| Introduction: Gastric cancer is the second leading cause of cancer death in the world,and it is also the highest incidence of cancer in Northeast Asia.Although many methods are used to improve the survival rate of patients with gastric cancer,the survival time of late-stage stomach cancer is still very short,and effective treatment is limited.Therefore,it is imperative to determine the new early diagnostic biomarkers and to carry out the new anticancer targeting therapy.It is found that non-coding RNA,especially microRNA(miRNA,miR),also plays an important regulatory role in tumor development,invasion and metastasis.miRNA is a kind of endogenous,small,non-coding,and single stranded RNA,about 19-25 nucleotide lengths.miRNA can identify specific target genes,mainly through combining with the 3’ untranslated region(3’-UTR)of target gene mRNA,promoting the degradation of target gene mRNA and/or inhibiting target protein translation at the post transcriptional level.It plays the role of tumor suppressor gene or oncogene in tumor cells,and then participates in many biological processes,such as proliferation,differentiation,apoptosis and metastasis.Studies have shown that many mi RNAs,such as mi R-122,miR-26 a,and miR-200 c,are dysregulated in gastric cancer.This indicates that miRNA can be used as a new molecular marker for a class of gastric cancer.miR-1236-3p,an intronic miRNA,is located in the Chr6p21.33 and embedded within the intron of the NELFE gene.Accumulating evidence predicts that miR-1236-3p may play as a tumor suppressor gene,and downregulation of miR-1236-3p has been specified in some cancers.In hepatocellular carcinoma,miR-1236-3p down-regulates AFP,leading to PTEN accumulation,which inhibits the PI3K/Akt pathway.In advanced serous ovarian carcinoma,miR-1236-3p inhibits the migration and invasion of cells by targeting ZEB1.Moreover,the down-regulation of miR-1236-3p was also reported in bladder cancer,lung cancer,and breast cancer,however,its biological function in GC is unclear so far.Through preliminary tests,we found that miR-1236-3p was significantly down-regulated in gastric cancer and may be a potential diagnostic marker and therapeutic target for gastric cancer.Objective: The aim of this study was to verify the expression of miR-1236-3p in human gastric cancer tissues and cell lines,study the relationship between miR-1236-3p expression and clinicopathological features,and perform mi R-1236-3p functional experiments to explore its biological functions,identify its target genes,and detect the effects of miR-1236-3p up-regulation or down-regulation on epithelial mesenchymal transition(EMT)and PI3K/Akt signaling pathway.It provides a new way and target for the study of the development mechanism of gastric cancer and gene therapy and target therapy of gastric cancer.Methods: 1.Quantitative real time PCR(qRT-PCR)was used to detect the expression level of miR-1236-3p in 83 cases of gastric carcinoma and adjacent tissues,analysis its difference of expression level and correlation with clinical pathological characteristics,at the same time,we also determine miR-1236-3p expression level in human gastric cancer cell lines MKN-45,SGC-7901,MGC-803 and a normal gastric mucosa epithelial cell line GES-1 by using qRT-PCR.2.Lentivirus transfection was used to over-express the level of miR-1236-3p in SGC-7901 cells,and inhibit the level of miR-1236-3p in MKN-45 cells.In vitro,the effect of miR-1236-3p on the proliferation of gastric cancer cells was detected by CCK-8;the effect of miR-1236-3p on the migration of gastric cancer cells was detected by wound healing assay and transwell migration assay;transwell invasion assay was performed to detect the effect of miR-1236-3p on the invasion of gastric cancer cells.In vivo,a model of nude mouse subcutaneous transplantation was established,and the effect of mir-1236-3p on the growth ability of transplanted tumor in nude mice was analyzed.3.We used online target gene prediction software to forecast the potential target genes of miR-1236-3p associated with metastasis,and further clarified the binding sites of mi R-1236-3p to its target gene by using the dual luciferase reporter assay system;qRT-PCR and Western blot were utilized to detect the influence mechanism of overexpression or inhibition of miR-1236-3p on target gene.4.The morphological changes of cells in the miR-1236-3p overexpression group and the inhibition group were observed.Western blot was performed to detect the expression changes of E-cadherin protein and N-cadherin protein in the cells of miR-1236-3p overexpression,inhibition,negative control groups and control groups.5.The expression of p-AKT(Ser473)and AKT in the cells of miR-1236-3p overexpression group,inhibition group and negative control group was detected by Western blot.Results: 1.The results of qRT-PCR showed that the expression level of miR-1236-3p in 83 gastric cancer tissues was significantly lower than that in paracancerous tissues(P<0.001).miR-1236-3p expression level in gastric carcinoma was closly related to the tumor TNM stage,differentiation and lymph node metastasis,the lower miR-1236-3p expression in patients with gastric cancer was,the later TNM stage(P=0.003)and the degree of differentiation(P=0.015)were;the higher mi R-1236-3p expression in patients with gastric cancer was,the