Influencing Factors And Mechanism Of Autophagic Degradation Of Tumor Suppressor Gene Rap1GAP

Objective: Degradation of Rap1 GAP cells by the molecular autophagy machinery.Cervical cancer is one of the important cancers threatening women’s life health at present,and high-risk human papillomavirus often leads to a high incidence of cancer.Accumulating data suggest that the impact of autophagy on tumors is also increasingly highlighted.Rap1GAP is a negative regulator of oncogene Rap1,and the main role is to convert Rap1 that is hyperfunctional when bound to GTP to Rap1 that is less active when bound to GDP,thereby inhibiting the activity of oncogene Rap1.Recently,it has been reported that Rap1 GAP can inhibit tumor cell growth,and decreased expression of Rap1 GAP has been found in gastric cancer and head and neck cancer,but the mechanism is still undefined.Rap1 GAP as a tumor suppressor gene plays an important role in the process of tumor occurrence and development.Our group previously showed that Rap1 GAP can be reduced by knockdown of E6 AP in HPV positive He La,thus starting to explore the pathway of Rap1 GAP degradation.Therefore,this trial will further explore the effect and mechanism of Rap1 GAP degradation pathway,and provide a new way and basis for the treatment and postoperative rehabilitation of cervical cancer.Methods:1.Detect Rap1 GAP protein half-life using the protein synthesis inhibitor CHX to inhibit protein synthesis.2.To calculate the ratio of LC3 Ⅱ / Ⅰ,the relative expression levels of p62 and the expression levels of LC3 Ⅱ and LC3 Ⅰ in He La and C33 A cells after knockdown of E6 AP were detected by lipofection assay.3.The relative expression levels of PI3 K and the phosphorylation levels of Akt in He La and C33 A cells after knockdown of E6 AP were determined by Western blot analysis.4.Screen the maximum concentration at which chloroquine does not affect cell viability by MTT method.5.The changes of relative expression levels of p62 in 293 T cells after autophagy inhibition by chloroquine were detected by immunoblotting assay,the expression levels of LC3 Ⅱ and LC3 Ⅰ,and the ratio of LC3 Ⅱ / Ⅰ was calculated.6.Change values of relative expression levels of Rap1 GAP in 293 T cells after autophagy inhibition by chloroquine were detected by immunoblotting method.Result:1.Detection of Rap1 GAP half-lifeUpon inhibition of cellular protein synthesis,the intracellular Rap1 GAP content gradually decreased over time.By simple linear regression analysis,Rap1 GAP protein half-life in He La cells was 50.78 h;Rap1GAP protein half-life in C33 A cells was 57.4 h.2.Knockdown of E6 AP activates the autophagic degradation pathway in HPV positive cervical cancer cellsAfter knockdown of E6 AP,the relative p62 expression was reduced to 0.4988 ±0.08509 of that in the non knockdown group(P < 0.05).The ratio of LC3 Ⅱ / Ⅰ,an autophagosomal membrane protein,was significantly increased,which was 1.415 ±0.1522-fold of that in the non knockdown group(P < 0.05).After knockdown of E6 AP,the ratio of autophagosomal membrane protein LC3 Ⅱ/ Ⅰ in C33 A cells was 1.042 ± 0.0518-fold of that in control cells(P > 0.05);The relative expression of p62 was 1.14 ± 0.1645-fold of that in the control group(P > 0.05).Downregulation of E6 AP can induce protein autophagy in HPV positive cells but not in HPV negative cells.3.Knockdown of E6 AP regulates PI3 K / Akt signaling to affect autophagy in HPV positive cervical cancer cellsAfter knockdown of E6 AP,the relative expression of PI3 K in He La cells was 1.028± 0.05385-fold of that in control cells(P > 0.05);The ratio of p Akt / Akt was reduced to0.6285 ± 0.0677-fold of that in the control group(P < 0.05).After knockdown of E6 AP,the relative PI3 K expression in C33 A cells was 1.111 ±0.0789-fold of that in control cells(P > 0.05);The ratio of p Akt / Akt was 1.039 ± 0.7659-fold of control(P > 0.05).Downregulation of E6 AP can induce autophagy in HPV positive cervical cancer cells through the PI3 K / Akt signaling pathway4.The maximum concentration at which chloroquine does not affect cell viability is20 μmol / L5.Effects on Rap1 GAP after inhibition of autophagy in 293 T cellsAfter 20 μmol / L chloroquine treatment of 293 T cells,the relative expression of LC3 Ⅱ/Ⅰ in the cells was significantly increased,which was 3.216 ± 0.6865-fold of that in the untreated group(P < 0.05);P62 increased to 1.212±0.02515-fold of that in the untreated group(P < 0.05).Inhibition of autophagy in 293 T cells by 20 μmol / L chloroquine decreased Rap1 GAP in the cells to 0.6396±0.018-fold of that in the untreated group(P < 0.05).Conclusion:In HR-HPV cells,the decline of E6 AP can regulate the autophagic degradation of Rap1 GAP through the PI3K/Akt pathway.