MicroRNA-23a参与调控胰腺癌发生发展过程的机制研究
发布时间：2018-10-30 07:33
【摘要】：背景:胰腺癌是最具侵袭性的癌症之一,经过大量的努力,胰腺癌的早期诊断有了很大的提高,但总体诊断率还是很低,胰腺癌的预后仍未得到明显的改善,五年生存率仅有8%。因此,对其肿瘤分子生物学的研究有助于进一步揭示其发生、发展机制,有助于早期诊断和治疗。miRNAs(MicroRNA)是近来肿瘤分子生物学研究的一个热点,虽然很多胰腺癌的研究提示miR-23a是一种致癌调节因子,但其潜在的分子机制依旧不明。PLK-1(Polo样激酶1)在多种细胞内有丝分裂进程中起到至关重要的作用,许多miRNAs抑制PLK-1的表达,从而抑制肿瘤的发展。前期的预实验研究结果显示miR-23a抑制胰腺癌细胞MIA-PaCa-2的增殖,靶基因预测软件推测miR-23a与PLK-1可能存在靶向关系。考虑到miRNAs和PLK-1的重要作用,有必要对其进一步的研究。目的:1、研究miR-23a和PLK1在人胰腺癌组织和细胞株中表达水平,并探讨两者的相关性。2、研究miR-23a对胰腺癌细胞增殖、迁移和侵袭、以及凋亡的影响。3、研究miR-23a靶向PLK-1抑制胰腺癌细胞增殖的相关机制。方法:1、荧光定量PCR检测miR-23a和PLK-1在对胰腺癌与癌旁组织和4种胰腺癌细胞株中的表达情况。构建miR-23a和PLK-1的过表达/干扰质粒,荧光素酶报告基因方法证明PLK-1是miR-23a直接调控的靶基因。2、选取人胰腺癌细胞株MIA-PaCa-2和Panc-1,CCK8法检测转染后细胞增殖能力的变化;Transwell实验检测转染后细胞侵袭能力的变化;流式细胞仪检测转染后细胞凋亡的变化。建立MIA-PaCa-2细胞的裸鼠皮下异种成瘤模型,验证miR-23a对胰腺癌细胞成瘤能力的影响。3、选取人胰腺癌细胞株MIA-PaCa-2,转染miR-23a、PLK-1以及共转染miR-23a和PLK-1,Transwell实验检测后细胞侵袭能力的变化;流式细胞仪检测转染后细胞凋亡的变化。荧光定量PCR检测体内成瘤试验中miR-23a对PLK-1不表达的影响。最后,Western Blot检测转染后细胞中PLK-1以及其下游信号因子Bax、Bcl2、cyclinB1,E-cadherin、Vimentin 蛋白表达变化。结果:1、20对组织中,胰腺癌组织中miR-23a表达高于相应癌旁组织的有19对,PLK-1表达高于相应癌旁组织的有11对。在人胰腺癌细胞株中,miR-23a和PLK-1表达水平均明显升高,两者的表达水平呈负相关。双荧光素酶实验证明PLK-1是miR-23a直接调控的靶基因。2、与对照组相比,MIA-PaCa-2细胞的miR-23a的过表达组细胞增殖能力减弱,细胞迁移和侵袭能力减弱,细胞凋亡明显增强,而Panc-1细胞结果与MIA-PaCa-2细胞相反。动物实验结果表明MIA-PaCa-2细胞的miR-23a的过表达组的肿瘤体积和瘤重明显减小,抑瘤率明显增加。3、MIA-PaCa-2细胞过表达PLK-1组与对照组相比,细胞迁移和侵袭能力增强,细胞凋亡明显减弱;共转染miR-23a和PLK-1组结果与对照组相似。裸鼠成瘤实验MIA-PaCa-2细胞转染miR-23a后,导致PLK-1表达的下调,进而导致其下游信号通路因子BCL-2,CyclinB1 and Vimentin表达的下调,以及Bax and E-cadherin表达的上调。而转染过表达PLK-1载体后,结果却相反。体内实验中,当miR-23a多表达时,PLK-1的表达明显抑制。结论:1、miR-23a在胰腺癌组织和胰腺癌细胞中高表达,在胰腺癌细胞株中,miR-23a与PLK-1的表达呈负相关性,PLK-1是miR-23a直接调控的靶基因。2、miR-23a抑制胰腺癌细胞MIA-PaCa-2增殖、迁移和侵袭,促进细胞凋亡,而Panc-1细胞却相反。3、miR-23a通过靶向PLK-1抑制胰腺癌细胞增殖、迁移和侵袭,促进细胞凋亡。
[Abstract]:Background: Pancreatic cancer is one of the most invasive cancers. After a great deal of effort, the early diagnosis of pancreatic cancer has improved greatly, but the overall diagnostic rate is still low, the prognosis of pancreatic cancer is still not significantly improved, and the five-year survival rate is only 8%. Therefore, the study of molecular biology of tumor helps to further reveal its genesis and development mechanism, which can be helpful for early diagnosis and treatment. MicroRNA is a hot spot in recent tumor molecular biology. Although many pancreatic cancer studies suggest that miR-23a is an oncogenic regulatory factor, its potential molecular mechanism remains unknown. PLK-1 (Polo-like kinase 1) plays an important role in the mitosis of various cells, and many miRNAs inhibit the expression of PLK-1, thus inhibiting the development of tumor. Pre-experimental results showed that miR-23a inhibited the proliferation of MIA-PaCa-2 in pancreatic cancer cells. The target gene prediction software estimated that miR-23a and PLK-1 might have targeted relationship. Considering the important role of miRNAs and PLK-1, it is necessary to further study it. Objective: To study the expression level of miR-23a and PLK1 in human pancreatic cancer tissues and cell lines and to investigate the correlation of miR-23a to pancreatic cancer cell proliferation, migration and invasion and apoptosis. Methods: 1. Fluorescent quantitative PCR was used to detect the expression of miR-23a and PLK-1 in pancreatic cancer and 4 pancreatic cancer cell lines. The expression/ interference plasmids of miR-23a and PLK-1 were constructed, and the luciferase reporter gene method proved that PLK-1 was a target gene directly regulated by miR-23a. Flow cytometry was used to detect the changes of apoptosis after transfection. A nude