MUC16在HPV16相关宫颈癌发生与演进中的作用

发布时间：2018-01-16 04:22

本文关键词：MUC16在HPV16相关宫颈癌发生与演进中的作用　出处：《新乡医学院》2014年硕士论文　论文类型：学位论文

【摘要】：背景高危型人乳头瘤病毒(high risk-human papillomaviruses, HR-HPVs)(如HPV16等)病毒癌基因E6的持续表达是宫颈癌最重要的致病因素。本课题组设计并筛选了靶向HPV16E6且沉默效应均达95%以上的短发夹RNA(short hairpin RNA, shRNA),经Agilent人基因组表达谱芯片分析,发现沉默E6表达后,粘蛋白抗原16(mucin antigen16, MUC16)基因表达下调(2-5倍)。MUC16是否是HPV16E6的直接作用靶点及调控机制尚不清楚。因此,深入研究E6与MUC16的关系,对探索HPV16相关肿瘤新的治疗方法具有重要意义。目的以HPV16E6阳性和阴性的宫颈癌细胞系、正常宫颈鳞状上皮、宫颈上皮内瘤变(cervical intraepithelial neoplasias, CIN)和宫颈鳞癌组织为研究对象,从细胞和组织水平分析HPV16E6和MUC16的关系,试图阐明MUC16在HPV16相关宫颈癌发生与演进中的作用,丰富HPV16E6的致癌机制。方法 1.以HPV16阳性(CaSku SiHa细胞)和阴性(C-33A细胞)的宫颈癌细胞为研究对象,采用Western blotting方法检测HPV16E6和MUC16的表达,并分析二者的关系。 2.以HPV16E6阳性和阴性的正常宫颈鳞状上皮、CIN和宫颈鳞癌组织为研究对象,采用免疫组化方法检测HPV16E6和MUC16的表达,并分析二者的关系。 3.利用生物信息学技术设计靶向MUC16mRNA的shRNA序列,采用分子克隆方法构建含靶向MUC16的shRNA的pGenesil-1真核表达重组质粒(命名为pGenesil-l-shRNA-MUC16)和含无关序列的对照质粒(命名为pGenesil-1-shRNA-vect)。 4.通过脂质体介导将上述质粒分别转染入HPV16E6阳性的宫颈癌CaSki细胞(分别命名为CaSki-shRNA-MUC16和CaSki-shRNA-vect细胞)。G418筛选抗性细胞。通过RT-PCR和Western blotting方法鉴定MUC16沉默效应。 5.通过Annexin V-FITC/PI结合流式细胞术、Caspase-3活性测定、Transwell迁移实验和Transwell侵袭实验,检测靶向沉默MUC16表达后HPV16E6阳性宫颈癌CaSki细胞凋亡和迁移侵袭能力的变化。结果 1. Western blotting结果显示：①C-33A细胞中HPV16E6无表达,CaSki细胞和SiHa细胞中可见HPV16E6表达；②C-33A细胞中MUC16的表达显著低于CaSki细胞和SiHa细胞(P0.01),CaSki细胞和SiHa细胞中的MUC16的表达差异无显著性(P0.05)。 2.免疫组化结果显示：(DHPV16E6阳性的正常宫颈鳞状上皮、CIN和宫颈鳞癌组织中MUC16的表达均显著高于HPV16E6阴性的正常宫颈鳞状上皮、CIN和宫颈鳞癌组织(相关系数分别为0.531、0.629和0.452,P0.01)。 3.含MUC16shRNA真核表达重组质粒的鉴定结果显示：目的片段插入位点和方向正确,与设计一致。 4.靶向MUC16mRNA的shRNA对宫颈癌CaSki细胞中MUC16表达的沉默效应 RT-PCR结果显示：与CaSki细胞和CaSki-shRNA-vect细胞相比,CaSki-shRNA-MUC16细胞中MUC16mRNA的表达显著降低(P0.01),沉默效率为：93.137%。而CaSki细胞和CaSki-shRNA-vect细胞中MUC16mRNA的表达差异无显著性(P0.05)。 Western blotting结果显示：与CaSki细胞和CaSki-shRNA-vect细胞相比,CaSki-shRNA-MUC16细胞中MUC16蛋白的表达显著降低(P0.01),沉默效率为：93.913%。而CaSki细胞和CaSki-shRNA-vect细胞中MUC16蛋白的表达差异无显著性(P0.05)。鉴于以上研究结果,后续实验中均以CaSki-shRNA-MUC16细胞为实验组,以CaSki-shRNA-vect细胞为对照组。 5.靶向沉默MUC16表达对HPV16E6阳性宫颈癌CaSki细胞凋亡和侵袭的影响：①Annexin V-FITC/PI结合流式细胞术检测细胞凋亡结果显示：CaSki-shRNA-MUC16细胞凋亡均数明显高于CaSki-shRNA-vect细胞(P0.01),凋亡细胞百分比分别为：5.598%和0.150%；②Caspase-3活性测定结果显示：与CaSki-shRNA-vect细胞相比,CaSki-shRNA-MUC16细胞中Caspase-3活性显著增加(P0.01)；③Transwell迁移和侵袭实验的结果均显示：与CaSki-shRNA-vect细胞相比,CaSki-shRNA-MUC16细胞穿过小室底膜的细胞数均显著减少(P0.01)。结论 1.在HPV16E6阳性和阴性宫颈癌细胞系和宫颈组织中MUC16与E6表达呈正相关。 2.靶向沉默MUC16的表达可促进HPV16E6阳性宫颈癌CaSki细胞的凋亡,并抑制其迁移侵袭能力。
[Abstract]:The background of high-risk human papillomavirus (high risk-human, papillomaviruses, HR-HPVs) (such as HPV16) for the expression of viral oncogene E6 is one of the most important pathogenic factors of cervical cancer. The research group design and screening of HPV16E6 targeting and silencing effect was more than 95% short hairpin RNA (short hairpin RNA, shRNA). The Agilent of human genome expression microarray analysis, found that silencing E6 expression after mucin antigen 16 (mucin antigen16 MUC16) gene expression (2-5 times).MUC16 is the direct target and the regulation mechanism of HPV16E6 is not clear. Therefore, to study the relationship between E6 and MUC16, is of great significance to explore the treatment a new method for HPV16 related tumors.
Objective to cervical cancer cell line HPV16E6 positive and negative, normal cervical squamous epithelium, cervical intraepithelial neoplasia (cervical intraepithelial, neoplasias, CIN) and cervical squamous cell carcinoma as the research object, analyze the relationship between HPV16E6 and MUC16 from the cell and tissue level, attempts to clarify the occurrence and evolution of the role of MUC16 in HPV16 associated cervical cancer. Rich HPV16E6 carcinogenic mechanism.
Method
1. cervical cancer cells with HPV16 positive (CaSku SiHa cells) and negative (C-33A cells) as the subjects, Western blotting method was used to detect the expression of HPV16E6 and MUC16, and the relationship between the two was analyzed.
2., we used HPV16E6 positive and negative normal cervical squamous epithelium, CIN and cervical squamous cell carcinoma as the research objects. Immunohistochemical method was used to detect the expression of HPV16E6 and MUC16, and the relationship between the two was analyzed.
