Chi-29b嵌合体在卵巢癌中的抗癌作用及机制研究

发布时间：2018-01-25 18:40

本文关键词： 卵巢癌适体 miRNA PTEN 嵌合体甲基化肿瘤干细胞动物模型　出处：《中南大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分Chi-29b嵌合体在卵巢上皮性癌细胞中的抗癌作用目的：建立一种可以将miRNA-29b分子通过肿瘤组织特异性转运方式运入肿瘤细胞,进而影响抑癌基因PTEN的重新表达,从而抑制卵巢癌生长的治疗方法。方法：构建可以靶向结合卵巢癌细胞表面MUC1蛋白的适体和miR-29b构成的嵌合体(Chi-29b)。Dicer酶体外消化Chi-29b,检测Chi-29b能否释放miR-29b。FACS法检测Chi-29b的细胞内化率。Western-blot法检测OVCAR-3细胞中DNMT1、DNMT3A、DNMT3B、PTEN蛋白表达水平,甲基化技术检测OVCAR-3细胞中PTEN启动子区的甲基化,RT-PCR检测OVCAR-3细胞中PTEN mRNA表达水平,Hoechst33342法检测细胞凋亡。结果:Dicer酶能有效裂解Chi-29b嵌合体,并释放miR-29b。Chi-29b嵌合体可以通过浓度依赖的方式特异性的转运到OVCAR-3细胞中；而且当嵌合体Chi-29b浓度为250nM时,达到细胞内化的高峰拐点(81%),500nM和1000nM时的Chi-29b内化数量更多。和对照组比较,Chi-29b嵌合体可以显著下调OVCAR-3细胞中DNMT1, DNMT3A, DNMT3B的蛋白表达水平(P0.001),诱导PTEN基因启动子甲基化低表达(P0.01),上调PTEN mRNA和蛋白表达水平(P0.001和P0.01),并且可以显著诱导OVCAR-3细胞凋亡(P0.001)。结论：嵌合体Chi-29b能通过MUC1的靶向作用以浓度依赖方式进入OVCAR-3细胞；OVCAR-3细胞中,Chi-29b通过甲基化机制上调PTEN基因和蛋白表达,诱导细胞凋亡。第二部分卵巢癌中Chi-29b嵌合体的抗化疗耐药效应及机制目的：确定在卵巢癌移植模型和化疗药物耐药的卵巢癌肿瘤模型中MUC1核酸适体-miR-29b嵌合体的抗肿瘤作用,并进一步探明相关的机制。方法：构建OVCAR-3-taxol细胞亚株,MTT法检测其IC50值；将OVCAR-3-taxol细胞分别和1mg/ml紫杉醇、500nM的Chi-29b、1mg/ml紫杉醇加500nM的Chi-29b共孵育,MTT法检测细胞增殖。裸鼠右后肢皮下注射OVCAR-3、OVCA-420、OVCAR-3-taxol细胞,建立相应的异种卵巢癌移植动物模型并分组；腹腔注射Chi-29b,评价肿瘤生长情况及其对ALDH1+细胞的影响,甲基化技术检测移植瘤细胞中PTEN启动子的甲基化,RT-PCR法检测PTENmRNA及ALDH1mRNA表达水平,Western-blot法检测PTEN、MAPK4、 MAPK10、IGF1、AKT、Bax及血管动蛋白的蛋白表达水平,Tunnel法检测细胞凋亡水平。结果：()VCAR-3-taxol细胞亚株的IC50值是5764±143μg/L；Chi-29b嵌合体能显著抑制体外OVCAR-3-taxol细胞增殖(P0.001)。在OVCAR-3移植瘤中,和对照组比较,腹腔内注射Chi-29b嵌合体能显著抑制肿瘤生长(p0.001),显著上调PTENmRNA表达(p0.001),显著下调PTEN甲基化和MAPK4、IGF1蛋白的表达水平(p0.001)；然而在OVCA-420移植瘤中,Chi-29b通过下调MAPK4、MAPK10和IGF1蛋白表达而抑制肿瘤生长(p0.01),但不影响PTEN基因的表达。在OVCAR-3-taxol移植瘤中,和对照组比较,腹腔注射Chi-29b嵌合体能显著抑制肿瘤生长和增加肿瘤细胞凋亡(p0.001),显著上调PTENmRNA、PTEN和Bax蛋白表达水平(p0.001),显著下调Akt、MAPK4、MAPK10和IGF1蛋白表达水平(p0.001)。OVCAR-3-taxol移植瘤中的ALDH1mRNA显著高于OVCAR-3移植瘤中的表达水平(p0.001)；腹腔内注射Chi-29b嵌合体能显著降低这两种移植瘤中的ALDH1mRNA表达水平(p0.001)和OVCAR-3-taxol移植瘤中的ALDH1+细胞数(p0.001)。结论：Chi-29b在卵巢癌及卵巢癌耐药的动物模型中均能发挥有效的抗肿瘤作用；卵巢癌耐药细胞的产生和ALDH1阳性细胞增加有关；Chi-29b通过抑制肿瘤干细胞活性发挥抗卵巢癌耐药的作用；Chi-29b通过调节PTEN-Akt-Bax及MAPK和IGF信号途径抑制卵巢癌的生长,并且可能和ALDH1有关。
[Abstract]:The anticancer effect of Chi-29b chimeras in epithelial ovarian cancer cells
Objective: to establish a therapeutic method that can transport miRNA-29b molecules into tumor cells through tumor tissue specific transport, thereby affecting the re expression of tumor suppressor gene PTEN, thereby inhibiting the growth of ovarian cancer.
Methods: construct chimera targeted binding aptamer and miR-29b ovarian cancer cell surface MUC1 protein A (Chi-29b).Dicer enzyme digestion in vitro Chi-29b, detection of Chi-29b can release miR-29b.FACS method to detect Chi-29b cell internalization rate of.Western-blot DNMT1 was detected in OVCAR-3 cells, DNMT3A, DNMT3B, PTEN protein expression, OVCAR-3 methylation detection cell PTEN in the promoter methylation level of PTEN, mRNA expression of OVCAR-3 cells was measured by RT-PCR, cell apoptosis was detected by Hoechst33342.
Results: Dicer enzyme can effectively cleaved Chi-29b chimeras, and the release of miR-29b.Chi-29b chimeras can be dependent on the concentration of specific transport into OVCAR-3 cells; and when the chimera Chi-29b concentration was 250nM, reached the peak of the cellular internalization of inflection point (81%), 500nM and 1000nM when the internalization of Chi-29b and control group in greater numbers. Comparison of Chi-29b chimeras can significantly reduce DNMT1, OVCAR-3 in DNMT3A cells, the expression level of DNMT3B protein (P0.001), inducible promoter methylation of PTEN gene low expression (P0.01), mRNA and upregulation of PTEN protein expression (P0.001 and P0.01), and significantly induced apoptosis in OVCAR-3 cells (P0.001).
