RNA干涉抑制宫颈癌 CaSki细胞株 HPV16 E6基因的研究

背景与目的：研究表明宫颈癌的发生发展与人乳头瘤病毒(human papilloma virus,HPV)E6、E7癌基因密切相关,于是人们采用核酶或HPV E6、E7反义寡核苷酸抑制其表达来治疗宫颈癌,虽然取得了一定的效果,但仍面临基因抑制效率低、维持时间短、工作量大、耗费多等问题。本研究采用最新的RNA干扰技术干扰宫颈癌CaSki细胞中HPV16 E6转录,以了解其能否特异性抑制HPV16 E6基因及其时效性如何。方法：设计合成针对HPV16 E6的荧光标记siRNA,借脂质体转染宫颈癌CaSki细胞,通过荧光照片计数荧光细胞占所有细胞的比例计算转染效率;测定转染后不同时间点的细胞凋亡率;RT-PCR测定HPV16 E6 mRNA变化,Western blot和流式细胞仪检测转染前后蛋白表达情况。结果：相差显微镜荧光照片显示细胞转染的效率为81％。HPV16 E6 siRNA转染细胞后24h、48h、5天凋亡率分别为7．7％、11．8％和37．4％,转染9天时凋亡率下降至12．6％。RT-PCR扩增结果显示,细胞转染前HPV16 E6 mRNA的量与siRNA阴性对照比较没有显著性差异,但转染后24h、48h、5天和9天分别减少了77％、83％、59％和41％;而作为内对照的β-actin mRNA在转染前后无变化。流式细胞仪定量测定HPV16 E6蛋白,结果显示转染后24h、48h和5天,蛋白表达抑制率分别为79．7％、80．4％和71．3％;9天时抑制率有下降,但仍可达57．4％。此结果与HPV16 E6 Western blot结果相吻合。以Lamin A/C作为内对照,不同的时间点Lamin A/C蛋白表达均无差异。结论：宫颈癌CaSki细胞中确实有RNA干扰现象存在,对外源性的HPV16病毒E6基因的干扰具有基因特异性和高效性。     

HPV16 E6、E7通过糖原合成酶激酶-3β通路影响宫颈鳞癌细胞生物学行为

目的:探讨人乳头瘤病毒16(HPV16)的致癌基因E6、E7对宫颈鳞癌细胞C33A生物学行为的影响及其机制。方法:构建HPV16 E6、E7及E6/E7重组质粒,流式细胞术检测转染前后细胞周期的变化;划痕实验和Transwell实验检测细胞迁移及侵袭能力;克隆形成实验检测细胞的增殖能力;qPCR和Western blot检测相关基因和蛋白表达水平。结果:HPV16 E7重组质粒可引起宫颈鳞癌细胞S期比例显著增加（P＜0.01）,HPV16 E6、E7和E6/E7重组质粒能显著增强宫颈鳞癌细胞C33A的迁移能力（P＜0.05）,同时细胞的克隆形成能力也显著增强（P＜0.05）。HPV16 E6重组质粒可显著上调细胞糖原合成酶激酶-3β(GSK3β) mRNA水平（P＜0.05）;HPV16 E6、E7重组质粒可在24 h内引起GSK3β磷酸化水平升高,同时调控下游靶基因CyclinD1和β-catenin在不同时间点的表达发生变化。结论:HPV16 E6、E7在较短时间（24 h内）可通过GSK3β蛋白磷酸化激活,发挥对GSK3β下游靶基因CyclinD1、β-catenin基因的调控作用,从而促进宫颈鳞癌细胞的增殖、迁移及侵袭。     

