Optimized thyroid transcription factor-1 core promoter-driven microRNA-7 expression effectively inhibits the growth of human non-small-cell lung cancer cells

Optimized thyroid transcription factor-1 core promoter-driven microRNA-7 expression effectively inhibits the growth of human non-small-cell lung cancer cells 优化的TTF-1核心启动子驱动miRNA-7表达有效抑制人非小细胞肺癌细胞的生长 1 2 equal first-author 1 2 equal 1 2 1 2 1 2 1 2 1 2 1 2 1 3 corresp corresp Lin XU, xulinzhouya@163.com Lin XU, xulinzhouya@163.com Ya ZHOU, zhouyazmc@163.com Ya ZHOU, zhouyazmc@163.com zhouyazmc@163.com https://orcid.org/0000-0003-2943-9220 4 corresp corresp Lin XU, xulinzhouya@163.com Lin XU, xulinzhouya@163.com Yong HAN, Chinahotfog@126.com Yong HAN, Chinahotfog@126.com Chinahotfog@126.com https://orcid.org/0000-0003-4497-6083 1 2 corresp Lin XU, xulinzhouya@163.com Lin XU, xulinzhouya@163.com xulinzhouya@163.com https://orcid.org/0000-0002-6889-0279 Special Key Laboratory of Gene Detection and Therapy & Base for Talents in Biotherapy of Guizhou Province, Zunyi 563000, China Special Key Laboratory of Gene Detection and Therapy & Base for Talents in Biotherapy of Guizhou Province, Zunyi 563000, China 贵州省基因检测与治疗特色重点实验室暨贵州省生物治疗人才基地，中国遵义市，563000 贵州省基因检测与治疗特色重点实验室暨贵州省生物治疗人才基地，中国遵义市，563000 Department of Immunology, Zunyi Medical University, Zunyi 563000, China Department of Immunology, Zunyi Medical University, Zunyi 563000, China 遵义医科大学免疫学教研室，中国遵义市，563000 遵义医科大学免疫学教研室，中国遵义市，563000 Department of Medical Physics, Zunyi Medical University, Zunyi 563000, China Department of Medical Physics, Zunyi Medical University, Zunyi 563000, China 遵义医科大学医学物理系，中国遵义市，563000 遵义医科大学医学物理系，中国遵义市，563000 Department of Physiology, Zunyi Medical University, Zunyi 563000, China Department of Physiology, Zunyi Medical University, Zunyi 563000, China 遵义医科大学生理学教研室，中国遵义市，563000 遵义医科大学生理学教研室，中国遵义市，563000 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 as opt TTF-1 promoter), and verified the binding efficiency of NF-1 on the opt TTF-1 promoter by electrophoretic mobility shift assay (EMSA). As expected, the opt TTF-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) 1α subcomplex 4 (NDUFA4)/protein kinase B (Akt) pathway. Consistently, opt TTF-1 promoter-driven miR-7 expression 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. 靶向基因治疗是一种很有前景的肺癌治疗方法。在前期工作中，我们报道了由甲状腺转录因子-1（TTF-1）启动子调控微小RNA-7（miR-7）的靶向表达可在体外和体内抑制人肺癌细胞的生长，但干预效率有待进一步提高。在本研究中，我们通过5’缺失分析鉴定了TTF-1的核心启动子（从−1299 bp到−871 bp），并筛选出潜在的结合转录因子核因子-1（NF-1）和激活蛋白-1（AP-1）。进一步分析的结果表明：NF-1的表达水平，而非AP-1的表达水平，与人非小细胞肺癌 （ NSCLC ） 细胞中TTF-1核心启动子的活性呈正相关。此外，NF-1的沉默可以降低由TTF-1核心启动子调控的miR-7的表达。重要的是，我们在TTF-1核心启动子的序列上优化了四个不同的序列（称为 opt TTF-1启动子）以形成额外的NF-1结合位点（TGGCA），并通过电泳迁移率实验（EMSA）分析验证了NF-1对 opt TTF-1启动子的结合效率。通进一步研究，结果显示 opt TTF-1启动子可更有效地驱动miR-7表达并在体外抑制人NSCLC细胞的生长，同时减少Ⅰ型辅酶脱氢酶1α亚复合物4（NDUFA4）/蛋白激酶B（Akt）通路转导。 opt TTF-1启动子驱动的miR-7表达同样也可有效地抑制人NSCLC异种移植模型中肿瘤细胞的体内生长和转移。最后，模型动物的心脏、肝脏和脾脏等重要组织和器官的生物学指标和组织学未见明显变化。总之，本研究揭示了优化的TTF-1启动子可更有效地调控miR-7表达来抑制人NSCLC细胞的生长，为开发基于miRNA的靶向基因治疗临床肺癌提供了新的实验基础。 Lung cancer Thyroid transcription factor-1 (TTF-1) Promoter MicroRNA-7 (miR-7) Nuclear factor-1 (NF-1) 肺癌 甲状腺转录因子-1（TTF-1） 启动子 微小RNA-7（miR-7） 核因子-1（NF-1） 1 Introduction 1 Lung cancer is a malignant tumor with the second highest incidence in the world. Although chemotherapy, radiotherapy, and surgery have made significant advances in recent years, the overall survival rate of patients with lung cancer is still very low ( Chan and Coward, 2013 Chan and Coward, 2013 ; Billiet et al., 2016 Billiet et al., 2016 ; Nasim et al., 2019 Nasim et al., 2019 ). Based on the improvement of biomedical techniques, some prolific genetic alterations and novel molecular changes in lung cancer were verified, including epidermal growth factor receptor ( EGFR ), anaplastic lymphoma kinase ( ALK ), c-ros oncogene 1 ( ROS1 ), B-Raf proto-oncogene ( BRAF ), and neuro trophin receptor kinase ( NTRK ), which aid the development of the pathological diagnosis and classification of lung cancers, as well as the therapeutic strategies of clinical lung cancer ( Wilson et al., 2017 Wilson et al., 2017 ; Zhuang et al., 2019 Zhuang et al., 2019 ; Dziadziuszko et al., 2021 Dziadziuszko et al., 2021 ; Umakanthan and Bukelo, 2021 Umakanthan and Bukelo, 2021 ). Furthermore, a variety of new anticancer therapies have been applied to compensate for the deficiencies of traditional treatments, such as gene therapy that is expected to provide an alternative treatment effect and minimize the side effects in lung cancer patients ( Toloza et al., 2006 Toloza et al., 2006 ; Lara-Guerra and Roth, 2016 Lara-Guerra and Roth, 2016 ; Zhao X et al., 2021 Zhao X et al., 2021 ; Du et al., 2022 Du et al., 2022 ). However, because of a series of shortcomings, such as the lack of sustained and stable expression of genes of interest in vivo and the weak targeting of such genes, its application in clinical lung cancer needs to be further explored. Notably, accumulating evidence has shown that targeted gene expression technology, as a key strategy of gene therapy, enables these therapeutic genes to be accurately expressed in tumor cells through specific promoters and unique delivery systems, thereby improving the intervention effect on lung cancer ( Wiggins et al., 2010 Wiggins et al., 2010 ; Rai et al., 2011 Rai et al., 2011 ; Amreddy et al., 2017 Amreddy et al., 2017 ; Lee et al., 2019 Lee et al., 2019 ). A promoter is the upstream regulatory element of a specific genetic region, which starts transcription by combining with certain specific transcription factors, and its activity directly affects the expression level of genes. In the progression of various tumors, sequence mutation, methylation level change, or the regulation of some trans-acting elements reduces the promoter activity, affecting the expression of functional genes ( Zhang BY et al., 2013 Zhang BY et al., 2013 ; Zhang EB et al., 2014 Zhang EB et al., 2014 ; Zhao et al., 2015 Zhao et al., 2015 ; Chen et al., 2022 Chen et al., 2022 ). Therefore, researchers’ aim is to find a promoter with high activity, or increase the promoter activity to improve the dilemma of genes of interest. Importantly, previous studies showed that tumor-specific promoters have high regulatory activity and targeting specificity to drive the expression of the genes of interest while reducing the damage to normal tissues ( Fukazawa et al., 2007 Fukazawa et al., 2007 ; Sher et al., 2009 Sher et al., 2009 ; Xie et al., 2009 Xie et al., 2009 ). In