| Introduction: Gastric cancer(GC)is one of the common digestive malignant tumors.It has the second highest incidence and the third highest cancer-related mortality in China.More than 90% of new cases are diagnosed as advanced GC.Although the effect of the surgery and drug treatment has been greatly improved in recent years,GC is still prone to metastasis easily.The 5-year survival rate of GC patients in China from 2010 to 2014 was only 35.9%,which was not significantly improved from 2000 to 2004 with 30.2%.Therefore,the novel biomarkers for early GC is of great significance for the treatment and prognosis of GC patients.A large number of studies reported that MicroRNA(miRNA)plays an important role in the occurrence and development of various tumors.miRNA is a type of non-coding single-stranded RNA encoded by an endogenous gene,approximately 20-24 nucleotides in length.It binds specifically with the 3’ non-coding region(3’-UTR)sequence of a target gene.It can inhibit target gene to translate and reduces gene expression at the post-transcription level,which plays an important role in various diseases such as inflammatory diseases,immune diseases,connective tissue diseases and malignant tumors.In addition,miRNAs play a role similar to oncogenes or tumor suppressor gene in a variety of tumors,and participate in the regulation of many biological characteristics such as tumor cell proliferation,invasion,and metastasis.MicroRNA-665(miR-665)is a small-molecule RNA generated from a single-stranded RNA precursor containing 72 ribonucleic acids located on human chromosome 14q32.2.MiR-665 has been found to regulate tumor cell proliferation,differentiation,apoptosis,invasion,migration,and chemotherapy resistance in a variety of cancers,including neuroblastoma,esophageal cancer,lung cancer,liver cancer,gynecological tumors,and prostate cancer,but the regulatory mechanism in GC is unclear.Objective: The purpose of this study is to investigate the expression level of miR-665 in GC tissues samples and cell lines,and the relationship between the expression level of mi R-665 and the biological characteristics of GC patients’ clinical patholgical characteristics.Identification of potential target genes of miR-665,and regulated mechanism of it on epithelial-mesenchymal transition.It could provide therapeutic target and basis for the molecular regulation mechanism of GC that miR-665 may participate in.Methods:1.We obtained the GSE93415 dataset from the GEO(Gene Expression Omnibus database)database and analyzed the differentially expressed miRNA;2.We detected miRNA-665 expression level in GC cells(MGC-803,MKN-45,HGC-27 and AGS)and normal gastric epithelial cells(GES-1)using real-time fluorescent quantitative PCR(RT-qPCR).At the same time,the expression level of miRNA-665 in 63 pairs of GC tissue samples and adjacent normal gastric tissues(more than 3 cm from the tumor margin)was detected and the relationship between the expression level of miR-665 and clinical pathological characteristics was analyzed;3.miRNA-665 mimics,miRNA-665 mimics NC,mi RNA-665 inhibitor,and miRNA-665 inhibitor NC were transfected into GC cells MGC-803 and HGC-27 using lipofectamine 3000(Invitrogen),respectively;4.In vitro experiments,the effects of miRNA-665 on the proliferation and colony forming ability of GC cells were detected by the CCK-8 assays and colony formation assays.Cell scratch healing assays and Transwell cell assays were used to examine the effect of miRNA-665 on GC cell invasion and migration ability;Western blot was used to detect the effect of mi RNA-665 on GC cell EMT markers(E-cadherin,N-cadherin and vimentin);5.Online biological websites were used to predict the potential target genes of miRNA-665,and the binding site between miRNA-665 and the CRIM1 by luciferase assays;6.pcDNA3.1-CRIM1 vector was transfected into MGC-803 and HGC-27 cells with miR-665 up-regulated in cell rescue assays,and the effects on the invasion,migration and EMT were measured.Results: 1.With P<0.05,|log2FC|≥1 as the cutoff value,a total of 110 abnormally expressed mi RNAs were found in the GSE93415 dataset,of which 19 mi RNAs were significantly down-regulated and 91 miRNAs were significantly up-regulated(P<0.05).2.RT-qPCR results showed that the expression level of miR-665 in four GC cell lines was lower than that in GES-1(P<0.05).Furthermore,the expression level of miR-665 in GC tissue samples was significantly lower than that in normal gastric tissue(P<0.05).Clinical and pathological analyses showed that the low expression of miR-665 was significantly associated with high TNM stage(P=0.007),distant metastasis(P=0.031),and poor differentiation(P=0.029).3.MiRNA-665 mimics,miRNA-665 mimics NC,miRNA-665 inhibitor,and miRNA-665 inhibitor NC were successfully transfected into MGC-803 and HGC-27 cells.4.The results of CCK-8 showed that compared with the control group,the OD values of HGC-27 and MGC-803 cells transfected with mi R-665 mimics decreased significantly after 48 h,72,and 96 h,while cells was transfected with mi R-665 nhibitor showed the exact opposite effect(P<0.05);The results of the colony formation assays are consistent with the above results,overexpression of miR-665 inhibited the proliferation of GC cells,while inhibition of miR-665 expression showed the opposite effect(P<0.05);The results of cell scratch healing assays showed that the speed of cell scratch healing in the miR-665 mimics group was significantly reduced,the cell healing rate of transfected with miR-665 inhibitor was significantly higher(P<0.05);The results of the Transwell assays showed that the number of MGC-803 and HGC-27 cells transfected with miR-665 mimics passed through the bottom membrane of the Transwell chamber was significantly higher,and transfection with miR-665 inhibitor in gastric cancer cells showing opposite results(P<0.05);Western blot results showed that epithelial-related marker protein the expression of E-cadherin was enhanced(P<0.05)and the expression of N-cadherin and Vimentin were decreased(P<0.05)in after-transfcted mi R-665 mimics GC cells.Decreasing the expression of miR-665 in GC cells can inhibit the expression of E-cadherin and promote the expression of N-cadherin and Vimentin(P<0.05).5.Through online bioinformatics databases(miRDB,TargetScan and miRWalk),CRIM1 was found as a potential target gene of mi R-665.The results of the luciferase assay showed that miR-665 mimics can effectively inhibit the luciferase activity of the CRIM1＿WT(wild type)group(p<0.01),but it didn’t inhibit the CRIM1＿Mut(mutant)luciferase activity,which proved that the miR-665 can bind with the 3’-UTR of the CRIM1.6.RT-qPCR and Western blot results showed that the expression level of CRIM1 mRNA and CRIM1 protein in GC cell lines was higher than that in GES-1(P<0.05).In addition,the expression level of CRIM1 mRNA and CRIM1 protein in GC tissue samples was significantly higher than that in matched normal gastric tissues(P<0.05).7.The results of cell rescue assays showed that overexpression of miR-665 can significantly reduce the effect of CRIM1 on the invasion,migration and EMT of GC cells MGC-803 and HGC-27(P<0.05).Conclusion: 1.In GSE93415 dataset,the expression level of miR-665 proves that it is a tumor suppressor in GC;2.MiR-665 expression level is significantly down-regulated in GC cells and tissue samples,and its expression level is significantly related to the stage,differentiation and distant migration of GC patients;3.MiR-665 mimics and mi R-665 inhibitor can be successfully transfected into GC cell lines MGC-803 and HGC-27,the expression level of miR-665 in the transfected cells was significantly up-regulated or down-regulated;4.MiR-665 significantly inhibited the proliferation,invasion,migration,and EMT progression of GC cells;5.The expression of CRIM1 mRNA and CRIM1 protein was significantly up-regulated in GC cells and tissue samples;6.miR-665 can bind with CRIM1 in HGC-27 and MGC-803 cells to inhibit the invasion,migration and EMT of GC cells. |
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