The Construction Of Adenovirus-mediates RNA Interference System And Combing With TNF-α To Induce The Apoptosis Of Hepatocarcinoma Cell

Object：Some previous studies have already verified that the Rho protein familyare highly-expressed in tumor cells and it also has close relationship with the presenceof the tumor, participated in the process of transferring and infiltrating. While theadenovirus-mediated RNA interference technology has a lot of advantage over theplasmid-mediated RNA interference technology, such as it can infect wide range ofhost cells, it processes high efficiency and stabilization,which can apply into animalexperiment more easily. The plasmid-mediated RNA interference technology hasovercome the disadvantage of the plasmid-mediated RNA interference technology,such as low transfection rate and unstabilization in the host cells, this technology willlead a new trend in the research of gene function and gene treatment. So, the purposeof this experiment is to construct the adenovirus-mediated RNA interference systemof RhoA. After that, we combine it with TNF-α to observe the apoptosis of HepG2cells. On one hand, this research can provide a platform for the further research of theRhoA gene. On the other hand, this research will help to find out the function of theRhoA gene in the future and combining with TNF-α can provide a new method for thetreatment of tumor.Method: Based on the already constructed interference plasmid, we haveconstructed the adenovirus-mediated RNA interference system.We use the well-constructed adenovirus to infect the HepG2cells, and test the effect of inhibitionof RhoA gene expression though RT-PCR and Western blot assay. And then, we useadenovirus-mediated RNA interference system, TNF-α, and adenovirus-mediatedRNA interference system combining with TNF-α respectively to affect the HepG2cells and then observe the proliferation of HepG2cells though MTT essay andobserve the apoptosis of HepG2cells by TUNEL reaction.Result: Though the enzyme cut assay and other test, we have constructed theadenovirus-mediated RNA interference system of RhoA successfully. Western blotessay shows that, though adenovirus-mediated RNA interference, the expression ofRhoA protein is reduced by76.48%. Semi-quantitative RT-PCR assay shows thatmRNA transcription rate declined by74.46%.All these results indicate thatadenovirus-mediated RNA can inhibit the expression of RhoA gene obviously. MTTeassy indicates that the adenovirus combining with TNF-α can effectively inhibit thegrowth and proliferation of HepG2cells.TUNEL essay shows that the adenovirusitself and the adenovirus combining with TNF-α both can induce the apoptosis ofHepG2cells.Conclusion: This research has successfully constructed the adenovirus-mediatedRNA interference system of RhoA. RNA interference combining with TNF-α,canenhance the the apoptosis of HepG2cells effect. By conduct such research, on onehand we lay a good foundation for the tumor treatment. On the other hand weprovided a promising targeting site for screening the new cure for tumor.