LINC00052通过miR-128和miR-485-3p调节NTRK3的表达影响肝癌细胞侵袭转移
发布时间：2019-04-29 18:12
【摘要】：目的:探讨LINC00052影响肝癌SMMC7721细胞侵袭转移的机制。方法:首先,通过一种能够随机插入并整合到细胞染色体中的捕获载体pU21质粒捕获、G418抗生素筛选成功转染pU21质粒的稳定细胞系、通过Transwell侵袭实验和细胞划痕实验筛选出与原始SMMC7721细胞株细胞侵袭和迁移能力发生变化的单克隆细胞株,通过5'RACE实验、分子生物学等方法确定被获载体pU21质粒捕获的基因;其次,构建该基因的过表达质粒和合成其干扰RNA检测该基因在肿瘤细胞侵袭、迁移、增殖等方面的功能。通过生物学分析寻找该基因发挥生物功能的靶基因,并用real-time PCR和Western blot实验筛选靶基因,采用Transwell侵袭实验、细胞划痕实验MTS实验及克隆形成实验研究靶基因在肿瘤细胞侵袭、迁移、增殖方面的功能;然后,通过碱基互补分析、虫荧光素酶活性等实验方法探讨该基因调节器靶基因的机制;最后,将捕获基因的过表达稳定细胞系和干扰稳定细胞系分别注入BALB/c裸鼠皮下或肝脏构建体外移植瘤模型和转移模型,通过免疫组织化学实验检测该基因是否在体内能影响肿瘤发生发展。结果:通过5'RACE、分子生物学等方法证明在A554细胞系中,被捕获载体pU21质粒捕获的基因为LINC00052(long inter-genic non-protein coding RNA 52)。用LINC00052的过表达质粒和合成的干扰RNA转染到肝癌SMMC7721细胞中,通过划痕实验和侵袭实验发现,LINC00052能够影响肝癌细胞的侵袭转移能力。通过染色体上基因间的位置关系,发现了LINC00052影响肝癌SMMC7721细胞侵袭转移的一个靶基因NTRK3(neurotrophic tyrosine kinase,receptor,type3)。LINC00052可以通过miR-128和miR-485-3p间接调节NTRK3的表达,进而影响肝癌SMMC7721细胞侵袭转移。体内实验表明,过表达LINC00052能够抑制移植瘤的生长及癌细胞的转移,而干扰LINC00052的表达后能够促进移植瘤的生长及癌细胞的转移,这与体外实验所取得的结果一致。结论:LINC00052可以通过调节miR-128和miR-485-3p的表达,miR-128和miR-485-3p直接影响NTRK3的表达,从而影响肝癌SMMC7721细胞侵袭转移。
[Abstract]:Objective: to investigate the effect of LINC00052 on the invasion and metastasis of hepatocellular carcinoma (SMMC7721) cells. Methods: first of all, the stable cell line transfected with pU21 plasmid was successfully screened by G418 antibiotics by a capture vector pU21, which could be inserted randomly into the cell chromosome and integrated into the cell chromosome, and the stable cell line was successfully transfected with G418 antibiotics. Monoclonal cell lines were screened by Transwell invasion test and cell scratch test, and the genes captured by pU21 plasmid were determined by 5'RACE assay and molecular biology. Secondly, the over-expression plasmid of the gene was constructed and its interference RNA was synthesized to detect the function of the gene in the invasion, migration and proliferation of tumor cells. The target genes were screened by real-time PCR and Western blot experiments. The invasion of target genes in tumor cells was studied by Transwell invasion test, cell scratch test MTS test and clone formation test, and the results showed that the target gene could play an important role in the invasion of tumor cells. Function of migration and proliferation; Then, the mechanism of the target gene of the gene regulator was studied by base complementary analysis and luciferase activity. Finally, the over-expression stable cell line and the interference stable cell line were injected into BALB/c nude mice subcutaneously or liver to construct tumor model and metastasis model in vitro, respectively. Whether the gene can affect the tumorigenesis and development in vivo was detected by immunohistochemical assay. Results: in A554 cell line, the gene captured by plasmid pU21 was LINC00052 (long inter-genic non-protein coding RNA 52), which was proved to be the gene in A554 cell line by means of 5-race, molecular biology and so on. The over-expression plasmid of LINC00052 and the synthetic interfering RNA were transfected into SMMC7721 cells. It was found that LINC00052 could affect the invasion and metastasis ability of HCC cells by scratch test and invasion test. A target gene NTRK3 (neurotrophic tyrosine kinase,receptor,type3 (LINC00052), which affects the invasion and metastasis of hepatocellular carcinoma SMMC7721 cells, was found through the location relationship of genes on chromosomes. LINC00052 can indirectly regulate the expression of NTRK3 through miR-128 and miR-485-3p. Furthermore, the invasion and metastasis of hepatocellular carcinoma SMMC7721 cells were affected. In vivo experiments showed that overexpression of LINC00052 could inhibit the growth of transplanted tumor and metastasis of cancer cells, while interfering with the expression of LINC00052 could promote the growth of transplanted tumor and metastasis of cancer cells, which was consistent with the results obtained in vitro. Conclusion: LINC00052 can directly affect the expression of NTRK3 by regulating the expression of miR-128 and miR-485-3p, and miR-128 and miR-485-3p can directly affect the invasion and metastasis of SMMC7721 cells.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