HPV18E6基因沉默对宫颈癌Hela细胞生长和凋亡的影响

目的：观察RNA干扰法沉默HPV18E6基因表达对宫颈癌Hela细胞牛长和凋亡的影响，探索宫颈癌基因治疗的新途径。方法：针对HPV18E6 mRNA序列合成一对60bp的编码siRNA的DNA模板和一对60bp的非特异性对照DNA模板，构建pSUPER—siRNA和pSUPER—com重组质粒，瞬时转染Hela细胞；采用RT—PCR法检测质粒转染后细胞HPV18E6基因表达的变化，用蛋白免疫印迹法检测转染后Hela细胞p53、p21、Bcl-2和Bax蛋白表达变化，以细胞计数法检测细胞生长情况，Hoechest／PI双荧光活细胞染色法检测细胞凋亡。结果：pSUPER—siRNA质粒转染能有效降低HPV18E6在mRNA水平的表达，转染后48小时，抑制效率达70％以上；转染后细胞053、p21和Bax蛋白表达显著增加，Bcl-2蛋白表达减少。RNA干扰法沉默HPV18E6基因表达后，Hela细胞增殖受到明显抑制，细胞凋亡率明显增加。结论：pSUPER—siRNA质粒转染可有效抑制HPV18E6在人宫颈癌Hela细胞中的表达，抑制Hela细胞生长并促进其凋亡。以HPV18E6为靶点的RNA干扰技术可望成为宫颈癌基因治疗的新途径。     

小干扰RNA抑制宫颈癌Hela细胞株HPV18 E6、E7基因的表达

目的:以人乳头瘤病毒(human papillomavirus,HPV)18型E6基因为靶点,研究小干扰RNA(small interference RNA,siRNA)对宫颈癌Hela细胞株HPV18基因组中恶性转化基因E6、E7的抑制作用及对细胞内P53蛋白表达的影响。方法:实验分细胞培养液阴性对照组(阴性对照组),无关序列siRNA对照组(无关序列对照组)及转染HPV18 E6-siRNA实验组(siRNA实验组)。设计并合成HPV18 E6-siRNA及无关序列siRNA,转染Hela细胞后,RT-PCR检测转染后48、120 h细胞内HPV18E6、E7mRNA的变化,Western blotting检测转染后48 h细胞内HPV18 E7和P53蛋白的变化。结果:siRNA转染Hela细胞的效率约为85%。siRNA转染后48 h,实验组细胞内HPV18E6、E7mRNA及E7蛋白含量降低,其含量分别为阴性对照组的33.33%、36.78%及33.84%;实验组细胞内P53蛋白含量增加,其含量为阴性对照组的2.194倍。siRNA转染后120 h,实验组细胞HPV18E6、E7mRNA含量恢复为阴性对照组的90.91%、101.60%。结论:HPV18 E6-siRNA体外能明显抑制宫颈癌Hela细胞HPV18E6、E7基因的表达,增加细胞内肿瘤抑制因子P53蛋白的水平。     

HPV16E7 siRNA表达载体抑制宫颈癌SiHa细胞E7基因的研究

目的：研究HPV16E7siRNA表达载体对宫颈癌SiHa细胞E7基因的抑制作用。方法：利用脂质体将HPV16E7 siRNA表达载体psiRNA-1、psiRNA-2、psiRNA-3及空载体psiRNA转染SiHa细胞,以荧光定量RT-PCR和流式细胞仪检测不同时间点E7 mRNA和蛋白的变化。结果：载体psiRNA-1、psiRNA-2和psiRNA-3均能抑制SiHa细胞E7基因 mRNA和蛋白的表达,其中载体psiRNA-1的抑制作用最强。在抗性克隆形成后1周和4周,对E7mR-NA和蛋白抑制率分别为92.15%、84.30%和65.69%、59.11%。而空载体对E7mR-NA和蛋白的表达均无明显抑制作用。结论：HPV16E7 siRNA表达载体能较长期地抑制宫颈癌SiHa细胞E7基因的表达。     

