siRNA抑制HPV18 E6基因及其对HeLa细胞凋亡的影响

目的研究特异小干扰RNA(sm all interfering RNA,siRNA)对宫颈癌HeLa细胞中人乳头瘤病毒(hum an pap-illom avirus,HPV)18型E6基因的抑制及其对细胞凋亡的影响。方法针对HPV18E6基因设计siRNA序列,经PCR方法体外扩增,得到含有U6启动子以及siRNA序列的PCR产物,利用L ipofectam ineTM2000脂质体转染HeLa细胞,在U6启动子的作用下于细胞内转录siRNA。针对转染后不同时间点采用四唑盐(MTT)比色法测定细胞活力,流式细胞仪PI染色法检测细胞凋亡率,RT-PCR测定HPV18E6 mRNA变化。结果转染siRNA后细胞活力受到显著抑制(P<0.05),光镜下出现明显的凋亡形态,72 h的凋亡率达到55.8%。RT-PCR结果显示,细胞转染24、48和72 h后HPV18E6 mRNA分别减少了57%、78%和40%,而siRNA阴性对照与未转染细胞相比差异不显著。结论siRNA可特异有效的干扰宫颈癌HeLa细胞内HPV18E6基因的表达,从而可诱导肿瘤细胞凋亡。     

siRNA沉默P21表达对细胞周期解偶联和细胞凋亡的影响

目的： 利用RNAi技术探讨P21蛋白表达对HeLa 细胞周期解耦联和细胞凋亡的影响。方法： 丝裂霉素刺激HeLa细胞后可诱导P21蛋白高表达，采用脂质体转染技术将p21 siRNA 载体转染至HeLa细胞48 h后给予MMC刺激，利用流式细胞术检测HeLa细胞的P21蛋白表达、 细胞倍体的形成和细胞凋亡的改变。结果： p21 siRNA 载体可有效干扰经MMC诱导的HeLa细胞中P21蛋白表达, MMC刺激后24 h和48 h细胞2倍体百分数明显少于对照组(P<0.01)，4倍体和8倍体细胞百分数明显多于对照组(P<0.01)。p21 siRNA沉默HeLa细胞p21后，凋亡细胞百分率明显高于空质粒对照组(P<0.01)。结论： p21 siRNA可有效沉默HeLa细胞P21蛋白表达，在P21蛋白低表达的情况下，HeLa细胞可通过p53非依赖途径诱导细胞死亡，可能与细胞周期解偶联和p53非依赖的细胞凋亡有关。     

RNA干扰对宫颈癌细胞中E6AP基因的影响及其意义

目的 探讨RNA干扰(RNAi)对宫颈癌细胞系HeLa细胞E6AP基因表达及细胞增殖和凋亡的影响.方法 实验分为3组:空白对照组(未经转染的HeLa细胞)、转染阴性对照的小干扰RNA(siRNA)组及转染特异性E6AP siRNA组.采用半定量RT-PCR技术、Western blot方法 检测E6AP mRNA、蛋白表达水平,用四甲基偶氮唑蓝比色(MTT)法检测细胞增殖状况,用流式细胞术检测细胞凋亡.结果 转染E6AP siRNA 24、48、72 h后,E6AP siRNA组E6AP mRNA表达水平与对照siRNA组比较下降33%、72%、70%.Western blot结果 显示,在转染48及72 h,E6AP蛋白表达下降38%、59%.MTT法检测显示,转染HeLa细胞24、48、72、96 h后细胞生长速度明显降低,E6AP siRNA组与空白对照组(F=101.38,P＜0.05)、对照siRNA组(F=38.64,P＜0.05)比较,差异均有统计学意义.E6AP siRNA作用24、48、72 h后E6AP siRNA组凋亡率明显高于对照siRNA组(F=41.48,P＜0.05)和空白对照组(F=86.36,P＜0.05),差异有统计学意义.结论 体外合成的siRNA能有效封闭宫颈癌细胞中E6AP基因的表达,抑制细胞增殖,促进细胞的凋亡,为宫颈癌的基因治疗提供理论依据.     

siRNA抑制HPV18 E6 基因对HeLA细胞凋亡的影响

目的 研究特异小干扰RNA (small interfering RNA, siRNA)对宫颈癌HeLa细胞中人乳头瘤病毒（human papillomavirus, HPV）18型 E6基因的抑制及其对细胞凋亡的影响。方法 针对HPV18 E6 基因设计siRNA序列，经PCR方法体外扩增，得到含有U6启动子以及siRNA序列的PCR产物，利用LipofectamineTM2000脂质体转染HeLa细胞，在U6启动子的作用下于细胞内转录siRNA。针对转染后不同时间点采用四唑盐（MTT）比色法测定细胞活力，流式细胞仪PI染色法检测细胞凋亡率，RT-PCR测定HPV18 E6 mRNA变化。结果 转染siRNA后细胞活力受到显著抑制（P＜0.05），光镜下出现明显的凋亡形态，72h 的凋亡率达到55.8%。RT-PCR结果显示，细胞转染24、48和72h后HPV18 E6 mRNA分别减少了57％、78％和40%，而siRNA阴性对照与未转染细胞相比差异不显著。结论 siRNA可特异有效的干扰宫颈癌HeLa 细胞内HPV18 E6基因的表达，从而可诱导肿瘤细胞凋亡。     

