NRSN2对骨肉瘤细胞增殖、分化的作用及其分子机制研究

发布时间：2018-01-19 20:34

本文关键词： 骨肉瘤 NRSN2 PI3K/AKT/mTOR信号通路 Wnt/β-catenin信号通路　出处：《新疆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:(1)通过检测NRSN2在骨肉瘤组织及骨肉瘤细胞中的表达差异,研究NRSN2与骨肉瘤发生之间的相关性。(2)通过沉默/过表达NRSN2骨肉瘤细胞系的体内体外实验,探讨NRSN2对骨肉瘤细胞增殖、分化的作用。(3)探讨NRSN2在骨肉瘤细胞增殖、分化中的分子机制研究,确定NRSN2调控作用是否通过P13K/AKT/m TOR及Wnt/β-catenin信号通路。方法:(1)收集18例骨肉瘤和旁骨肉瘤组织标本,采用实时定量PCR及免疫组织化学法检测标本中NRSN2的mRNA及蛋白表达水平,分析NRSN2在骨肉瘤和旁骨肉瘤组织标本中的表达差异。(2)采用实时定量PCR及Western blot检测体外培养的U20S、Saos2、MG63骨肉瘤细胞系中NRSN2的m RNA和蛋白表达水平,分析NRSN2在骨肉瘤细胞系中的表达差异。(3)选择U20S、Saos2骨肉瘤细胞系(NRSN2相对高表达),构建沉默NRSN2的U20S、Saos2骨肉瘤细胞系,Western blot验证沉默效果;体外培养沉默NRSN2的骨肉瘤细胞系,采用CCK8法检测骨肉瘤细胞增殖、分化能力,通过软琼脂细胞克隆形成实验,测量克隆细胞数,沉默NRSN2的U2OS骨肉瘤细胞系接种裸鼠皮下,进行异种皮下肿瘤形成实验,测量瘤体重量,进一步分析沉默NRSN2在体内体外对骨肉瘤细胞增殖、分化的作用。(4)选择MG63骨肉瘤细胞系(NRSN2相对低表达),构建过表达NRSN2的MG63骨肉瘤细胞系,Western blot验证NRSN2过表达效果;体外培养过表达NRSN2的骨肉瘤细胞系,利用CCK8法检测骨肉瘤细胞增殖、分化能力,通过软琼脂细胞克隆形成实验,测量克隆细胞数,过表达NRSN2的骨肉瘤细胞系接种裸鼠皮下,进行异种皮下肿瘤形成实验,测量瘤体重量,分析过表达NRSN2在体内体外对骨肉瘤细胞增殖、分化的作用。(5)沉默NRSN2的U20S、Saos2骨肉瘤细胞系中,采用Wesrtern blot检测PI3K/AKT/m TOR及Wnt/β-catenin信号通路相关蛋白Akt、p-Akt、mTOR、p-mTOR、GSK3β、p-GSK3β、nu-β-catenin的蛋白表达水平,进一步采用实时定量PCR分别检测Wnt/β-catenin信号通路目标蛋白CCND1和c-myc的mRNA表达水平,分析沉默NRSN2对PI3K/AKT/m TOR及Wnt/β-catenin信号通路的调控。(6)过表达NRSN2的MG63骨肉瘤细胞系中,采用Wesrtern blot检测Akt、p-Akt、mTOR、p-m TOR、GSK3β、p-GSK3β、β-catenin的蛋白表达水平,采用实时定量PCR检测Wnt/β-catenin信号通路目标蛋白CCND1和c-myc的m RNA表达水平,加入β-catenin抑制剂IWR-1-endo后采用CCK8法检测骨肉瘤细胞增殖、分化能力,分析过表达NRSN2对PI3K/AKT/mTOR及Wnt/β-catenin信号通路的调控。结果:(1)18例骨肉瘤组织标本中NRSN2的mRNA及蛋白表达水平均高于旁骨肉瘤组织标本,差异有统计学意义(P0.01),提示:NRSN2与骨肉瘤相关。(2)U20S、Saos2、MG63骨肉瘤细胞系中NRSN2的mRNA和蛋白表达水平均高于hFOB1.19组,NRSN2与骨肉瘤细胞增殖、分化相关,差异有统计学意义(均P0.01)。U20S和Saos2骨肉瘤细胞系相对高于MG63骨肉瘤细胞系,差异有统计学意义(P0.05),U20S和Saos2骨肉瘤细胞系间差异无统计学意义(P0.05)。(3)沉默NRSN2明显抑制骨肉瘤细胞系U2OS、Saos2的增殖、分化能力(P0.01),重新上调NRSN2生物学作用后重新提高U2OS、Saos2骨肉瘤细胞系增殖、分化能力(P0.01),沉默NRSN2组克隆细胞数少于对照组(均P0.01),沉默NRSN2组裸鼠皮下形成的瘤体重量小于对照组(P0.01),未转染和重新上调NRSN2组作为对照组。(4)过表达NRSN2使MG63骨肉瘤细胞系增殖、分化能力增强(P0.01),重新下调NRSN2生物学作用后MG63骨肉瘤细胞系增殖、分化能力下降(P0.01),过表达NRSN2组克隆细胞数多于对照组(均P0.01),过表达NRSN2组裸鼠皮下形成的瘤体重量大于对照组(P0.01),空质粒和重新下调NRSN2组作为对照组。(5)沉默NRSN2的U2OS骨肉瘤细胞系中p-Akt、p-m TOR、p-GSK3β、nu-β-catenin的蛋白表达水平,CCND1及c-myc的mRNA表达水平均低于对照组(P0.01),未转染组和重新上调NRSN2组作为对照组。(6)过表达NRSN2的MG63骨肉瘤细胞系中p-Akt、p-mTOR、p-GSK3β、nu-β-catenin的蛋白表达水平,CCND1及c-myc的mRNA表达水平均高于对照组(P0.01),加入β-catenin信号通路抑制剂IWR-1-endo后,MG63骨肉瘤细胞系增殖、分化能力比对照组明显受到抑制(P0.01),空质粒组作为对照组。结论:(1)通过检测骨肉瘤组织及U20S、Saos2、MG63骨肉瘤细胞系中NRSN2的mRNA和蛋白表达水平,确定NRSN2与骨肉瘤之间的相关性。(2)通过沉默/过表达NRSN2骨肉瘤细胞系的体内体外实验研究,进一步证实NRSN2对骨肉瘤细胞增殖、分化有促进作用。(3)通过NRSN2的分子机制研究,阐明NRSN2调控PI3k/AKT/mTOR及Wnt/β-catenin信号通路促进骨肉瘤细胞增值、分化。
[Abstract]:Objective: (1) through the detection of NRSN2 in osteosarcoma tissue and osteosarcoma cells in the differential expression, to study the correlation between NRSN2 and osteosarcoma. (2) in vivo and in vitro silencing / NRSN2 osteosarcoma cell line expression of NRSN2, on the proliferation of osteosarcoma cells, differentiation (3. Study) NRSN2 on the proliferation of osteosarcoma cells, molecular mechanism of differentiation, determine whether the regulation of NRSN2 by P13K/AKT/m TOR and Wnt/ -catenin signaling pathway. Methods: (1) collected 18 cases of bone