HPV18 E6E7 反义荧光真核表达载体的构建及其在宫颈癌HeLa 细胞中的表达

 目的 构建HPV18型E6E7反义荧光真核表达载体,并观察其对宫颈癌HeLa细胞中HPV18 E6和E7基因表达的影响,探索反义技术用于治疗临床HPV感染及宫颈癌的可能性。方法 以HPV18型全基因质粒为模板,PCR法扩增HPV18型E6E7区716bp片段,利用基因重组技术将目的片段反向插入荧光真核表达载体pEGFP-C1,EcoR I酶切并测序鉴定;采用脂转法将重组质粒pEGFP-HPV18 E6E7as（EGFP-18AS）转染宫颈癌HeLa细胞株,通过RT-PCR及western blot检测细胞中E6、E7 mRNA和蛋白的表达。结果 成功构建HPV18E6E7反义荧光真核表达载体EGFP-18AS,经脂质体转染HeLa细胞,48h后在荧光倒置显微镜下可见明显的绿色荧光,且细胞中E6、E7 mRNA及蛋白表达水平均明显下调。结论 反义荧光真核表达载体可以有效的抑制HPV18E6、E7癌基因的表达,为治疗HPV感染和宫颈癌提供了一种新的方法。     

化学合成siRNA对宫颈癌细胞SiHa中E6基因表达的抑制作用

背景与目的宫颈癌的发生发展与人乳头瘤病毒(human papilloma virus)HPV16 E6、E7癌基因密切相关,人们曾采用核酶或E6、E7反义寡核苷酸抑制其表达,然而,上述两种方法存在费时、费力、抑制效率不高和维持时间短的问题.本研究拟探讨化学合成siRNA对HPV16阳性SiHa细胞E6基因表达的抑制作用以及对靶细胞的影响.方法化学合成3条靶向HPV16 E6基因的siRNA,用脂质体法转染SiHa细胞,同时设立脂质体对照.采用半定量逆转录-聚合酶链反应(RT-PCR)检测siRNA作用后E6 mRNA水平的变化;应用四甲基偶氮唑蓝(MTT)比色法、流式细胞技术(FCM)、免疫组化以及透射电镜技术(TEM)分别对细胞增殖活性、细胞周期、p53蛋白水平以及细胞超微结构的改变进行检测分析.结果3条siRNA均能有效抑制E6 mRNA的转录表达,以siRNA1作用效果最为明显.MTT检测结果显示20、50和80 nmol/L终浓度转染后细胞增殖活性均有所下降,其中以50 nmol/L转染时增殖活性下降最明显.流式细胞术检测结果显示转染后G0/G1期细胞数占生长周期细胞数的比例增多,即表现为G1期阻滞,S期细胞的比例降低;免疫组化法检测对照组细胞P53蛋白灰度平均值为0.43±0.03,转染72 h后细胞p53蛋白灰度平均值为0.75±0.06,差异有显著性(P〈0.05);转染72 h后透射电镜下可见细胞细胞质浓缩,胞质中出现空泡.结论化学合成siRNA可有效沉默SiHa细胞E6基因的转录表达,进而导致细胞生物学行为的改变.     

RNA干涉宫颈癌细胞抑制HPV16 E6基因表达对FHIT基因表达的影响

目的:研究HPV16 E6基因被RNA干涉抑制后FHIT基因表达的变化,探讨两者在宫颈癌组织中的相关性。方法:合成针对HPV16 E6基因的特异性si RNA,脂质体介导转染宫颈癌CaSki细胞,RT-PCR测定转染前后HPV16E6、FHIT基因mRNA表达;Western blot和流式细胞仪检测转染前后HPV16E6、FHIT蛋白表达情况。结果:将HPV16 E6 si RNA转染CaSki细胞后48h,HPV16E6mRNA表达水平较转染前降低了80.3%,P<0.01;FHIT mRNA的表达水平较转染前升高了41.7%,P<0.01。转染前后HPV16E6蛋白表达水平分别为26.7±3.3和8.2±1.1,t=14.224,P=0.005;FHIT蛋白表达水平分别为73.5±5.6和109.9±5.1,t=22.018,P=0.002。结论:抑制HPV16 E6基因的表达可使宫颈癌组织中FHIT基因的表达增加。     

