Optimized TTF-1 Core Promoter-operated MiRNA-7 Expression Inhibits The Growth Of Human Lung Cancer Cells

This study consists of two parts as described as below.The first part is that the construction of optimized TTF-1 core promoter-operated miRNA-7 expression vector.The second part is that the effect of optimized TTF-1 core promoter operating miR-7expression on the growth of human lung cancer.Objective:Part One To construct the eukaryotic expression vector of different lengths of TTF-1 promoter operated the miR-7 expression,and screen out the core sequence of the TTF-1 promoter by Real-time PCR and biological databases.To construct the optimized TTF-1 core promoter-operated miRNA-7 expression vector and investigate the biological effects in vitro.Part Two To observe the effect of the optimized TTF-1 core promoter-operated miRNA-7 expression vector on the growth of human lung cancer cells in vivo by using nude mice model of human lung cancer through related techniques including Real-time PCR,immunofluorescence（Ki-67 and TUNEL）,western blot and HE staining.Methods:Part One We constructed the eukaryotic expression vector of different lengths of TTF-1 promoter operated the miR-7 expression based on the constructed p-EGFP-N1-TTF-1-miR-7 vector,the recombinant plasmids and p-Cont were transiently transfected into human lung cancer 95D cells in vitro.The relative expression level of miR-7 in each transfection group was determined by Real-time PCR,and the proliferation of 95D cells in each transfection group was determined by CCK-8 assay.Further,we use Signalscan,Matrix and Patch biological databases to predict the transcription factors bound to the core sequence of the TTF-1 promoter,the human lung cancer cell line 95D cells,A549 cells,human normal bronchial epithelial B2 cells,human breast cancer MB231 cells and human colon cancer SW620cells were transiently transfected with p-Tcor-miR-7 plasmids in vitro,the expression of miR-7 and related transcription factors were detected by Real-time PCR.we further construct the optimized TTF-1 core promoter-operated miRNA-7 expression vector(termed as p-^optTcor-miR-7)with more binding sites of NF-1,and the EMSA assay was used to verify the level of pivotal transcription factor protein binding to the TTF-1^optpromoter,then we transiently transfected p-^optTcor-miR-7 into human lung cancer cell line 95D cells in vitro,the expression of miR-7 was determined by Real-time PCR assay.Then we transiently transfected p-^optTcor-miR-7 into six different human cell lines,including human lung cancer cell line 95D cells,A549cells,human breast cancer MB231 cells,human colon cancer cell line SW620 cells,HCT116 cells and human normal bronchial epithelial cell B2 cells in vitro,the expression of miR-7 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.Flow cytometry was used to detect the apoptosis ratio and cell cycle,and the tumor growth cycle dependent kinases and the tumor metastasis-associated factors in lung cancer 95D cells were determined by Real-time PCR.Finally,Western blot was used to detect the expression levels of Akt,p-Akt,Erk,p-Erk and NDUFA-4.Part Two Human lung cancer tumor-bearing mouse model was established,human lung cancer cell line 95D cells were injected subcutaneously into left flank of Balb/c nude mice（n=5）.11 days later,the plasmid of p-^opt2Tcor-miR-7（100mg）,p-Tcor-miR-7（100mg）or p-Cont（100mg）were respectively injected through tail vein in nude mice five times every three days.The tumor volume of the three groups of tumor bearing nude mice were recorded by dynamic detection every 3 days,and the subcutaneous tumor growth curves were drawn to calculate the tumor inhibition rate.3 days after last injection,all mice were killed,the major organs and tumor tissue were obtained.The tumor tissue and major organ tissue were stained by HE staining.Moreover,the tumor growth cycle dependent kinases and tumor metastasis-associated factors were detected by Real-time PCR respectively.The expression of Ki-67 was analyzed by immunofluorescence assay,and the apoptosis of tumor cells was detected by TUNEL method.Morevoer,the expression of associated signal pathway,including p-Akt,p-Erk,and NDUFA-4 were detected by western blot assay.The expression level of miR-7 and distribution of plasmid copies in major organs of nude mice model were detected by Real-time PCR.Lastly,the expression levels of TC,TG,GLU,AST and ALT were detected to preliminary assess the safety of p-^opt2Tcor-miR-7 vector.Results:Part One The optimized vector were successfully constructed,in which the length of TTF-1 promoter was 1612bp,1212bp and 853bp（termed as p-T1612-miR-7,p-T1212-miR-7,p-T853-miR-7）.The recombinant plasmids and p-Cont vector were transiently transfected into human lung cancer 95D cells in vitro,and the Real-time PCR assay indicated that the expression levels of miR-7 was significantly up-regulated in the p-T1212-miR-7 transfected group compared with the p-Cont control group（P<0.05）.The CCK-8 assay showed that proliferation of 95D cells was significantly decreased in the p-T1212-miR-7 transfected group compared with the p-Cont group（P<0.05）,while the proliferation of 95D cells was constant in the p-T853-miR-7 group（P>0.05）.Therefore,The-1212 bp to-853bp sequence（total359bp）of the TTF-1 promoter was defined as the core sequence of the TTF-1promoter,and the prediction analysis of potential transcription factors bound to the core sequence of the TTF-1 promoter by using Signalscan,Matrix,and Patch biological databases yielded two transcription factors:NF-1 and AP-1,the binding sites are-1166bp-1162bp（TGGCA）and-1141bp-1137bp（CCAAG）,and then we transiently transfected p-T1212-miR-7（renamed to p-Tcor-miR-7）into human lung cancer cell line 95D cells,A549 cells,human