Study On The Association Between Transcriptional Coactivator HADA3 And Human Papillomavirus Oncoprotein E6

Human cervical carcinoma represents the second most common cancer in female worldwide and it is regarded to be closely associated with infection of high-risk human papillomavirus(HPV).The malignant transforming ability of high-risk HPV is mostly dependent on the activity of crucial viral oncoprotein E6.E6 protein binds to the key tumor suppressor protein P53,targets it for ubiquitination and proteosomal degradation, finally brings on immortalization and malignant transformation of human epithelial cells.And E6-associated protein(E6AP) is essential during the progression of carcinogenesis induced by virus oncoprotein.Although E6-induced degradation of P53 has been recognized as a crucial event,it is not enough to elucidate completely the mechanisms by which oncoprotein E6 functions.Recent studies have shown that the mutated E6 that is incapable of interacting with and targeting P53 for degradation still has the ability of leading to abnormal cellular transformation.There may be an additional mechanism that is independent of direct P53 degradation in the HPV infected cells.Recent studies have implicated a number of additional cellular targets of E6,such as E6BP,hDlg,Bak and so on,but the detailed mechanism is still unclear.Thus,it is likely that it's a complicated network to concurrently alter multiple cellular controls of a cell,leading to malignant transformation.Thus,the devotion of investigation at the field of transcriptional coactivator may make sense of the mechanisms by which oncoprotein E6 functions.The hADA3 protein is the human homologue of the yeast transcriptional coactivator yADA3(yeast alteration/deficiency in activation).The previous studies in yeast demonstrated that ADA3,as a crucial component of coactivator,is believed to be crucial for a variety of transcriptional pathways.Although little is known about hADA3 function in mammalian systems,it's very likely that evolutionarily conserved hADA3, similar to its yeast counterpart,would act as a coactivator in multiple mammalian transcriptional pathways.For example,the studies showed that hADA3 appeared to be required for P53-mediated transcriptional activation of a variety of target promoters and P53-mediated G1 cell cycle arrest.Another research showed that hADA3 is a crucial coactivator for transactivation function of estrogen receptor.As we all know,P53 and estrogen receptor are both crucial transcriptional factors.Thus,it is very likely that hADA3 may be involved in the pathway of E6-induced cellular malignant transformation that is independent of direct P53 degradation.In addition,it's unknown whether E6AP is involved in hADA3 degradation.In this study,we investigated the association between viral oncoprotein E6 and cellular protein hADA3,as well as ubiquitin ligase E6AP and hADA3,using RNA interference technique.As a novel E6 target protein,given the likely role of the evolutionarily conserved hADA3 in multiple transcriptional pathways,inhibition of hADA3 by E6 may perturb numerous cellular pathways during HPV oncogenesis. PARTâ… sequence-targeting siRNA induces gene silencing of HPV16 E6 and E6APObjective:To explore whether gene silencing of HPV16 E6 and E6AP can be induced respectively by sequence-targeting siRNA in SiHa cells.Methods:The siRNAs targeting HPV 16 E6 and E6AP were designed,transcripted and synthesized in vitro,transfected into cervical cancer cell line SiHa.Then the mRNA of E6 and E6AP expression were observed to choose the optimal siRNA.Results:E6 mRNA was reduced by E6-C siRNA,optimal concentration of which was 30uM and optimal transfection time was 48 hours.E6AP mRNA was reduced by E6AP-B siRNA,optimal concentration of which was 40uM and optimal transfection time was 48 hours.Conclusions:1,The HPV 16 E6 sequence-targeting siRNA,which was transcripted and synthesized in vitro,could effectively induce gene silencing of viral genes E6 in SiHa cells.2,The E6AP sequence-targeting siRNA,which was transcripted and synthesized in vitro,could effectively induce gene silencing of E6AP in SiHa cells. PARTâ…¡HPV16 E6-induced and E6AP-dependent inhibition of the transcriptional coactivator hADA3 in human cervical carcinoma cellsObjective:To determine whether hADA3 is involved in the pathway of E6-induced cellular malignant transformation and to determine involvement of E6AP in hADA3 inhibition.Methods:1,We detected E6 and hADA3 expression as well as cellular proliferation and apoptosis in SiHa cells after E6 siRNA was transfected into the cells.2,We detected E6AP and hADA3 expression as well as cellular proliferation and apoptosis in SiHa cells after E6AP siRNA was transfected into the cells.Results:1,E6 mRNA and protein expression were simultaneously reduced by E6 siRNA. Knockdown of E6 gene can retrieve hADA3 expression and at least partly reduce cell proliferation and enhance cell apoptosis.2,E6AP mRNA and protein expression were simultaneously reduced by E6AP siRNA.The expression of hADA3 protein was substantially increased in SiHa cells treated by E6AP siRNA.Meanwhile,cellular proliferation was significantly inhibited and apoptotic rate was significantly increased.Conclusions:1,HPV 16 E6 inhibited expression of transcriptional coactivator hADA3 in human cervical carcinoma cells. 2,E6AP-dependent ubiquitination and proteosomal degradation is involved in the process of hADA3 inhibition in SiHa cells.