Study On The Relationship Between PKR/EIF2α Signal Transduction Passage And Cervical Carcinoma

ObjectiveTo identify the effects to the expressions and location of PKR,p-PKR,p-EIF2a in PKR/EIF2a signal transduction passage by HPV (16/18) E6 protein in every grades of cervical lesions tissue,the relations between the difference of their expressions and grades of cervical lesions, their effects on the prognosis of cervical carcinoma paitients; Design and construct two lentiviral vectors which contain shRNA interfering sequence aiming at the target of HPV18E6 oncogene and NC sequence (HPV18E6-RNAi-LV,NC-GFP-LV); Based on the transduction with HPV18E6-RNAi-LV and NC-GFP-LV into HeLa cell to interfere the expression of HPV18E6 oncogene and NC sequence, the target molecule which may regulate the activation of PKR/EIF2a signal transduction passage is determined, which can provide the basis of exploring methods to activate effectly PKR/EIF2a signal transduction passage in next experiments.MethodsPart 1:The expressions and location of HPV(16/18) E6, PKR, p-PKR, p-EIF2a in human cervical carcinoma tissue of 63 cases, cervical intraepithelial neoplasia (CINⅠ～CINⅢ) of 114 cases and normal cervical epithelium of 15 cases are detected by immunohistochemical technique, analyze the effects on the activation of PKR/EIF2a signal transduction passage by HPV(16/18)E6 protein, their roles in generation and progression of cervical carcinoma,their effects to prognosis of cervical carcinoma patients.Part 2:Based on the information of target gene,recombination plasmid vector which contains shRNA is constructed and apprased;Extract and purify recombination plasmid;Recombination plasmid and packaging plasmid co-transduce the 293T cell to package the lentiviral vector,collects the lentiviral vector solution,concentrates and purifies it; The lentiviral vector solution after concentrating and purifying transducer 293T cellto determine the concentration of the lentiviral vector solution.Part 3:Before and after the transduction into HeLa cell with HPV18E6-RNAi-LV and NC-GFP-LV, the expressions of mRNA and protein (including phosphating pattern) of HPV18E6,PKR,EIF2a,NF-κBp65,STAT1,MAPK,JNK1 are measured with RT-PCR and Western Blot, the target molecule which may regulate the activation of PKR/EIF2a signal transduction passage is determined; Before and after the transduction, the activation and invading ability of HeLa cell are determined with MTT and Transwell cell methods.ResultsPart 11.HPV (16/18) E6 protein mainly locates in nuclei.PKR,p-PKR,p-EIF2aprotein mainly locate in cytoplasm;2.With the rise of the grades of cervical lesions,the positive-expression rate of HPV (16/18) E6 oncoprotein and PKR increase and correlated significantly with the grades of cervical lesions (p< 0.05); With the rise of the grades of cervical lesions,the positive-expression rates of p-PKR,p-EIF2a increase firstly,then descend; In cervical carcinoma group,the positive-expression rate of PKR is much higher than that of p-PKR (p<0.01);3.The development and progression of later clinical stages cervical carcinoma is quicker than that of earlier clinical stages cervical carcinoma (p<0.01), the development and progression of cervical carcinoma which has positive-expression of HPV (16/18) E6 oncoprotein is quicker than that of negative-expression of HPV (16/18) E6 oncoprotein (p< 0.05). the development and progression of cervical carcinoma which has negative-expression of p-PKR,p:EIF2a is quicker than that of positive-expression of p-PKR,p-EIF2a (p<0.05, p<0.05).Part 21.Indexing shRNA of interfering sequence aiming at the target of HPV18E6 oncogene with human genes in BLASTER,there is no homogeneous.The second constructure of the transcripts of interfering sequence aiming at the target of HPV18E6 oncogene and NC sequence can conform short hairpin;2. The sequencing of positive clones of containing shRNA interfering sequence aiming at the target of HPV18E6 oncogene and NC sequence prove that the positive clones are the lentiviral vectors;3. After packaging and collecting and concentrating the lentiviral vector successfully, the concentration of lentiviral vector solution is determined, the concentration of HPV18E6-RNAi-LV is 3E+8 TU/ml,the concentration of NC-GFP-LV is 2E+9 TU/ml.Part 3 1.The expressions of HPV18E6,NF-κBp65 mRNA in HPV18E6-RNAi-LV group are lower than those in NC-GFP-LV group and control group (p<0.05, p <0.05); The expression of PKR mRNA in HPV18E6-RNAi-LV group are higher than that in NC-GFP-LV group and control group (p<0.05); The expression of EIF2a,STAT1,MAPK,JNK1 mRNA in every group are no significant difference (p>0.05);2.The expressions of HPV18E6,NF-κBp65 protein in HPV18E6-RNAi-LV group are lower than those in NC-GFP-LV group and control group (p<0.05, p <0.05); The expression of p-STAT1,PKR protein in HPV18E6-RNAi-LV group are higher than those in NC-GFP-LV group and control group (p<0.05, p <0.05); The expression of p-PKR,p-EIF2a protein in HPV18E6-RNAi-LV group are higher than those in NC-GFP-LV group and control group (p<0.01, p <0.01); The expression of STAT1,MAPK,p-MAPK,JNK1,p-JNK1 protein in every group are no significant difference (p>0.05);3. The restraining rate of cell activation and aggression are 39.6% and 56.2% in HPV18E6-RNAi-LV group, which are higher than those in NC-GFP-LV group and control group(p<0.05, p<0.05), there are no significant difference between NC-GFP-LV group and control group (p>0.05)Conclusions1.HPV(16/18) E6 can dephosphorylate p-PKR,p-EIF2a to restrain activation of PKR/EIF2a signal transduction passage,prevent apoptosis of abnormal cells, promote the development and progression of cervical lesions; Effective activation of PKR/EIF2a signal transduction passage can restrain the development and progression of cervical carcinoma,improve the prognosis of cervical carcinoma paitients;2. The lentiviral vectors of HPV18E6-RNAi-LV and NC-GFP-LV are constructed successfully,which provides practical tool for later study;3,That reduce the expression of HPV18E6 oncogene can increase the expression of PKR and recover the activation of PKR/EIF2a signal transduction passage, restrain the activation and aggression of HeLa cell and promote apoptosis of HeLa cell which prove that HPV (16/18) E6 protein can inactivate PKR/EIF2a signal transduction passage; It is possible that ruclear factor NF-κBp65 becomes the candidate to regulate the activation of PKR/EIF2a signal transduction passage,which provides experimental basis for exploring the methods of activating effectively PKR/EIF2a signal transduction passage later.