addition, some studies confirmed that promoter activity could be improved by screening the core promoter, adding enhancement elements, or adding transcription factor-binding sequences, and eventually enhanced the expression of its target genes ( Linnerth-Petrik et al., 2012 Linnerth-Petrik et al., 2012 ; Danino et al., 2015 Danino et al., 2015 ; Zhang et al., 2015 Zhang et al., 2015 ; Even et al., 2017 Even et al., 2017 ). Therefore, the means to enhance promoter activity is not only crucial for targeted gene expression, but is also a vital issue for the clinical gene therapy of lung cancer. In our previous study, we constructed a eukaryotic vector of thyroid transcription factor-1 (TTF-1)‍ promoter-operated microRNA-7 (miR-7) expression, and found that the TTF-1 promoter could regulate miR-7 expression and inhibit the growth of human lung cancer cells both in vivo and in vitro, indicating that TTF-1 promoter-operated miR-7 expression may be one of the promising strategies for the targeted gene therapy of lung cancer ( Lei et al., 2017 Lei et al., 2017 ). However, this intervention effect in lung cancer needs to be further improved. In this study, we screened the TTF-1 core promoter by a 5' deletion assay and further identified the key transcription factor that binds to the core sequence of TTF-1 promoter. Then, we optimized the core sequence of TTF-1 promoter to form more binding sites of key transcription factors. Finally, we observed the effect of miR-7 expression operated by optimized TTF-1 core promoter on the growth and metastasis of human lung cancer cells in vitro and in vivo, and explored the related molecular mechanism, which might provide a preliminary experimental basis for promoting the development of new clinical lung cancer gene therapy strategies. 2 Results 1 2.1 Screening and optimization of TTF-1 core promoter 2 The schematic of the research program was shown in Fig. 1 Fig. 1 a. Firstly, 5' deletion assay and bioinformatic analysis were performed to screen out the TTF-1 core promoter and putative transcription factor ( Fig. 1 Fig. 1 a, upper). Secondly, the site mutation technique was employed to optimize the TTF-1 core promoter, and then the efficiency of optimized TTF-1 core promoter-driven miR-7 expression was estimated in a murine xenograft model of human lung cancer ( Fig. 1 Fig. 1 a, lower). As shown in Fig. 1 Fig. 1 b, the 5' deletion assay was designed in the distinct TTF-1 promoter with different lengths. The data showed that, compared with the p-Cont (pEGFP-N1-basic) group, the expression of miR-7 in the p-T1229-miR-7 (pEGFP-N1-basic-TTF-1 promoter with 1229 bp-miR-7) group was the most upregulated ( P < 0.01), but not significantly increased in the p-T1612-miR-7 (pEGFP-N1-basic-TTF-1 promoter with 1612 bp-miR-7) with a longer promoter sequence or p-T871-miR-7 (pEGFP-N1-basic-TTF-1 promoter with 871 bp-miR-7) with a shorter promoter sequence transfection group ( P > 0.05; Fig. 1 Fig. 1 c). Moreover, cell counting kit-8 (CCK-8) analysis further showed that the proliferation of human lung cancer 95D cells in the p-T1229-miR-7 transfection group was strongly inhibited ( P < 0.01; Fig. 1 Fig. 1 d), but the inhibition effect in the p-T1612-miR-7 or p-T871-miR-7 transfection group was not significantly changed ( P > 0.05; Fig. 1 Fig. 1 d). These results indicated that the sequence from -‍1229 bp to -‍871 bp (359 bp in total) was the core sequence of the TTF-1 promoter. Then, the SignalScan, MatrixCatch, and Patch biological databases were used to predict and analyze the putative transcription factors on the core sequence of the TTF-1 promoter. Two transcription factors, nuclear factor-1 (NF-1) and activator protein-1 (AP-1), were screened out by cross comparison, whose binding sites were TGGCA and CCAAG, respectively ( Fig. 2 Fig. 2 a). Next, to explore the relationship among the activity of TTF-1 core promoter regulating miR-7 expression and potential transcription factors, the related vectors were transiently transfected into five different cell lines in vitro. The results showed that, among the five cell lines, the expression levels of miR-7 in human non-small-cell lung cancer (NSCLC) 95D and A549 cells were higher ( P < 0.01; Fig. 2 Fig. 2 b). Unexpectedly, the expression of transcription factor NF-1, but not AP-1, in 95D and A549 cells was also significantly higher than that in other cell lines ( P < 0.05; Fig. 2 Fig. 2 c), displaying a positive connection with the miR-7 expression pattern. Next, to further verify whether NF-1 was the key transcription factor regulating the activity of TTF-1 core promoter in NSCLC cells, we used RNA interference (RNAi) to reduce the expression of NF-1 in human NSCLC 95D cells. The results showed that RNAi-1 in different RNAi sequences inhibited NF-1 messenger RNA (mRNA) most significantly ( P < 0.01; Fig. 2 Fig. 2 d). After downregulation of NF-1 by RNAi-1 sequence, the expression of miR-7 was also downregulated ( P < 0.05; Fig. 2 Fig. 2 e). Meanwhile, we mutated the binding site of NF-1 and obtained a similar phenomenon ( P < 0.05; Figs. 2 2 f and 2 2 g). These results suggested that NF-1 is a key transcription factor that binds to the core sequence of TTF-1 promoter to regulate the expression of miR-7 in human NSCLC cells. Next, considering NF-1 as one of the key factors in regulating the activation of TTF-1 core promoter, we envisioned whether the activity of TTF-1 core promoter could be enhanced by increasing the NF-1-binding sites to recruit more NF-1. Therefore, based on above bioinformatic analysis, two or four distinct sequences without any putative transcription factor-binding sites in the TTF-1 core promoter were optimized to form more NF-1-binding sites (TGGCA) (Figs. 2 2 h and 2 2 i). Interestingly, compared with that in the control group, the electrophoretic mobility shift assay (EMSA) showed that more NF-1 transcription factors were bound to the p- opt1 Tcor-miR-7 (pEGFP-N1-basic-TTF-1 core promoter with 3 NF-1-bounding sites-miR-7) group with two artificial NF-1-binding sites ( Fig. 2 Fig. 2 j). Of note, more NF-1 transcription factors were bound to the core sequence of the p- opt2 Tcor-miR-7 (pEGFP-N1-basic-TTF-1 core promoter with 5 NF-1-bounding sites-miR-7) group with four artificial NF-1-binding sites compared with other groups ( Fig. 2 Fig. 2 j). Finally, we further confirmed that the expression of miR-7 driven by the TTF-1 core promoter in the p- opt2 Tcor-miR-7 transfection group was upregulated to the greatest extent ( P < 0.01; Fig. 2 Fig. 2 k). Combining these data demonstrated that miR-7 could be more effectively operated by the optimized TTF-1 core promoter. 