lower lymph node metastasis rate(P=0.018)was.2.Compared with normal gastric mucosal epithelial cell line GES-1,the expression level of miR-1236-3p in gastric cancer cell lines SGC-7901,MKN-45 and MGC-803 were significantly lower than that in human gastric mucosal epithelial cell line(P<0.05),relatively speaking,miR-1236-3p expression in gastric cancer cell line SGC-7901 was lower,and it is higher expressed in MKN-45 cell line.Overexpressed the level of miR-1236-3p in SGC-7901 cells and inhibited the expression of miR-1236-3p in MKN-45 cells for the subsequent cell biological-behavior assays.3.CCK-8 assay results showed that overexpression of miR-1236-3p was significantly inhibited the proliferation of gastric cancer cells(P<0.01),inhibiting the expression of miR-1236-3p can significantly promote the proliferation of gastric cancer cells(P<0.01).4.Wound healing assay showed that mi R-1236-3p could reduce the migration ability of gastric cancer cells.Scratches of SGC-7901 cells overexpressed with miR-1236-3p were still distant 48 h later,while the control group had narrowed the scratches.Scratches of MKN-45 cells transfected with miR-1236-3p were narrower than that of the control group after 48 h.Transwell migration assay showed that SGC-7901 cells overexpressed with miR-1236-3p were significantly reduced compared with the control group after 48 h,with the P value <0.01,and MKN-45 cells transfected with miR-1236-3p group were increased significantly compared with the control group after 48 h,P<0.05.The two groups of experiments showed that miR-1236-3p had inhibitory effect on the migration ability of gastric cancer cells.5.Transwell invasion assay showed that mi R-1236-3p could reduce the invasiveness of gastric cancer cells.Overexpression of miR-1236-3p significantly inhibited invasive numbers of SGC-7901 cells compared with the control group after 48 h,with the P value <0.001.Inhibition of miR-1236-3p enhanced invasive numbers of MKN-45 cells dramatically compared with the control group after 48 h,and the P value,with the P value <0.01.6.The nude mice subcutaneous tumor model showed that the tumor volume of mi R-1236-3p overexpression group was significantly decreased(P<0.01),and the tumor weight was significantly reduced(P<0.01).7.By using the online target gene prediction software Targetscan,mi RDB and miRanda,we predicted that MTA2 may be the target gene of miR-1236-3p.qRT-PCR and Western blot were used to detect the MTA2 expression in miR-1236-3p overexpression group and inhibition group.The results showed that the expression of MTA2 at mRNA level and protein level were all decreased,and the mechanism of miR-1236-3p acting on target gene MTA2 was post transcriptional level inhibition.Combined with the results of the double luciferase reporter gene system,MTA2 was a tumor metastasis related target gene for miR-1236-3p.8.Western blot was used to detect the expression of E-cadherin and N-cadherin in the cells of miR-1236-3p overexpression,inhibition group,negative control groups and control groups.We found that when mi R-1236-3p was overexpressed,E-cadherin protein level increased(P<0.001),but the level of N-cadherin protein decreased(P<0.001).After miR-1236-3p inhibition,E-cadherin protein level decreased(P<0.05),but N-cadherin protein level increased(P<0.001).At the same time,we observed the morphological changes in miR-1236-3p overexpression group and inhibition group.The results showed that,upregulation of miR-1236-3p resulted in morphological changes from an extended morphology to more organized cell-cell contacts in SGC-7901 cells,and downregulation of miR-1236-3p had the opposite result in MKN-45 cells.9.The expression of p-AKT(Ser473)and AKT in miR-1236-3p overexpression group,inhibition group and control group was detected by Western blot.The expression of p-AKT(Ser473)and AKT in the cells of miR-1236-3p overexpression group,inhibition group and control group was detected by Western blot.The results showed that the expression level of p-AKT protein in miR-1236-3p overexpression group was significantly lower than that in control group,P value <0.001,while the expression level of p-AKT protein in miR-1236-3p inhibition group was significantly higher than that in control group,P value <0.001.In each group,there was no significant difference in AKT protein expression.Conclusion: 1.the expression of miR-1236-3p in gastric cancer cell lines and gastric cancer tissues is significantly reduced.The low expression of miR-1236-3p is closely related to TNM stage,differentiation degree and lymph node metastasis.2.miR-1236-3p serves as tumor suppressor gene in gastric cancer,and it can inhibit the proliferation,invasion and migration of gastric cancer cells.3.The mechanism of miR-1236-3p inhibiting the invasion and metastasis of gastric cancer is to directly regulate the expression of target gene MTA2,and inhibit the development of gastric cancer by regulating the target gene.4.miR-1236-3p directly inhibits the epithelial mesenchymal transition(EMT)process in gastric cancer cells and inhibits the PI3K/Akt signaling pathway.The discovery of the mi R-1236-3p/MTA2 regulatory mechanism reveals a new mechanism about gastric cancer metastasis and provides a new potential therapeutic target for gastric cancer. |
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