Conclusion: chimeric Chi-29b can enter OVCAR-3 cells in a concentration dependent manner through the targeted action of MUC1. In OVCAR-3 cells, Chi-29b can upregulate PTEN gene and protein expression and induce cell apoptosis through methylation.
Anti chemotherapeutic resistance and mechanism of Chi-29b chimerism in second parts of ovarian cancer
Objective: to determine the antitumor effect of MUC1 aptamer -miR-29b chimeras in ovarian cancer transplantation models and chemotherapeutic drug resistant ovarian cancer models, and further explore the related mechanisms.
Methods: the OVCAR-3-taxol cell line, detect the IC50 value of MTT; OVCAR-3-taxol cells were 1mg/ml and paclitaxel, 500nM Chi-29b, 1mg/ml paclitaxel plus 500nM Chi-29b co incubation, cell proliferation was detected by MTT. The right hind nude mice subcutaneous injection of OVCAR-3, OVCA-420, OVCAR-3-taxol cells, to establish xenograft transplanted ovarian cancer animal model and the corresponding group; intraperitoneal injection of Chi-29b, the growth of tumor and its effects on ALDH1+ cells, the methylation of PTEN promoter methylation detection of tumor cells in the PTENmRNA and RT-PCR method to detect the expression level of ALDH1mRNA, detection of PTEN, Western-blot MAPK4, MAPK10, IGF1, AKT, Bax and angiomotin protein the expression, detect the apoptosis rate of Tunnel method.
Results: (IC50) VCAR-3-taxol cell line was 5764 + 143 g/L; Chi-29b chimera could significantly inhibit the proliferation of OVCAR-3-taxol cells in vitro (P0.001). In the OVCAR-3 transplantation tumor, compared with control group, intraperitoneal injection of Chi-29b chimeras can significantly inhibit tumor growth (p0.001), up regulate the expression of PTENmRNA (p0.001), significant downregulation of PTEN methylation and MAPK4 expression level of IGF1 protein (p0.001); however, in the OVCA-420 transplantation tumor in Chi-29b by downregulating MAPK4 expression of MAPK10 and IGF1 protein and inhibit tumor growth (P0.01), but did not affect the expression of PTEN gene. In the OVCAR-3-taxol transplantation tumor, compared with control group, intraperitoneal injection of Chi-29b chimera could significantly inhibit the growth of tumor cells and increase the apoptosis of tumor (p0.001), the significant increase in PTENmRNA, PTEN and Bax protein expression (p0.001), Akt MAPK4, MAPK10 was significantly down regulated, and the expression level of IGF1 protein (p0.001) ALDH1mRNA in.OVCAR-3-taxol xenograft was significantly higher than that in OVCAR-3 transplanted tumor (p0.001). Intraperitoneal injection of Chi-29b chimeras could significantly reduce ALDH1mRNA expression level (p0.001) and ALDH1+ cell number (p0.001) in two kinds of transplanted tumors.
Conclusion: Chi-29b can exert potent antitumor effects in animal models of ovarian cancer and ovarian cancer resistance; increase production and the positive cells of ALDH1 resistant ovarian cancer cells; Chi-29b by inhibiting the tumor stem cells play anti multidrug resistance in ovarian carcinoma; Chi-29b through inhibition of ovarian cancer and the regulation of PTEN-Akt-Bax and MAPK IGF signal the way of growth, and may be associated with ALDH1.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.31

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