MiR-146a-5p Targets PRSS1 To Inhibit Gastric Cancer MGC803 Cell Proliferation

[Objective] Gastric carcinoma(GC)is one of the most common malignant tumors,which seriously threatens human life and health.The group previously identified gastric cancer-associated differentially expressed protein serine protease 1(PRSS1)using isotope-labeled quantitative proteomics isotopically labeled quantitative Proteomics technology.In the present study,we explored the molecular mechanisms that regulate the expression of PRSS1 and its affect the proliferation of GC MGC803 cells.[Methods] 1.Immunohistochemistry and Western blot were used to detect the expression of PRSS1 protein in GC tissues and GC SGC7901,BGC823,MGC803 cells and immortalized gastric mucosal epithelial GES-1 cells.The clinicopathological significance of PRSS1 expression and its relationship with the prognosis of patients were analyzed.2.We use bioinformatics methods to predict miRNAs targeted to PRSS1.Firstly,we used miRwalk(zmf.umm.uni-heidelberg.de)software to predict miRNAs that bind to PRSS1.Secondly,miRanda(www.microrna.org)software was used to observe the mirSVR and PhastCons score of PRSS1-miRNA to obtain the best miRNA with specificity and stability for PRSS1 targeted binding.Then,the target sites and sequences of miRNA and PRSS1 binding were predicted by TargetScan(V7.2)software.3.The expression of miR-146a-5p in GC was analyzed by the GEO2 R database.MiR-146a-5p and PRSS1 mRNA expression levels were detected in GES-1 and GC SGC7901,BGC823,MGC803 cells by real-time fluorescent quantitative PCR(qRT-PCR).And we analyze the correlation between PRSS1 mRNA and miRNA expression levels.4.Lipofectamine 2000 reagent was used to transiently transfect mimic NC,miR-146a-5p mimic,inhibitor NC and miR-146a-5p inhibitor to gastric cancer MGC803 cell line.We observed the effect of miR-146a-5p expression on the proliferation of GC MGC803 cells through CCK8 and Clone formation assays.Western blot and immunocytochemistry were used to detect the effect on the expression of PCNA protein.5.The PRSS1-WT wild-type vector and PRSS1-Mut mutant vector were constructed,and the targeted binding of miR-146a-5p to PRSS1 3’-UTR was detected using the dual luciferase reporter system.6.Western blot and qRT-PCR were used to detect the effects of miR-146a-5p on the expression level of PRSS1 protein and mRNA in GC MGC803 cells.We analyzed the effects of miR-146a-5p on the regulation of PRSS1 expression.[Results] 1.PRSS1 protein is mainly localized in the cytoplasm.The positive expression rates of PRSS1 in adjacent normal gastric mucosa tissues,well differentiated,moderately differentiated,poorly differentiated gastric adenocarcinoma tissues and lymph node metastatic cancer tissues are 35.00%,66.67%,73.68%,80.00% and 88.57%,respectively.Compared with normal gastric mucosa adjacent to cancer,PRSS1 expression was significantly increased in well differentiated,moderately differentiated,poorly differentiated gastric adenocarcinoma and lymph node metastatic cancer tissues(P<0.05).The expression of PRSS1 in GC tissues is associated with tumor size,TNM staging and lymph node metastasis(P=0.007,0.001 and 0.004,respectively).Patients with high expression of PRSS1 have a poor prognosis(P<0.05).Western Blot analysis showed that PRSS1 was highly expressed in GC cell lines(P <0.001).2.We comprehensively analyzed the prediction results of miRwalk,miRanda and TargetScan software and found that there was a binding site between mir-146a-5p and 45-51 bases of 3’ UTR of PRSS1,and the binding sequence is 5’-guucuc-3 ’.It is concluded that miR-146a-5p is a target miRNA molecule for PRSS1.3.miR-146a-5p is lowly expressed in GC tissues(P <0.05).qRT-PCR confirmed that compared with gastric immortalized GES-1 cells,miR-146a-5p expression was significantly reduced in GC cell lines(P <0.001).And miR-146a-5p expression was negatively correlated with PRSS1 mRNA expression in GES-1 and GC cells.4.The results of CCK8 and Clone formation assays showed that the proliferation ability(P = 0.0092)and colony forming ability(P <0.01)of MGC803 cells in miR-146a-5p mimic group was significantly reduced,and the proliferation abiliy(P = 0.0343)and colony forming ability(P <0.01)of miR-146a-5p inhibitor group was significantly enhanced.Western blot and immunocytochemical analysis revealed that PCNA expression levels of proliferation-related proteins were significantly down-regulated in MGC803 cells in the miR-146a-5p mimic group(P <0.05,P = 0.0026,respectively),and the miR-146a-5p inhibitor group PCNA expression level was significantly up-regulated in MGC803 cells(P <0.05,P <0.0001,respectively).5.Targetscan software predicted that there is a binding site between mir-146a-5p and 45-51 bases of 3’ UTR of PRSS1,and the binding sequence is 5’-guucuc-3’.We confirmed that PRSS1-WT vector and miR-146a-5p mimic co-transfected into 293 T cells significantly reduced luciferase activity(P=0.0019),while PRSS1-MUT vector and miR-146a-5p during co-transfection,there was no significant change in luciferase activity(P > 0.05),by dual luciferase reporter gene experiment.6.We further confirmed the luciferase results by qRT-PCR and WB analysis.Our data shown that miR-146a-5p mimic significantly down-regulates the expression of PRSS1 mRNA(P <0.05)and protein in MGC803 cells(P <0.0001),while miR-146a-5p inhibitor significantly up-regulates the expression of PRSS1 mRNA(P <0.01)and protein(P <0.001).Therefore,PRSS1 is a direct target gene of miR-146a-5p,miR-146a-5p negatively regulates the expression of PRSS1 and inhibits the proliferation of GC MGC803 cells.[Conclusions] 1.PRSS1 is highly expressed in GC tissues,and its expression is associated with tumor size,TNM staging and lymph node metastasis of GC tissues.2.MiR-146a-5p targets PRSS1 and inhibits the proliferation of GC MGC803 cells.