mouse subcutaneous xenograft model of MIA-PaCa-2 cells was established to verify the effect of miR-23a on the tumorigenicity of pancreatic cancer cells. Flow cytometry was used to detect the changes of apoptosis after transfection. The effect of miR-23a on the expression of PLK-1 in vivo was detected by fluorescence quantitative PCR. Finally, the expression of PLK-1 and its downstream signal factors Bax, Bcl2, cyclin B1, E-cadherin and Vimentin in transfected cells were detected by Western blot. Results: The expression of miR-23a in pancreatic cancer tissues was higher than that of the adjacent tissues, and the expression of PLK-1 was higher than that of the adjacent tissues. In human pancreatic cancer cell line, the expression level of miR-23a and PLK-1 was significantly increased, and the expression level of miR-232a and PLK-1 was negatively correlated. The results of double luciferase assay showed that PLK-1 was the target gene directly regulated by miR-23a. Compared with the control group, the expression of miR-23a in MIA-PaCa-2 cells weakened, cell migration and invasion ability weakened, cell apoptosis was enhanced, and the results of Panc-1 cells were opposite to MIA-PaCa-2 cells. The results of animal experiment showed that the tumor volume and tumor weight of miR-23a in MIA-PaCa-2 cells decreased significantly, and the tumor suppressor rate was increased. The results of miR-23a and PLK-1 group were similar to those in the control group. After Mia-PaCa-2 cells transfected with miR-23a in nude mice, the down-regulation of the expression of PLK-1 resulted in down-regulation of the downstream signal pathway factor BCL-2, CyclinB1 and Vimentin and up-regulation of Bax and E-cadherin expression. However, after the expression of PLK-1 vector was transfected, the results were reversed. In vivo experiments, the expression of PLK-1 was significantly inhibited when miR-23a was expressed. Conclusion: 1. miR-23a is highly expressed in pancreatic cancer tissues and pancreatic cancer cells. In pancreatic cancer cell lines, the expression of miR-23a is negatively correlated with the expression of PLK-1, and PLK-1 is a target gene directly regulated by miR-23a. The miR-23a inhibits the proliferation, migration and invasion of human pancreatic cancer cells MIA-PaCa-2, and promotes apoptosis. and the Panc-1 cell is opposite. 3, miR-23a inhibits proliferation, migration and invasion of pancreatic cancer cells by targeting PLK-1, and promotes apoptosis.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.9
[Abstract]:Background: Pancreatic cancer is one of the most invasive cancers. After a great deal of effort, the early diagnosis of pancreatic cancer has improved greatly, but the overall diagnostic rate is still low, the prognosis of pancreatic cancer is still not significantly improved, and the five-year survival rate is only 8%. Therefore, the study of molecular biology of tumor helps to further reveal its genesis and development mechanism, which can be helpful for early diagnosis and treatment. MicroRNA is a hot spot in recent tumor molecular biology. Although many pancreatic cancer studies suggest that miR-23a is an oncogenic regulatory factor, its potential molecular mechanism remains unknown. PLK-1 (Polo-like kinase 1) plays an important role in the mitosis of various cells, and many miRNAs inhibit the expression of PLK-1, thus inhibiting the development of tumor. Pre-experimental results showed that miR-23a inhibited the proliferation of MIA-PaCa-2 in pancreatic cancer cells. The target gene prediction