is marked with no unstable markers . In cancI-H CRCs . MoreoveI-H CRCs .MLH1, MSH2, MSH6, and PMS2); mutations in MLH1 or MSH2 gene lead to an increased risk (70–80%) of developing cancer, while mutations in the MSH6 or PMS2 gene have a comparatively lower risk (25–60%) of cancer development [MLH1 silencing by aberrant DNA methylation [MSI is rarely found in polyps, except in Lynch syndrome ,110. Furelopment  On the chylation ,113.BRAFV600E mutations, on the contrary, Lynch syndrome arises from MMR genes germline mutations and lacks mutated BRAF [Furthermore, according to modeling studies and absence of colon cancer in individuals with biallelic germline mutations in MMR genes suggest that lack of MMR activity is insufficient to trigger polyp formation ,115. Relted BRAF ,118. BRAF-mutated CRC correlates with poor prognosis and overall survival (OS) in comparison to BRAF wild-type disease [BRAF-mutated CRC patients had worst OS as compared to patients carrying RAS (KRAS and NRAS) mutations [BRAF is a negative prognostic factor in stage II and III disease [BRAF V600E mutation is associated with worst OS, but not disease recurrence [BRAF-mutated metastatic CRC [BRAF-mutated (BRAF codons 594 and 596) CRCs have better prognosis as compared to BRAF-mutated CRCs; BRAF 594 and 496 tumors are microsatellite stable, rectal, non-mucinous with no peritoneal spread and have a significantly longer OS as compared to V600E BRAF-mutated tumors [BRAF-mutated tumors, non-V600E BRAF-mutated tumors are present in the younger population, lower grade tumors and the median OS is significantly longer compared with both V600E BRAF-mutant and BRAF wild-type patients [Clinically,  disease . Furtherutations . Also, m disease . On the currence . Moreoveatic CRC . It has d tumors . Similarpatients ,126.DNA methylation is the addition of a methyl group to cytosine in the 5′-position that is catalyzed by DNA methyltransferases via covalent linkage within a CG dinucleotide sequence within the promoter region, termed CpG transcription ,128. In APC, MLH1, MGMT, SFRP1, SFRP2, CDKN2A, TIMP3, VIM, SEPT, CDH1 and HLTF. Additionally, there is a distinct subset of CRCs, known as the CpG island methylator phenotype (CIMP) [BRAFV600E mutations [MSI, KRAS and BRAF mutations [MLH1, p16, MINT1, MINT2 and MINT31. Additional markers include CACNA1G, CRABP1, IGF2, NEUROG1, RUNX3, SOCS1, HIC1 and IGFBP3 for positive CIMP identification [Many genes are identified to be methylated and silenced in CRC, some commonly methylated ones include e (CIMP) ; CIMP tuutations . CIMP isutations ,136. DNAutations ,137. Thefication ,139.PTEN, a TSG, shows reduced methylation rates, TWIST1 gene is silenced by promoter methylation in CRC. Aside from all of the above-mentioned mechanisms of DNA hypermethylation, global DNA hypomethylation frequently occurs at repetitive sequences including LINE-1 repeats, retrotransposons, introns and gene deserts [Although, upregulated expression of the DNA methyltransferases (DNMT3B or DNMT1) is associated with CIMP, the underlying mechanism(s) that promote CIMP are still unknown . One of  deserts ,145.EGFR expression is present in various cancers including CRC [EGFR expression is present in 25–77% of CRC cancers [The epidermal growth factor receptor (EGFR) belongs to the ErbB/HER family and consists of four members; ErbB1 (EGFR/HER1), ErbB2 (Neu/HER2), ErbB3 (HER3), and ErbB4 (HER4) ,147. Actding CRC ; increas cancers ,152. Act cancers ,154. KRAS and BRAF; the RAS/RAF/MAPK pathway is involved in regulating cell proliferation, differentiation, apoptosis and senescence [RAS provokes cellular proliferation, thus, promoting cell survival and tumor invasion and metastasis [KRAS mutations include codons 12 and 13 on exon 2 and codon 61 on exon 3; codon 12 being the most frequently affected through missense mutations [KRAS along with NRAS and HRAS are oncogenes belonging to the RAS family [KRAS is commonly mutated in sporadic CRCs (35–45%) [KRAS mutations occur after APC mutations [The MAPK pathway includes nescence . Activatnescence ,156. PI3tastasis . KRAS muutations  includinutations , of whicutations . KRAS alS family . KRAS is(35–45%) ,162,163 (35–45%) ,165,166;utations . BRAF, a member of RAF family of serine/threonine kinases regulates cellular responses to growth signals through the RAS-RAF-MAP kinase pathway [BRAF are found in approximately 5–10% of metastatic CRC, however, they are rare in Lynch syndrome forms of CRC [BRAF mutations were identified in 40% of MSI-H and 4% of MSI-L tumors [BRAF mutations include the hotspot mutation, V600E  and is found to correlate with poor prognosis in CRC patients [On the other hand,  pathway . Activats of CRC ,168. MorL tumors . The majpatients ,171.KRAS and TP53 as well as COX-2 expression regulate VEGF-VEGFR activity alteration, thus promoting cancer growth and migration [Angiogenesis, the development of new blood vessels, is involved in tumor initiation, growth, and metastasis and involves several factors including vascular endothelial growth factors (VEGFs). In CRC, VEGF levels and VEGFR