靶向阻断LRP提高肺腺癌细胞对紫杉醇的敏感性

目的利用小干扰RNA(siRNA)技术沉默肺腺癌细胞肺耐药相关蛋白基因(LRP),探讨其对肺腺癌紫杉醇耐药株(A549/TXL20)紫杉醇(TXL)敏感性的影响。方法逐步增加药物浓度法建立A549紫杉醇耐药细胞株(A549/TXL20),用小干扰RNA技术沉默LRP在A549/TXL20细胞中的表达,以MTT法检测紫杉醇对A549/TXL20细胞的半数抑制浓度(IC50);以q PCR检测细胞中LRP mRNA的表达,Western blot检测细胞中LRP蛋白的水平,裸鼠腋窝皮下注射转染后的A549/TXL20细胞,建立裸鼠移植瘤模型,观察LRP靶向siRNA对人肺腺癌耐药细胞株A549/TXL20移植瘤耐药的影响。结果肺腺癌紫杉醇耐药株(A549/TXL20)对紫杉醇的敏感性明显增强(P0.01),A549/TXL20细胞中LRP mRNA及蛋白表达显著增加(P0.01);siRNA沉默A549/TXL20细胞LRP基因后,与空白组和空质粒组比较,LRP mRNA及蛋白表达均被抑制(P0.01);小干扰RNA可提高荷瘤裸鼠对紫杉醇的敏感性。结论 siRNA可有效沉默A549/TXL20肺腺癌耐药细胞株LRP基因的表达,提高耐药的肺腺癌细胞对紫杉醇的敏感性。     

特异siRNA抑制宫颈癌细胞中人乳头状瘤病毒16型E6表达的研究

目的 优化HPV-16 E6癌基因特异的U6质粒表达的siRNA，抑制HPV癌基因表达及其对子宫颈癌细胞生长繁殖的影响。方法 选择4个分别针对HPV-16 E6 mRNA外显子和内含子序列为靶序列，合成DNA链，构建表达HPV-16 E6短发卡样dsRNA的重组pSilencer1．0-U6载体，导入HPV-16DNA阳性的宫颈癌细胞株CaSki中，观察该细胞中HPV-16 E6、E7基因表达水平及其蛋白含量的变化，并观察细胞生长被抑制的情况。结果 4种HPV-16 E6 siRNA均能降低宫颈癌细胞CaSki的生长速率。通过细胞生长曲线观察到HPV-16 E6 shRNA表达质粒导入细胞0-96h内，可降低细胞生长速度。荧光定量RT-PCR检测HPV-16 E6 siRNA可使宫颈癌细胞株CaSki中HPV-16 E6、E7基因转录的mRNA水平降低，其中针对E6 mRNA内含子的重组shRNA只抑制E6基因的表达水平。Western blot分析表明，4个HPV-16 E6 siRNA作用72h后，未能检测到宫颈癌细胞中HPV-16 E6蛋白。结论 HPV-16 E6 siRNA能使宫颈癌细胞CaSki生长缓慢；选择针对E6内含子的siRNA作用位点，特异性抑制E6表达；而针对E6外显子的siRNA作用位点，可抑制E6和E7基因的表达，是用于治疗HPV阳性宫颈癌细胞的理想靶位。     

靶向XBP1S的RNA干扰质粒对细胞增殖的影响

目的 构建靶向人XBP1S的siRNA真核表达载体(pSUPER-XBP1S)并观察其对人HeLa细胞和HepG2细胞增殖能力的影响.方法 设计并合成针对XBP1S基因的siRNA,退火成互补双链后克隆至真核表达载体pSUPER构建重组质粒,并将其转染入HeLa细胞和HepG2细胞中.采用RT-PCR检测转染前后XBP1S在HeLa细胞和HepG2细胞中的转录,Western印迹检测转染前后XBP1S蛋白的表达;MTT法、细胞计数检测重组质粒对HeLa细胞和HepG2细胞增殖能力的影响.结果 重组质粒能有效地抑制HeLa细胞和HepG2细胞中XBP1S基因的转录和表达;转染HeLa细胞和HepG2细胞后,细胞增殖抑制率及细胞增殖数与对照组比较,差异有统计学意义(P<0.05).结论 成功构建了靶向人XBP1S的siRNA表达载体pSUPER-XBP1S,并且有效的抑制了HeLa细胞和HepG2细胞中XBP1S的转录和表达,有效抑制了细胞的增殖能力.     

