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。     

p53和HPV—16、18E6蛋白在宫颈癌的表达

目的探讨宫颈癌中抑癌基因p53蛋白与HPV-16、18癌基因E6蛋白的表达及其意义.方法采用SP免疫组织化学方法,检测68例宫颈癌和8例慢性宫颈炎上皮组织中p53和HPV-16、18E6蛋白的表达.结果68例宫颈癌中有17例p53蛋白和60例HPV-16、18E6蛋白呈阳性表达,阳性率分别为25.0%和88.2%.p53蛋白阳性表达与组织学类型有关,其阳性率在宫颈腺癌中为83.3%和宫颈鳞癌中为17.2%,有极显著性差异(P＜0.01);p53蛋白表达也与临床分期相关,该蛋白阳性表达率随着临床分期上升而增高.p53表达同肿瘤分化程度,淋巴结转移无关(P＞0.05).HPV-16、18E6蛋白在宫颈癌组织中的表达同肿瘤分化程度、淋巴结转移、组织学类型和临床分期无关(P＞0.05).在慢性宫颈炎上皮组织中均无p53和HPV-16、18E6蛋白表达.结论p53蛋白阴性和弱阳性表达以及HPV-16、18E6蛋白过度表达与宫颈癌发生有关,但在宫颈腺癌和临床分期升高的宫颈癌组织中,p53蛋白阳性表达的增强可能是p53基因突变的结果,或者与其他细胞蛋白结合,阻止了HPV-16、18E6蛋白对p53蛋白的降解作用,使p53蛋白稳定性增加和阳性表达水平提高.     

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。     

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。     

HPV16E7基因沉默对宫颈癌细胞系CaSki细胞增殖的影响

[目的]探讨脂质体转染HPVl6E7siRNA对人宫颈癌CaSki细胞增殖的影响。[方法]人工合成抑制HPVl6E7基因的siRNA片段,通过脂质体转染到CaSki细胞内,显微镜下观察其形态学变化;流式细胞术检测各组细胞周期变化;RT-PCR检测HPVl6E7mRNA的表达;Westernblot检测HPVl6E7蛋白表达。[结果]转染siRNA后的CaSki细胞,细胞增殖受到显著抑制．HPVl6E7mRNA表达显著下降,HPVl6E7蛋白水平显著下降。[结论]应用RNA干扰靶向抑制HPVl6E7基因可以显著抑制CaSki细胞增殖。     

膀胱移行细胞癌HPV-16/18感染与p53基因改变的关系

目的初步探讨膀胱移行细胞癌(TCC)中HPV-16／18感染与p53基因改变的关系。方法选择已确定为HPV-16／18感染的18例膀胱TCC患者(其中HPV-16／18E7片段阳性者13例，HPV-16／18E7阴性者5例)，采用微卫星方法检测肿瘤组织内与p53基因紧密连锁的TP53位点杂合性缺失(LOH)情况，并采用免疫组织化学方法检测p53蛋白表达情况。结果18例TCC组织中TP53位点杂合率为83．33％(15／18)，其LOH率达46．67％(7／15)；p53蛋白阳性率为50．00％(9／18)。其中，HPV-16／18E7 DNA阳性和p53基因改变[包括TP53位点LOH和(或)p53蛋白阳性]并存者11例，占61.11％(11／18)；仅HPV-16／18E7 DNA阳性或p53基因改变[包括TP53位点LOH和(或)p53蛋白阳性]者5例，占27．78％(5／18)；两者均阴性者2例，占11．11％(2／18)。结论HPV-16／18感染可能通过p53基因改变在TCC的发生发展过程中发挥作用，这种p53基因改变可以是LOH和(或)蛋白异常表达。     