靶向HPV16-E 6的siRNA对宫颈癌细胞的影响

目的：构建并筛选出HPV16-E6基因特异的最有效的小干扰RNA表达载体，观察其对宫颈癌细胞中HPV16-E6基因表达的长期影响，探讨巧基因在宫颈癌发生过程中的分子作用机制，为临床HPV感染及宫颈癌治疗探索新方法。方法：应用Fugene 6为转染试剂，将HPV16-E6 siRNA转染入CaSki细胞，Western blot方法检测HPV16-E6的表达；MTT分析检测HPV16-E6 siRNA对细胞增殖活性的影响；流式细胞术检测HPV16-E6 siRNA对细胞周期和细胞凋亡的影响。结果：siRNA抑制宫颈癌CaSki细胞中HPV16-E6的表达；siRNA抑制宫颈癌CaSki细胞增殖；siRNA抑制宫颈癌CaSki细胞的细胞周期行进，诱导CaSki细胞凋亡；结论：在CaSki细胞中，靶向HPV16-E6的siRNA能够有效抑制细胞增殖，诱导细胞周期阻滞于G0／G1期，并诱导细胞凋亡。     

HPV16编码蛋白与宫颈癌细胞内CALCA和TFPI-2表达的关系

目的：研究人乳头瘤病毒16（HPV16）编码蛋白的表达与宫颈癌细胞内CALCA和TFPI-2基因表达水平的关系,探讨依赖于HPV感染的宫颈癌发病机制。方法：设计与合成HPV16编码基因E6和E7序列特异性短发夹RNA（shRNA）寡聚核苷酸片段,构建表达shRNA的pRNAi载体,其中携带绿色荧光蛋白（GFP）报告基因,以此感染HPV16阳性的SiHa宫颈癌细胞,采用荧光成像和实时荧光定量PCR（qPCR）分析等方法,评价载体转染效率、抑制效率及候选基因转录表达水平的变化。结果：在激光共聚焦显微镜下,观察到释放绿色荧光的细胞群,转染成功;根据定量qPCR分析,HPV16编码基因E6和E7的mRNA表达水平明显下降,shRNA表达载体转染产生抑制效率;CDK4和BCL-2 mRNA表达水平明显下降,推测抑制HPV基因转录后,可能抑制宫颈癌细胞生长,引起细胞周期停滞;抑制E7基因表达后,CALCA和TFPI-2 mRNA表达水平明显上升,而抑制E6基因表达后,对上述基因表达水平的影响不明显。结论：HPV16编码蛋白质E7可能引起宫颈癌细胞内CALCA和TFPI-2基因表达下调,此为揭示依赖于HPV感染的宫颈癌发病机制提供了依据。     

New highly potent and specific E6 and E7 siRNAs for treatment of HPV16 positive cervical cancer

Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16-cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21(WAF1/CIP1), as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.     

HPV16型-E6、E7在食管鳞癌组织与非癌组织中的表达

背景与目的:越来越多的证据表明高危型人乳头状瘤病毒(humanpapillomavirus,HPV),特别是HPV16型与多种肿瘤发生、发展密切相关。本研究旨在分析人乳头状瘤病毒HPV16型E6、E7在食管正常组织、不典型增生组织和癌组织中的表达,以探讨HPV16型在食管鳞癌发生、发展中的生物学意义。方法:采用PicTureTM免疫组织化学方法对70例食管癌切除标本上、下切缘的正常粘膜上皮组织、43例不典型增生组织以及18例癌组织中的HPV16型E6、E7蛋白表达情况进行研究。结果:E6蛋白阳性率在正常食管粘膜上皮组织中为59.3%,在上皮不典型增生组织中为88.4%,在癌组织中为83.3%;E7蛋白在上述3种组织中的阳性率分别为62.1%、90.7%和88.9%。与正常食管粘膜上皮相比,上皮不典型增生组织和癌组织中E6、E7蛋白表达均有显著性差异(P<0.05)。HPV16型E6、E7蛋白在正常食管粘膜组织中同时表达即同步率为25.7%,而在上皮不典型增生组织和癌组织中二者同步率分别为88.3%和83.3%。结论:HPV16型E6、E7蛋白与食管鳞癌发生、发展密切相关,二者协同作用可能是食管鳞癌发生、发展的重要因素之一。     

