Targeted gene therapy has become a promising approach for lung cancer treatment.In our previous work,we reported that the targeted expression of microRNA-7(miR-7)operated by thyroid transcription factor-1(TTF-1)promot...Targeted gene therapy has become a promising approach for lung cancer treatment.In our previous work,we reported that the targeted expression of microRNA-7(miR-7)operated by thyroid transcription factor-1(TTF-1)promoter inhibited the growth of human lung cancer cells in vitro and in vivo;however,the intervention efficiency needed to be further improved.In this study,we identified the core promoter of TTF-1(from-1299 bp to-871 bp)by 5’deletion assay and screened out the putative transcription factors nuclear factor-1(NF-1)and activator protein-1(AP-1).Further analysis revealed that the expression level of NF-1,but not AP-1,was positively connected with the activation of TTF-1 core promoter in human non-small-cell lung cancer(NSCLC)cells.Moreover,the silencing of NF-1 could reduce the expression level of miR-7 operated by TTF-1 core promoter.Of note,we optimized four distinct sequences to form additional NF-1-binding sites(TGGCA)in the sequence of TTF-1 core promoter(termed asTTF-1 promoter),and verified the binding efficiency of NF-1 on theTTF-1 promoter by electrophoretic mobility shift assay(EMSA).As expected,theTTF-1 promoter could more effectively drive miR-7 expression and inhibit the growth of human NSCLC cells in vitro,accompanied by a reduced transduction of NADH dehydrogenase(ubiquinone)1αsubcomplex 4(NDUFA4)/protein kinase B(Akt)pathway.Consistently,TTF-1 promoter-driven miR-7expression could also effectively abrogate the growth and metastasis of tumor cells in a murine xenograft model of human NSCLC.Finally,no significant changes were detected in the biological indicators or the histology of some important tissues and organs,including heart,liver,and spleen.On the whole,our study revealed that the optimized TTF-1 promoter could more effectively operate miR-7 to influence the growth of human NSCLC cells,providing a new basis for the development of microRNA-based targeting gene therapy against clinical lung cancer.展开更多
基金the National Natural Science Foundation of China(Nos.32160178,82160503,31760258,and 81960509)the Project of the Guizhou Provincial Department of Science and Technology(Nos.QKH-JC-2018-1428,QKHZC-2020-4Y156,and QKH-JC-ZK-2022-624)+1 种基金the Collaborative Innovation Center of Chinese Ministry of Education(No.2020-39)the Program for Excellent Young Talents of Zunyi Medical University(No.15ZY-001),China。
文摘Targeted gene therapy has become a promising approach for lung cancer treatment.In our previous work,we reported that the targeted expression of microRNA-7(miR-7)operated by thyroid transcription factor-1(TTF-1)promoter inhibited the growth of human lung cancer cells in vitro and in vivo;however,the intervention efficiency needed to be further improved.In this study,we identified the core promoter of TTF-1(from-1299 bp to-871 bp)by 5’deletion assay and screened out the putative transcription factors nuclear factor-1(NF-1)and activator protein-1(AP-1).Further analysis revealed that the expression level of NF-1,but not AP-1,was positively connected with the activation of TTF-1 core promoter in human non-small-cell lung cancer(NSCLC)cells.Moreover,the silencing of NF-1 could reduce the expression level of miR-7 operated by TTF-1 core promoter.Of note,we optimized four distinct sequences to form additional NF-1-binding sites(TGGCA)in the sequence of TTF-1 core promoter(termed asTTF-1 promoter),and verified the binding efficiency of NF-1 on theTTF-1 promoter by electrophoretic mobility shift assay(EMSA).As expected,theTTF-1 promoter could more effectively drive miR-7 expression and inhibit the growth of human NSCLC cells in vitro,accompanied by a reduced transduction of NADH dehydrogenase(ubiquinone)1αsubcomplex 4(NDUFA4)/protein kinase B(Akt)pathway.Consistently,TTF-1 promoter-driven miR-7expression could also effectively abrogate the growth and metastasis of tumor cells in a murine xenograft model of human NSCLC.Finally,no significant changes were detected in the biological indicators or the histology of some important tissues and organs,including heart,liver,and spleen.On the whole,our study revealed that the optimized TTF-1 promoter could more effectively operate miR-7 to influence the growth of human NSCLC cells,providing a new basis for the development of microRNA-based targeting gene therapy against clinical lung cancer.
