| Purpose:construct the HPV16E6 over-expression adenovirus vector,transfect it into the cervical cancer Me180 cell,compare the invasion and migration ability among the Me180 cell HPV16E6 over-expression group,blank vector group and blank control group,as well as the expression of correlation factor E-cadherin and methylation status of its promoter,and study the influence of the HPV16E6 over-expression on the invasion and migration ability of cervical cancer Me180 cell and its mechanism.Method:1. Construct the HPV16E6 adenovirus:sub-clone the HPV16E6 gene to adenovirus shuttle plasmid,p HBAd-MCMV-GFP,judge it with double enzyme digestion of Eco R I and Bam H I,amplify it with PCR technique,and combine the product with p HBAd-MCMV-GFP to transform into the Escherichia coli,and check if the sequence is correct;get the HPV16E6 over-expression adenovirus vector plasmid and backbone plasmid p HBAd-BHG,amplify and purify the recombinant adenovirus in 293 A cell using the Lipofiter TM transfection reagent.2.Virus transfection effect verification : divide the over-expression HPV16E6 adenovirus-infected cervical cancer Me180 cell, blank adenovirus-infected cervical cancer Me180 cell and cervical cancer Me180 cell without adenovirus transfection into three groups:HPV16E6 over-expression group, blank vector group and blank control group.Infect the Me180 cell using the over-expression HPV16E6 gene adenovirus and blank adenovirus vector and observe the green fluorescence expression after 36 hours;observe the fluorescent effect and use the Western-Blot test to verify the HPV16E6 expression of the three groups to verify the transfection effect.3. Cell migration and invasion ability : detect the influence of over-expression HPV16E6 gene on cervical cancer Me180 cell migration and invasion ability using the Traswell migration and invasion test.4. Mechanism research:use the Western-Blot and RT-q PCR test to detect the E-cadherin and m RNA expression in the three groups,analyze the influencing mechanism of HPV16E6 gene on Me180 cell migration and invasion ability,and use the methylation-specific PCR to detect the E-cadherin methylation status in the three groups.Results:1. through the agarose gel electrophoresis test,it was confirmed that the shuttle vector with HPV16E6 sequence was constructed successfully; the fluorescence microscope images and Western blot showed that the over-expression HPV16E6 adenovirus had be successfully transfected to cervical cancer ME180 cell with high transfection efficiency. 2. In the Transwell migration and invasion experiment, after the over-expression HPV16E6 adenovirus was transfected to cervical cancer Me180 cells, the cell migration and invasion ability was greatly improved compared to the blank control group(p<0.05),while that of the blank vector group had no significant change compared to the blank control group(p>0.05).3. In the RT-q PCR and Western Blot experiment,the m RNA and protein of E-cadherin in the over-expression HPV16E6 group was significantly decreased compared to the blank control group(p<0.05),while that of the blank vector group had no significant change compared to the blank control group(p>0.05). 4. The methylation-specific PCR showed that through the rough quantitative- grey value estimate,the E-cadherin promoter region of the over-expression HPV16E6 group was in the complete methylation status with significantly higher methylation level than the blank control group(p<0.05), while that of the blank vector group had no significant difference from the blank control group(p>0.05).Conclusion:1. HPV16E6 gene can strengthen the cervical cancer cell line Me180 cell migration and invasion ability through down-regulating the expression of E-cadherin lead to promote the invasion and migration of tumor.2. In terms of the epigenetics,HPV16E6 gene can inhibit the E-cadherin expression through enhancing the methylation level of E-cadherin promoter region,which may become the breakthrough to reverse the cervical cancer invasion and migration. |
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