CAJ | 학술논문

目的探讨人乳头状瘤病毒18型E6蛋白(HPV18E6)与信号转导和转录激活因子1(STAT1)、蛋白激酶R(PKR)/真核细胞翻译启始因子2α(eIF2α)、核因子-κBp65(NF-κBp65)、丝裂酶原激活的蛋白激酶(MAPK)/cJun氨基末端激酶(JNK)信号转导的关系以及可能的分子机制.方法构建靶向HPV18E6癌基因及无关序列(NC序列)的短发夹结构RNA(shRNA)干扰序列的慢病毒载体(HPV18E6-RNAi-LV,NC-GFP-LV),转染宫颈癌HeLa细胞,在沉默HPV18E6癌基因表达的基础上,以RT-PCR、Western blot法分别在核酸、蛋白水平(包括磷酸化型)检测各组HPV18E6、STAT1、PKR、eIF2α、NF-κBp65、MAPK、JNK的表达,以transwell侵袭试验及MTT法检测各组HeLa细胞侵袭能力及对卡铂敏感性的差异.结果 HPV18E6癌基因的表达水平能影响NF-κBp65、PKR基因的核酸及蛋白表达水平,并影响磷酸化蛋白p-STAT1、p-PKR、p-eIF2α的磷酸化水平;HPV18E6-RNAi-LV转染组细胞侵袭抑制率及对卡铂的敏感程度明显高于其他组(P＜0.05或P＜0.01).结论 HPV18E6通过降低PKR表达,并使p-STAT1、p-PKR、p-eIF2α去磷酸化的方式抑制PKR/eIF2α信号传导通路激活,维持HeLa细胞增殖活性及侵袭能力,也可抑制凋亡.HPV18E6与MAPK/JNK信号转导关系尚不明确.
목적 탐토인유두상류병독18형E6단백(HPV18E6)여신호전도화전록격활인자1(STAT1)、단백격매R(PKR)/진핵세포번역계시인자2α(eIF2α)、핵인자-κBp65(NF-κBp65)、사렬매원격활적단백격매(MAPK)/cJun안기말단격매(JNK)신호전도적관계이급가능적분자궤제.방법 구건파향HPV18E6암기인급무관서렬(NC서렬)적단발협결구RNA(shRNA)간우서렬적만병독재체(HPV18E6-RNAi-LV,NC-GFP-LV),전염궁경암HeLa세포,재침묵HPV18E6암기인표체적기출상,이RT-PCR、Western blot법분별재핵산、단백수평(포괄린산화형)검측각조HPV18E6、STAT1、PKR、eIF2α、NF-κBp65、MAPK、JNK적표체,이transwell침습시험급MTT법검측각조HeLa세포침습능력급대잡박민감성적차이.결과 HPV18E6암기인적표체수평능영향NF-κBp65、PKR기인적핵산급단백표체수평,병영향린산화단백p-STAT1、p-PKR、p-eIF2α적린산화수평;HPV18E6-RNAi-LV전염조세포침습억제솔급대잡박적민감정도명현고우기타조(P＜0.05혹P＜0.01).결론 HPV18E6통과강저PKR표체,병사p-STAT1、p-PKR、p-eIF2α거린산화적방식억제PKR/eIF2α신호전도통로격활,유지HeLa세포증식활성급침습능력,야가억제조망.HPV18E6여MAPK/JNK신호전도관계상불명학.
Objective To explore the relationship of signal transduction among human papillomavirus 18 E6 oncoprotein (HPV18E6), signal transducers and activators of transcription 1 (STAT1), protein kinase R( PKR )/α subunit of eukaryotic initiation factor 2 ( eIF2α ), nuclear factor-kappa Bp65 ( NF-κBp65 ), mitogen-activated protein kinase( MAPK)/c-Jun N-terminal kinase(JNK) ,and possible molecular mechanism. Methods Construct two lentiviral vectors which contain shRNA interfering sequence aiming at the targets 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 expressions of mRNA and protein( including phosphating patem)of HPV18E6, STATI, PKR, eIF2α, NF-κBp65, MAPK, JNK are measured with RT-PCR and Western blot, the difference of proliferation and sensitivity to carboplatin of HeLa cell are determined with Transwell cell methods and MTT among every groups. Results The expression of HPV18E6 oncogene can affect the expression level of mRNA and protein of NF-κBp65 and PKR genes, also affect phosphating levels of phosphating protein p-STAT1, p-PKR and p-eIF2α;the restraining rates of proliferation and sensitivity to carboplatin of HeLa cell are higher in HPV18E6-RNAi-LV group than the other groups( P＜0. 05 or P＜0.01 ). Conclusion HPV18E6 oncoprotein not only reduces the expression of PKR but dephosphorylates p-STAT1, pPKR and p-eIF2α to restrain activation of PKR/eIF2α signal transduction passage, maintain the proliferation and invading ability of HeLa cell and restrain apoptosis. The signal transduction among HPV18E6, MAPK/JNK are not clear.