| OBJECTIVES:Tongue squamous cell carcinoma(TSCC)was the most frequent kind of oral squamous cell carcinoma(OSCC).It was famous for its high ability of metastasis and proliferation.TSCC usually leads to disfunction of speech,mastication and deglutition.Although recent development has been achieved in the therapeutic managements of TSCC,the mortality of TSCC is still high.It is necessary to understand the molecular pathways involving in TSCC progression to find the novel therapeutic strategies for TSCC.MicroRNAs(miRNAs)is a kind of nonprotein-coding RNAs.It regulates the expression of genes by binding 3'UTR of their target mRNAs.Existing studies have demonstrated that miRNAs plays an important role in tumor development.Emerging evidences have showed that deregulated expression miRNA is correlated with cancer initiation,progression and promotion through modulating tumor suppressor gene or oncogene.Previous studies showed that miR-137 played an important role in the tumor development.However,the role of miR-137 in the TSCC was still uncovered.In this study,we demonstrated that the expression of miR-137 was downregulated in TSCC tissues and cells.In addition,we identified SP1 as a direct target gene of miR-137 in SCC1 cell.SP1 over-expression rescued the inhibitory effects exerted by miR-137 on cell proliferation and EMT.These results suggested that miR-137 exerted a suppressive role in TSCC by targeting SP1 Tongue squamous cells carcinoma(TSCC)is the most frequent type of oral malignancy.This study is to investigate the correlation between the miR-137 and tongue squamous cell on cell proliferation,migration and invasion and also study their preliminary mechanism of molecular regulatory.MATERIALS AND METHODS:First Part:The expression and epigenetically regulation of miR-137 in TSCC cell.Total RNA from cells or tissues was isolated.The relative expression of miR-137 and SP1 was confirmed with the use of qRT-PCR.The expression level of miR-137 and SP1 was measured with the use of the 2-AACt method,where GAPDH was used as an internal control for SP1 and miR-137 respectively.Cells process with the 5-aza-2'-deoxycytidine(5-Aza-CdR;Sigma-Aldrich)or trichostatin A(TSA;Sigma-Aldrich)for totally 24 hours.The expression of miR-137 was measured by qRT-PCR.Second Part:To study the effect of miR-137 on the biological behaviour of TSCC cell lines(SCC1)by targeting gene SP1.The effect of cell proliferation,invasion and migration after miR-137 mimics and scramble transfected TSCC cell lines(SCC1)was confirmed using kinds of assays such as MTT,colony formation,invasion and wound-healing and so on.Quantitative RT-PCR assay the express of E-cadherin,N-catenin,Snail,Vimentin protein in tongue squamous carcinoma tissues after TSCC transfected miR-137 mimics and scramble.TargetScan explored the potential target genes of miR-137.We used real-time fluorescent quantitative PCR(qRT-PCR)to determine the expression of SP1 after tongue squamous cell carcinoma(TSCC)cell line transfected the miR-137 mimics and scramble.By applicating of bioinformatics and molecular biology technology,cloning 3 'UTR region of SP1 gene and connected to pGL3 vector which includes the luciferase reporter gene.The vectors constructed was named pGL3-REPORT-SP1-3 'UTR.Validate the targeting relation of miR-137 and SP1 gene by the use of dual luciferase report gene experiment.RESULTS:First Part:?We measured miR-137 expression in 25 TSCC tissues and the matched normal tissues by real-time fluorescent quantitative PCR(qRT-PCR).The expression of miR-137 was downregulated in TSCC tissues compared to the normal tissues.Besides,we demonstrated that miR-137 expression was downregualted in TSCC cell lines compared to immortalized NOK16B cell line and normal oral keratinocyte cell(NHOK).(P<0.001)?Cells dealed with the 5-aza-2'-deoxycytidine(5-Aza-CdR;Sigma-Aldrich)respectively at 0.5,1.5,and 3?mol/l or 300 nmol/1 trichostatin A(TSA;Sigma-Aldrich)for totally 24 hours.We treated SCC1 and UM1 cells with the DNA methylation inhibitor and also with histone deacetylase inhibitor trichostatin A(TSA).We found that miR-137 expression was upregulated in both SCC1 and UM1 cells after disposed with TSA or AZA.Second Part:?The expression of SCC1 cell lines miR-137 in miR-137 mimic group and scramble group was 0.6327±0.12?32.725±0.34(p<0.001).? It was demonstratedby MTT experiment that after the tongue squamous cell carcinoma(TSCC)cell line SCC1 transfected the miR-137 mimics,the inhibition of cell proliferation obviously higher than the control group,and the difference showed statistically significant(p<0.05).In the colony formation assay,after the transfection of tongue squamous cell carcinoma(TSCC)cell line,the cell colony count of miR-137 mimics group was obviously lower than that in scramble group.And their difference was statistically significant(P<0.05),means that the miR-137 expression can significantly reduce the cell proliferation ability of TSCC cell line SCC1.?Over-expression of miR-137 promoted the expression of E-cadherin in SCCl cell.Moreover,the expression of N-cadherin,Snail and Vimentin in SCC1 cell in scramble group suppressed obviously by the ectopic expression of miR-137.These results suggested that the miR-137 inhibits SCC1 ectomesenchymal transformation,it showed that their difference was statistically significant(P<0.05).? In other vitro experiments the result showed that the cells that the tongue squamous cell carcinoma(TSCC)cell line SCC1 transfected miR-137 in mimics group was obviously lower than that scramble group,and their difference showed statistically significant(P<0.05),we demonstrated that over-expression of miR-137 inhibited SCC1 cell invasion.In addition,the wound-healing analyssis showed that ectopic expression of miR-137 suppressed SCC1 cell migration.?In qRT-PCR experiments,the luciferase relative expression in the pGL3-REPORT-SP1-3 'UTR and miR-137 mimics transfection group,pGL3-REPORT-SP1-3 'UTR and scramble transfection group,pGL3-REPORT-MUT 3'UTR and miR-137 mimics transfection group,pGL3-REPORT-MUT 3'UTR and scramble transfection group were 0.97±0.07?0.47±0.06?0.97±0.08?0.98±0.09,suggesting that the ectopic expression of miR-137 suppressed the luciferase activity of the wild type SP1 3' UTR vector,the difference showed statistically significant(P<0.05).Western blot detection demonstrated the SP1 protein expression of SCC1 cell in miR-137 group decreased obviously than that in scramble group.The cell proliferation ability was obviously higher in miR-137 mimics and SP1 group than the control and scramble group,and the difference showed statistically significant(p<0.05)determined by MTT experiment.qRT-PCR detected that SP1 and miR-137 mimics the promoted the expression of E-cadherin in SCC1 cell.Moreover,it suppressed the expression of N-cadherin,Snail and Vimentin in SCC1 cell.These results suggested that miR-137 regulated the TSCC cell epithelial mesenchymal transition(EMT).These results suggest that miR-137 regulated the SCC1 ectomesenchymal transformation of epithelial cells by SP1,and the difference was statistically significant(P<0.05).CONCLUSIONS:1.The expression of miR-137 was downregualted in TSCC tissues and cell lines compared with the normal control group.MiR-137 expression was downregulated in TSCC cell lines compared to immortalized NOK16B cell line and normal oral keratinocyte cell culture(NHOK).2.MiR-137 expression was epigenetically regulated in TSCC cell.3.MiR-137 suppressed TSCC cell proliferation,colony formationepithelial,mesenchymal transition(EMT),cell invasion and migration.4.MiR-137 targeted SP1 in TSCC cell.SP1 regulated the SCClcell proliferation and EMT. |
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