2.2 Effects of opt TTF-1 promoter-driven miR-7 expression on the growth and metastasis of human NSCLC cells in vitro 2 In order to observe the effects of optimized TTF-1 promoter-driven miR-7 expression on human NSCLC in vitro, relative vectors were transiently transfected into human NSCLC cells. The results showed that, compared with the p-Tcor-miR-7 (pEGFP-N1-basic-TTF-1 core promoter-miR-7) transfection group, the cell growth and clone formation ability of human NSCLC cells in the p- opt2 Tcor-miR-7 (termed as opt TTF-1 promoter) transfection group were dramatically suppressed ( P < 0.05; Figs. 3 Figs. 3 a‍–‍3d and S1). Consistently, the expression of cell growth-related molecules cyclin-dependent kinase 1 (CDK1), CDK2, CDK4, and CDK6 was significantly decreased ( P < 0.01; Fig. 3 Fig. 3 e). In addition, the expression of metastasis-related molecules, including matrix metalloproteinase 2 (MMP2), MMP3, MMP9, C-X-C chemokine receptor type 4 (CXCR4), and E-cadherin, was also dramatically suppressed ( P < 0.01; Fig. 3 Fig. 3 f), accompanied by an attenuation of migration and invasion ability ( P < 0.05; Figs. 3 Figs. 3 g‍–‍3j). Further analysis showed that the proportions of G0/G1 and G2/M phases in the p- opt2 Tcor-miR-7 group were significantly upregulated, while the cell proportion of S phase was significantly downregulated ( P < 0.05; Figs. 3 3 k and 3 3 l), indicating that optimized TTF-1 promoter-driven miR-7 expression blocked the cycle progression of human NSCLC 95D cells. The above data suggested that opt TTF-1 promoter-driven miR-7 expression was more effective in decreasing the growth and metastasis of human NSCLC cells in vitro. 2.3 Effects of opt TTF-1 promoter-driven miR-7 expression on the transduction of NDUFA4 pathway in vitro 2 Many studies have evidenced that miR-7 could regulate biological processes such as tumor growth and metastasis by protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) signaling pathways ( Liu et al., 2014 Liu et al., 2014 ; Cao et al., 2016 Cao et al., 2016 ). In our previous work, we also found that miR-7 could affect the growth of human lung cancer cells through the target molecule NADH dehydrogenase (ubiquinone) 1α subcomplex 4 (NDUFA4) and related signaling pathways, such as Akt and Erk ( Lei et al., 2017 Lei et al., 2017 ). Therefore, to further investigate the molecular mechanism of human NSCLC cell growth affected by opt TTF-1 promoter-driven miR-7 expression, the relative protein expression levels were analyzed. We found no difference in the expression of Akt or Erk among these groups ( P > 0.05; Figs. 4 4 a and 4 4 b); however, the expression of NDUFA4 in the p- opt2 Tcor-miR-7 transfection group was significantly decreased ( P < 0.05; Figs. 4 4 a and 4 4 b), which was consistent with our previous findings ( Lei et al., 2017 Lei et al., 2017 ). As expected, the expression of phospho-Akt (p-Akt) and phospho-Erk (p-Erk) also decreased significantly ( P < 0.05; Figs. 4 4 a and 4 4 b). Moreover, the energy metabolism and growth-related molecule analyses revealed that the total adenosine 5'-monophosphate (AMP)‍-activated protein kinase (AMPK) did not change, but phospho-AMPK (p-AMPK) and growth-related molecules CDK1 and CDK4 were significantly downregulated ( P < 0.05; Figs. 4 4 c and 4 4 d). In addition, we confirmed the expression changes of p-Akt, p-Erk, and NDUFA4 in the three groups by immunofluorescence, and similar results were obtained ( Fig. 4 Fig. 4 e). Overall, our data indicated that opt TTF-1 promoter-driven miR-7 expression decreased the growth of human NSCLC cells in vitro by regulating the expression of NDUFA4 and related pathways. 2.4 Effects of opt TTF-1 promoter-driven miR-7 expression on the tumorigenicity of NSCLC in vivo 2 In order to further explore the intervention effect of opt TTF-1 promoter-driven miR-7 expression on human NSCLC in vivo, a xenotransplantation model of human NSCLC in nude mice was established ( Fulzele et al., 2006 Fulzele et al., 2006 ; Huang et al., 2017 Huang et al., 2017 ). Once the tumor formation was complete, tumor-bearing nude mice were injected with p-Cont, p-Tcor-miR-7, or p- opt2 Tcor-miR-7 recombinant vectors through the tail vein every 3 d, for a total of five times ( Fig. 5 Fig. 5 a). Then, the growth changes of the tumors were observed accordingly. As shown in Figs. 5 5 b and 5 5 c, the tumor growth in the p- opt2 Tcor-miR-7 injection group was significantly slower than that in the p-Tcor-miR-7 injection group ( P < 0.05). Moreover, hematoxylin and eosin (HE) staining showed that the tumor tissues in the p- opt2 Tcor-miR-7 injection group showed extensive necrosis, accompanied with decreased tumor weight ( P < 0.05; Figs. 5 5 d and 5 5 e). In our previous research, we found that TTF-1 promoter-operated miR-7 expression could effectively inhibit the growth of tumors in vivo, with the tumor inhibition rate of about 34% ( Lei et al., 2017 Lei et al., 2017 ). Importantly, our data showed that the tumor inhibition rate of the p- opt2 Tcor-miR-7 injection group was significantly enhanced, with an inhibition rate increased by 126% (inhibition rate 34% vs. 77%) ( P < 0.01; Fig. 5 Fig. 5 f). Moreover, the expression level of miR-7 in tumor tissues in the p- opt2 Tcor-miR-7 injection group was significantly higher than that in the p-Tcor-miR-7 injection groups ( P < 0.05; Fig. 5 Fig. 5 g). Meanwhile, the immunofluorescence assay further showed that the proliferation ability of tumor cells was seriously weakened ( P < 0.05; Figs. 5 5 h and 5 5 i). Conversely, the apoptosis of tumor cells was significantly increased ( P < 0.05; Fig. S2). Finally, to confirm the effect of opt TTF-1 promoter-operated miR-7 expression on the growth and metastasis of tumor cells in vivo, the relative molecules in tumor tissues were analyzed. Consistent with these findings in vitro, the expression of growth-related molecules, including CDK2, CDK4, and CDK6, and metastasis-related molecules, including MMP2, MMP3, MMP9, CXCR4, and E-cadherin, was significantly reduced in the p- opt2 Tcor-miR-7 injection group ( P < 0.05; Figs. 5 5 j and 5 5 k). Combining these data demonstrated that the opt TTF-1 promoter could effectively drive the expression of miR-7 in tumor tissues, which subsequently inhibited the