software estimated that miR-23a and PLK-1 might have targeted relationship. Considering the important role of miRNAs and PLK-1, it is necessary to further study it. Objective: To study the expression level of miR-23a and PLK1 in human pancreatic cancer tissues and cell lines and to investigate the correlation of miR-23a to pancreatic cancer cell proliferation, migration and invasion and apoptosis. Methods: 1. Fluorescent quantitative PCR was used to detect the expression of miR-23a and PLK-1 in pancreatic cancer and 4 pancreatic cancer cell lines. The expression/ interference plasmids of miR-23a and PLK-1 were constructed, and the luciferase reporter gene method proved that PLK-1 was a target gene directly regulated by miR-23a. Flow cytometry was used to detect the changes of apoptosis after transfection. A nude mouse subcutaneous xenograft model of MIA-PaCa-2 cells was established to verify the effect of miR-23a on the tumorigenicity of pancreatic cancer cells. Flow cytometry was used to detect the changes of apoptosis after transfection. The effect of miR-23a on the expression of PLK-1 in vivo was detected by fluorescence quantitative PCR. Finally, the expression of PLK-1 and its downstream signal factors Bax, Bcl2, cyclin B1, E-cadherin and Vimentin in transfected cells were detected by Western blot. Results: The expression of miR-23a in pancreatic cancer tissues was higher than that of the adjacent tissues, and the expression of PLK-1 was higher than that of the adjacent tissues. In human pancreatic cancer cell line, the expression level of miR-23a and PLK-1 was significantly increased, and the expression level of miR-232a and PLK-1 was negatively correlated. The results of double luciferase assay showed that PLK-1 was the target gene directly regulated by miR-23a. Compared with the control group, the expression of miR-23a in MIA-PaCa-2 cells weakened, cell migration and invasion ability weakened, cell apoptosis was enhanced, and the results of Panc-1 cells were opposite to MIA-PaCa-2 cells. The results of animal experiment showed that the tumor volume and tumor weight of miR-23a in MIA-PaCa-2 cells decreased significantly, and the tumor suppressor rate was increased. The results of miR-23a and PLK-1 group were similar to those in the control group. After Mia-PaCa-2 cells transfected with miR-23a in nude mice, the down-regulation of the expression of PLK-1 resulted in down-regulation of the downstream signal pathway factor BCL-2, CyclinB1 and Vimentin and up-regulation of Bax and E-cadherin expression. However, after the expression of PLK-1 vector was transfected, the results were reversed. In vivo experiments, the expression of PLK-1 was significantly inhibited when miR-23a was expressed. Conclusion: 1. miR-23a is highly expressed in pancreatic cancer tissues and pancreatic cancer cells. In pancreatic cancer cell lines, the expression of miR-23a is negatively correlated with the expression of PLK-1, and PLK-1 is a target gene directly regulated by miR-23a. The miR-23a inhibits the proliferation, migration and invasion of human pancreatic cancer cells MIA-PaCa-2, and promotes apoptosis. and the Panc-1 cell is opposite. 3, miR-23a inhibits proliferation, migration and invasion of pancreatic cancer cells by targeting PLK-1, and promotes apoptosis.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.9
【参考文献】
相关期刊论文 前7条
1 Milena Ilic;Irena Ilic;;Epidemiology of pancreatic cancer[J];World Journal of Gastroenterology;2016年44期
2 周燕;唐运莲;甘润良;黄s，
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