activity is enhanced and is associated with poor prognosis . Elevateigration ,174. TheMolecular profiling has helped in developing biomarkers that can potentially improve clinical outcomes in CRC patients and significantly enhance the survival of metastatic patients. Several molecular aberrations are defined for candidate biomarkers that have been tested in completed and ongoing trials in conjunction with targeted therapies and immunotherapies.CTNNB1 by inhibiting GSK3. Moreover, non-steroidal anti-inflammatory drugs (NSAIDs) and celecoxib, the selective COX-2, block CTNNB1-dependent transcription in CRC [There are intense efforts to develop synthetic modulators of Wnt signaling including small molecules, peptides, and blocking antibodies to inhibit Wnt pathway . As apprn in CRC ,177 and n in CRC ,179. Recn in CRC ,181. On n in CRC . In compn in CRC ,184. In n in CRC . In addin in CRC ,187.SMAD4 expression levels correlate with prognosis and response to 5-fluorouracil (5-FU) [On the other hand, no definite clinical role for TGF-β signaling pathway has been identified thus far; however, studies show that l (5-FU) ,189,190.l (5-FU) . They all (5-FU) . Remarkal (5-FU) ,192.MSI status has been demonstrated to be a reliable biomarker of immunotherapy response in the metastatic setting. Currently, checkpoint inhibitors are investigated in various solid tumors with promising responses ,194. PemRAS, BRAF, or PIK3CA mutations, loss of PTEN, HER-2 amplification, and altered VEGF and VEGFR signaling are resistant to anti-EGFR therapy due to continuous activation of EGFR downstream signaling pathways [Targeting the EGFR pathway generally involves use of anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors (TKIs) aimed at intracellular kinases. Cetuximab was the first monoclonal antibody introduced to target EGFR. Cetuximab provokes EGFR internalization and degradation once bound to the external domain of EGFR . Multiplpathways . In thespathways . In the pathways ,207. PRIpathways . In addipathways . Althougpathways .KRAS-mutated CRC. However, a novel agent, AMG 510, which is a small molecule that targets KRASG12 C mutation, was introduced. AMG 510 specifically and irreversibly blocks KRASG12 C by locking it in the inactive GDP-bound state [KRAS-modulating agents targeting G12C  and G12D mutations are also under investigations [On the other hand, there are no approved specific targeted therapies for nd state . In addiigations ,213.BRAF mutations are more frequent in melanoma and papillary thyroid carcinoma in comparison with CRC, a few studies analyzed the effects of BRAF inhibitors (vemurafenib or dabrafenib and trametinib) in patients with metastatic CRC. Although downstream MAPK activity was inhibited, the PFS or OS of patients did not improve [BRAF and EGFR inhibitors in trials using vemurafenib in combination with IRI and cetuximab in BRAF-mutant CRC patients showed positive results [BRAF-mutant CRC patients [BRAF-V600E-mutated CRC, when treated with a triplet regimen , produced a better response rate in comparison with the double regimens (dabrafenib + panitumumab or trametinib + panitumumab). The ongoing BEACON trial, which employs the triplet regimen of encorafenib, binimetinib (a MEK inhibitor), and cetuximab, has shown less toxicity and higher survival rates [Although  improve ,215,216. results ,219,220.patients . Furtherpatients  found thal rates .KRAS-mutant patients were also found to benefit from bevacizumab [Anti-VEGF/VEGFR therapies are necessary to target steps in tumor metastasis. Bevacizumab (Avistin), a humanized IgG monoclonal antibody, was the first FDA-approved VEGF-targeted agent for metastatic CRC. According to several trials, bevacizumab showed to improve both progression-free survival and OS in metastatic CRC ,224. Furacizumab ,226. Moracizumab .This brief review provides information about candidate biomarkers that can aid in improving the diagnosis and help with the early detection of CRC cases, thus ameliorating the prognosis of CRC patients. Although there are marked advances in CRC investigations, the role of molecular classification in therapeutic interventions remains unclear. The use of molecular alterations in predicting risk for CRC shows promise, while further studies are needed to determine if aberrantly methylated CpGs or other molecular alterations can be used as reliable and accurate indicators of risk for polyps or CRCs. Moreover, it is important to analyze the efficacy of multi-kinase/BRAF-inhibitor, sorafenib, in addition to other specific inhibitors of the EGFR as well as PI3K signaling pathway, in the treatment of CRC to further identify novel therapeutic targets. On the other hand, we believe that using new drug combinations and specific  targets will be the future avenue for efficient treatment of CRC patients with limited risk of toxicity and adverse side effects. Thus, understanding the underlying mechanisms of CRC cell genetic alteration and subsequent consequences can help pave the way for the development of novel diagnostic and therapeutic strategies.