tumorigenicity of NSCLC in vivo. 1;2; 2.5 Effects of opt TTF-1 promoter-driven miR-7 expression on the NDUFA4 pathway in vivo 2 Next, to investigate the changes of related signaling pathways in vivo, the relevant molecules were analyzed in the tumor tissues of tumor-bearing mice. The results showed that, compared with those in the p-Tcor-miR-7 group, the expression levels of NDUFA4, p-Akt, and p-Erk in the p- opt2 Tcor-miR-7 group were significantly decreased ( P < 0.05; Figs. 6 6 a and 6 6 b). Besides, p-AMPK, CDK1, and CDK4 were also significantly downregulated ( P < 0.05; Figs. 6 6 c and 6 6 d). To confirm these findings, we detected the expression levels of p-Akt and p-Erk in tumor tissues by immunohistochemistry (Figs. 6 6 e and 6 6 f), and the expression of NDUFA4 by immunofluorescence ( P < 0.05; Figs. 6 6 g and 6 6 h), and obtained similar results. Overall, these findings showed that opt TTF-1 promoter-driven miR-7 expression affects the tumorigenicity of NSCLC in vivo, which is related to the altered expression of NDUFA4 pathway. 2.6 Ideal targeting efficacy and safety of opt TTF-1 promoter-driven miR-7 expression 2 Many studies have shown that the biological changes of organs or tissues have an important reference value for the wide application of targeted gene therapy ( Naldini, 2015 Naldini, 2015 ; Lux and Scharenberg, 2017 Lux and Scharenberg, 2017 ; Bucher et al., 2021 Bucher et al., 2021 ). Therefore, we analyzed the distribution of p- opt2 Tcor-miR-7 plasmid copies in the main organs of mice. As shown in Fig. 7 Fig. 7 a, the p- opt2 Tcor-miR-7 plasmid was distributed in all the main organs. Importantly, the distributions of plasmid copy numbers in tumor and lung tissues were significantly higher than those in other major organs ( P < 0.01; Fig. 7 Fig. 7 a). Consistently, the expression levels of miR-7 were significantly increased in lung and tumor tissues ( P < 0.01), but not in other organs ( Fig. 7 Fig. 7 b), indicating rational targeting efficacy. Furthermore, we observed the potential influence of optimized TTF-1 promoter-driven miR-7 expression on the major organs of the murine tumor-bearing model. The data revealed no significant differences in the weights or indexes of main organs, including heart, liver, spleen, lung, kidney, brain, and intestine, between the three groups ( P > 0.05; Figs. 7 7 c and 7 7 d). In addition, the results of HE staining showed no obvious pathological changes in these major organs (Fig. S3). Finally, we detected some key biological indexes such as aspartate aminotransferase (AST), alanine transaminase (ALT), and serum total cholesterol (TC) in the sera, which were used to assess the functional changes of various organs. As expected, these biochemical indexes showed no significant changes among these groups ( P > 0.05; Fig. 7 Fig. 7 e). Combining these data suggested that opt TTF-1 promoter-driven miR-7 expression exhibits an ideal targeting efficacy and safety. 3 Discussion 1 In recent years, the expression of target genes driven by specific gene promoters has become an important strategy of gene therapy for many kinds of cancers ( Powell et al., 2015 Powell et al., 2015 ; Massaro et al., 2020 Massaro et al., 2020 ; Pruller et al., 2021 Pruller et al., 2021 ). The TTF-1, as a pedigree-specific oncogene, is dominantly expressed in lung cancer, but not in other types of cancer or tissues ( Kim et al., 2018 Kim et al., 2018 ; Guan et al., 2021 Guan et al., 2021 ). Importantly, in our previous work, we reported that TTF-1 promoter-operated miR-7 expression could reduce the growth and metastasis of human lung cancer cells, indicating the potential value of TTF-1 promoter in targeted gene therapy against lung cancer ( Lei et al., 2017 Lei et al., 2017 ). However, the intervention efficiency needed to be further improved. In the present study, we further identified and optimized the core sequence of TTF-1 promoter. Notably, we could extend our previous findings by demonstrating that the optimized TTF-1 promoter could more effectively drive miR-7 expression and abrogate the growth of human NSCLC cells in vivo with an inhibition rate increased by 126% (inhibition rate 34% vs. 77%), indicating that the reasonable optimization of TTF-1 promoter might be much more valuable for successive application in gene therapy against clinical lung cancer. In addition, it was noticed that one recent study reported TTF-1 as mainly expressed in lung adenocarcinoma ( Umakanthan et al., 2021 Umakanthan et al., 2021 ). Therefore, the potential effects of TTF-1 promoter-driven miR-7 expression in other types of lung cancer still need to be further explored, which might be valuable for the clinical application of this approach in lung cancer patients. MiR-7 is a unique member of the microRNA (miRNA) family that plays an important role in the progression of various diseases, including lung cancer, through multiple targets ( Zhao JJ et al., 2015 Zhao JJ et al., 2015 ; Chen et al., 2021 Chen et al., 2021 ; Zhao JJ et al., 2021 Zhao JJ et al., 2021 ). For example, miR-7 can inhibit the growth and migration of NSCLC cells in vivo by targeting BCL-2, an important anti-apoptotic gene ( Xiong et al., 2011 Xiong et al., 2011 ; Du et al.,2021 Du et al.,2021 ). In addition, Li JR et al. (2014) Li JR et al. (2014) found that the restoration of miR-7 expression inhibited the tumorigenicity of lung cancer cells in vivo. Moreover, highly expressed miR-7 targeting the epidermal growth factor EGFR promoted lung tumor regression and attenuated drug resistance in vivo ( Rai et al., 2011 Rai et al., 2011 ). Consistently, our previous work also showed that the overexpression of miR-7 could reduce the growth and metastasis of human lung cancer cells through NDUFA4 ( Lei et al., 2017 Lei et al., 2017 ). In this study, we further reported that the optimized TTF-1 promoter-driven miR-7 expression could more effectively reduce the growth and metastasis of human NSCLC cells both in vivo and in vitro, accompanied by a reduced expression of NDUFA4 and altered transduction of Akt, Erk, and AMPK pathways. Therefore, combined with these works, we further highlighted the important role of miR-7 as a tumor suppressor gene in the development of lung cancer through regulating multiple targets and signaling pathways, which verifies its potential for gene therapy against lung cancer. As a GTPase-activating protein, NF-1 negatively regulates Ras signal transduction under normal