VEGF siRNA增强白血病HL60细胞对阿糖胞苷药物的敏感性

目的 探讨VEGF siRNA是否能增强自血病细胞HL60对化疗药物阿糖胞苷的敏感性.方法 体外转录合成VEGF siRNA,转染HL60细胞,采用CCK8法检测细胞增殖的情况,RT-PCR方法检测细胞血管内皮细胞生长因子(VEGF)mRNA的表达水平,ELISA方法定量检测细胞培养液VEGF蛋白的表达水平,用流式细胞仪检测细胞的凋亡.结果 VEGF siRNA可以提高HL60细胞对阿糖胞苷的敏感性,VEGF siRNA与阿糖胞苷联用能显著抑制HL60细胞的存活,抑制率为82.9%(F=121.63,P<0.01).VEGF siRNA能降低VEGF mRNA121(F=17.17,P<0.01)和VEGF蛋白(F=3290.49,P<0.01)的表达水平.但是VEGF siRNA没有促进阿糖胞苷诱导的细胞凋亡水平.结论 VEGF siRNA通过抑制细胞的增殖来增强HL60细胞对阿糖胞苷的敏感性.     

E-钙黏蛋白siRNA促进胃癌细胞SGC7901耐药与增殖

目的研究E-钙黏蛋白siRNA对胃癌细胞SGC7901化疗敏感性和增殖能力的影响,并对其相关机制进行初步探讨。方法用Lipofectamine 2000将E-钙黏蛋白siRNA及阴性对照序列转入细胞SGC7901,用MTT法检测细胞对药物的敏感性并观察细胞增殖能力。Western印迹法检测细胞E-钙黏蛋白、PTEN、AKT、p-AKT、BAX和BCL-2蛋白表达。结果 SGC7901细胞转染E-钙黏蛋白siRNA后显著抑制细胞E-钙黏蛋白蛋白表达(P<0.05)。转染E-钙黏蛋白siRNA后细胞对CDDP、5-FU、紫杉醇和ADR的IC50分别为(mg/L):4.375±0.199、6.855±0.780、2.530±0.259和0.368±0.042;阴性对照组分别为(mg/L):2.882±0.166、4.058±0.946、1.483±0.225和0.228±0.012,转染E-钙黏蛋白siRNA细胞对CDDP、5-FU、紫杉醇和ADR的IC50显著高于阴性对照组(P<0.05)。转染E-钙黏蛋白siRNA细胞增殖能力显著高于阴性对照组(P<0.05)。转染E-钙黏蛋白siRNA72 h后PTEN和BA...     

Cytogenetic and molecular genetic changes in malignant transformation of immortalized esophageal epithelial cells

The purpose of this study was to evaluate the extent to which the expression of p53, c-myc, bcl-2, ras genes and chromosomes, along with activity of hTERT, impacts on the malignant transformation of immortalized esophageal epithelial cells. The SHEE cell line was established from an embryonic esophageal epithelial cell induced by transduction of E6E7 genes of human papillomavirus type 18 (HPV18E6E7). In cells of the 85th passage (SHEE85), the malignant transformation of SHEE was confirmed by morphology, cell proliferative index and tumor formation in SCID mice. C-myc, p53, bcl-2 and ras genes were assayed by the multi-PCR method with house-keeping gene GAPDH as control. The modal number of chromosomes was analyzed and its expression of subunit of telomerase, hTERT, was assessed by RT-PCR. Expression of HPV18E6E7 was assayed by Western blotting. The results showed that cells of SHEE85 were atypical and exhibited proliferative status with a proliferation index of 45.70%. Tumors formed in SCID mice with invasion of adjacent tissue. The karyotype belonged to hypotriploid and displayed expression of hTERT. C-myc, k-ras, bcl-2 and p53 (expression of phosphoprotein) were positive in SHEE85. Expression of HPV18E6E7 was positive. Taken together, SHEE85 cells were in fully malignant transformation and their molecular mechanism involved the expression of cellular genes, such as p53, bcl-2, c-myc and ras, and aberrance of chromosomes. It is probable that all of these changes were related with HPV18E6E7.     