Lasiopodomys brandtii was determined using Illumina NovaSeq platform. The assembled genome was 16,557 bp in length and included 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. The total nucleotide composition frequencies present clearly the A-T skew (59.5%), which mostly in D-loop and PCGs regions. Whole mt genome phylogenetic analysis revealed a closely related among Lasiopodomys, Proedromys, and Microtus with high support. It would provide further evolutionary research for the subfamily Arvicolinae.In this study, the complete mitochondrial (mt) genome sequence of  Lasiopodomys brandtii), known as the colonial rodent species of the typical temperate grassland, which is distributed Inner Mongolia of China, east of the Republic of Mongolia, southern of Russia  based on molecular phylogenetic , which was collected from the field of Huairou, Beijing, China . The total genomic DNA was extracted from the L. brandtii muscle tissue using TIANamp Genomic DNA Extraction Kit , sequenced using Illumina NovaSeq 6000 , assembled using NOVOPlasty version 3.6  genome is 16,557 bp (GenBank accession number: MN614478), contains 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and a control region (D-loop). Among these genes, All PCGs began with ATN codons , except for ND1 with GTG as the alternative initiation codons. The typical stop codon TAA has been assigned to 10 PCGs, BESIDES, ND1, and ND6 are terminated with TAG, COX3 has TAC stop codon. The total nucleotide composition frequencies of L. brandtii mt genome were A (32.1%), T (27.4%), C (26.4%), and G (14.0%), clearly emerge the A/T skew, which was generally apparently in the D-loop and PCGs.In this study, L. brandtii, we used complete mitochondrial genome sequence include eight related species (two sequences of L. mandarinus) and Cricetulus kamensis as the outgroup derived from NCBI. The phylogenetic analysis was used BEAST version 1.8.3 software to construct a Bayesian inference with 50,000,000 generations using default parameters . In summary, this study confirms the mt genome sequence would be an important marker for the Lasiopodomys and related genus evolutionary.In order to investigate the phylogenetic position of le clade . Both of


Sodium–glucose co-transporter 2 (SGLT2) inhibitors are a new family of antidiabetic drugs that reduce blood glucose independent of insulin. In this review, we present the advantages and adverse effects of SGLT2 inhibitors plus insulin therapy as a treatment regimen for patients with type 2 diabetes (T2D). Compared with placebo, SGLT2 inhibitors plus insulin therapy could significantly decrease fasting blood glucose and HbA1c, thereby reducing the daily required dose of insulin. A reduction in body weight and improvements in insulin resistance and β-cell function have also been widely reported with this therapy, and other potential advantages, including the reduction in blood pressure, adverse cardiovascular outcomes, and visceral adipose tissue volume, have been revealed. SGLT2 inhibitors cause a greater reduction than dipeptidyl peptidase-4 (DPP-4) inhibitors in body weight and the risk of cardiovascular disease. Furthermore, compared with glucagon-like peptide-1 (GLP-1) agonists, SGLT2 inhibitors reduce blood pressure, and heart failure. As this therapy is an oral preparation, an improvement in patient compliance is also achieved. Despite these advantages, however, combination therapy with SGLT2 inhibitors and insulin has several risks. Although no difference has been found in the incidence of hypoglycemic events and urinary tract infection between the administration of this combination and that of placebo, the risk of genital tract infections was reported to increase with the combination therapy. Additionally, bone adverse effects, euglycemic diabetic ketoacidosis, and volume depletion—and osmotic diuresis—related adverse effects have been observed. Altogether, we could conclude that SGLT2 inhibitors plus insulin therapy is an efficient treatment option for patients with T2D, especially those requiring high daily insulin doses and those with insulin resistance, obesity, and a high risk of cardiovascular events. However, careful monitoring of the adverse effects of this combination is also warranted. The incidence of type 2 diabetes (T2D) is continuously increasing. Owing to acute complications, such as ketoacidosis and hypertonic coma, or chronic complications, such as nephropathy, vasculopathy, neuropathy, and retinopathy, the health and quality of life of patients with T2D have been severely affected, thereby increasing the health burden. Although numerous oral antidiabetic drugs are available, including sulfonylureas, meglitinides, metformin, thiazolidinediones, glucagon-like peptide-1 (GLP-1) agonists, and dipeptidyl peptidase-4 (DPP-4) inhibitors, maintaining long-term optimal blood glucose control has been difficult in most patients with T2D, even those administered with antidiabetics plus insulin . ChronicSodium–glucose co-transporter 2 (SGLT2) inhibitors are a new family of antidiabetic drugs that reduce blood glucose independent of insulin sensitivity and secretion. As a result, these inhibitors differ from other oral antidiabetic drugs. Currently, there are four types of SGLT2 inhibitors available in Europe, America, and Japan, namely, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, the first three of which are also available in China. Three other types of SGLT2 inhibitors, namely, tofogliflozin, ipragliflozin, and luseogliflozin are also available in Japan. Among the different SGLT2 inhibitors, empagliflozin is 2,500-fold more selective for SGLT2 than SGLT1; whereas tofogliflozin, dapagliflozin, ipragliflozin, and canagliflozin are 1,875, 1,200, 550, and 250-fold, respectively, more selective for SGLT2 than SGLT1 . DapagliThe insulin-independent hypoglycemic mechanism indicates that theoretically, SGLT2 inhibitors might be effective in patients with any stage of diabetes and particularly effective in those with severe insulin resistance and receiving high-dosage insulin therapy. Previously, SGLT2 inhibitors plus insulin therapy was reported to improve glycemic control, reduce daily insulin requirements, and mitigate insulin-related weight gain. However, the side effects of SGLT2 inhibitors