下调CCNO基因表达对宫颈癌Hela细胞生物学行为的影响
发布时间：2018-10-16 18:11
【摘要】：背景：宫颈癌是最常见的妇科恶性肿瘤之一,居女性癌症患者死亡原因的第4位。近年来发病率逐渐增高,尤其是发展中国家。除HPV感染外,宿主与细胞生长分化相关的基因表达异常在宫颈癌的发病机制中同样具有重要作用。近年来,临床前基础研究表明宫颈癌基因治疗是很有潜力的治疗手段,其中包括在细胞周期控制和凋亡信号中具有重要作用的p53基因治疗、宫颈癌基因治疗中研究较多的Bax基因治疗、HPVE2和p21基因以及针对癌基因的反义RNA技术治疗和宫颈癌治疗的理想候选基因FHIT和PTEN基因的研究。我们课题组前期研究利用RNA-Seq技术,发现细胞周期蛋白家族中的cyclin O在宫颈癌组织中的表达水平明显高于癌旁组织,提示其编码基因CCNO可能与宫颈癌的发生发展相关。目的：研究下调CCNO基因表达对宫颈癌Hela细胞株体外增殖、细胞周期及凋亡的影响,为研究宫颈癌生物治疗提供理论依据。材料：病理组织切片来源于北京协和医学院妇产科宫颈癌患者,包括癌灶组织及癌旁组织样本,剔除术前接受放射治疗、化学治疗以及未能获取满意存档石蜡组织标本者；Hela细胞株,为人类宫颈腺癌细胞株,购自上海细胞库(TCHu187,中国上海细胞典藏委员会)。方法1.应用免疫荧光检测CCNO在宫颈癌组织中的表达情况；2.设计靶向CCNO小干扰RNA (CCNO-siRNA),将其转染入体外培养的宫颈癌Hela细胞株细胞中,沉默细胞内源性CCNO基因表达；3.应用Western blotting方法检测CCNO-siRNA的转染效率,确定CCNO基因被沉默,可用于后续实验；4.应用CCK-8方法检测CCNO对宫颈癌细胞增殖的影响；5.应用流式细胞分析技术检测CCNO对细胞周期转化、细胞凋亡的影响。结果1.免疫荧光检测宫颈癌中CCNO基因表达高于癌旁组织；2.转染CCNO-siRNA可特异高效地下调宫颈癌Hela细胞CCNO蛋白表达水平；3.在转染72 h、96 h后,CCK-8法显示CCNO-siRNA转染实验组生长速率明显低于对照组,差异有统计学意义(P0.05,P0.01)；4.细胞周期检测显示,Hela细胞转染CCNO-siRNA实验组G1期细胞较对照组增多均7%左右(P0.05),而S期细胞比例则减少12%左右(P0.05)；5.细胞凋亡检测显示,Hela细胞转染CCNO-siRNA实验组较对照组凋亡细胞增加约17%左右(P0.05)。结论1. CCNO在宫颈癌组织中的表达高于癌旁组织；2.靶向CCNO-siRNA可抑制宫颈腺癌Hela细胞中内源性CCNO的表达；3. CCNO对体外培养的宫颈腺癌Hela细胞具有促进细胞增殖、促进细胞周期转化和抑制凋亡作用。
[Abstract]:Background: cervical cancer is one of the most common gynecological malignancies and is the fourth leading cause of death in female cancer patients. In recent years, the incidence of disease has gradually increased, especially in developing countries. In addition to HPV infection, the abnormal expression of host genes associated with cell growth and differentiation also plays an important role in the pathogenesis of cervical cancer. In recent years, preclinical basic studies have shown that gene therapy for cervical cancer is a potential therapy, including p53 gene therapy, which plays an important role in cell cycle control and apoptosis signal. Bax gene therapy, HPVE2 and p21 gene, antisense RNA therapy for oncogene and ideal candidate genes FHIT and PTEN for cervical cancer therapy were studied. Our previous study using RNA-Seq technique showed that the expression of cyclin O in the cyclin family was significantly higher than that in the adjacent tissues, suggesting that the CCNO encoding gene might be related to the occurrence and development of cervical cancer. Aim: to study the effect of down-regulation of CCNO gene expression on proliferation, cell cycle and apoptosis of cervical cancer Hela cell line in vitro. Materials: histopathological sections were obtained from cervical cancer patients in obstetrics and gynecology of Peking Union Medical College, including samples of cancer focus and adjacent tissues. Those receiving preoperative radiotherapy, chemotherapy and failing to obtain satisfactory archived paraffin tissue specimens were excluded. Hela cell line, a human cervical adenocarcinoma cell line, was purchased from Shanghai Cell Bank (TCHu187, Shanghai Cell Collection Committee). Method 1. Immunofluorescence was used to detect the expression of CCNO in cervical carcinoma. 2. Small interfering RNA (CCNO-siRNA) targeting CCNO was designed and transfected into cervical cancer Hela cell line in vitro, and the expression of endogenous CCNO gene was silenced. 3. Western blotting method was used to detect the transfection efficiency of CCNO-siRNA and to confirm that the CCNO gene was silenced and could be used in subsequent experiments. 4. CCK-8 assay was used to detect the effect of CCNO on the proliferation of cervical cancer cells. Flow cytometry was used to detect the effect of CCNO on cell cycle transformation and apoptosis. Result 1. The expression of CCNO gene in cervical carcinoma was higher than that in adjacent tissues by immunofluorescence. Transfection of CCNO-siRNA can specifically and efficiently down-regulate the expression of CCNO protein in cervical cancer Hela cells. CCK-8 assay showed that the growth rate of the experimental group transfected with CCNO-siRNA was significantly lower than that of the control group at 72 h or 96 h after transfection, and the difference was statistically significant (P0.05, P0.01). Cell cycle analysis showed that compared with the control group, the G1 phase of Hela cells transfected with CCNO-siRNA increased by about 7% (P0.05), while the percentage of S phase cells decreased by 12% (P0.05); Apoptosis assay showed that Hela cells transfected with CCNO-siRNA increased by about 17% compared with control group (P0.05). Conclusion 1. The expression of CCNO in cervical cancer tissues was higher than that in paracancerous tissues. Targeted CCNO-siRNA could inhibit the expression of endogenous CCNO in cervical adenocarcinoma Hela cells. CCNO can promote cell proliferation, promote cell cycle transformation and inhibit apoptosis of cervical adenocarcinoma Hela cells cultured in vitro.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.33