physiological conditions, and its abnormal expression contributes to the development of various cancers ( Li C et al., 2014 Li C et al., 2014 ; Philpott et al., 2017 Philpott et al., 2017 ; Su et al., 2019 Su et al., 2019 ). Furthermore, recent studies have shown that the expression of NF-1 in NSCLC is significantly upregulated and enhances the abilities of cell proliferation and migration ( Qian et al., 2018 Qian et al., 2018 ). In the present study, we screened out NF-1, but not AP-1, as the putative transcription factor binding to the TTF-1 core promoter, and its expression level was closely related to the activation of TTF-1 promoter-driven miR-7 expression in NSCLC cells. Moreover, the silencing of NF-1 could impair the activation of TTF-1 core promoter. These data indicated that NF-1 is a critical transcription factor binding to the TTF-1 core promoter in human NSCLC. Notably, based on bioinformatic analysis, we replaced several distinct sequences without any putative transcription factor-binding sites with NF-1-binding site for optimization, and successively verified the appreciable binding capacity of the optimized TTF-1 core promoter. Meanwhile, we found that in the TTF-1 core promoter, four NF-1-binding sites showed stronger activity than the two NF-1-binding sites. These data indicated the new value of NF-1 in gene therapy against cancer. However, it must be pointed out that some transcription factors documented can regulate the activity of TTF-1 promoter, such as nuclear factor I (NFI), hepatocyte nuclear factor-3β (HNF‍-‍3β), and small mothers against decapentaplegic homolog 2 (Smad2) ( Ikeda et al., 1996 Ikeda et al., 1996 ; Nakazato et al., 2000 Nakazato et al., 2000 ; Li et al., 2013 Li et al., 2013 ). Moreover, we also noticed that the activity of TTF-1 promoter was elevated in the absence of the region from -‍1612 bp to -‍1229 bp sites, indicating that some other factors might negatively regulate its activity. Therefore, successive research work on the identification of other transcription factors, which were not investigated in current study, and the potential connections among these factors might be rather valuable for the optimization of TTF-1 promoter, ultimately benefiting the development of gene therapy against clinical lung cancer. The safety of gene therapy strategies, including the distribution of exogenous DNA, the function of vital organs, and histopathological changes, is essential for the potential application of cancer gene therapy ( Chandler et al., 2021 Chandler et al., 2021 ; Li et al., 2021 Li et al., 2021 ; Sheikh et al., 2021 Sheikh et al., 2021 ). In our previous work, we described that the remote subcutaneous injection of TTF-1 promoter-operated miR-7 expression plasmid had no significant effects on tissues or organs ( Lei et al., 2017 Lei et al., 2017 ). In the present study, we further observed that the opt TTF-1 promoter-driven miR-7 expression plasmid was injected through the distal tail vein rather than tissue locally, mainly distributed in lung and tumor tissues, while only trace amounts were found in other major organs. Besides, some studies reported that naked plasmid DNA injected through the tail vein was mainly distributed in liver, but not in lung ( Sebestyén et al., 2006 Sebestyén et al., 2006 ; Kizzire et al., 2013 Kizzire et al., 2013 ). Meanwhile, the expression of miR-7 driven by opt TTF-1 promoter was mainly upregulated in tumors and lungs, but not in other tissues or organs. We proposed that these phenomena might be related to the different dosages and time courses in distinct experimental settings, as well as the regulatory mechanism of TTF-1 promoter activity including differential intrinsic expression of NF-1 in distinct tissues and organs, which remains to be elucidated. Finally, the function or pathological structure of several major organs was not significantly affected, indicating the valuable application of opt TTF-1 promoter in successive research work on clinical NSCLC patients. 4 Conclusions 1 In summary, for the first time, our study revealed that opt TTF-1 promoter-driven miR-7 expression could significantly inhibit the growth of human NSCLC cells both in vitro and in vivo, which was related to the decreased expression of NDUFA4 and related signaling pathways. Our current study further expands the application prospect of targeted gene expression technology in lung cancer, providing a new basis for the development of miRNA-based targeting gene therapy against clinical lung cancer. Methods and materials Detailed methods are provided in the electronic supplementary materials of this paper. References Amreddy N , Babu A , Muralidharan R , et al. , 2017 . Polymeric nanoparticle-mediated gene delivery for lung cancer treatment . Top Curr Chem (Cham) , 375 ( 2 ): 35 . https://doi.org/10.1007/s41061-017-0128-5 https://doi.org/10.1007/s41061-017-0128-5 Polymeric nanoparticle-mediated gene delivery for lung cancer treatment Billiet C , Peeters S , Decaluwé H , et al. , 2016 . Postoperative radiotherapy for lung cancer: is it worth the controversy? Cancer Treat Rev , 51 : 10 - 18 . https://doi.org/10.1016/j.ctrv.2016.10.001 https://doi.org/10.1016/j.ctrv.2016.10.001 Postoperative radiotherapy for lung cancer: is it worth the controversy? Bucher K , Rodríguez-Bocanegra E , Dauletbekov D , et al. , 2021 . Immune responses to retinal gene therapy using adeno-associated viral vectors-implications for treatment success and safety . Prog Retin Eye Res , 83 : 100915 . https://doi.org/10.1016/j.preteyeres.2020.100915 https://doi.org/10.1016/j.preteyeres.2020.100915 Immune responses to retinal gene therapy using adeno-associated viral vectors-implications for treatment success and safety Cao Q , Mao ZD , Shi YJ , et al. , 2016 . MicroRNA-7 inhibits cell proliferation, migration and invasion in human non-small cell lung cancer cells by targeting FAK through ERK/MAPK signaling pathway . Oncotarget , 7 ( 47 ): 77468 - 77481 . https://doi.org/10.18632/oncotarget.12684 https://doi.org/10.18632/oncotarget.12684 MicroRNA-7 inhibits cell proliferation, migration and invasion in human non-small cell lung cancer cells by targeting FAK through ERK/MAPK signaling pathway Chan BA , Coward JIG , 2013 . Chemotherapy advances in small-cell lung cancer . J Thorac Dis , 5 ( S5 ): S565 - S578 . https://doi.org/10.3978/j.issn.2072-1439.2013.07.43 https://doi.org/10.3978/j.issn.2072-1439.2013.07.43 Chemotherapy advances in small-cell