should be considered.In this review, we aimed to elucidate the benefits and risks of SGLT2 inhibitors plus insulin therapy on patients with T2D and identify patients that may benefit from the use of SGLT2 inhibitors as a first-line therapy.n = 1,000, MD−1.35%, 95% confidence interval (CI)−2.36 to −0.34; p = 0.009]. Additionally, a minor but significant reduction in fasting blood glucose (FBG) level  was observed  vs. −0.73%  after 18 weeks and 0.58%  vs. 0.73%  after 52 weeks. Additionally, a significant decrease in the level of FBG was observed after 18 weeks  and 52 weeks    Table 1p < 0.001Recent head-to-head studies reported that GLP-1 agonists cause superior reduction in the level of HbA1c relative to SGLT2 inhibitors –17. Howen = 813, MD 4.85 U/24 h, 95% CI 2.29–7.42, p = 0.002) in the placebo-controlled trials  after a group administered with SGLT2 inhibitors plus insulin was compared with a group administered with placebo plus insulin –13 Tabl. Harris p < 0.001 and WMD 1.86 IU/day, 95% CI 0.45–3.27 IU/day, p = 0.010, respectively). However, the differences in the insulin-reducing effects between the SGLT2 and DPP-4 inhibitors were not statistically significant -induced hepatic steatosis and weight gain were inhibited in rats . After tDeFronzo et al. employed a glucose clamp test to measure insulin resistance in patients with T2D after 2 weeks of treatment with SGLT2 inhibitors. Compared with the placebo group, muscle glucose uptake was significantly increased in the SGLT2 inhibitor group, indicating an improvement in peripheral insulin resistance . FerrannA study using a rat model of T2D with pancreatectomy found that chronic glycosuria induced by phlorizin improved insulin resistance and restored β-cell function . In SGLTp < 0.001] and hospitalization due to heart failure by 35% . In this trial, ~48% of patients were administered with insulin. The HR for primary composite outcome in the subgroup of insulin users was 0.93 (95% CI 0.75–1.13)  and heart failure . No statistically significant difference in cardiovascular outcomes was found between the different SGLT2 inhibitors  0.62, 95% CI 0.49–0.77, 75–1.13) . A 2016 hibitors . Clinican = 22,830) to users of other hypoglycemic drugs , the study found that compared with other hypoglycemic drugs, the SGLT2 inhibitors decreased major adverse cardiovascular events , cardiovascular disease mortality , and hospital events due to heart failure   . A compa 0.0001) .None of the current studies sought to directly compare combination therapy with SGLT2 inhibitors and insulin with monotherapy with SGLT2 inhibitors on cardiovascular outcomes. Therefore, whether SGLT2 inhibitors plus insulin combination therapy could affect the notable improvement in cardiovascular outcomes achieved with SGLT2 inhibitor monotherapy remains unclear. Nonetheless, SGLT2 inhibitors plus insulin therapy has definitive cardiovascular benefits compared with those of monotherapy with insulin or combination therapy with insulin and other drugs.In the trials performed with dapagliflozin and canagliflozin combined with insulin, the albumin-to-creatinine ratio was significantly improved , 12. CanThe kidneys play a key role in glucose metabolism. After glucose filtration by the glomerulus, glucose is reabsorbed in the proximal tubule. Thereafter, it is transported through the phospholipid bilayer of the cell membrane via glucose transporters. SGLTs are a class of transporters found in the small intestinal mucosa and proximal convoluted tubules of the kidney. Of these transporters, SGLT2 is predominant . SGLT2 iIn patients with T2D, the expression level of SGLT2 is enhanced in renal cells . BlockinBody weight loss is not only due to the calorie loss induced by glycosuria and negative energy balance  but alsoCurrent studies have analyzed the potential mechanisms whereby SGLT2 inhibitors improve insulin resistance and β-cell function in patients with T2D  Figure .+ T cells infiltrate the tissue and interact with macrophages to form an incommensurate feedforward loop, causing fat inflammation and insulin resistance   . Howeverp = 0.28); however, the risk of GTI was higher in the former group  than in the latter group  and urinary tract infection (UTI) are the major adverse effects of SGLT2 inhibitors. A meta-analysis reported that UTI and GTI were more common with the administration of SGLT2 inhibitors than with placebo  . In a meer group .Many studies have shown that the risk of ketoacidosis might increase in patients administered with SGLT2 inhibitors , 82, a pThe FDA has added the increased risk of fracture and decreased bone mineral density (BMD) as adverse effects on the canagliflozin label. Both animal studies and clinical trials have revealed that canagliflozin might affect bone microarchitecture, increase bone resorption, and reduce total hip BMD –85, whicStudies on other types of SGLT2 inhibitors, such as ertugliflozin and dapagliflozin, did not identify their detrimental effects on bone turnover and BMD –88. HoweA decrease in estimated glomerular filtration rate (eGFR) level occurred during the initial treatment with canagliflozin; this decrease was weakened over time and was recognized to be unrelated to the severe renal adverse events . Other rIn this review, we have summarized the findings of current studies on SGLT2 inhibitors. We have reported the advantages and adverse effects of SGLT2 inhibitors plus insulin therapy for the treatment of T2D . SGLT2 iSGLT2 inhibitors combined with insulin might serve as a promising therapy for the treatment of T2D. SGLT2 inhibitors have several advantages, and when combined with insulin, a significant decrease in HbA1c and FBG levels is achieved, enabling a reduction in the daily administered with insulin dose. Additionally, these inhibitors reduce body weight via glycosuria-induced calorie loss and negative energy balance, thereby increasing fat and sugar utilization and β-oxidation of fatty acids. SGLT2 inhibitors polarize M2 macrophages, affect adipocyte-derived hormones, and promote the expression of different β-cell-related factors, consequently improving insulin resistance and β-cell function. Other potential advantages of these inhibitors include a reduction in blood pressure, cardiovascular events, and visceral adipose tissue