抑制ATM表达对60Co照射下宫颈癌细胞损伤修复机制的影响

目的：探讨抑制毛细血管扩张-共济失调突变基因（ataxia-telangiectasia mutated，ATM）表达对人宫颈癌细胞株HeLa在^60Co照射下细胞损伤修复机制的影响。方法：电穿孔法将人ATM基因siRNA的重组真核表达质粒pSup．ATM转染宫颈癌HeLa细胞；G418筛选建立稳定转染株；RT-PCR、Western-blot、免疫荧光法检测ATM基因表达的抑制情况；Western-blot法检测^60Co照射前后各组细胞ATM、p53Ser15磷酸化、CHK2Thr 68磷酸化、p53、CHK2蛋白的表达水平；流式细胞术检测^60Co照射后各组的细胞周期变化。结果：ATM基因在HeLa^ATM细胞中表达明显低于在HeLa、HeLa^ms细胞中的表达水平，成功地建立了ATM低表达的宫颈癌细胞模型。HeLa^ATM细胞在^60Co照射后p53^Ser15磷酸化、CHK2^Thr68磷酸化、p53蛋白表达均显著降低，细胞周期呈现为G2期延长，G1／G2倒置。结论：抑制ATM基因表达可显著抑制宫颈癌细胞对^60Co辐射损伤的修复。这一机制可能与HeLa^ATM细胞中ATM蛋白表达缺失，ATM依赖性的细胞损伤修复通路受阻有关。     

人乳头瘤病毒16型E6、E7反义RNA对人宫颈癌SiHa细胞的恶性逆转作用

背景与目的:人乳头瘤病毒16型(human papillomavirus 16,HPV16)的早期基因E6、E7可分别诱导p53的泛素化降解和pRb的高磷酸化失活,与宫颈癌的发生发展关系密切.本研究通过真核表达载体介导HPV16型E6、E7基因反义RNA在SiHa细胞中的表达,探讨反义RNA能否降低E6、E7基因的表达和诱导细胞凋亡.方法:利用pEGFP构建HPV16型E6、E7基因反义RNA的真核表达载体并转染SiHa细胞,利用RT-PCR和Western blot技术检测转染后E6、E7基因的mRNA和蛋白的变化,利用MTT法检测SiHa细胞转染后细胞增殖活性,利用流式细胞仪和激光共聚焦显微镜检测转染后细胞的凋亡情况.结果:转染携带HPV16型E6、E7反义基因的质粒后,SiHa细胞的E6、E7基因的mRNA和蛋白表达均明显下调;MTT结果显示转染后细胞的增殖活性(0.50±0.05)与转染空载体细胞(1.01±0.06)和未转染细胞(1.28±0.06)比较明显降低(P＜0.05);流式细胞仪检测结果显示转染后细胞凋亡率(59.3±11.3)%与转染空载体细胞(9.4±1.8)%和未转染细胞(2.1±0.4)%比较有显著性差异(P＜0.05).激光共聚焦显微镜结果示转染后细胞凋亡明显增多.结论:HPV16型E6、E7基因反义RNA可降低宫颈癌细胞中E6、E7癌基因的表达,诱导宫颈癌细胞凋亡.     

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。     

特异性siRNA抑制宫颈癌细胞CaSKi中HPV16 E6基因的表达

目的：研究RNAi技术抑制宫颈癌细胞CaSKi中HPV16 E6癌基因的表达以及对细胞生物学特性的影响。方法：构建真核细胞表达特异性shRNA的pGENESIL-1/E6（PS6）重组质粒,脂质体转染细胞后用半定量RT-PCR技术检测CaSKi细胞中E6mRNA水平的变化,然后用western blotting检测RNA干扰前后p53和p21蛋白的表达来验证RNAi效应。用透射电子显微镜观察CaSKi/PS6细胞形态变化;应用流式细胞仪检测细胞的凋亡和细胞周期的变化,最后利用细胞计数和裸鼠成瘤实验测定CaSKi/PS6细胞增殖能力。结果：成功构建了真核细胞表达特异性siRNA的PS6重组质粒;在RNA干扰24、48和72小时后CaSKi/PS6细胞中E6mRNA分别下降了21.50%、52.60%和65.60%;CaSKi/PS6细胞出现凋亡的特征性改变;RNA干扰24h、48h和72h后CaSKi/PS6细胞的凋亡率分别为27.94%、31.95%和56.63%,细胞生长周期停滞于S期。结论：应用RNA干扰技术能有效地、特异地抑制宫颈癌细胞中HPV16 E6基因的表达,为RNA干扰应用于研究宫颈癌发病分子机制、临床治疗和干预性预防提供了新的方法。     