3. the use of bioinformatics techniques to design shRNA sequence targeting MUC16mRNA, constructed by molecular cloning method pGenesil-1 eukaryotic expression recombinant plasmid containing the target MUC16 shRNA (named pGenesil-l-shRNA-MUC16) and containing unrelated sequence control plasmid (named pGenesil-1-shRNA-vect).
4., these plasmids were transfected into HPV16E6 positive cervical cancer CaSki cells (named CaSki-shRNA-MUC16 and CaSki-shRNA-vect cells respectively) by liposome mediated.G418, and the resistant cells were screened by.G418. The MUC16 silence effect was identified by RT-PCR and Western blotting.
5. through Annexin V-FITC/PI combined with flow cytometry, Caspase-3 activity assay, Transwell migration test and Transwell invasion test, we detected the change of apoptosis and migration and invasion ability of HPV16E6 positive cervical cancer CaSki cells after silencing MUC16 expression.
Result
1. Western blotting showed that HPV16E6 expression of C-33A cells, the expression of CaSki showed HPV16E6 cells and SiHa cells; the expression of MUC16 in C-33A cells was significantly lower than that of CaSki cells and SiHa cells (P0.01), the differential expression of CaSki cells and SiHa cells in MUC16 had no significant difference (P0.05).
2. immunohistochemical results showed that: (DHPV16E6 positive normal cervical squamous epithelium, CIN and cervical squamous cell carcinoma MUC16 expression was significantly higher than that of HPV16E6 negative normal cervical squamous epithelium, CIN and cervical squamous cell carcinoma (correlation coefficient were 0.531,0.629 and 0.452, P0.01).
Identification of recombinant plasmid containing 3. MUC16shRNA eukaryotic expression
The results showed that the insertion site and direction of the target fragment were correct and consistent with the design.
The silence effect of 4. target MUC16mRNA shRNA on the expression of MUC16 in CaSki cells of cervical cancer
RT-PCR results showed that compared with CaSki cells and CaSki-shRNA-vect cells, the expression of MUC16mRNA in CaSki-shRNA-MUC16 cells was significantly decreased (P0.01), and the silence efficiency was 93.137%., but there was no significant difference in MUC16mRNA expression between CaSki cells and CaSki-shRNA-vect cells (P0.05).
Western blotting results showed that: compared with CaSki cells and CaSki-shRNA-vect cells, the expression of MUC16 protein in CaSki-shRNA-MUC16 cells decreased significantly (P0.01), silencing efficiency: 93.913%. and MUC16 expression of CaSki cells and CaSki-shRNA-vect cells in protein had no significant difference (P0.05).
In view of the above results, CaSki-shRNA-MUC16 cells were used as the experimental group in the follow-up experiments, and the CaSki-shRNA-vect cells were used as the control group.
5. target expression on apoptosis and invasion of HPV16E6 positive cervical cancer CaSki cells to silence MUC16 Annexin V-FITC/PI: combined with flow cytometry to detect the apoptosis results showed that apoptosis of CaSki-shRNA-MUC16 cells was significantly higher than that of CaSki-shRNA-vect cells (P0.01), the percentage of apoptotic cells were 5.598% and 0.150%; the Caspase-3 activity determination results show: compared with CaSki-shRNA-vect cells, the activity of Caspase-3 in CaSki-shRNA-MUC16 cells increased significantly (P0.01); the Transwell migration and invasion experiment results showed that: compared with CaSki-shRNA-vect cells, the number of cells in CaSki-shRNA-MUC16 cells through cell basement membrane were significantly decreased (P0.01).
conclusion
1. there was a positive correlation between MUC16 and E6 expression in HPV16E6 positive and negative cervical cancer cell lines and cervix tissues.
2. the expression of target silencing MUC16 can promote the apoptosis of HPV16E6 positive cervical cancer CaSki cells and inhibit its migration and invasion ability.

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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