| MicroRNAs(miRNAs),a new class of endogenous,noncoding and single-stranded RNAs,were recently discovered in both animals and plants.They trigger translational repression and/or mRNA degradation mostly through complementary binding to the 3'-untranslated regions of target mRNAs.Studies have shown that miRNAs can regulate a wide array of biological processes such as cell proliferation,differentiation and apoptosis.Aberrant expression and metabolism of miRNAs are associated with human diseases including malignancy.Alterations of miRNAs expression may play various roles in the pathogenesis of many human cancers.Some miRNAs have been shown to possess oncogenic or tumor suppressor activity,influencing cell transformation,tumor progression or metastasis.Accumulating evidences suggest that miRNAs could potentially be widely used in the diagnosis,prognosis and therapy of human cancer.High-throughput techniques have been used to screen miRNAs differentially expressed between human nonmalignant and malignant samples and found a number of miRNAs deregulated in numerous human tumors including lung,breast,liver, esophageal,colon and prostate cancers.Among these miRNAs,miR-141,a member of the miR-200 family,is over-expressed in ovarian cancer,colorectal cancer cells and cholangiocarcinoma cells,and down-regulated in prostate,hepatocellular and renal cell carcinoma.Recent studies suggested that miR-141 might play a role in the proliferation of cholangiocarcinoma cells and the invasiveness of renal carcinoma cells.Gastric cancer is the second leading cause of male cancer-related death and the third leading cause for female cancer-related death worldwide.In year 2008,the estimated new cases of stomach cancer is 21,500 and the estimated death reaches 10,880 in United States.However,little is known about the role of miRNAs in gastric cancer.Recently,it was reported that miR-106b-25 cluster,miR-21 and miR-27a were up-regulated in gastric tumor tissues.Moreover,intestinal-type and undifferentiated gastric cancers had distinct miRNAs expression profiles,suggesting that miRNAs were involved in the gastric carcinogenesis.In this study,we screened differentially expressed miRNAs between gastric tumor tissue and adjacent non-tumor tissue through microarray technology,and found that miR-141 was one of the most substantially down-regulated miRNAs.Therefore,we aimed to examine the expression of miR-141 in stomach cancer tissue and cell lines using quantitative Real-time PCR;and explore the effect of miR-141 on cancer progression through gastric cancer cell model in vivo.We hope our study could provide convincing evidence on miR-141's association with gastric tumorigenesis,and facilitate the search of suitable candidates for cancer targeting therapy.1 Materials and Methods1.1 Tissue samples were obtained from 35 patients undergoing gastrectomy for gastric cancer in Sir Run Run Shaw Hospital,Zhejiang University School of Medicine, Hangzhou,P.R.China.Written informed consent was obtained before collection.Tumor samples were resected at surgery.Non-tumor samples from the macroscopic tumor margin were isolated at the same patients and used as the matched adjacent non-neoplastic tissue.Microarray technology was applied to screen differentially expressed miRNAs between gastric tumor tissue and adjacent non-tumor tissue.The expression of miR-141 was analyzed using Real-time PCR.Then,we investigated the association of clinical and pathological characteristics of cancer samples with miR-141 expression through independent sample t test and one-way ANOVA. 1.2 Expression of miR-141 in 5 gastric cancer cell lines originated from various differentiation stages was analyzed using Real-time PCR.The proliferation of these cells was also evaluated for the selection of suitable cell lines.1.3 MiR-141 in MGC-803 cell lines was over-expressed by miR-141 precursor molecule transfection.Phase contrast microscopy and MTT analyses were performed to investigate the effect of miR-141 over-expression on cell proliferation.1.4 Potential target genes of miR-141 were predicted by several bioinformatic programs,and their association with gastric pathogenesis was preliminarily investigated.2 Results2.1 Our microarray results showed that compared with the adjacent non-tumor tissue,miR-141 expression in tumor tissue was down-regulated by approximately 12.0 folds.Real-time PCR analysis displayed that the decrease of miR-141 was clearly found in 80%out of the 35 cases with a median change about 2.1 folds(P<0.01).However, we failed to observe any association of miR-141 expression with clinical and pathological parameters of gastric cancer.2.2 Gastric cancer cell line MGC-803 was found to be a suitable in vivo cell model. The expression of miR-141 in MGC-803 cells transfected with miR-141 precursor was up-regulated by approximately 7×10~4 folds compared with that in cells transfected with pre-miR negative control oligonucleotides.Under the observation of fluorescence microscopy,almost all cells transfected with pre-miR negative control showed red color, suggesting the miR-141 precursor transfection was successful and it over-expressed miR-141 level in MGC-803 cells.2.3 The phase-contrast microscopy and MTT assays showed that 48 h and 72 h after transfection,the proliferation of MGC-803 cells transfected with miR-141 precursor was significantly inhibited relative to that of cells transfected with pre-miR negative control(48 h,P<0.05;72 h,P<0.01).2.4 Using bioinformatic algorithms,we predicted FGFR2 and ZEB2 as potential targets of miR-141 involved in gastric carcinogenesis.However,quantative Real-time PCR analysis failed to show any significant changes of FGFR2 and ZEB2 mRNA expression in MGC-803 cells with miR-141 over-expression.3 Conclusions3.1 MiRNA microarray and quantitative Real-time PCR analyses showed that miR-141 was down-regulated in gastric tumor tissue compared with adjacent non-tumor tissue,suggesting that down-regulation of miR-141 is associated with the pathogenesis of stomach cancer.3.2 MiR-141 over-expression significantly inhibited the proliferation of MGC-803 cells,suggesting that the down-regulation of miR-141 may play a role in gastric cancer cell proliferation.3.3 Whether FGFR2 and ZEB2 were truly target genes of miR-141 which were associated with gastric tumorigenesis remains unclear.Further investigations are warranted to elucidate the inhibitory and targeting relations between these two genes with miR-141.
|
PDF Full Text Request