volume. SGLT2 inhibitors exhibit better glycemic control and cause higher body weight loss, higher blood pressure reduction, and lower cardiovascular events than do DPP-4 inhibitors. However, these inhibitors exhibit similar insulin-reducing effects. Although SGLT2 inhibitors caused less HbA1c reduction and, controversially, body weight loss than did GLP-1 agonists, their reduction of blood pressure and heart failure was more significant than those of the GLP-1 agonists. Further, as SGLT2 inhibitors are administered orally, therapy compliance can be improved.The main risk of treatment with SGLT2 inhibitors combined with insulin therapy is mild GTIs; however, a good response is achieved with therapy. No difference in the incidence of hypoglycemic events and UTI was noted by treatment with the inhibitors compared to placebo. As euglycemic diabetic ketoacidosis, bone adverse effects, and other adverse effects have been reported, further studies are warranted. According to the findings of recent studies, treatment with SGLT2 inhibitors plus insulin is a suitable regimen for patients with T2D, especially those requiring a high insulin dose daily and those with insulin resistance, obesity, or high risks of cardiovascular disease. Large-scale and long-term clinical studies are, however, needed to confirm such findings.All authors contributed equally to the writing, revision, and editing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Herpesvirus egress mechanisms are strongly associated with intracellular compartment remodeling processes. Previously, we and other groups have described that intracellular compartments derived from the Golgi apparatus are the maturation sites of Epstein-Barr virus (EBV) virions. However, the mechanism by which these virions are released from the host cell to the extracellular milieu is poorly understood. Here, I adapted two independent induction systems of the EBV lytic cycle in vitro, in the context of Rab GTPase silencing, to characterize the EBV release pathway. Immunofluorescence staining revealed that p350/220, the major EBV glycoprotein, partially co-localized with three Rab GTPases: Rab8a, Rab10, and Rab11a. Furthermore, the knockdown of these Rab GTPases promoted the intracellular accumulation of viral structural proteins by inhibiting its distribution to the plasma membrane. Finally, the knockdown of the Rab8a, Rab10, and Rab11a proteins suppressed the release of EBV infectious virions. Taken together, these findings support the hypothesis that mature EBV virions are released from infected cells to the extracellular milieu via the secretory pathway, as well as providing new insights into the EBV life cycle. Epstein–Barr virus (EBV) is a ubiquitous human gammaherpesvirus that infects the majority of the world population. EBV infections are persistent, but mostly asymptomatic. However, they are also associated with various lymphoid and epithelial malignancies, such as Burkitt’s lymphoma (BL), Hodgkin’s disease, gastric carcinoma, and nasopharyngeal carcinoma . SimilarDuring a productive infection, the newly synthesized viral genomes are packaged into capsids in the nucleoplasm, which then bud through the inner nuclear membrane into the perinuclear space, acquiring their primary envelope. This envelope is then lost by fusion with the outer nuclear membrane, leading to the release of the nucleocapsids into the cytoplasm. In the cytoplasm, these de-enveloped nucleocapsids acquire a series of tegument proteins and then their secondary envelope by budding into different cellular compartments, producing the mature virions . While aUltimately, herpesviruses are released by fusion of the enveloped virions with the plasma membranes (PM). However, effective viral release depends not only on these final fusion events, but also on the transport mechanisms that allow the mature virions to reach the PM. A few studies have characterized the alphaherpesviruses release mechanism. Using live-cell fluorescence microscopy, Hogue and colleagues demonstrated that vesicles containing pseudorabies virus (PRV) were associated with small Rab GTPases, such as Rab6a, Rab8b, and Rab11a, involved in the secretory pathway-mediated vesicular transport. These vesicles frequently appeared near patches of LL5b, component of a complex that anchors microtubule plus ends to the PM, suggesting that their intracellular trafficking was microtubule-mediated . AdditioHere, I adapted two independent induction-systems of the EBV lytic cycle in vitro ,36,37,38+) cells were derived from a Japanese BL patient [−) were isolated from African BL-derived EBV-positive Daudi cells, by limiting dilution [+ and Daudi− cells were maintained in RPMI-1640 medium containing 10% FBS and antibiotics at 37 °C in a 5% CO2 atmosphere. Human embryonic kidney 293 cells, which were modified to be an inducible system of EBV lytic infection (i293/2089), were maintained in Dulbecco’s modified eagle medium (DMEM) containing 10% FBS, antibiotics, 1 µg/mL puromycin, and 100 µg/mL hygromycin at 37 °C with a 5% CO2 atmosphere. This cell line carries a BACmid coding for the complete EBV genome (B95.8 strain), green fluorescent protein (GFP), and hygromycin B resistance gene [EBV-positive Akata , were cultured for 48 h in the presence of 1% goat anti-human IgG F(ab’)2 polyclonal antibody  [5 cells/well), were cultured for 48 h with 100 nM 4-hydroxytamoxifen  [The EBV lytic cycle was induced in AkataCA, USA) ,41. i293MO, USA) ,38. Lyti+ or i293/2089 cells were transfected with siRNAs encoding the corresponding target sequences: 5′-GAGUCAAAAUCACACCGGA-3′ (siRab8a), 5′-GGACGACAAAAGAGUUGUA-3′ (siRab10), or 5′-GAGAUUUACCGCAUUGUUU-3′ (siRab11a) . As a control, a siRNA encoding a mock-sequence (Thermo Fisher Scientific) was used. Transfection was performed using the Neon Transfection System (Thermo Fisher Scientific) for Akata+ cells and the TransIT-TKO reagent  for i293/2089 cells, according to the manufacturers’ instructions. Rab GTPases downregulation was confirmed by Western blotting. Transfected Akata+ cells were used to evaluate distribution of gp350/220 by immunofluorescence staining. Transfected i293/2089 cells were used for analysis of the EBV virion secretion.To knockdown Rab8a, Rab10, or Rab11a gene expression, AkataWestern blotting was performed