lung cancer Chandler RJ , Venturoni LE , Liao J , et al. , 2021 . Promoterless, nuclease-free genome editing confers a growth advantage for corrected hepatocytes in mice with methylmalonic acidemia . Hepatology , 73 ( 6 ): 2223 - 2237 . https://doi.org/10.1002/hep.31570 https://doi.org/10.1002/hep.31570 Promoterless, nuclease-free genome editing confers a growth advantage for corrected hepatocytes in mice with methylmalonic acidemia Chen HZ , Guo MM , Yue DX , et al. , 2021 . MicroRNA-7 negatively regulates Toll-like receptor 4 signaling pathway through FAM177A . Immunology , 162 ( 1 ): 44 - 57 . https://doi.org/10.1111/imm.13252 https://doi.org/10.1111/imm.13252 MicroRNA-7 negatively regulates Toll-like receptor 4 signaling pathway through FAM177A Chen SP , Wang Y , Li DM , et al. , 2022 . Mechanisms controlling microRNA expression in tumor . Cells , 11 ( 18 ): 2852 . https://doi.org/10.3390/cells11182852 https://doi.org/10.3390/cells11182852 Mechanisms controlling microRNA expression in tumor Danino YM , Even D , Ideses D , et al. , 2015 . The core promoter: at the heart of gene expression . Biochim Biophys Acta , 1849 ( 8 ): 1116 - 1131 . https://doi.org/10.1016/j.bbagrm.2015.04.003 https://doi.org/10.1016/j.bbagrm.2015.04.003 The core promoter: at the heart of gene expression Du X , Xiao JJ , Fu XF , et al. , 2021 . A proteomic analysis of Bcl-2 regulation of cell cycle arrest: insight into the mech anisms. J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 10 ): 839 - 855 . https://doi.org/10.1631/jzus.B2000802 https://doi.org/10.1631/jzus.B2000802 A proteomic analysis of Bcl-2 regulation of cell cycle arrest: insight into the mech Du X , Zhang JQ , Liu L , et al. , 2022 . A novel anticancer prop A novel anticancer prop erty of Lycium barbarum polysaccharide in triggering ferroptosis of breast cancer cells . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 23 ( 4 ): 286 - 299 . https://doi.org/10.1631/jzus.B2100748 https://doi.org/10.1631/jzus.B2100748 erty of Lycium barbarum polysaccharide in triggering ferroptosis of breast cancer cells Dziadziuszko R , Krebs MG , de Braud F , et al. , 2021 . Updated integrated analysis of the efficacy and safety of entrectinib in locally advanced or metastatic ROS1 fusion-positive non-small-cell lung cancer . J Clin Oncol , 39 ( 11 ): 1253 - 1263 . https://doi.org/10.1200/JCO.20.03025 https://doi.org/10.1200/JCO.20.03025 Updated integrated analysis of the efficacy and safety of entrectinib in locally advanced or metastatic ROS1 fusion-positive non-small-cell lung cancer Even DY , Kedmi A , Ideses D , et al. , 2017 . Functional screening of core promoter activity . In: Gould D (Ed.), Mammalian Synthetic Promoters . Humana, New York , p. 77 - 91 . https://doi.org/10.1007/978-1-4939-7223-4_7 https://doi.org/10.1007/978-1-4939-7223-4_7 Functional screening of core promoter activity Fukazawa T , Maeda Y , Durbin ML , et al. , 2007 . Pulmonary adenocarcinoma-targeted gene therapy by a cancer- and tissue-specific promoter system . Mol Cancer Ther , 6 ( 1 ): 244 - 252 . https://doi.org/10.1158/1535-7163.MCT-06-0408 https://doi.org/10.1158/1535-7163.MCT-06-0408 Pulmonary adenocarcinoma-targeted gene therapy by a cancer- and tissue-specific promoter system Fulzele SV , Chatterjee A , Shaik MS , et al. , 2006 . Inhalation delivery and anti-tumor activity of celecoxib in human orthotopic non-small cell lung cancer xenograft model . Pharm Res , 23 ( 9 ): 2094 - 2106 . https://doi.org/10.1007/s11095-006-9074-6 https://doi.org/10.1007/s11095-006-9074-6 Inhalation delivery and anti-tumor activity of celecoxib in human orthotopic non-small cell lung cancer xenograft model Guan L , Zhao X , Tang L , et al. , 2021 . Thyroid transcription factor-1: structure, expression, function and its relationship with disease . Biomed Res Int , 2021 : 9957209 . https://doi.org/10.1155/2021/9957209 https://doi.org/10.1155/2021/9957209 Thyroid transcription factor-1: structure, expression, function and its relationship with disease Huang QM , Zeng YM , Lin HH , et al. , 2017 . Transfection with Livin and Survivin shRNA inhibits the growth and proliferation of non-small cell lung cancer cells . Mol Med Rep , 16 ( 5 ): 7086 - 7091 . https://doi.org/10.3892/mmr.2017.7490 https://doi.org/10.3892/mmr.2017.7490 Transfection with Livin and Survivin shRNA inhibits the growth and proliferation of non-small cell lung cancer cells Ikeda K , Shaw-White JR , Wert SE , et al. , 1996 . Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells . Mol Cell Biol , 16 ( 7 ): 3626 - 3636 . https://doi.org/10.1128/MCB.16.7.3626 https://doi.org/10.1128/MCB.16.7.3626 Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells Kim JH , Kim HS , Kim BJ , et al. , 2018 . Prognostic impact of TTF-1 expression in non-squamous non-small-cell lung cancer: a meta-analysis . J Cancer , 9 ( 22 ): 4279 - 4286 . https://doi.org/10.7150/jca.26830 https://doi.org/10.7150/jca.26830 Prognostic impact of TTF-1 expression in non-squamous non-small-cell lung cancer: a meta-analysis Kizzire K , Khargharia S , Rice KG , 2013 . High-affinity PEGylated polyacridine peptide polyplexes mediate potent in vivo gene expression . Gene Ther , 20 ( 4 ): 407 - 416 . https://doi.org/10.1038/gt.2012.47 https://doi.org/10.1038/gt.2012.47 High-affinity PEGylated polyacridine peptide polyplexes mediate potent in vivo gene expression Lara-Guerra H , Roth JA , 2016 . Gene therapy for lung cancer . Crit Rev Oncog , 21 ( 1-2 ): 115 - 124 . https://doi.org/10.1615/CritRevOncog.2016016084 https://doi.org/10.1615/CritRevOncog.2016016084 Gene therapy for lung cancer Lee AY , Cho MH , Kim S , 2019 . Recent advances in aerosol gene delivery systems using non-viral vectors for lung cancer therapy . Expert Opin Drug Deliv , 16 ( 7 ): 757 - 772 . https://doi.org/10.1080/17425247.2019.1641083 https://doi.org/10.1080/17425247.2019.1641083 Recent advances in aerosol gene delivery systems using non-viral vectors for lung cancer therapy Lei LY , Chen C , Zhao JJ , et al. , 2017 . Targeted expression of miR-7 operated by TTF-1 promoter inhibited the growth of human lung cancer through the NDUFA4 pathway . Mol Ther Nucleic Acids , 6 : 183 - 197 . https://doi.org/10.1016/j.omtn.2016.12.005 https://doi.org/10.1016/j.omtn.2016.12.005 Targeted expression of miR-7 operated by TTF-1 promoter inhibited the growth of human lung cancer through the NDUFA4 pathway Li C , Wu X , Zhang W , et al. , 2014 . AEG-1 promotes metastasis through