| This study consists of two parts as described as below.The first part is to examine the effect of TTF-1 promoter operating miR-7 expression on the growth of human lung cancer in vivo.The second part is to determine the possible mechanism of TTF-1 promoter operating miR-7 expression on the growth of human lung cancer.Objective:Part One To observe the possible effects of TTF-1 promoter operating miR-7expression on the growth of human lung cancer cells in vivo using nude mice model of human lung cancer through related techniques including HE staining,immunofluorescence(Ki-67 and TUNEL),in situ hybridization and Real-time PCR assay.Part Two To investigate the possible molecular mechanism of TTF-1promoter operating miR-7 expression on the growth of human lung cancer cells using cDNA microarray technology,Real-time PCR and Western blot,as well as overexpression vector and molecular cloning technique.Methods:Part One Establishment of human lung cancer model using nude mice.Human lung cancer cell line 95 D cells were injected subcutaneously into right flank of Balb/c nude mice(n=8).7 days later,the plasmid of p-T-miR-7(100mg)or p-Cont(100mg)was remote given by subcutaneous injection into the left flank of nude mice five times every three days.The tumor size was measured every 3 days.3 days after last injection,all mice were killed.The tumor tissue and lung tissue were obtained andstained by HE staining.The expression of Ki-67 was detected using immunofluorescence assay.And the apoptosis of cells was analyzed through TUNEL method.The expression level of miR-7 was detected by Real-time PCR assay and in situ hybridization.Moreover,the cell growth cycle-associated factors(CDK2,CDK3,CDK4 and CDK6)and metastasis-associated factors(MMP-2,MMP-9,E-cadherin and CXCR4)were detected by Real-time PCR assay.Lastly,the transduction of related signal pathway,including Akt/ Erk,were detected by western blot assay.Part Two Tumor RNAs were extracted for cDNA gene chip detection and differentially expressed genes were analyzed and screened.Further,the putative target genes of miR-7 were predicted by biological information technology and identified by Real-time PCR assay.Therefore,we selected NDUFA4,a putative target gene of miR-7,and verified its expression in tumor mass using immunohistochemistry technique and Western blot assay.Furthermore,we constructed an eukaryotic expression vector of NDUFA4(termed as p-NDUFA4),and then we transiently transfected p-NDUFA4 into human lung cancer cell line 95 D cells in vitro.The expression of NDUFA4 was determined by Real-time PCR assay.The proliferation,clone forming ability and migration of cells were analyzed by CCK-8 assay,scratch assay and clone formation assay respectively.In addition,the expression of cell growth-related molecules including CDK2,CDK3,CDK4 and CDK6,as well as metastasis-related molecules including CXCR4,E-Cadherin,MMP-2,MMP-3 and MMP-9,were determined by Real-time PCR assay.Finally,we also analyzed the expression of NDUFA4,Akt,phosphor-Akt,Erk and phosphor-Erk by Western blot assay.In addition,to further explore whether downregulation of NDUFA4 was contributed to the suppressive effect of TTF-1 promoter operating miR-7 expression on human lung cancer cells,we transiently co-transfected p-T-miR-7 and p-NDUFA4 into lung cancer cell line 95 D cells and observed the possible change on the growth,metastasis and clone forming ability of cells by CCK-8 assay,scratch assay and clone formation assay respectively.Moreover,the expression of NDUFA4,cell growth-related molecules including CDK2,CDK3,CDK4 and CDK6,as well asmetastasis-related molecules including CXCR4,E-Cadherin,MMP-2,MMP-3 and MMP-9,were determined by Real-time PCR assay.Finally,we also analyzed the expression of NDUFA4,Akt,phosphor-Akt,Erk and phosphor-Erk by western blot assay.Results:Part One The human lung cancer model of 95 D cells in nude mice were regularly established,And the growth of tumor in p-T-miR-7 injection group was significantly decreased when compared with that in p-Cont group(P<0.05).The expression level of miR-7 in tumor tissue in p-T-miR-7 injection group was significantly higher than that in the p-Cont group(P<0.05).To confirm this data,we further detected the expression of miR-7 in tumor mass by using in situ hybridization and obtained similar result(P<0.05).HE staining of lung tissue in nude mice showed that,the counts of pulmonary metastatic nodules was significantly reduced in p-T-miR-7 injection group.HE staining further showed that there were large areas of necrosis in tumor mass in p-T-miR-7 injection group.Moreover,mmunofluorescence