LINC00052通過miR-128和miR-485-3p調(diào)節(jié)NTRK3的表達(dá)影響肝癌細(xì)胞侵襲轉(zhuǎn)移
發(fā)布時(shí)間：2019-04-29 18:12
【摘要】：目的:探討LINC00052影響肝癌SMMC7721細(xì)胞侵襲轉(zhuǎn)移的機(jī)制。方法:首先,通過一種能夠隨機(jī)插入并整合到細(xì)胞染色體中的捕獲載體pU21質(zhì)粒捕獲、G418抗生素篩選成功轉(zhuǎn)染pU21質(zhì)粒的穩(wěn)定細(xì)胞系、通過Transwell侵襲實(shí)驗(yàn)和細(xì)胞劃痕實(shí)驗(yàn)篩選出與原始SMMC7721細(xì)胞株細(xì)胞侵襲和遷移能力發(fā)生變化的單克隆細(xì)胞株,通過5'RACE實(shí)驗(yàn)、分子生物學(xué)等方法確定被獲載體pU21質(zhì)粒捕獲的基因;其次,構(gòu)建該基因的過表達(dá)質(zhì)粒和合成其干擾RNA檢測該基因在腫瘤細(xì)胞侵襲、遷移、增殖等方面的功能。通過生物學(xué)分析尋找該基因發(fā)揮生物功能的靶基因,并用real-time PCR和Western blot實(shí)驗(yàn)篩選靶基因,采用Transwell侵襲實(shí)驗(yàn)、細(xì)胞劃痕實(shí)驗(yàn)MTS實(shí)驗(yàn)及克隆形成實(shí)驗(yàn)研究靶基因在腫瘤細(xì)胞侵襲、遷移、增殖方面的功能;然后,通過堿基互補(bǔ)分析、蟲熒光素酶活性等實(shí)驗(yàn)方法探討該基因調(diào)節(jié)器靶基因的機(jī)制;最后,將捕獲基因的過表達(dá)穩(wěn)定細(xì)胞系和干擾穩(wěn)定細(xì)胞系分別注入BALB/c裸鼠皮下或肝臟構(gòu)建體外移植瘤模型和轉(zhuǎn)移模型,通過免疫組織化學(xué)實(shí)驗(yàn)檢測該基因是否在體內(nèi)能影響腫瘤發(fā)生發(fā)展。結(jié)果:通過5'RACE、分子生物學(xué)等方法證明在A554細(xì)胞系中,被捕獲載體pU21質(zhì)粒捕獲的基因?yàn)長INC00052(long inter-genic non-protein coding RNA 52)。用LINC00052的過表達(dá)質(zhì)粒和合成的干擾RNA轉(zhuǎn)染到肝癌SMMC7721細(xì)胞中,通過劃痕實(shí)驗(yàn)和侵襲實(shí)驗(yàn)發(fā)現(xiàn),LINC00052能夠影響肝癌細(xì)胞的侵襲轉(zhuǎn)移能力。通過染色體上基因間的位置關(guān)系,發(fā)現(xiàn)了LINC00052影響肝癌SMMC7721細(xì)胞侵襲轉(zhuǎn)移的一個(gè)靶基因NTRK3(neurotrophic tyrosine kinase,receptor,type3)。LINC00052可以通過miR-128和miR-485-3p間接調(diào)節(jié)NTRK3的表達(dá),進(jìn)而影響肝癌SMMC7721細(xì)胞侵襲轉(zhuǎn)移。體內(nèi)實(shí)驗(yàn)表明,過表達(dá)LINC00052能夠抑制移植瘤的生長及癌細(xì)胞的轉(zhuǎn)移,而干擾LINC00052的表達(dá)后能夠促進(jìn)移植瘤的生長及癌細(xì)胞的轉(zhuǎn)移,這與體外實(shí)驗(yàn)所取得的結(jié)果一致。結(jié)論:LINC00052可以通過調(diào)節(jié)miR-128和miR-485-3p的表達(dá),miR-128和miR-485-3p直接影響NTRK3的表達(dá),從而影響肝癌SMMC7721細(xì)胞侵襲轉(zhuǎn)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.7
本文編號：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.7
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1 熊冬梅;LINC00052通過miR-128和miR-485-3p調(diào)節(jié)NTRK3的表達(dá)影響肝癌細(xì)胞侵襲轉(zhuǎn)移[D];重慶醫(yī)科大學(xué);2016年
，本文編號：2468421
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