miR-143对膀胱癌细胞生物学行为和化疗药物敏感性的影响及其机制研究

发布时间：2018-05-18 13:20

本文选题：膀胱癌 + miR-143　；参考：《南京医科大学》2015年博士论文

【摘要】：背景:miR-143是microRNA的一种,已被证实在多种肿瘤组织中低表达,并可以起到抑癌基因的作用,体外细胞实验中证实IGF-IR3'-UTR区存在miR-143调控位点,高表达的miR-143能作用于IGF-IR来抑制肿瘤细胞增殖、侵袭、转移。microRNA在基因调控中所起的作用受到越来越多的重视,有关在膀胱癌细胞中miR-143调控IGF-IR影响吉西他滨化疗敏感性的研究尚未见报道。我们的研究提示miR-143可能在膀胱癌细胞的IGF-IR基因调控机制中起到重要的作用,探索miR-143介导的IGF-IR基因调控作用对膀胱癌细胞生物学行为和化疗药物敏感性的影响十分重要。目的:明确miR-143在膀胱癌组织标本和细胞中的表达水平及其与膀胱癌细胞生长的相关性,探讨miR-143影响膀胱癌恶性生物学行为和对化疗药物敏感性的分子机制。方法:1、RT-PCR方法检测23例膀胱癌及其相对应的癌旁正常膀胱组织标本中miR-143的表达水平,并检测膀胱癌细胞株T24和5637细胞中miR-143的表达水平,分析miR-143表达与膀胱癌分期的相关性。2、RT-PCR方法检测23例膀胱癌及其相对应的癌旁正常膀胱组织标本{中IGF-IR的表达水平,分析miR-143与IGF-IR表达的相关性。构建miR-143过表达慢病毒系统,转染膀胱癌细胞。RT-PCR方法检测过表达miR-143的T24和5637细胞中IGF-IR的表达水平。3、CCK-8法检测过表达miR-143和干扰IGF-IR对5637细胞增殖的影响。同时检测过表达miR-143和干扰IGF-IR对吉西他滨敏感性影响。4、Western blotting 实验分析过表达 miR-143 和干扰 IGF-IR 后 p-AKT、AKT、p-ERK和ERK的蛋白表达情况。结果:1、23对膀胱癌组织标本中,miR-143在膀胱癌组织中表达低于癌旁正常膀胱组织,并且其表达水平与肿瘤的分期密切相关,在膀胱癌细胞株T24和5637中miR-143表达降低。2、miR-143和IGF-IR在膀胱癌组织标本中的表达呈负相关,在T24和5637细胞中高表达的miR-143能明显抑制IGF-IR表达。3、在5637细胞中过表达miR-143和干扰高表达IGF-IR均能抑制细胞的生长。过表达miR-143和干扰高表达IGF-IR的5637细胞对吉西他滨的敏感性增加。4、上调miR-143能抑制5637细胞中P-AKT和P-ERK的表达。干扰5637细胞中IGF-IR表达可以调节AKT和ERK信号通路的激活来抑制P-AKT和P-ERK的表达。结论:1、miR-143在膀胱癌组织和细胞中广泛低表达,与膀胱癌的恶性生物学行为密切相关。2、miR-143抑制膀胱癌细胞的增殖能力,并且可以通过靶向IGF-IR影响AKT和ERK信号通路起到抑癌基因的作用。3、miR-143和吉西他滨对膀胱癌细胞生长有协同抑制作用,miR-143可以增加膀胱癌细胞对化疗药物(吉西他滨)的敏感性。
[Abstract]:Background: miR-143 is a kind of microRNA, which has been proved to be low expressed in many kinds of tumor tissues and can act as a tumor suppressor gene. In vitro, it has been proved that there are miR-143 regulatory sites in the IGF-IR3'-UTR region, and that high expression miR-143 can act on IGF-IR to inhibit the proliferation of tumor cells. More and more attention has been paid to the role of invasion, metastasis, microRNAs in gene regulation. The effect of miR-143 on the chemosensitivity of gemcitabine in bladder cancer cells has not been reported. Our results suggest that miR-143 may play an important role in the regulation of IGF-IR gene in bladder cancer cells. It is important to explore the effects of miR-143 mediated IGF-IR gene regulation on the biological behavior and chemosensitivity of bladder cancer cells. Objective: to investigate the expression of miR-143 in bladder cancer tissues and its correlation with the growth of bladder cancer cells, and to explore the molecular mechanism of miR-143 affecting malignant biological behavior and chemosensitivity of bladder cancer. Methods the expression of miR-143 in 23 cases of bladder cancer and its adjacent normal bladder tissues was detected by using 1: 1 RT-PCR, and the expression of miR-143 in bladder cancer cell lines T24 and 5637 was also detected. To analyze the correlation between the expression of miR-143 and the stage of bladder cancer. 2RT-PCR was used to detect the expression of IGF-IR in 23 cases of bladder cancer and its adjacent normal bladder tissues, and to analyze the correlation between miR-143 and IGF-IR expression. MiR-143 overexpression lentivirus system was constructed. The expression level of IGF-IR in T24 and 5637 cells overexpressed with miR-143 was detected by RT-PCR. The effect of overexpression of miR-143 and interference of IGF-IR on the proliferation of 5637 cells was detected by the method of CCK-8. The effect of overexpression of miR-143 and interference of IGF-IR on the sensitivity of gemcitabine. 4Western blotting assay was used to analyze the expression of p-AKTT-AK p-ERK and ERK after IGF-IR interference and overexpression of miR-143. Results the expression of miR-143 in bladder cancer tissues was lower than that in adjacent normal bladder tissues, and the expression level was closely related to the stage of the tumor. The expression of miR-143 and IGF-IR in bladder cancer cell lines T24 and 5637 were negatively correlated. Overexpression of miR-143 in T24 and 5637 cells significantly inhibited the expression of IGF-IR. 3. Overexpression of miR-143 and interference of overexpression of IGF-IR in 5637 cells inhibited the growth of T24 and 5637 cells. Overexpression of miR-143 and overexpression of IGF-IR increased the sensitivity of 5637 cells to gemcitabine. Upregulation of miR-143 inhibited the expression of P-AKT and P-ERK in 5637 cells. Interfering with the expression of IGF-IR in 5637 cells can inhibit the expression of P-AKT and P-ERK by regulating the activation of AKT and ERK signaling pathway. Conclusion the low expression of 1: 1 miR-143 in bladder cancer tissues and cells is closely related to the malignant biological behavior of bladder cancer. It can inhibit the proliferation of bladder cancer cells. Moreover, targeting IGF-IR could inhibit the growth of bladder cancer cells by targeting AKT and ERK signaling pathway. 3miR-143 and gemcitabine had synergistic inhibitory effects on the growth of bladder cancer cells. MiR-143 could increase the sensitivity of bladder cancer cells to chemotherapeutic drugs (gemcitabine).
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.14

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