HPV16-E6/E7 Oncogene Mutation and p53 Expression among Indonesian Women with Cervical Cancer

Objective: To characterize HPV16 E6/E7 mutation and its association with p53 expression among Indonesian women with cervical cancer. Methods: This is a cross-sectional study involving 31 Indonesian women with pathologically proven cervical cancer and HPV16 infection. Data about the clinical characteristics of the study population were obtained from the medical records. Biopsy specimen of the cervical cancer mass from each study participant was obtained for DNA isolation. The ORFs of E6 and E7 genes were amplified using specific primer designed according to K02718/HPV16R gene sequence obtained from GenBank. Sequencing was performed using software program MEGA10. HPV16 E6 and E7 prototype sequences for nucleotide alignment (HPv16. P, GenBank Access code: NC_001526) was selected from European variant. The sequence of nucleotide and amino acid was aligned using software program BioEdit. p53 expression was evaluated through immunohistochemistry and quantified using immunoreactivity score (IRS). Results: Twelve subjects (38.7%) present with E6 and E7 mutation. Median age, parity, stage and histologic type of the tumour did not associate with E6/E7 mutation. E6 and E7 mutation rate was 25.8% (8/31) and 12.9% (4/31), respectively. Seven single nucleotide changes were identified within the E6 and E7 oncogenes, including four non-synonymous and three synonymous mutations. E6 T27C was the most prevalent mutation (16.1%). Nonsynonymous mutations were more prevalent within E7 gene (9.6%) (N29T, N29S, and R77C). Median IRS did not differ between HPV16-E6/E7 variants and wildtype (p value = 0.990). There was no association between E6/E7 mutations and p53 expression in Indonesian women with cervical cancer (PR 1.4, 95% CI: 0.29-6.77, p value = 0.704). Conclusions: HPV16 E6 mutation was more prevalent than E7 mutation among Indonesian women. There was no association between E6/E7 mutation and p53 expression level.     

Enhanced specificity of HPV16 E6E7 siRNA by RNA-DNA chimera modification

Although efforts have been made to develop new drugs for infectious and neoplastic diseases utilizing synthetic small interfering RNA(siRNAs), those intrinsically have undesirable effects, including silencing of unintended genes (off-target effect) and nonspecific cytotoxicity. Off-target effects can be avoided by DNA substitution in the guide strand (GS) seed region of nucleotide positions 1-8 and its complementary part of the passenger strand plus the 3' overhang, which is designated as a double-strand RNA-DNA chimera (dsRDC). In this study, we found that the specificity of potent siRNAs targeting human papillomavirus 16 (HPV16) E6 and E7 oncogenes, which we previously reported, could be enhanced by short dsRDC modification (first six nucleotides from the 5' end of the GS and its complementary nucleotides of the passenger strand). Such dsRDC modification reduced nonspecific cytotoxicity in two of three siRNAs (497 and 752), although not in the other (573), which correlated with their off-target effects. In addition, silencing activity was marginally impaired in two dsRDCs (497 and 573) and moderately in one (752). Finally, dsRDC-497 induced E6E7-specific growth suppression of cervical cancer cells as well as E6E7-immortalized human keratinocytes. Our results show that dsRDC modification enhances the specificity of E6E7 siRNA, which is required for use in in vivo settings.     