sarcoma and adjacent osteosarcoma tissues, the expression level of mRNA protein and NRSN2 by real-time quantitative PCR and immunohistochemistry in the sample, analysis of differential expression of NRSN2 in osteosarcoma and adjacent bone sarcoma tissues. (2) using real-time quantitative PCR and Western blot cultured in vitro detection of U20S, Saos2, m RNA and NRSN2 MG63 protein expression in osteosarcoma cell lines of water Flat, differential expression of NRSN2 in osteosarcoma cell lines. (3) U20S, Saos2 osteosarcoma cell lines (NRSN2 relatively high expression), construction of NRSN2 silencing U20S, Saos2 osteosarcoma cell line, Western blot NRSN2 to verify the silence effect; silence of osteosarcoma cell lines cultured in vitro and the ability of using CCK8 the detection method of osteosarcoma cell proliferation, differentiation, cell clone formation assay in soft agar cloning, measuring cell number, U2OS osteosarcoma cell line was inoculated subcutaneously in nude mice. NRSN2, subcutaneous tumor xenograft formation experiment, measuring tumor weight, further analysis of the silencing of NRSN2 in vitro and in vivo on osteosarcoma cell proliferation and differentiation. (4) MG63 osteosarcoma cell lines (NRSN2 low expression), construct MG63 osteosarcoma cell lines expressing NRSN2, Western blot to verify the expression of NRSN2 in vitro effect; overexpression of human osteosarcoma cell line NRSN2, using the CCK8 method The ability to detect proliferation and differentiation of osteosarcoma cells, forming experiment by soft agar cloning, cloning of measuring cell number, over expression of osteosarcoma cell line NRSN2 were inoculated subcutaneously in nude mice subcutaneous tumor xenograft formation, experiment, measurement of tumor weight, analyzed the expression of NRSN2 in vivo in vitro proliferation of osteosarcoma cells, differentiation. (5) NRSN2 silencing U20S, Saos2 in osteosarcoma cell lines, using Wesrtern blot and Wnt/ TOR detection of PI3K/AKT/m beta -catenin signaling pathway related protein Akt, p-Akt, mTOR, p-mTOR, GSK3 beta, p-GSK3 beta, beta -catenin expression level of nu- protein were determined by real-time PCR detection of Wnt/ beta -catenin signaling pathway target CCND1 c-myc and protein expression levels of mRNA, NRSN2 analysis of the silence on the regulation of PI3K/AKT/m TOR and Wnt/ -catenin signaling pathway. (6) the expression of NRSN2 MG63 in osteosarcoma cell lines, using Wesrtern blot to detect A KT, p-Akt, mTOR, P-M, TOR, GSK3 beta, p-GSK3 beta, beta -catenin protein expression level, using M RNA real-time quantitative PCR detection of Wnt/ beta -catenin signaling pathway protein CCND1 and c-myc expression levels, detection of osteosarcoma cell proliferation by CCK8 method, adding beta -catenin inhibitors after IWR-1-endo differentiation ability analysis the expression of NRSN2 on the regulation of PI3K/AKT/mTOR and Wnt/ beta -catenin signaling pathway. Results: (1) 18 cases of mRNA and NRSN2 protein in osteosarcoma tissues the expression level was higher than that of adjacent osteosarcoma tissues, the difference