MiR-338-3p Influences Gastric Cancer Progression By Targeting PTP1B

Gastric cancer(GC)is one of the most common malignant tumors in China,with high morbidity and mortality.Since GC is usually asymptomatic in its early stages,many people are diagnosed with advanced cancer at the beginning.Peritoneal metastasis,which could lead to bowel obstruction and formation of malignant ascites,is the most common cause of mortality among patients with advanced gastric cancer.Once peritoneal metastasis occurs,the median survival time is only 6.3 months.Therefore,a better understanding of molecular mechanisms underlying tumor development,especially metastasis,is necessary for early diagnoses and more effective therapeutic strategies.Protein-tyrosine phosphatase 1B(PTP1B),encoded by the PTPN1 gene in human,belongs to non-receptor-type classical PTP family.It acts as a switch by regulating the phosphorylation of protein tyrosine.PTP1B could inhibit the transduction of insulin and the leptin receptors,leading to the development of type 2 diabetes and obesity.In the last decades,more and more studies have established that PTP1B was a key player in cancer and served as both tumor suppressor and tumor promoter depending on the cellular context.MicroRNAs(miRNAs)are short non-coding RNAs molecules,with 19-23 nucleotide in length.miRNAs mainly function in negative regulation of gene expression by binding to their target sites,usually in the 3'-untranslated regions(UTRs)of mRNAs.In the initiation and progression of human cancers,miRNAs can act as either oncomiRs or tumor suppressors via interacting with oncogenes or tumor-suppressor genes.Based on these backgrounds,we focused on GC and investigated how miR-338-3p affected the progression of GC by inhibiting its target gene PTP1BIn this study,we first evaluated the expression level of PTP1B in GC and found that PTP1B protein levels were remarkably higher in GC tissues compared with those of the noncancerous counterparts.We then tested the biological function of PTP1B in GC cells.Results showed that PTP1B could promote the migration and suppress the apoptosis of GC cells,playing a role of the oncogene in GC.In this process,we found an interesting phenomenon that PTP1B mRNA levels and protein levels were inconsistently expressed in human GC tissues.Hence,we predicted an important impact of post-transcriptional regulation on the expression of PTP1B protein levels in GC tissues.It is well known that miRNAs are widely implicated in post-transcriptional regulation in cancer,so we supposed that PTP1B protein levels in GC might be regulated by miRNAs.We searched two databases with different algorithms to identify potential miRNAs which could target PTP1B and finally selected miR-338-3p as the candidate based on the comparison of free energy and literature review.We next examined the expression level of miR-338-3p in GC tissues and found that miR-338-3p was significantly down-regulated in GC tissues,which was negatively correlated with the changes of PTP1B.Then,we confirmed that miR-338-3p could inhibit PTP1B protein expression in GC cell lines.Luciferase reporter assays further demonstrated that this inhibition effect was owed to the direct combination of the seed sequence of miR-338-3p and its corresponding sites on the 3'-UTR of PTP1B mRNA.In terms of cell function,Transwell migration assays and scratch-wound assays were performed to evaluate migration of GC cells.Results showed that miR-338-3p suppressed GC cell migration by targeting PTP1B.Flow cytometry and WB of cleaved Caspase-3 were also performed to evaluated apoptosis of GC cells.The evaluation showed that miR-338-3p could promote GC cell apoptosis by silencing PTP1B.In addition,we further investigated the downstream signaling pathways of miR-338-3p/PTP1B regulatory axis.We found that PTP1B overexpression could activate PI3K/AKT/mTOR and Ras/Raf/MAPK pathways in GC cells,suggesting that miR-338-3p/PTP1B regulatory axis exerted its biological function in GC through influencing these pathways.Finally,we established an orthotopic xenograft model and a peritoneal metastasis model of GC and further demonstrated that miR-338-3p/PTP1B regulatory axis could indeed affect tumor growth and metastasis of GC in vivoIn summary,our study found that miR-338-3p/PTP1B regulatory axis played an important role in the progression of GC.It provided new insight into the molecular therapeutics of GC and peritoneal metastasis.