本文關(guān)鍵詞：MUC16在HPV16相關(guān)宮頸癌發(fā)生與演進(jìn)中的作用　出處：《新鄉(xiāng)醫(yī)學(xué)院》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人乳頭瘤病毒16型 宮頸鱗癌 E6基因 粘蛋白抗原16 凋亡 遷移 侵襲

【摘要】：背景高危型人乳頭瘤病毒(high risk-human papillomaviruses, HR-HPVs)(如HPV16等)病毒癌基因E6的持續(xù)表達(dá)是宮頸癌最重要的致病因素。本課題組設(shè)計(jì)并篩選了靶向HPV16E6且沉默效應(yīng)均達(dá)95%以上的短發(fā)夾RNA(short hairpin RNA, shRNA),經(jīng)Agilent人基因組表達(dá)譜芯片分析,發(fā)現(xiàn)沉默E6表達(dá)后,粘蛋白抗原16(mucin antigen16, MUC16)基因表達(dá)下調(diào)(2-5倍)。MUC16是否是HPV16E6的直接作用靶點(diǎn)及調(diào)控機(jī)制尚不清楚。因此,深入研究E6與MUC16的關(guān)系,對(duì)探索HPV16相關(guān)腫瘤新的治療方法具有重要意義。 目的以HPV16E6陽性和陰性的宮頸癌細(xì)胞系、正常宮頸鱗狀上皮、宮頸上皮內(nèi)瘤變(cervical intraepithelial neoplasias, CIN)和宮頸鱗癌組織為研究對(duì)象,從細(xì)胞和組織水平分析HPV16E6和MUC16的關(guān)系,試圖闡明MUC16在HPV16相關(guān)宮頸癌發(fā)生與演進(jìn)中的作用,豐富HPV16E6的致癌機(jī)制。 方法 1.以HPV16陽性(CaSku SiHa細(xì)胞)和陰性(C-33A細(xì)胞)的宮頸癌細(xì)胞為研究對(duì)象,采用Western blotting方法檢測HPV16E6和MUC16的表達(dá),并分析二者的關(guān)系。 2.以HPV16E6陽性和陰性的正常宮頸鱗狀上皮、CIN和宮頸鱗癌組織為研究對(duì)象,采用免疫組化方法檢測HPV16E6和MUC16的表達(dá),并分析二者的關(guān)系。 3.利用生物信息學(xué)技術(shù)設(shè)計(jì)靶向MUC16mRNA的shRNA序列,采用分子克隆方法構(gòu)建含靶向MUC16的shRNA的pGenesil-1真核表達(dá)重組質(zhì)粒(命名為pGenesil-l-shRNA-MUC16)和含無關(guān)序列的對(duì)照質(zhì)粒(命名為pGenesil-1-shRNA-vect)。 4.通過脂質(zhì)體介導(dǎo)將上述質(zhì)粒分別轉(zhuǎn)染入HPV16E6陽性的宮頸癌CaSki細(xì)胞(分別命名為CaSki-shRNA-MUC16和CaSki-shRNA-vect細(xì)胞)。G418篩選抗性細(xì)胞。通過RT-PCR和Western blotting方法鑒定MUC16沉默效應(yīng)。 5.通過Annexin V-FITC/PI結(jié)合流式細(xì)胞術(shù)、Caspase-3活性測定、Transwell遷移實(shí)驗(yàn)和Transwell侵襲實(shí)驗(yàn),檢測靶向沉默MUC16表達(dá)后HPV16E6陽性宮頸癌CaSki細(xì)胞凋亡和遷移侵襲能力的變化。 結(jié)果 1. Western blotting結(jié)果顯示：①C-33A細(xì)胞中HPV16E6無表達(dá),CaSki細(xì)胞和SiHa細(xì)胞中可見HPV16E6表達(dá)；②C-33A細(xì)胞中MUC16的表達(dá)顯著低于CaSki細(xì)胞和SiHa細(xì)胞(P0.01),CaSki細(xì)胞和SiHa細(xì)胞中的MUC16的表達(dá)差異無顯著性(P0.05)。 2.免疫組化結(jié)果顯示：(DHPV16E6陽性的正常宮頸鱗狀上皮、CIN和宮頸鱗癌組織中MUC16的表達(dá)均顯著高于HPV16E6陰性的正常宮頸鱗狀上皮、CIN和宮頸鱗癌組織(相關(guān)系數(shù)分別為0.531、0.629和0.452,P0.01)。 3.含MUC16shRNA真核表達(dá)重組質(zhì)粒的鑒定 結(jié)果顯示：目的片段插入位點(diǎn)和方向正確,與設(shè)計(jì)一致。 4.靶向MUC16mRNA的shRNA對(duì)宮頸癌CaSki細(xì)胞中MUC16表達(dá)的沉默效應(yīng) RT-PCR結(jié)果顯示：與CaSki細(xì)胞和CaSki-shRNA-vect細(xì)胞相比,CaSki-shRNA-MUC16細(xì)胞中MUC16mRNA的表達(dá)顯著降低(P0.01),沉默效率為：93.137%。而CaSki細(xì)胞和CaSki-shRNA-vect細(xì)胞中MUC16mRNA的表達(dá)差異無顯著性(P0.05)。 Western blotting結(jié)果顯示：與CaSki細(xì)胞和CaSki-shRNA-vect細(xì)胞相比,CaSki-shRNA-MUC16細(xì)胞中MUC16蛋白的表達(dá)顯著降低(P0.01),沉默效率為：93.913%。而CaSki細(xì)胞和CaSki-shRNA-vect細(xì)胞中MUC16蛋白的表達(dá)差異無顯著性(P0.05)。 鑒于以上研究結(jié)果,后續(xù)實(shí)驗(yàn)中均以CaSki-shRNA-MUC16細(xì)胞為實(shí)驗(yàn)組,以CaSki-shRNA-vect細(xì)胞為對(duì)照組。 5.靶向沉默MUC16表達(dá)對(duì)HPV16E6陽性宮頸癌CaSki細(xì)胞凋亡和侵襲的影響：①Annexin V-FITC/PI結(jié)合流式細(xì)胞術(shù)檢測細(xì)胞凋亡結(jié)果顯示：CaSki-shRNA-MUC16細(xì)胞凋亡均數(shù)明顯高于CaSki-shRNA-vect細(xì)胞(P0.01),凋亡細(xì)胞百分比分別為：5.598%和0.150%；②Caspase-3活性測定結(jié)果顯示：與CaSki-shRNA-vect細(xì)胞相比,CaSki-shRNA-MUC16細(xì)胞中Caspase-3活性顯著增加(P0.01)；③Transwell遷移和侵襲實(shí)驗(yàn)的結(jié)果均顯示：與CaSki-shRNA-vect細(xì)胞相比,CaSki-shRNA-MUC16細(xì)胞穿過小室底膜的細(xì)胞數(shù)均顯著減少(P0.01)。 結(jié)論 1.在HPV16E6陽性和陰性宮頸癌細(xì)胞系和宮頸組織中MUC16與E6表達(dá)呈正相關(guān)。 2.靶向沉默MUC16的表達(dá)可促進(jìn)HPV16E6陽性宮頸癌CaSki細(xì)胞的凋亡,并抑制其遷移侵襲能力。
[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.

【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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