本文编号：2468421
[Abstract]:Objective: to investigate the effect of LINC00052 on the invasion and metastasis of hepatocellular carcinoma (SMMC7721) cells. Methods: first of all, the stable cell line transfected with pU21 plasmid was successfully screened by G418 antibiotics by a capture vector pU21, which could be inserted randomly into the cell chromosome and integrated into the cell chromosome, and the stable cell line was successfully transfected with G418 antibiotics. Monoclonal cell lines were screened by Transwell invasion test and cell scratch test, and the genes captured by pU21 plasmid were determined by 5'RACE assay and molecular biology. Secondly, the over-expression plasmid of the gene was constructed and its interference RNA was synthesized to detect the function of the gene in the invasion, migration and proliferation of tumor cells. The target genes were screened by real-time PCR and Western blot experiments. The invasion of target genes in tumor cells was studied by Transwell invasion test, cell scratch test MTS test and clone formation test, and the results showed that the target gene could play an important role in the invasion of tumor cells. Function of migration and proliferation; Then, the mechanism of the target gene of the gene regulator was studied by base complementary analysis and luciferase activity. Finally, the over-expression stable cell line and the interference stable cell line were injected into BALB/c nude mice subcutaneously or liver to construct tumor model and metastasis model in vitro, respectively. Whether the gene can affect the tumorigenesis and development in vivo was detected by immunohistochemical assay. Results: in A554 cell line, the gene captured by plasmid pU21 was LINC00052 (long inter-genic non-protein coding RNA 52), which was proved to be the gene in A554 cell line by means of 5-race, molecular biology and so on. The over-expression plasmid of LINC00052 and the synthetic interfering RNA were transfected into SMMC7721 cells. It was found that LINC00052 could affect the invasion and metastasis ability of HCC cells by scratch test and invasion test. A target gene NTRK3 (neurotrophic tyrosine kinase,receptor,type3 (LINC00052), which affects the invasion and metastasis of hepatocellular carcinoma SMMC7721 cells, was found through the location relationship of genes on chromosomes. LINC00052 can indirectly regulate the expression of NTRK3 through miR-128 and miR-485-3p. Furthermore, the invasion and metastasis of hepatocellular carcinoma SMMC7721 cells were affected. In vivo experiments showed that overexpression of LINC00052 could inhibit the growth of transplanted tumor and metastasis of cancer cells, while interfering with the expression of LINC00052 could promote the growth of transplanted tumor and metastasis of cancer cells, which was consistent with the results obtained in vitro. Conclusion: LINC00052 can directly affect the expression of NTRK3 by regulating the expression of miR-128 and miR-485-3p, and miR-128 and miR-485-3p can directly affect the invasion and metastasis of SMMC7721 cells.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
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1 熊冬梅;LINC00052通过miR-128和miR-485-3p调节NTRK3的表达影响肝癌细胞侵袭转移[D];重庆医科大学;2016年
，本文编号：2468421
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