as previously . Inducti+ cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized with PBS containing 0.05% Triton X-100 for 10 min and blocked in PBS containing 1% bovine serum albumin (BSA) for 20 min, all at room temperature (r.t.). Cells were then incubated with mouse anti-EBV gp350/220 monoclonal antibody (clone C-1) [2 and/or Alexa Fluor 488-labeled anti-rabbit IgG F(ab’)2  diluted at 1:1000 in PBS for 1 h at r.t. After washing in PBS, the nuclei were counterstained with Hoechst 33342 . Preparations were observed with a 60 × oil-immersion objective (NA = 1.3) using a confocal laser scanning microscope  and acquired using the FV10-ASW software (Olympus). Images of four randomly selected independent fields containing between 5 and 10 gp350/220-positive cells (per condition) were captured. Line scan imaging and measurements of the integrated fluorescent intensities in defined regions of interest were performed using the FV10-ASW software (Olympus).Akataone C-1) , rabbit − cells (2 × 105 cells/condition) for 1 h at 37 °C. Cells were then washed in culture media and cultured for 48 h. The percentage of GFP-positive cells was then measured by flow cytometry using a FACSCalibur (BD).i293/2089 cell supernatants were harvested after EBV lytic cycle induction and incubated with Daudip < 0.05.The results are representative of three independent experiments. Statistical differences were assessed by the Student’s t-test and determined significant when + cells by cross-linking their cell-surface IgG molecules [In living cells, synthesized proteins are properly distributed to the distinct organelles, the PM, or even to the extracellular milieu via secretion. The TGN is a key station of the constitutive secretory pathway, which is responsible for these sorting processes in all cell types . To undeolecules ,41 and tolecules ,45, knowolecules , suggestLooking at Rab8a, a small GTPase that regulates secretory vesicle transport from the TGN to the basolateral PM of epithelial cells, neuronal dendrites, and cilia ,47, was,+ cells and the distribution of EBV glycoprotein was evaluated by immunofluorescence. The knockdown of the target proteins was confirmed by Western blot analysis . These results indicate that EBV infectious virions are released from the cell compartments where they acquire their final envelopment via the secretory pathway.To confirm the role of the secretory pathway in the release of infectious EBV virions, the EBV lytic cycle was induced in i293/2089 transfected with siRNAs targeting each individual Rab protein C. Two da virions A,B, as eAccumulating evidence indicates that herpesvirus subfamilies share a mechanism for the maturation and egress of their progeny virions ,3,4. HowThis work characterized the mechanism of EBV virion release into the extracellular milieu. Immunofluorescence staining revealed that small GTPases involved in the cellular secretory pathway, such as Rab8a, Rab10, and Rab11a, partially co-localized with a viral glycoprotein . FurtherBecause Rab GTPases control vesicle budding and fusion events, and because of their motility via the recruitment of effectors, such as sorting adaptors, tethering factors, enzymes, and motor proteins, they have been used to understand the dynamics of intracellular membrane trafficking . HoweverTrafficking of intracellular organelles is tightly regulated by the microtubule cytoskeleton. Various microtubule-binding proteins, responsible for microtubule-dependent intracellular membrane dynamics, were identified. Interestingly, microtubules were reported to be responsible for the intracellular trafficking of nascent alphaherpesviruses-containing vesicles ,30, sugg+ T cells to polarize the distribution of viral antigens towards the VS [The efficiency of EBV infection in epithelial cells is significantly enhanced in the context of co-cultures ,35,55, ss the VS . Howevers the VS . We alsoTaken together, the findings suggest that mature EBV virions, with secondary envelopes derived from the Golgi apparatus, are released into the extracellular milieu via the secretory pathway, as well as providing new insights into the EBV life cycle in general.


Paeonia suffruticosa Andr., Paeonia lactiflora Pall., or Paeonia veitchii Lynch and has been used in traditional medical applications for more than 2,000 years. Paeoniflorin is a monoterpenoid glycoside with various effects on liver diseases. Recent studies have revealed that paeoniflorin demonstrates a wide range of activities, including hepatic protection, cholestasis alleviation, liver fibrosis attenuation, nonalcoholic fatty liver disease prevention, and hepatocellular carcinoma inhibition involved in multiple pathways. Moreover, anti-inflammation, antioxidation, and immune regulation with the regulation of TLR4-NF-κB, ROCK/NF-κB, HO-1, mitochondria-dependent as well as HMGB1‐TLR4 signaling pathways are correlated with hepatic protection in liver injury and nonalcoholic fatty liver disease. Antioxidative mechanisms, anti-inflammation, and hepatic transporter regulation involved in NOX4, PI3K/Akt/Nrf2, NF‐κB, NTCP, BSEP, as well as MRP2 signals are mainly relevant to the anticholestatic effect of paeoniflorin. The inhibition of hepatic stellate cell activation and alleviation of extracellular matrix deposition via vast signals such as mTOR/HIF-1α, TGF-β1/Smads, and JAK2/STAT6 are primarily involved in the antifibrotic effect of paeoniflorin. The regulation of macrophages also contributes to the alleviation effect on liver fibrosis. In addition, the reduction of invasion, metastasis, and adhesion and the induction of apoptosis-related targets, including Bax, Bcl-2, and caspase-3, are related to its effect on hepatocellular carcinoma. The literature indicates that paeoniflorin might have potent efficacy in complex liver diseases and demonstrates the profound medicinal value of paeoniflorin.Paeoniflorin is derived from  Current4), concanavalin A (Con-A), D-galactosamine , bacillus Calmette-Guérin (BCG), and lipopolysaccharide (LPS), that can induce liver injury  excretion  also confirmed this result. Paeoniflorin at doses from 50 to 800 μM most likely contributed to the alleviation of Con-A-induced inflammation in HHSECs. Preincubation with paeoniflorin caused a concentration-dependent downregulation of