，
本文编号：2275211
[Abstract]:Background: cervical cancer is one of the most common gynecological malignancies and is the fourth leading cause of death in female cancer patients. In recent years, the incidence of disease has gradually increased, especially in developing countries. In addition to HPV infection, the abnormal expression of host genes associated with cell growth and differentiation also plays an important role in the pathogenesis of cervical cancer. In recent years, preclinical basic studies have shown that gene therapy for cervical cancer is a potential therapy, including p53 gene therapy, which plays an important role in cell cycle control and apoptosis signal. Bax gene therapy, HPVE2 and p21 gene, antisense RNA therapy for oncogene and ideal candidate genes FHIT and PTEN for cervical cancer therapy were studied. Our previous study using RNA-Seq technique showed that the expression of cyclin O in the cyclin family was significantly higher than that in the adjacent tissues, suggesting that the CCNO encoding gene might be related to the occurrence and development of cervical cancer. Aim: to study the effect of down-regulation of CCNO gene expression on proliferation, cell cycle and apoptosis of cervical cancer Hela cell line in vitro. Materials: histopathological sections were obtained from cervical cancer patients in obstetrics and gynecology of Peking Union Medical College, including samples of cancer focus and adjacent tissues. Those receiving preoperative radiotherapy, chemotherapy and failing to obtain satisfactory archived paraffin tissue specimens were excluded. Hela cell line, a human cervical adenocarcinoma cell line, was purchased from Shanghai Cell Bank (TCHu187, Shanghai Cell Collection Committee). Method 1. Immunofluorescence was used to detect the expression of CCNO in cervical carcinoma. 2. Small interfering RNA (CCNO-siRNA) targeting CCNO was designed and transfected into cervical cancer Hela cell line in vitro, and the expression of endogenous CCNO gene was silenced. 3. Western blotting method was used to detect the transfection efficiency of CCNO-siRNA and to confirm that the CCNO gene was silenced and could be used in subsequent experiments. 4. CCK-8 assay was used to detect the effect of CCNO on the proliferation of cervical cancer cells. Flow cytometry was used to detect the effect of CCNO on cell cycle transformation and apoptosis. Result 1. The expression of CCNO gene in cervical carcinoma was higher than that in adjacent tissues by immunofluorescence. Transfection of CCNO-siRNA can specifically and efficiently down-regulate the expression of CCNO protein in cervical cancer Hela cells. CCK-8 assay showed that the growth rate of the experimental group transfected with CCNO-siRNA was significantly lower than that of the control group at 72 h or 96 h after transfection, and the difference was statistically significant (P0.05, P0.01). Cell cycle analysis showed that compared with the control group, the G1 phase of Hela cells transfected with CCNO-siRNA increased by about 7% (P0.05), while the percentage of S phase cells decreased by 12% (P0.05); Apoptosis assay showed that Hela cells transfected with CCNO-siRNA increased by about 17% compared with control group (P0.05). Conclusion 1. The expression of CCNO in cervical cancer tissues was higher than that in paracancerous tissues. Targeted CCNO-siRNA could inhibit the expression of endogenous CCNO in cervical adenocarcinoma Hela cells. CCNO can promote cell proliferation, promote cell cycle transformation and inhibit apoptosis of cervical adenocarcinoma Hela cells cultured in vitro.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.33
，
本文编号：2275211
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