was statistically significant (P0.01), suggested that NRSN2 associated with osteosarcoma. (2) U20S, Saos2, expression the level of mRNA and NRSN2 MG63 protein in osteosarcoma cell lines were higher than that of group hFOB1.19, NRSN2 and osteosarcoma cell proliferation, differentiation, the difference was statistically significant (P0.01).U20S and Saos2 osteosarcoma cell line of MG63 is higher than that of osteosarcoma cell lines, difference There was statistical significance (P0.05), there were no significant differences between U20S and Saos2 in osteosarcoma cell line (P0.05). (3) silencing NRSN2 inhibited osteosarcoma cell line U2OS, Saos2 proliferation, differentiation ability (P0.01), to improve the biological function of NRSN2 up-regulated U2OS, Saos2 proliferation of osteosarcoma cells, differentiation capacity (P0.01), silent group NRSN2 cell clone number is less than the control group (P0.01), the tumor weight of NRSN2 silencing groups nude mice formed less than that of the control group (P0.01), transfected and re raised NRSN2 group as the control group. (4) overexpression of NRSN2 to proliferation of human osteosarcoma MG63 cells, enhance the ability of differentiation (P0.01), to the down-regulation of NRSN2 biological effects of proliferation of line MG63 osteosarcoma cells, differentiation ability (P0.01), NRSN2 overexpression group cloned cells more than the control group (P0.01), expression of tumor weight in NRSN2 group nude mice formed greater than that of the control group (P0.01), empty The new plasmid and down-regulation of NRSN2 group as the control group. (5) p-Akt, U2OS osteosarcoma cell line NRSN2 in P-M silencing TOR, p-GSK3 beta, beta -catenin expression level of nu- protein, the expression level of CCND1 and c-myc mRNA were lower than the control group (P0.01), non transfection group and up-regulated NRSN2 group as a control group. (6) the over expression of p-Akt and MG63 in osteosarcoma cell line NRSN2 in p-mTOR, p-GSK3 beta, beta -catenin expression level of nu- protein, the expression level of CCND1 and c-myc mRNA were higher than the control group (P0.01), the addition of the beta -catenin signaling pathway inhibitor IWR-1-endo, MG63 proliferation of osteosarcoma cells, differentiation ability than the control group was significantly inhibited (P0.01), empty plasmid group as the control group. Conclusion: (1) through the detection of osteosarcoma tissue and U20S, Saos2, mRNA and protein expression level of NRSN2 MG63 in osteosarcoma cell lines, to determine the correlation between NRSN2 and osteosarcoma. (2) through the silence / expression In vivo and in vitro studies of NRSN2 osteosarcoma cell line further confirm that NRSN2 can promote the proliferation and differentiation of osteosarcoma cells. (3) through the molecular mechanism of NRSN2, it is clarified that NRSN2 regulates PI3k/AKT/mTOR and Wnt/ beta -catenin signaling pathway to promote the proliferation and differentiation of osteosarcoma cells.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R738

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