摘要
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）／c—Jun氨基末端激酶（JNκ）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、Westernblot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MTr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、PκR基因的核酸及蛋白表达水平，并影响磷酸化蛋白p-STAT1、P—PκR、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PκR表达，并使p-STAT1、p-PκR、P-eIF2α去磷酸化的方式抑制PκR／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。
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 （ JNκ）, 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 patern） of HPV18E6, STAT1, PκR, eIF2ct, 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 MTI＂ among every groups. Results The expression of HPV18E6 oncogene can affect the expression level of mRNA and protein of NF-κBp65 and PκR genes, also affect phosphating levels of phosphating protein p-STAT1, p-PκR 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 PκR but dephosphorylates p-STAT1, p- PKR 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/ JNκ are not clear.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2011年第7期597-602,共6页
Chinese Journal of Microbiology and Immunology
