| Objective:To investigate the underlying miRNA from expression profiling of microRNAs in laryngeal squamous cell carcinoma(LSCC).To predict target gene of miR-195 by bioinformatics methods and to verify the relationship between them.To study the expression and correlation of miR-195 and target gene DCUN1D1 in fresh specimens and adjacent normal mucosa of laryngeal squamous cell carcinoma,and to evaluate the connection of miR-195 and its target gene DCUN1D1 expression with clinicopathologic parameters and their prognostic value in LSCC.To probe the effects of miR-195 on cell proliferation,cloning,migration,invasion,apoptosis and cell cycle of laryngeal squamous cell carcinoma in vitro,and to expound the effects of miR-195 on human laryngeal squamous cell carcinoma Hep-2 cells xenograft tumor in invo,and to clarify their biological functions.To detect the expression of key molecules in tumor metastasis and invasion and to explore the molecular mechanism of miR-195 and DCUN1D1 in the invasion and metastasis of laryngeal squamous cell carcinoma.Methods:The bioinformatics was used in prediction of modulation between miR-195and DCUN1D1 which was verifed by dual luciferase reporter gene assay.Quantitative RT-PCR and immunohistochemistry were used to detect mRNA and protein levels in LSCC specimens and adjacent normal mucosa.To evaluate the relationship between miR-195 and DCUN1D1 expression with clinical pathologic parameters and postoperative follow-up data,and to evaluate their clinical significance and prognosis.The gene transfection technology was used to explore miR-195 mimics on the biological function of laryngeal squamous cell carcinoma in vitro by MTS assay,colony formation assay,wound healing assay,transwell assay and flow cytometry.The tumor inhibition effect of miR-195 was observed in nude mice transplanted tumor model.SiRNA DCUN1D1 was transfected into laryngeal squamous cell carcinoma cells by RNA interference technique,and the effect of siRNA DCUN1D1 on the proliferation of laryngeal squamous cell carcinoma cells was observed by MTS method.The effect of miR-195,miR-195+DCUN1D1 and siRNA DCUN1D1 on the migration and invasion of laryngeal squamous cell carcinoma cells was observed by transwell method.The changes of key molecules in tumor invasion and metastasis were detected by western-blot method.Results:There were significant differences in the changes of the fluorescence activity of the wild type(Wt DCUN1D1 3'UTR group)compared with that of the control group(P<0.01).There was no significant difference in the fluorescence activity of the mutant(Mut DCUN1D1 3'UTR)group compared with the control group(P>0.05).Chi square test results showed that the proportion of low expression of miR-195 was significantly higher in T3+T4 stage,N+stage and clinical advanced stage(III+IV)(P<0.05).On the contrary,the proportion of low expression of miR-195 were significantly lower in T1+T2 stage,N0 stage and clinical early stage(I+II)(P<0.05).There was no significant difference in the expression of miR-195 with age,sex,primary sites,degree of differentiation,distant metastasis and preoperative smoking(P>0.05).Rank sum test showed that the intensity of miR-195 expression were significantly lower in T3+T4 stage,N+stage,M+stage and clinical advanced stage(III+IV)(P<0.05).There was no significant difference in the intensity of miR-195expression with age,sex,primary sites,degree of differentiation and preoperative smoking(P>0.05).Chi square test results showed that the proportion of high expression of DCUN1D1 was significantly higher in T3+T4 stage,N+stage and clinical advanced stage(III+IV)(P<0.05).On the contrary,the proportion of high expression of DCUN1D1 was significantly lower in T1+T2,N0 stage and clinical early stage(I+II)(P<0.05).There was no significant difference in the expression of DCUN1D1 with age,sex,degree of differentiation,distant metastasis and preoperative smoking(P>0.05).Rank sum test showed that the intensity of DCUN1D1 expression were significantly higher in T3+T4 stage,N+stage and clinical advanced stage(III+IV)(P<0.05).There was no significant difference in the intensity of DCUN1D1 expression with age,sex,primary sites,degree of differentiation,distant metastasis and preoperative smoking(P>0.05).The survival analysis(Kaplan-Meier method)and through the Log-rank test,the average survival time of patients with high expression of miR-195 was longer than that of patients with low expression of miR-195(?~2=4.782,P=0.029),and the average time of patients with low expression of DCUN1D1 was longer than that of patients with high expression of DCUN1D1(?~2=7.278,P=0.007).We further classify the expression of miR-195 as well as DCUN1D1 and the results showed that the average survival time of high expression of miR-195 with low expression of DCUN1D1 was longer than the low expression of miR-195 with high expression of DCUN1D1(?~2=4.972,P=0.026).Univariate Cox regression model showed that the degree of differentiation(RR=2.014,95%CI:1.065~3.810,P=0.031),T stage(RR=3.344,95%CI:1.184~9.449,P=0.023),lymph node metastasis(RR=2.809,95%CI:1.474~5.350,P=0.002),miR-195(RR=0.476,95%CI:0.241~0.942,P=0.033),DCUN1D1(RR=2.588,95%CI:1.264~5.297,P=0.009)were the independent prognostic factors in patients with laryngeal squamous cell carcinoma.Multivariate Cox regression model showed that miR-195(RR=0.358,95%CI:0.134~0.959,P=0.041),DCUN1D1(RR=4.253,95%CI:1.243~14.557,P=0.021)were the independent prognostic factors in patients with laryngeal squamous cell carcinoma.In vitro,after transfection into Hep-2 and TU-177 cells by miR-195 mimics,the proliferation,colony formation,migration,invasion of miR-195 group was significantly inhibited(P<0.05).The percentage of G0/G1 cells and early apoptotic cells of miR-195 group was increased(P<0.05).In vivo,the tumor growth rate of miR-195 agomir group was slower than Control agomir group and Blank group(P<0.01).After transfection into Hep-2 and TU-177 cells by DCUN1D1 siRNA3,the cell growth was significantly inhibited(P<0.05).After transfection into Hep-2 and TU-177 cells by miR-195,miR-195+DCUN1D1,DCUN1D1 siRNA3,The number of migrating cells of miR-195 group and DCUN1D1 siRNA3 group was significantly lower than that of Control group(P<0.01).The number of migrating cells of miR-195+DCUN1D1 group was significantly higher than that of miR-195 group and DCUN1D1 siRNA3 group(P<0.01).The number of invasion cells of miR-195 group and DCUN1D1 siRNA3 group was significantly lower than that of Control group(P<0.01).The number of invasion cells of miR-195+DCUN1D1 group was significantly higher than that of miR-195 group and DCUN1D1 siRNA3 group(P<0.01).miR-195 and DCUN1D1 siRNA3 can increase the expression of E-cadherin and can decrease the expression of?-catenin,MMP-2 and MMP-9(P<0.05),however miR-195+DCUN1D1 can inhibit these effect.Conclusions:DCUN1D1 is a target gene of miR-195,miR-195 was significantly down-regulated,and its target gene DCUN1D1 was significantly up-regulated in laryngeal squamous cell carcinoma.The expression of miR-195 and DCUN1D1 can be used as independent prognostic factors for the patients with laryngeal squamous cell carcinoma.MiR-195 has inhibitory effect on growth of human laryngeal squamous cell carcinoma in vitro and in vivo.MiR-195 is involved in the invasion and metastasis of laryngeal squamous cell carcinoma by modulating its target gene DCUN1D1,which provides a new molecular marker for diagnosis,early treatment and prognosis evaluation of LSCC. |
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