DRR1在HPV16相关宫颈癌发生与演进中的作用

发布时间：2018-06-13 12:24

本文选题：人乳头瘤病毒 + 宫颈鳞癌　；参考：《新乡医学院》2014年硕士论文

【摘要】：背景宫颈癌是女性常见的恶性肿瘤之一,感染高危型人乳头瘤病毒(high risk-human papillomaviruses,HR-HPVs)是其发生与演进的重要基础,E7的持续表达是HR-HPVs致癌的关键,其可通过结合并降解pRb蛋白等,导致细胞异常增殖。本课题组经Agilent人基因表达谱芯片分析,发现沉默E7表达后,肾细胞癌下调基因1(down-regulated in renal cell carcinoma1, DRR1)表达上调3-11倍。DRR1广泛表达于正常组织,但在许多癌细胞系和原发性肿瘤中表达明显降低,DRR1作为肿瘤抑制基因可能在癌变过程中发挥重要作用。目的以HPV16E7(?)日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,探讨DRR1在HPV16相关宫颈癌发生与演进中的作用,以期丰富HPV16的致癌机制,为探讨HPV16相关肿瘤新的治疗措施提供实验和理论依据。方法 1.以HPV16E7日性宫颈癌CaSki和SiHa细胞及HPV16E7阴性宫颈癌C33-A细胞为研究对象,采用Western blotting方法检测E7和DRRl的表达,分析HPV16E7表达与DRR1表达的关系。 2.以HPV16E7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,通过免疫组织化学(immunohistochemistry, IHC)方法检测E7和DRR1的表达,分析HPV16E7与DRR1表达的关系。 3.常规培养HPV16E7阴性的宫颈癌C33-A细胞至对数生长期,提取总RNA,体外逆转录合成cDNA第一条链,PCR扩增DRR1基因编码序列,构建真核表达重组质粒pcDNA3.1-DRR1并鉴定,经脂质体介导将pcDNA3.1-DRR1重组质粒转染对数生长期的宫颈癌CaSki细胞,通过MTT绘制细胞生长曲线、平板克隆形成实验、细胞周期分布,Annexin V-FITC/PI结合流式细胞术等方法观察稳定表达DRR1对HPV16E7阳性宫颈癌CaSki细胞增殖和凋亡能力的影响。结果 1. Western blotting结果显示：①C33-A细胞中HPV16E7无表达,CaSki细胞和SiHa细胞中可见HPV16E7表达。②C33-A细胞中DRR1的表达显著高于CaSki细胞和SiHa细胞(P0.01),CaSki细胞和SiHa细胞中DRRl的表达无显著差异(P0.05)。 2.IHC结果显示：HPV16E7阳性正常宫颈鳞状上皮、CIN和宫颈鳞癌组织中DRR1的表达均分别显著低于HPV16E7阴性的正常宫颈鳞状上皮组织、CIN和宫颈鳞癌组织,相关系数分别为-0.452,-0.485及-0.444(P0.01)。 3.①MTT比色法绘制细胞生长曲线的结果显示：与CaSki-vect细胞相比,CaSki-DRR1细胞生长速度明显减慢(P0.01),倍增时间显著延长(P0.01)。②平板克隆形成实验结果显示：CaSki-DRR1细胞克隆形成均数明显低于CaSki-vect细胞(P0.01),且克隆体积明显较小。③流式细胞术检测细胞周期分布的结果显示：与CaSki-vect细胞相比,CaSki-DRR1细胞G1期阻滞(P0.01),但S期和G2/M期细胞百分比均数均明显减少(P0.01)。④Annexin V-FITC/PI结合流式细胞术检测细胞凋亡结果显示：CaSki-DRR1细胞凋亡均数明显高于CaSki-vect细胞(P0.01),凋亡细胞百分比分别为：12.116%和0.016%。结论 1. HPV16E7阳性和阴性宫颈癌细胞系和宫颈组织中,E7表达与DRRl表达负相关。 2.稳定表达DRR1可显著抑制HPV16E7P日性宫颈癌CaSki细胞的增殖能力,促进其凋亡。
[Abstract]:Background cervical cancer is one of the most common malignant tumors in women. High risk-human papillomaviruses (HR-HPVs) is an important basis for its occurrence and evolution. The continuous expression of E7 is the key to the carcinogenesis of HR-HPVs, which can lead to abnormal cell proliferation by combining and degrading pRb protein. This group is treated by Agilent people. Gene expression profile chip analysis showed that after the expression of silent E7, the expression of down regulated gene 1 (down-regulated in renal cell carcinoma1, DRR1) was 3-11 times higher than that of normal tissue, but the expression in many cancer cell lines and primary tumors was significantly reduced, and DRR1 as a tumor suppressor gene may play a role in the process of cancer. Important role.
Objective HPV16E7 (?) diurnal and negative cervical cancer cell lines, normal cervical squamous epithelium, cervical intraepithelial neoplasia (cervical intraepithelial neoplasias, CIN) and cervical squamous cell carcinoma were studied to explore the use of DRR1 in the occurrence and evolution of HPV16 related cervical cancer in order to enrich the carcinogenic mechanism of HPV16 and to explore the HPV16 associated swelling. The new treatment of tumor provides experimental and theoretical basis.
Method