normal bronchial epithelial cell line B2cells,human breast cancer MB231 cells and human colon cancer SW620 cells in vitro,the Real-time PCR assay indicated that miR-7 was significantly up-regulated in the human lung cancer 95D cells and A549 cells.Meanwhile,the expression level of NF-1 was aslo up-regulated,and when the expression level of NF-1 was decreased in other transfection groups,the expression level of miR-7 was aslo decreased correspondingly.The expression level of AP-1 had no corresponding trend with the expression level of miR-7,so NF-1 was determined as the pivotal transcription factor that regulating miR-7 expression positively.we further constructed the optimized TTF-1 core promoter-operated miRNA-7 expression vector(termed as p-^optTcor-miR-7)with more binding sites of NF-1（Respectively are:-1068bp-1064bp sequence:TGGCT,-998bp-994bp sequence:ATGCA,-952bp-948bp sequence:GGGCA,-926bp-922bp sequence:TGGCC）,then we transiently transfected p-^optTcor-miR-7 into human lung cancer cell line 95D cells in vitro,the Real-time PCR assay showed that the expression level of miR-7 was significantly up-regulated in the p-^opt2Tcor-miR-7 transfected group compared with the p-Cont group（P<0.05）,and CCK-8 assay showed that the proliferation of 95D cells was significantly decreased in the p-^opt2Tcor-miR-7 transfected group compared with the p-Cont control group（P<0.05）.Therefore,the p-^opt2Tcor-miR-7 plasmid was selected as the best eukaryotic expression vector to regulate the expression of miR-7.The Real-time PCR assay indicated that the expression of miR-7 in human lung cancer cell line 95D cells and A549 cells was significantly higher than that in other four types of cancer cells（P<0.05）.The CCK-8 assay indicated that the proliferation of 95D cells was significantly decreased in the p-^opt2Tcor-miR-7 group compared with the p-Cont control group and the p-Tcor-miR-7 transfected group（P<0.05）,scratch assay,clone formation assay and transwell assay showed that the migration,invasion,and clonogenic abilities of 95D cells were significantly reduced in the p-^opt2Tcor-miR-7group compared with the p-Cont control group and the p-Tcor-miR-7 transfected group（P<0.05）,and the flow cytometry assay showed that there was no significant difference of the proportion of cells apoptosis in the p-^opt2Tcor-miR-7 groups compared with the p-Cont control group and the p-Tcor-miR-7 transfected group（P>0.05）,while the proportion of cells in the G0/G1 and G2/M phases was significantly up-regulated in p-^opt2Tcor-miR-7 group compared with the p-Cont group and the p-Tcor-miR-7 group,whereas the proportion of cells in the S phase was significantly down-regulated（P<0.05）.Moreover,the Real-time PCR assay showed that the expression of the tumor growth cycle dependent kinases and the tumor metastasis-associated factors were significantly down-regulated in the p-^opt2Tcor-miR-7 group compared with the p-Cont group（P<0.05）.Finally,western blot indicated that the expression of NDUFA4 protein and the level of p-Akt and p-Erk were significantly increased in p-^opt2Tcor-miR-7 group compared with the p-Cont group and the p-Tcor-miR-7 group（P<0.05）.Part Two Human lung cancer model using nude mice was successfully established.And the growth of tumor in p-^opt2Tcor-miR-7 group significantly decreased compared with that in p-Cont group and the p-Tcor-miR-7 group（P<0.05）,and the tumor inhibition rate was significantly increased（P<0.05）.HE staining of tumor tissue showed that there were large areas of tumor necrosis in p-^opt2Tcor-miR-7 group.The Real-time PCR assay indicated that the expression level of miR-7 in tumor tissue in p-^opt2Tcor-miR-7 group was significantly higher compared with that in p-Cont group and the p-Tcor-miR-7 group（P<0.05）.Next,the expression of tumor growth cycle dependent kinases and tumor metastasis-associated factors in tumor tissue was significantly down-regulated in p-^opt2Tcor-miR-7 group compared with p-Cont group and the p-Tcor-miR-7 group（P<0.05）.In addition,immunofluorescence assay further showed that the Ki67 of tumor cells decreased obviously in p-^opt2Tcor-miR-7 group compared with that in p-Cont group and the p-Tcor-miR-7 group,conversely,the local apoptosis of tumor cells increased significantly in p-^opt2Tcor-miR-7 group compared with that in p-Cont group and the p-Tcor-miR-7 group.Western blot indicated that the expression level of p-Akt,p-Erk and NDUFA-4 in tumor tissue were decreased significantly in p-^opt2Tcor-miR-7 group compared with that in p-Cont group and the p-Tcor-miR-7 group（P<0.05）,which was consistent with the results at the cellular level.Importantly,there were no significant difference on the morphology and histology of major organ tissues including heart,liver,spleen,kidney,brain,intestine and lymph nodes between p-^opt2Tcor-miR-7 group and p-Cont group（P>0.05）.Besides,the p-^opt2Tcor-miR-7 vector is distributed in the main organs of mice,including heart,liver and so on.The plasmid copy numbers distributed highest in lung and tunor tissue,this is similar to the previous data.Most importantly,in the p-^opt2Tcor-miR-7 group,except for the lung and heart tissue,the expression of miR-7 in other major organs has no change significantly compared with the p-Cont group.Finally,we also found that there were no significant change on the biochemistry indicators such as TC,TG,GLU,AST and ALT between p-^opt2Tcor-miR-7 group,p-Tcor-miR-7 group and p-Cont group（P>0.05）.Conclusion:1.The optimized core sequence of TTF-1 promoter could effectively regulate miRNA-7 expression and inhibit the growth of human lung cancer cells in vitro.2.Optimized core sequence of TTF-1 promoter-operated miRNA-7 expression significantly inhibited the tumor growth of human lung cancer in nude mice model.