Human papillomavirus 16 E6, E7 siRNAs inhibit proliferation and induce apoptosis of SiHa cervical cancer cells

Objective:To evaluate the effects of HPVI6 E6/E7 siRNAs on cervical cancer SiHa cells.Methods:The expressions of the E6,E7,p53 and Rb genes were assayed by RT-PCR and Western-bloting respectively.The proliferation and apoptosis of the cells were evaluated by MTT and flow cytometry.Results:HPV 16 E6 and E7 oncogenes were selectivly downregulated by HPV 16 E6 and E7 siRNAs,which sustained at least 96 h by single dose siRNA.Furthermore,reduction of E6 and E7 oncogenes expression upregulated the expressions ...     

Vaccination of full-length HPV16 E6 or E7 protein inhibits the growth of HPV16 associated tumors

Cervical cancer is the second most common cancer in women worldwide. Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Two HPV16 proteins, E6 and E7, are consistently expressed in tumor cells. Most therapeutic vaccines target one or both of these proteins. Taking the advantages of safety and no human leukocyte antigen restriction, protein vaccine has become the most popular form of HPV therapeutic vaccines. Here we demonstrate that immunization with full-length HPV16 E6 or E7 protein elicited specific immunological effect and inhibition of TC-1 cell growth using TC-1 mouse model. HPV16 E6 and E7 genes were cloned into pET-28a(+) and introduced into E. coli Rosetta. Expression of the genes was induced by IPTG. Proteins were purified by Ni-NTA agarose and they were detected by SDS-PAGE and Western blotting. C57BL/6 mice were vaccinated with 1.5 nmol HPV16 E6 or E7 protein. Then they were implanted with 1x10(5) TC-1 cells. No tumor was detected in any mouse vaccinated with E7 protein. Forty days later, the tumor-free mice and control mice were challenged with 2x10(5) TC-1 cells. All control mice developed tumors 6 days later, but E7 immunized mice were tumor free until 90 days. Tumor growth was slow in the E6 immunized mice, but 83% of the mice developed tumors and the survival percentage was not significantly different from the control. An adoptive immune model was used to demonstrate the therapeutic effect. Results showed that the development of TC-1 cells was obviously reduced by transfusion of T-cells but not serum from mice immunized with E7 protein. T-cells from E7 immunized mice also induced the lysis of TC-1 cells in the cytotoxic T lymphocyte assay. These findings show that immunization with HPV16 E6 or E7 protein was able to elicit specific protective immunity against TC-1 tumor growth.     

新疆哈萨克族食管癌组织中HPV16 E6、E7基因的检测与p53的关系

目的探讨人乳头状瘤病毒(HPV)16型感染在新疆哈萨克族(哈族)食管癌发病中的作用,并分析HPV16感染与p53过表达之间的关系.方法采用聚合酶链(PCR)技术,检测41例食管鳞状细胞癌组织中HPV16 E6与E7基因表达差异;用LSAB免疫组织化学方法分析p53蛋白的表达.结果癌组织中HPV16 E6与E7基因阳性检出率分别为34.15%(14/41)和63.41%(26/41);用免疫组化检出p53蛋白阳性率分别在HPV16 E6阳性组(57.1%)与HPV16 E6阴性组(14.8%)间、HPV16 E7阳性组(42.3%)与HPV16 E7阴性组(6.7%)间均存在显著性差异(P＜0.05);用PCR检出HPV16 E6、E7基因在食管癌的高分化组、中低分化组中的检出率分别为7.69%(1/13)、46.43%(13/28)和38.46%(5/13)、75.00%(21/28),差别均有统计学意义(P＜0.05).结论提示p53基因突变与HPV16感染在哈族食管癌的发病过程中存在相互促进作用;另外,HPV16 E6与E7基因和哈萨克族食管癌病理组织学分级的生物学行为密切相关.     