downstream AKR1C2 and NF1 in liver cancer . Oncol Res , 22 ( 4 ): 203 - 211 . https://doi.org/10.3727/096504015X14386062091352 https://doi.org/10.3727/096504015X14386062091352 AEG-1 promotes metastasis through downstream AKR1C2 and NF1 in liver cancer Li C , Brant E , Budak H , et al. , 2021 . CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 4 ): 253 - 284 . https://doi.org/10.1631/jzus.B2100009 https://doi.org/10.1631/jzus.B2100009 CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement Li JR , Zheng YJ , Sun GY , et al. , 2014 . Restoration of miR-7 expression suppresses the growth of Lewis lung cancer cells by modulating epidermal growth factor receptor signaling . Oncol Rep , 32 ( 6 ): 2511 - 2516 . https://doi.org/10.3892/or.2014.3519 https://doi.org/10.3892/or.2014.3519 Restoration of miR-7 expression suppresses the growth of Lewis lung cancer cells by modulating epidermal growth factor receptor signaling Li Y , Eggermont K , Vanslembrouck V , et al. , 2013 . NKX2-1 activation by SMAD2 signaling after definitive endoderm differentiation in human embryonic stem cell . Stem Cells Dev , 22 ( 9 ): 1433 - 1442 . https://doi.org/10.1089/scd.2012.0620 https://doi.org/10.1089/scd.2012.0620 NKX2-1 activation by SMAD2 signaling after definitive endoderm differentiation in human embryonic stem cell Linnerth-Petrik NM , Santry LA , Yu DL , et al. , 2012 . Adeno-associated virus vector mediated expression of an oncogenic retroviral envelope protein induces lung adenocarcinomas in immunocompetent mice . PLoS ONE , 7 ( 12 ): e51400 . https://doi.org/10.1371/journal.pone.0051400 https://doi.org/10.1371/journal.pone.0051400 Adeno-associated virus vector mediated expression of an oncogenic retroviral envelope protein induces lung adenocarcinomas in immunocompetent mice Liu ZL , Jiang ZM , Huang JY , et al. , 2014 . miR-7 inhibits glioblastoma growth by simultaneously interfering with the PI3K/ATK and Raf/MEK/ERK pathways . Int J Oncol , 44 ( 5 ): 1571 - 1580 . https://doi.org/10.3892/ijo.2014.2322 https://doi.org/10.3892/ijo.2014.2322 miR-7 inhibits glioblastoma growth by simultaneously interfering with the PI3K/ATK and Raf/MEK/ERK pathways Lux CT , Scharenberg AM , 2017 . Therapeutic gene editing safety and specificity . Hematol Oncol Clin North Am , 31 ( 5 ): 787 - 795 . https://doi.org/10.1016/j.hoc.2017.05.002 https://doi.org/10.1016/j.hoc.2017.05.002 Therapeutic gene editing safety and specificity Massaro G , Hughes MP , Whaler SM , et al. , 2020 . Systemic AAV9 gene therapy using the synapsin I promoter rescues a mouse model of neuronopathic Gaucher disease but with limited cross-correction potential to astrocytes . Hum Mol Genet , 29 ( 12 ): 1933 - 1949 . https://doi.org/10.1093/hmg/ddz317 https://doi.org/10.1093/hmg/ddz317 Systemic AAV9 gene therapy using the synapsin I promoter rescues a mouse model of neuronopathic Gaucher disease but with limited cross-correction potential to astrocytes Nakazato M , Chung HK , Ulianich L , et al. , 2000 . Thyroglobu lin repression of thyroid transcription factor 1 (TTF-1) gene expression is mediated by decreased DNA binding of nuclear factor I proteins which control constitutive TTF-1 expression. Mol Cell Biol , 20 ( 22 ): 8499 - 8512 . https://doi.org/10.1128/MCB.20.22.8499-8512.2000 https://doi.org/10.1128/MCB.20.22.8499-8512.2000 Thyroglobu Naldini L , 2015 . Gene therapy returns to centre stage . Nature , 526 ( 7573 ): 351 - 360 . https://doi.org/10.1038/nature15818 https://doi.org/10.1038/nature15818 Gene therapy returns to centre stage Nasim F , Sabath BF , Eapen GA , 2019 . Lung cancer . Med Clin North Am , 103 ( 3 ): 463 - 473 . https://doi.org/10.1016/j.mcna.2018.12.006 https://doi.org/10.1016/j.mcna.2018.12.006 Lung cancer Philpott C , Tovell H , Frayling IM , et al. , 2017 . The NF1 somatic mutational landscape in sporadic human cancers . Hum Genomics , 11 : 13 . https://doi.org/10.1186/s40246-017-0109-3 https://doi.org/10.1186/s40246-017-0109-3 The NF1 somatic mutational landscape in sporadic human cancers Powell SK , Rivera-Soto R , Gray SJ , 2015 . Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy . Discov Med , 19 ( 102 ): 49 - 57 . Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy Pruller J , Hofer I , Ganassi M , et al. , 2021 . A human Myogenin promoter modified to be highly active in alveolar rhabdomyosarcoma drives an effective suicide gene therapy . Cancer Gene Ther , 28 ( 5 ): 427 - 441 . https://doi.org/10.1038/s41417-020-00225-0 https://doi.org/10.1038/s41417-020-00225-0 A human Myogenin promoter modified to be highly active in alveolar rhabdomyosarcoma drives an effective suicide gene therapy Qian B , Wang DM , Gu XS , et al. , 2018 . LncRNA H19 serves as a ceRNA and participates in non-small cell lung cancer development by regulating microRNA-107 . Eur Rev Med Pharmacol Sci , 22 ( 18 ): 5946 - 5953 . https://doi.org/10.26355/eurrev_201809_15925 https://doi.org/10.26355/eurrev_201809_15925 LncRNA H19 serves as a ceRNA and participates in non-small cell lung cancer development by regulating microRNA-107 Rai KM , Takigawa N , Ito S , et al. , 2011 . Liposomal delivery of microRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells . Mol Cancer Ther , 10 ( 9 ): 1720 - 1727 . https://doi.org/10.1158/1535-7163.MCT-11-0220 https://doi.org/10.1158/1535-7163.MCT-11-0220 Liposomal delivery of microRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells Sebestyén MG , Budker VG , Budker T , et al. , 2006 . Mech anism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules. J Gene Med , 8 ( 7 ): 852 - 873 . https://doi.org/10.1002/jgm.921 https://doi.org/10.1002/jgm.921 Mech Sheikh S , Ernst D , Keating A , 2021 . Prodrugs and prodrug-activated systems in gene therapy . Mol Ther , 29 ( 5 ): 1716 - 1728 . https://doi.org/10.1016/j.ymthe.2021.04.006 https://doi.org/10.1016/j.ymthe.2021.04.006 Prodrugs and prodrug-activated systems in gene therapy Sher YP , Tzeng TF , Kan SF , et al. , 2009 . Cancer targeted gene therapy of BikDD inhibits orthotopic lung cancer growth and improves long-term survival . Oncogene , 28 ( 37 ): 3286 - 3295 . https://doi.org/10.1038/onc.2009.187 https://doi.org/10.1038/onc.2009.187 Cancer targeted gene therapy of BikDD inhibits orthotopic lung cancer growth and improves long-term survival Su JC , Ruan SL , Dai SK , et al. , 2019 . NF1 regulates apoptosis in ovarian cancer cells by targeting MCL1 via miR-142-5p . Pharmacogenomics , 20 ( 3 ): 155 - 165 . https://doi.org/10.2217/pgs-2018-0161 https://doi.org/10.2217/pgs-2018-0161 NF1 regulates apoptosis in ovarian cancer cells by targeting MCL1 via miR-142-5p Toloza EM , Morse MA , Lyerly HK , 2006 . Gene therapy for lung cancer . J Cell Biochem , 99 ( 1 ): 1 - 22 . https://doi.org/10.1002/jcb.20851 https://doi.org/10.1002/jcb.20851 Gene therapy for lung cancer Umakanthan S , Bukelo MM , 2021 . Concise genetic profile of lung carcinoma . Postgrad Med J , 0 : 1 - 5 . https://doi.org/10.1136/postgradmedj-2021-139860 https://doi.org/10.1136/postgradmedj-2021-139860 Concise genetic profile of lung carcinoma Umakanthan S , Rao AVC , Mohammed W , 2021 . Role of immunohistochemistry markers in neoplastic lung lesions . J Cancer Res Ther , 17 ( 6 ): 1382 - 1388 . https://doi.org/10.4103/jcrt.JCRT_187_19 https://doi.org/10.4103/jcrt.JCRT_187_19 Role of immunohistochemistry markers in neoplastic lung lesions Wiggins JF , Ruffino L , Kelnar K , et al. , 2010 . Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34 . Cancer Res , 70 ( 14 ): 5923 - 5930 . https://doi.org/10.1158/0008-5472.CAN-10-0655 https://doi.org/10.1158/0008-5472.CAN-10-0655 Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34 Wilson C , Nimick M , Nehoff H , et al. , 2017 . ALK and IGF-1R as independent targets in crizotinib resistant lung cancer . Sci Rep , 7 : 13955 . https://doi.org/10.1038/s41598-017-14289-w https://doi.org/10.1038/s41598-017-14289-w ALK and IGF-1R as independent targets in crizotinib resistant lung cancer Xie XM , Hsu JL , Choi MG , et al. , 2009 . A novel hTERT promoter-driven E1A therapeutic for ovarian cancer . Mol Cancer Ther , 8 ( 8 ): 2375 - 2382 . https://doi.org/10.1158/1535-7163.MCT-09-0056 https://doi.org/10.1158/1535-7163.MCT-09-0056 A novel hTERT promoter-driven E1A therapeutic for ovarian cancer Xiong SD , Zheng YJ , Jiang P , et al. , 2011 . MicroRNA-7 inhibits the growth of human non-small cell lung cancer A549 cells through targeting BCL-2 . Int J Biol Sci , 7 ( 6 ): 805 - 814 . https://doi.org/10.7150/ijbs.7.805 https://doi.org/10.7150/ijbs.7.805 MicroRNA-7 inhibits the growth of human non-small cell lung cancer A549 cells through targeting BCL-2 Zhang BY , Wang O , Qin JC , et al. , 2013 . cis -Acting elements and trans -acting factors in the transcriptional regulation of Raf kinase inhibitory protein expression . PLoS ONE , 8 ( 12 ): e83097 . https://doi.org/10.1371/journal.pone.0083097 https://doi.org/10.1371/journal.pone.0083097 cis-Acting elements and trans-acting factors in the transcriptional regulation of Raf kinase inhibitory protein expression Zhang EB , Yin DD , Sun M , et al. , 2014 . P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression . Cell Death Dis , 5 ( 5 ): e1243 . https://doi.org/10.1038/cddis.2014.201 https://doi.org/10.1038/cddis.2014.201 P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression Zhang R , Wang Q , Zhang L , et al. , 2015 . Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B . Front Med , 9 ( 1 ): 90 - 99 . https://doi.org/10.1007/s11684-015-0390-2 https://doi.org/10.1007/s11684-015-0390-2 Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B Zhao JJ , Wang KL , Liao ZY , et al. , 2015 . Promoter mutation of tumor suppressor microRNA-7 is associated with poor prognosis of lung cancer . Mol Clin Oncol , 3 ( 6 ):‍ 1329 - 1336 . https://doi.org/10.3892/mco.2015.648 https://doi.org/10.3892/mco.2015.648 Promoter mutation of tumor suppressor microRNA-7 is associated with poor prognosis of lung cancer Zhao JJ , Chu FY , Xu HL , et al. , 2021 . C/EBPα/miR-7 controls CD 4+ T-cell activation and function and orchestrates experimental autoimmune hepatitis in mice . Hepatology , 74 ( 1 ): 379 - 396 . https://doi.org/10.1002/hep.31607 https://doi.org/10.1002/hep.31607 C/EBPα/miR-7 controls CD4+ T-cell activation and function and orchestrates experimental autoimmune hepatitis in mice Zhao X , Yang J , Huang RY , et al. , 2021 . The role and its mechanism of intermittent fasting in tumors: friend or foe? Cancer Biol Med , 18 ( 1 ): 63 - 73 . https://doi.org/10.20892/j.issn.2095-3941.2020.0250 https://doi.org/10.20892/j.issn.2095-3941.2020.0250 The role and its mechanism of intermittent fasting in tumors: friend or foe? Zhuang XB , Zhao C , Li JY , et al. , 2019 . Clinical features and therapeutic options in non-small cell lung cancer patients with concomitant mutations of EGFR , ALK , ROS1 , KRAS or BRAF . Cancer Med , 8 ( 6 ): 2858 - 2866 . https://doi.org/10.1002/cam4.2183 https://doi.org/10.1002/cam4.2183 Clinical features and therapeutic options in non-small cell lung cancer patients with concomitant mutations of EGFR, ALK, ROS1, KRAS or BRAF Shipeng CHEN and Lian GUAN performed the experiments, analyzed the data, and wrote this paper. Xu ZHAO, Jing YANG, and Longqing CHEN designed the paper. Mengmeng GUO, Juanjuan ZHAO, and Chao CHEN edited the manuscript. Ya ZHOU, Yong HAN, and Lin XU conceived, designed, and wrote the paper. All authors have reviewed and approved the paper, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by 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), the Collaborative Innovation Center of Chinese Ministry of Education (No. 2020-39), and the Program for Excellent Young Talents of Zunyi Medical University (No. 15ZY-001), China. In addition, we express sincere gratitude to the professional organization American Journal Experts (AJE) for the service of polishing this article. 10.1631/jzus.B2200116 1673-1581（2022）11-0915-16 This work was supported by 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), the Collaborative Innovation Center of Chinese Ministry of Education (No. 2020-39), and the Program for Excellent Young Talents of Zunyi Medical University (No. 15ZY-001), China. 2022-03-08 2022-07-22 2022-11-15 2022-12-27 2022-10-09 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2022 浙江大学出版社 Shipeng CHEN, Lian GUAN, Xu ZHAO, Jing YANG, Longqing CHEN, Mengmeng GUO, Juanjuan ZHAO, Chao CHEN, Ya ZHOU, Yong HAN, and Lin XU declare that they have no conflict of interest. All animal experiments were performed according to the guidelines for the Care and Use of Laboratory Animals of Zunyi Medical University. The experimental procedures were approved by the ethical guidelines of Zunyi Medical University Laboratory Animal Care and Use Committee (permit number 2018016). Compliance with ethics guidelines Supplementary information 1 Figs. S1‒S3; Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 11 23