1. the expression of E7 and DRRl was detected by Western blotting method, and the relationship between HPV16E7 expression and DRR1 expression was analyzed by Western blotting method with CaSki and SiHa cells of HPV16E7 and C33-A cells of HPV16E7 negative cervical cancer as the research object.
2. HPV16E7 positive and negative normal cervical squamous epithelium, CIN and cervical squamous cell carcinoma were studied. The expression of E7 and DRR1 was detected by immunohistochemistry (immunohistochemistry, IHC), and the relationship between HPV16E7 and DRR1 expression was analyzed.
3. the HPV16E7 negative cervical cancer C33-A cells were cultured to the logarithmic growth period, the total RNA was extracted, the first chain of cDNA was synthesized by reverse transcriptase in vitro, the encoding sequence of DRR1 gene was amplified by PCR, the eukaryotic expression recombinant plasmid pcDNA3.1-DRR1 was constructed and the recombinant plasmid of pcDNA3.1-DRR1 was transfected into the logarithmic long term cervical cancer CaSki cells via liposome. The effect of stable expression of DRR1 on the proliferation and apoptosis of HPV16E7 positive cervical cancer cell CaSki cells was observed by MTT plotting cell growth curve, cell clone formation experiment, cell cycle distribution and Annexin V-FITC/PI combined with flow cytometry.
Result
The results of 1. Western blotting showed that there was no expression of HPV16E7 in C33-A cells, and the expression of HPV16E7 in CaSki and SiHa cells. The expression of DRR1 in C33-A cells was significantly higher than that of CaSki and SiHa cells (P0.01).
2.IHC results showed that the expression of DRR1 in HPV16E7 positive normal cervical squamous epithelium, CIN and cervical squamous cell carcinoma were significantly lower than that of HPV16E7 negative normal cervical squamous epithelium, CIN and cervical squamous cell carcinoma, and the correlation coefficients were -0.452, -0.485 and -0.444 (P0.01), respectively.
3. the results of cell growth curve drawn by MTT colorimetric assay showed that the growth rate of CaSki-DRR1 cells was significantly slower than that of CaSki-vect cells (P0.01), and the doubling time was significantly prolonged (P0.01). The results of the formation of clones in the flat panel showed that the number of CaSki-DRR1 cells was significantly lower than that of CaSki-vect cells (P0.01), and the clone volume was obvious. The results of cell cycle distribution detected by flow cytometry showed that compared with CaSki-vect cells, the G1 phase block of CaSki-DRR1 cells (P0.01), but the percentage of S and G2/M cells decreased significantly (P0.01). 4. The apoptotic results of Annexin V-FITC/PI combined flow cytometry showed that the apoptotic average of CaSki-DRR1 cells was obvious. The percentage of apoptotic cells was higher than that of CaSki-vect cells (P0.01): 12.116% and 0.016%. respectively.
conclusion
1. there was a negative correlation between E7 expression and DRRl expression in HPV16E7 positive and negative cervical cancer cell lines and cervical tissues.
2. stable expression of DRR1 can significantly inhibit the proliferation of HPV16E7P cervical cancer CaSki cells and promote its apoptosis.
【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

【参考文献】