The stability of the human papillomavirus E6 oncoprotein is E6AP dependent

Human papillomavirus (HPV) E6 oncoproteins target numerous cellular proteins for ubiquitin-mediated degradation. In the case of p53 this is mediated by the E6AP ubiquitin ligase. However, there are conflicting reports concerning how central E6AP is to the global function of the HPV-16 and HPV-18 E6 oncoproteins. To investigate this further we have analysed the effects of E6AP removal upon the stability of endogenously expressed E6 protein. We show that when E6AP is silenced in HPV-positive cells, E6 protein levels are dramatically decreased in a proteasome-dependent manner. Further, we show that when E6AP is depleted in HeLa cells, E6 has a greatly decreased half-life. In addition, overexpression of E6AP stabilises ectopically expressed HPV-16 and HPV-18 E6 in a manner that is independent of its ubiquitin ligase activity. These results demonstrate that the stability of HPV E6 is critically dependent upon the presence of E6AP.     

Functional consequences of directed mutations in human papillomavirus E6 proteins: abrogation of p53-mediated cell cycle arrest correlates with p53 binding and degradation in vitro

Clinical and epidemiological studies have implicated the involvement of human papillomavirus (HPV) infection in cervical tumorigenesis. We have previously shown that expression of high-risk (HPV16) E6 can abrogate an important cell cycle checkpoint mediated by p53. Sublethal DNA damage causes p53 accumulation and G1 arrest in normal cells, but not in cells with mutant or absent p53, or in cells that express HPV16-E6. To investigate the functional consequences of low-risk (HPV11) E6 expression and to evaluate regions of E6 believed to mediate interaction with p53, we generated several E6 expression constructs, including HPV11-E6, and fourdifferent E6 mutants. HPV16E6 deltaD and HPV16E6 deltaB had short deletions of nucleotides encoding amino acids previously implicated in p53 degradation and binding, respectively. HPV16E6HL and HPV11E6LH had the putative p53 binding domain exchanged between the high- and the low-risk types. Unlike HPV16-E6, HPV11-E6 and the mutant E6 proteins were not able to bind or degrade p53 in in vitro assays. When expressed in RKO cells, HPV11-E6 or the mutant E6 proteins did not prevent p53 accumulation or interfere with p53-dependent WAF1/CIP1 mRNA expression, allowing p53-mediated G, cell cycle arrest after DNA damage. These findings demonstrate that low-risk and high-risk E6 proteins differ in their effects on p53-mediated cell cycle control and that rather subtle mutations of high-risk E6 can alter its ability to abrogate this important cellular response.     

沉默ClC-3氯通道基因对HeLa细胞周期分布的影响

目的:探讨沉默HeLa细胞的ClC-3氯通道基因后细胞周期分布的变化及其作用机制。方法:依照siRNA设计原则构建沉默ClC-3基因的ClC-3 siRNA并转染HeLa细胞;实验分为空白对照组(control组)、转染试剂对照组(Lipo组)、阴性对照组(negative siRNA组)和ClC-3 siRNA组。采用real-time PCR检测ClC-3 siRNA的沉默效率;流式细胞术检测细胞周期分布情况;Western blot检测ClC-3蛋白及相关细胞周期蛋白(cyclin)D1、细胞周期蛋白依赖激酶(cyclin-dependent kinase,CDK)4、CDK6、P21和P27等表达。结果:CIC-3 siRNA成功沉默HeLa细胞的ClC-3基因。和其它组相比,ClC-3 siRNA组的细胞周期被阻抑在G_0/G_1期。CIC-3 siRNA组的cyclin D1、CDK4和CDK6蛋白表达水平明显下降,P21和P27蛋白表达水平明显上升。结论:沉默HeLa细胞ClC-3氯通道基因可影响cyclin D1、CDK4、CDK6、P21和27蛋白的表达水平胆抑HeLa细胞周期停滞在G_0/G_1期。     

Degradation of p53 only is not sufficient for the growth stimulatory effect of human papillomavirus 16 E6 oncoprotein in human embryonic fibroblasts

Certain types of human papillomavirus (HPV), such as types 16 and 18, are thought to be responsible for the development of cervical carcinomas. The E6 and E7 genes of these viruses have transforming activities in various cultured cells and their mRNAs and proteins are expressed in almost all cervical carcinoma cells. Inactivation of the tumor suppressor p53 protein by the E6 gene is believed to be critical for transformation by these oncogenic HPVs. To determine whether degradation of the p53 protein is, in fact, sufficient for cellular transformation by the E6 gene, the E6 gene of HPV16 was introduced into human embryonic fibroblasts (HEF) using recombinant murine retrovirus and examined whether reduction of the p53 protein could substitute for the E6 function. It was found that HEF cells transfected with the E6 gene showed an increased saturation density and degraded the p53 protein. However, when expression of the p53 protein in normal HEF cells was suppressed by the antisense oligonucleotide of the p53 gene, growth stimulation was not observed. These results show that the E6 gene stimulates growth of HEF cells, but that this activity involves some other E6 gene-mediated functions than degradation of the p53 protein. © 1994 Wiley-Liss, Inc.