IL-8. Furthermore, paeoniflorin was able to inhibit IL-8 release by 52.8% at a dose of 800 μM. The mechanism might be closely related to blocking IL-8 secretion via the downregulation of ERK1/2 and Akt phosphorylation , bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2), were restored by paeoniflorin treatment (A series of studies from Zhao's group indicated that paeoniflorin exerts a dose-dependent (50–200 mg/kg) protective effect on alpha-naphthylisothiocyanate (ANIT)-induced cholestasis in rats by decreasing serum ALT, AST, TBIL, DBIL, total bile acid (TBA), γ-glutamyltranspeptidase (γ-GT), and alkaline phosphatase (ALP). Moreover, the extremely suppressed bile flow induced by ANIT was also increased by paeoniflorin treatment. The mechanism of this activity is partially related to attenuating oxidative stress with reactive oxygen species (ROS) inhibition by suppressing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 expression and the mitochondria-dependent pathway . In addiresponse . Anotherresponse ]. Therefreatment .Liver fibrosis is the process of chronic liver injury caused by hepatitis B and C, alcohol consumption, fatty liver disease, cholestasis, and autoimmune hepatitis . Hepatic4-induced liver fibrosis model, paeoniflorin was proven to effectively attenuate serum ALT, AST, HA, IV-C, and liver tissue Hyp at the doses of 20, 40, 80, and 200 mg/kg  has been the most common chronic liver disease worldwide in recent years. More than 40% of the population is affected in some countries. NAFLD has attracted concern worldwide since becoming a public health burden . NAFLD ivia the lipid metabolism pathway. On the other hand, paeoniflorin treatment also exerted insulin sensitizing effects via IRS/Akt/GSK3β and antioxidation  is believed to be the most common and malignant type of tumor. HCC is the third most common cancer-related cause of death due to poor prognosis. Over 700,000 HCC cases are diagnosed every year . The sitin vitro study, paeoniflorin at the doses of 6.25–200 μM was found to significantly inhibit the growth of HepG2 and Bel-7402 cell lines. Proteolysis could reduce the invasion, metastasis, and adhesion of HCC cell lines. In addition, paeoniflorin was able to decrease MMP-9 and ERK levels and increase E-cad expression in HepG2 and Bel-7402 cells , Sichuan Science and Technology Program (2019YJ0492), China Postdoctoral Science Found Grant (2017M622987), and Chengdu University of TCM Found Grant (QNXZ2018025).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Subchromosomal deletions and duplications could currently be detected by noninvasive preliminary screening (NIPS). However, NIPS is a screening test that requires further diagnosis. Here we report a fetus with an autosomal abnormality revealed by NIPS and conventional karyotype combined with copy number variations sequencing (CNV-seq) confirmed the fetus with an unbalanced translocation.This was the fourth pregnancy of a 30-year-old woman who underwent 2 spontaneous abortions and gave birth to a child with a normal phenotype. The woman and her husband were healthy and nonconsanguineous. NIPS indicated a repeat of about 19-Mb fragment at the region of 16q22.1-q22.4 at 17-week gestation.The combination of traditional karyotype and CNV-seq could better locate the abnormal chromosomal region and further identify the source of fetal chromosomal abnormalities. Simultaneously, we evaluated the fetal morphology by ultrasound examination. The karyotype of the fetus was 46,XX,der(7)t and CNV-seq results showed an approximately 20.96-Mb duplication in 16q22.1-q24.3 (69200001-90160000) and an approximately 3.86-Mb deletion in 7p22.3-p22.2 (40001-3900000). Prenatal ultrasound revealed the fetal micrognathia. The paternal karyotype was 46,XY, t  , while the maternal was normal. The fetus inherited an abnormal chromosome 7 from its father.No treatment for the fetus.Pregnancy was terminated.To our knowledge, the occurrence of de novo partial trisomy 16q (16q22.1-qter) and partial monosomy 7p (7p22.2-pter) has not previously been reported up to now. Here, we present the perinatal findings of such a case and a review of the literatures. CNV-seq combined with karyotype is a useful tool for chromosomal abnormalities indicated by NIPS. However, due to low production of cell-free fetal DNA from placental origin or low sequencing depth, NIPS yields false negative and false positive results in the detection of CNVs. Therefore, further diagnostic means is needed. CNV-seq is a high-resolution whole-genome screening technology that can be used to analyze the presence of CNVs. In this study, we report a fetus with a 19-Mb duplication on chromosome 16 indicated by NIPS, who was conformed to be a paternal balanced translocator through amniocentesis karyotype, CNV-seq, and the genetic analysis of proband family members. Meanwhile, we analyze the ultrasonic abnormal phenotype possibly related to the result of CNV-seq in molecular biology genetics.Noninvasive preliminary screening (NIPS), also known as noninvasive prenatal screening, is based on the analysis of cell-free fetal DNA in maternal blood. Since its introduction in 2011, NIPS for fetal aneuploidy has rapidly become a first-level screening test in clinical practice. The main advantages of NIPS are its good sensitivity and specificity to trisomy 21, 13, and 18.2This was the fourth pregnancy of a 30-year-old gravida 4, para 1, healthy woman who had experienced 2 spontaneous abortions and delivered a phenotypically normal child. The woman and her husband were healthy and nonconsanguineous. When the second missed abortion occurred, the doctor recommended that the couple have the peripheral blood karyotype test. However, the couple refused because they had decided not to have children any more. However, an unplanned pregnancy happened. NIPS indicated a repeat of about 19-Mb fragment at the region of 16q22.1-q22.4 at 17-week gestation. After consultation, she opted for prenatal karyotyping by amniocentesis. Amniotic cells cultured by G-banding showed an additional unidentified substance on chromosome 7  of the 51 positive cases (0.63%) were true positive. Therefore, all abnormal NIPS results are required further prenatal diagnosis. Karyotype analysis is a classical, commonly used approach in prenatal diagnosis, and specific chromosomal structures such as balanced reciprocal translocations can be identified at relatively low cost. However, due to the low resolution, this method has obvious limitations in detecting CNVs. In contrast, CNV-seq has a higher resolution and can effectively detect chromosomal microdeletions and microduplications. However, CNV-seq fails to discover low levels of chromosomal mosaicism and balanced reciprocal translocations. Here, the fetus with 16q duplication and 7p deletion due to paternal balanced translocation was detected by traditional karyotyping combined with CNV-seq. The occurrence of concurrent partial trisomy 16q (16q22.1-qter) and partial monosomy 7p (7p22.2-pter) has not previously been reported up to now.More recently, some laboratories have expanded NIPS to detect CNVs.