GFP/HPV-16E6 fusion protein induces apoptosis in MCF-7 and 293T cells using a transient expression system

Since mucosal high-risk human papillomavirus (HPV) E6 can target and degrade the tumor suppressor p53, it is recognized as a major causative agent of cervical cancer. However, to date the distribution of high-risk HPV-E6 protein remains elusive. Thus, in the present study we used a mammalian green fluorescent protein (GFP) expression system to express a GFP/HPV-16E6 fusion protein (GFP-16E6) in wild-type (wt) p53 cells, such as MCF-7 and 293T cells to investigate the trafficking and localization of E6 and p53. Following transfection, we observed that the overexpressed GFP-16E6 was a nuclear protein, and that endogenous wt p53 localized to the nucleus together with GFP-16E6. Strikingly, p53 levels were not decreased but increased in 24?h transfected with pGFP-16E6. Furthermore, we observed significant apoptosis induced by GFP-16E6, which proved to be dependent on p53 expression.     

Human papillomavirus type 16 E6 induces cervical cancer cell migration through the p53/microRNA-23b/urokinase-type plasminogen activator pathway

Deregulation of microRNA (miRNA or miR) expression in human cervical cancer is associated frequently with human papillomavirus (HPV) integration. miR-23b is often downregulated in HPV-associated cervical cancer. Interestingly, urokinase-type plasminogen activator (uPA), the miR-23b target, is detected in cervical cancer, but not in normal cervical tissues. Thus, the importance of miR-23b and uPA in HPV-associated cervical cancer development is investigated. In this study, the high-risk subtype HPV-16 E6 oncoprotein was found to decrease the expression of miR-23b, increase the expression of uPA, and thus induce the migration of human cervical carcinoma SiHa and CaSki cells. uPA is the target gene for miR-23b as the miR repressed uPA expression and interacted with the 3'-untranslated region of uPA mRNA. The tumor suppressor p53 is known to be inactivated by HPV-16 E6. A consensus p53 binding site is detected in the promoter region of miR-23b, whereas p53 trans-activated and also interacted with the miR's promoter. Therefore, p53 is believed to mediate the HPV-16 E6 downregulation of miR-23b. From the above, miR-23b/uPA are confirmed to be involved in HPV-16 E6-associated cervical cancer development.     

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 ...     

HPV16 E6小干扰RNA对人宫颈癌裸鼠移植瘤的抑制作用

目的探讨HPV16 E6小干扰RNA(siRNA)对人宫颈癌移植瘤的抑制作用。方法建立人宫颈癌CaSki细胞裸鼠皮下接种模型,将HPV16 E6 siRNA注入瘤体内(实验组),同时设对照组,动态观察并测定肿瘤的生长。采用原位末端标记(TUNEL)法检测肿瘤细胞凋亡情况;免疫组化法测定E6、p53蛋白的表达;检测血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)含量;光镜观察肝肾脏结构的改变。结果HPV16 E6 siRNA处理后,肿瘤生长明显受到抑制,实验组肿瘤体积[(0.21±0.06)cm3]及瘤重[(0.13±0.04)g]均明显降低,与对照组差异有统计学意义(P<0.05);实验组肿瘤细胞凋亡[(29.8±1.4)%]明显增加,E6和p53蛋白表达均下调;实验组和对照组血清ALT、AST含量无明显差异,实验组肝肾脏结构无明显异常。结论HPV16 E6 siRNA能够抑制宫颈癌移植瘤生长,且对肝脏无毒副作用,可为官颈癌的治疗提供一个新的特异性基因治疗方法。     

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。     

Overexpression of human papillomavirus type 16 oncoproteins enhances hypoxia-inducible factor 1 alpha protein accumulation and vascular endothelial growth factor expression in human cervical carcinoma cells.