Immunotherapeutic Effects of Dendritic Cells Pulsed with a Coden-optimized HPV 16 E6 and E7 Fusion Gene in Vivo and in Vitro

Background: : Cervical cancer is the second most common cause of cancer related death of women. PersistentHPV infection, especially with high-risk types such as HPV16 and HPV18, has been identified to be the primarycause of cervical cancer. E6 and E7 are the major oncoproteins of high-risk HPVs, which are expressed exclusivelyin HPV infected tissues, and thereby represent ideal therapeutic targets for immunotherapy of cervical cancer.Materials and Methods: In this work, we used recombinant adenovirus expressing coden-optimized HPV16 E6and E7 fusion protein (Ad-ofE6E7) to prime dendritic cells (DC-ofE6E7), to investigate the ability of primed DCvaccine in eliciting antitumor immunity in vitro and vivo. Results: Our results indicated that DC-ofE6E7 vaccineco-culturing with splenocytes could strongly induce a tumor-specific cytotoxic T lymphocyte (CTL) responseand kill the TC-1 cells effectively in vitro. Moreover, DC-ofE6E7 vaccine induced protective immunity againstthe challenge of TC-1 cancer cells in vivo. Conclusions: The results suggested that the HPV16 ofE6E7 primedDC vaccine has potential application for cervical cancer immunotherapy.     

Diagnostic Performance of HPV E6/E7 mRNA and HPV DNA Assays for the Detection and Screening of Oncogenic Human Papillomavirus Infection among Woman with Cervical Lesions in China

Background: Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide and it is responsible for most cases of cervical uterine cancer. Although HPV infections of the cervix do not always progress to cancer, 90% of cervical cancer cases have been found to be associated with high risk HPV (HRHPV) infection. HPV DNA testing is widely used, along with Papanicolaou (Pap) testing, to screen for cervical abnormalities. However, there are no data on the prevalence of genotype-specific HPV infections assessed by measuring HPV E6/E7 mRNA in women representative of the Chinese population across a broad age range. Materials and Methods: In the present study, we compared the results with the CervicGen HPV RT-qDx assay, which detects 16 HR-HPV genotypes (Alpha-9: HPV 16, 31, 33, 35, 52, and 58; Alpha-7: HPV 18, 39, 45, 51, 59, and 68; and Alpha-5, 6: HPV 53, 56, 66, and 69), and the REBA HPV-ID assay, which detects 32 HPV genotypes based on the reverse blot hybridization assay (REBA) for the detection of oncogenic HPV infection according to cytological diagnosis. We also investigated the prevalence and genotype distribution of HPV infection with a total of 324 liquid-based cytology samples collected in western Shandong province, East China. Results: The overall HPV prevalences determined by HPV DNA and HPV E6/E7 mRNA assays in this study were 79.9% (259/324) and 55.6% (180/324), respectively. Although the positivity of HPV E6/E7 mRNA expression was significantly lower than HPV DNA positivity, the HPV E6/E7 mRNA assay showed greater specificity than the HPV DNA assay (88.6% vs. 48.1%) in normal cytology samples. The prevalence of Alpha-9 (HPV 16, 31, 33, 35, 52, and 58) HPV infection among these women accounted for up to 80.3% and 76.1% of the high-grade lesions detected in the HPV mRNA and DNA tests, respectively. The HR-HPV genotype distribution, based on HPV DNA and E6/E7 mRNA expression by age group in patients with cytologically confirmed lesions, was highest in women aged 40 to 49 years (35.9% for cytologically confirmed cases, Pearson correlation r value=0.993, p<0.001) for high-grade lesions. Among the oncogenic HR-HPV genotypes for all age groups, there was little difference in the distribution of HPV genotypes between the HPV DNA (HPV -16, 53, 18, 58, and 33) and HPV E6/E7 mRNA (HPV -16, 53, 33, 58, and 18) assays. HPV 16 was the most common HPV genotype among women with highgrade lesions. Conclusions: Our results suggest that the HPV E6/E7 mRNA assay can be a sensitive and specific tool for the screening and investigation of cervical cancer. Furthermore, it may provide useful information regarding the necessity for early cervical cancer screenings and the development of additional effective HPV vaccines, such as one for HPV 53 and 58. Additionally, gaining knowledge of HPV distribution may also inform us about ecological changes in HPV after the vaccination.