assay further showed that the proliferation of tumor cells decreased significantly in p-T-miR-7 injection group(P<0.05).On the contrary,the apoptosis of cells increased obviously(P<0.05).In addition,the expression of cell growth-related molecules including CDK2,CDK3,CDK4 and CDK6,as well as metastasis-related molecules including CXCR4,E-Cadherin,MMP-2,MMP-3 and MMP-9 in tumor mass were significantly decreased in p-T-miR-7 injection group compared with in p-Cont group(P<0.05).Moreover,the expression levels of phosphor-Akt and phosphor-Erk were decreased significantly in p-T-miR-7 injection group in vivo,which was consistent with the finding in vitro(P<0.05).Part Two Gene expression profile showed that there were a large number of differentially expressed genes in tumor tissues between p-T-miR-7 injection group and p-Cont group.Whole-genome array showed that,compared with p-Cont group,the expression of NDUFA4 in the p-T-miR-7 injection group decreased 4.13 times fold.Real-time PCR assay showed that the expression of NDUFA4 was significantlydecreased in tumor tissue in p-T-miR-7 injection group compared with that in the p-Cont group(P<0.05).In addition,the expression of NDUFA4 was also significantly downregulated in p-T-miR-7 transfected 95 D cells(P<0.05),which further suggested that NDUFA4 may be a new target molecule of miR-7.Importantly,western blotting assay showed that the expression level of NDUFA4 protein was significantly decreased in tumor tissue in p-T-miR-7 injection group compared with that in p-Cont group(P<0.05).Moreover,we also performed immunohistochemistry assay to detect the expression of NDUFA4 protein in tumor tissue and obtained similar result.Furthermore,we constructed an eukaryotic expression vector of NDUFA4(termed as p-NDUFA4),and then we transiently transfected p-NDUFA4 into human lung cancer cell line 95 D cells in vitro.Expectedly,Real-time PCR assay showed that the expression level of NDUFA4 significantly increased in p-NDUFA4 transfection group compared with in p-Cont group.Importantly,the proliferation of 95 D cells was elevated(P<0.05).Moreover,scratch assay further showed that the migration ability of cells was also enhanced(P<0.05).And,the clone forming ability of cells was also promoted(P<0.05).At the same time,the expression of cell growth-related molecules including CDK2,CDK3,CDK4 and CDK6,as well as metastasis-related molecules including CXCR4,E-Cadherin,MMP-2,MMP-3 and MMP-9 was significantly increased in p-NDUFA4 transfection group compared with in p-Cont group(P<0.05).Finally,western blot assay showed that the expression level of NDUFA4 protein was significantly increased in p-NDUFA4 transfection group(P<0.05).Importantly,the expression of phosphor-Akt and phosphor-Erk were increased obviously(P<0.05).Then,to further explore whether downregulation of NDUFA4 was contributed to the suppressive effect of TTF-1 promoter operating miR-7 expression on human lung cancer cells,we transiently co-transfected p-T-miR-7 and p-NDUFA4 into lung cancer cell line 95 D cells and observed the possible change on the growth and metastasis of cells.Notably,we found that the proliferation of cells in p-T-miR-7 and p-NDUFA4co-transfection group was elevated obviously compared with in p-T-miR-7transfection group(P<0.05).Moreover,clone formation assays showed that the cloneformation ability of cells also increased significantly in p-T-miR-7 and p-NDUFA4co-transfection group compared with in p-T-miR-7 transfection group(P<0.05).Further,the migration ability of cells in p-T-miR-7 and p-NDUFA4 co-transfection group also enhanced(P<0.05).Consistently,Real-time PCR assay showed that the expression of NDUFA4,cell growth-related molecules including CDK2,CDK3,CDK4 and CDK6,as well as metastasis-related molecules including CXCR4,E-Cadherin,MMP-2,MMP-3 and MMP-9,were elevated significantly in p-T-miR-7and p-NDUFA4 co-transfection group compared with those in p-T-miR-7 transfection group(P<0.05).Finally,western blot showed that the expression of NDUFA4 protein and the level of phosphor-Akt and phosphor-Erk,were significantly increased in p-T-miR-7 and p-NDUFA4 co-transfection group compared with in p-T-miR-7transfection group(P<0.05).Conclusion:1.TTF-1 promoter operating miR-7 expression attenuated the growth of human lung cancer in vivo,which was related to altered transduction of AKT/ERK signaling pathway.2.NDUFA4 was directly targeted by miR-7;NDUFA4 overexpression reversed the suppressive effect of miR-7 expression operated by TTF-1 promoter on the growth human lung cancer cells. |
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