–6 Prenatal diagnosis of trisomy 16 or trisomy 16q is uncommon. Since the first case was reported, 47 patients with partial trisomy 16q or trisomy 16q have been reported. We reviewed 47 cases and summarized that the most common malformations of partial trisomy 16q were low birth weight, hypotonia, failure to thrive, high/prominent forehead, broad/depressed nasal bridge, low set/dysplastic ears, micrognathia, foot deformity, congenital heart disease and mental/psychomotor retardation  have been reported in the literature.–34 The clinical picture of the distal duplication of 16q tends to be closer to that of complete duplication(16q) and to be severer than the proximal duplication, since the heterochromatic region 16q11-q12 has no effect on the phenotype. Houlston RS described an infant with a de novo karyotype of 46,XX,der(15)t had multiple malformations such as intrauterine growth retardation, dislocation of the left knee and hip, stiffness of the elbow joints, choanal atresia, high forehead, narrow palpebral fissures, antimongoloid slant, midface hypoplasia, micrognathia, high-arched palate, anal stenosis, blind ending sinus in the coccygeal area, small 5th fingers, delayed motor development. In our case, the fetal ultrasound also showed microjaw deformity. Therefore, we speculated that micrognathia might be caused by partial trisomy 16q. In the present case, the region of 16q22.1–16q24.3 contains >150 Online Mendelian Inheritance in Man (OMIM) genes including 57 morbidity-associated genes (Table https://decipher.sanger.ac.uk/browser). Chromatin licensing and DNA replication factor 1 (CDT1) (OMIM: 605525), located at 16q24.3, is required for DNA replication at multiple stages of development and mitosis. CDT1 has been indicated to be associated with Meier-Gorlin syndrome 4. Patients with this syndrome usually have distinctive facial features, full lips, low-set/rotated ears, micrognathia, short stature, patellar aplasia/hypoplasia, narrow nose with a high nasal bridge and abnormalities in sexual development. So we hypothesized that this phenotype might be related to CDT1. In addition, ankyrin repeat domain 11 (OMIM: 611192) at 16q24.3 has been shown to be associated with KBG syndrome. Patients typically have facial deformities, upper and middle incisors, skeletal  deformities, and developmental delays. The gene may also be involved in the phenotype of partial trisomy 16q.In most cases, trisomy 16q is due to a malsegregation of maternal balanced translocation and rarely to paternal balanced translocation.y 16q 16q2-qter ha The most facial features in these patients include a broad nasal root, a prominent forehead a prominent glabella and arched eyebrows, micrognathia, metopic ridging or craniosynostosis, cleft palate, cardiac defects, and mild hypotonia. Micrognathia is also present, but no gene is found to be associated with this phenotype in this region. The region of 7p22.2-pter contains about 30 OMIM genes including 11 morbidity-associated genes  activator 1(OMIM: 614506), located in 7p22.3, may have a key role in the protein stability of ATM, which affects neural stem cell differentiation. Breast cancer 1-associated ATM activator 1have been indicated to be associated a severe phenotype known as rigidity and multifocal seizure syndrome, characterized by intractable seizures, hypertonia, autonomic instability, and early death. Mutations in integrator complex subunit 1(OMIM: 611345), located at 7p22.3, have been indicated to be associated the distinctive phenotype as manifested absent or severely limited speech, an abnormal gait, hypotonia, and cataracts. Whether haploinsufficiency of any OMIM gene necessarily leads to clinical phenotype in humans requires us to further summarize and track more cases. In this case, the fetus had trisomy 16q and monomer 7p. The genetic abnormalities of these 2 chromosome alterations may interact to making the individual phenotypic consequences. Through data collection, we can indirectly infer possible phenotypes of the fetus. With the exception of the micromaxillary deformity suggested by ultrasound, the detailed features and mental state were not appreciated because the woman's parents eventually terminated the pregnancy and refused autopsy.In addition to partial chromosome 16 duplication, the fetus also had a 3.86-Mb deletion in 7p22.3p22.2. Andrea C. Yu reported 5 patients with microdeletions at 7p22.3p22.2.4In conclusion, precise definitions of subtle chromosome variations should be detected by genome-wide diagnosis such including CNV-seq or chromosome microarray analysis. Meanwhile, ultrasonography can evaluate the malformations of fetus in time. In this report, CNV-seq combined with amniocentesis karyotype is a useful tool to discover the genomic imbalance indicated by NIPS. Through literature analysis, we also analyzed the phenotypes that may be caused by partial trisomy 16q and partial monosomy 7p. The reasonable prenatal screening and prenatal diagnosis can discover some serious birth defects in time, provide accurate prenatal consultation for pregnant women, guide the next pregnancy, and so as to effectively improve the quality of the birth population.Conceptualization: Huihui Xie, Tong Liu.Formal analysis: Huihui Xie.Funding acquisition: Jing-Fang Zhai.Project administration: Jing-Bo Zhang, Jing-Fang Zhai.Supervision: Jing-Fang Zhai.Validation: Jing-Fang Zhai.Visualization: Huihui Xie, Tong Liu, Ying Liu.Writing – original draft: Huihui Xie.