PURPOSE: Human papillomavirus (HPV)-16 oncoproteins, E6 and E7, are associated with enhanced tumor angiogenesis in human cervical cancers. The purpose of this study was (a) to investigate whether expression of HPV-16 E6 and E7 oncoproteins induces hypoxia-inducible factor 1 alpha (HIF-1 alpha) and vascular endothelial growth factor expression in cervical cancer cells; and (b) to assess the effect of resveratrol on 16 E6- and E7-induced HIF-1 alpha and VEGF gene expression. EXPERIMENTAL DESIGN: Human cervical cancer cell lines C-33A and HeLa were transiently cotransfected with pSG5-HPV-16 E6 or 16 E7 constructs along with HIF-1 alpha small interfering RNA (siRNA) or nonspecific siRNA. The expression of HIF-1 alpha/VEGF was measured using real-time PCR, Western blot analysis, or ELISA. The in vitro angiogenic activity induced by 16 E6- and E7-transfected cells was examined. The effect of resveratrol on oncoprotein-induced HIF-1 alpha/VEGF expression and in vitro angiogenesis was investigated. RESULTS: HPV-16 E6- and E7-transfected cervical cancer cells express increased HIF-1 alpha protein and VEGF expression. These stimulatory effects were abrogated by cotransfection with either HIF-1 alpha siRNA or treatment with resveratrol. Blocking extracellular signal-regulated kinase 1/2 (ERK 1/2) and phosphoinositide-3-kinase by PD98059 and LY294002, respectively, abolished 16 E6- and E7-induced HIF-1 alpha and VEGF expression. Functionally, we showed that HPV-16 E6- and E7-transfected cervical cancer cells stimulated in vitro capillary or tubule formation, and these angiogenic effects could be abolished either by cotransfection with HIF-1 alpha siRNA or by treatment with resveratrol. CONCLUSION: HPV-16 oncoproteins contribute to enhanced angiogenesis in cervical cancer cells via HIF-1 alpha-dependent VEGF expression. Resveratrol suppresses 16 E6- and E7-induced HIF-1 alpha-mediated angiogenic activity and, thus, is a promising chemotherapeutic agent for human cervical cancer.     

Plasmid-based E6-specific siRNA and co-expression of wild-type p53 suppresses the growth of cervical cancer in vitro and in vivo

The E6 protein of the oncogenic HPV-16 functions by interfering with the normal cell cycle control mechanisms, particularly those controlled by p53. In this study, we developed a dual expression plasmid that coexpressed-E6-specific siRNA and wild type p53, and to evaluate its effects on cervical cancer growth. We found that simultaneous expression of pSi-E6-P53 caused a robust suppression of tumor growth when compared to the controls either E6-specific siRNA or p53 alone. In conclusion, our findings demonstrate that a combined strategy of co-expressed E6-specific siRNA and p53 synergistically and more effectively suppressed cervical tumor growth when compared with single treatment.     

HPV E6,p53与宫颈癌的关系及其在基因治疗中的应用

目前宫颈癌的发病率仍居全世界妇女常见恶性肿瘤的第二位.高危型人乳头瘤病毒（high-risk Human Papillomavirus,HR-HPV）感染是宫颈癌及其癌前病变最主要的诱因之一.抑癌基因p53能够有效地抑制细胞增殖和促进细胞凋亡,人类近半数的肿瘤都与p53基因的突变和失活有关,HPV E6蛋白降解p53蛋白,是宫颈癌发生的前提.选择性杀伤p53功能缺失的肿瘤细胞,同时保护正常细胞,发展HPV疫苗及联合用药是临床肿瘤免疫学家进行宫颈癌基因治疗的目标.