SiRNA穩(wěn)定抑制肝癌細(xì)胞hTERT基因的表達(dá)

【關(guān)鍵詞】 肝腫瘤
Stable inhibition of hTERT gene by siRNA in hepatocarcinoma cells
　　【Abstract】 AIM: To elucidate the timeefficiency relation of stable inhibition of hepatocarcinoma cell proliferation by RNA interference in vitro. METHODS: The stable screeninginhibition technique of RNAi was adopted to suppress the expression of hTERT stably. After small hairpin RNA (shRNA) targeting hTERT gene was designed, recombinant vector pGenesilshRNAhTERT was constructed and transfected into hepatocarcinoma SMMC7721 cells, which were stably selected by G418 to establish hepatocarcinoma cell lines stablely expressing pGenesilshRNAhTERT. Realtime RTPCR, MTT and PCRTRAP were utilized to detect the alterations of hTERT mRNA expressions, telomerase activity and cell proliferation. RESULTS: The effectiveness of RNAi existed continually and stably in hepatocarcinoma SMMC7721 cells expressing stably pGenesilshRNAhTERT. In the cell lines expressing stably pGenesilshRNAhTERT, hTERT mRNA was obviously suppressed, and the telomerase activities were significantly decreased. Hepatocarcinoma SMMC7721 cell proliferation significantly inhibited in pGenesilshRNAhTERT group compared to that in negative control group. CONCLUSION: RNAi may continually and stably suppress hTERT mRNA expression and carcinoma cell proliferation, which is a potential new approach for antitumor gene therapy.
　　【Keywords】 RNA interference; liver neoplasms; telomerase
　　【摘要】 目的： 利用RNA干擾穩(wěn)定篩選抑制技術(shù)，抑制人端粒酶逆轉(zhuǎn)錄酶（hTERT）基因表達(dá)，探討靶向hTERT基因RNAi與抑制肝癌細(xì)胞增殖的時(shí)效關(guān)系. 方法： 設(shè)計(jì)靶向hTERT基因的小干擾RNA，構(gòu)建重組表達(dá)質(zhì)粒pGenesilshRNAhTERT并導(dǎo)入肝癌SMMC7721細(xì)胞株，經(jīng)G418篩選，建立穩(wěn)定表達(dá)siRNAhTERT的細(xì)胞株. 采用realtime RTPCR、MTT和PCRTRAP法同時(shí)檢測(cè)pGenesilshRNAhTERT穩(wěn)定抑制組和未處理SMMC7721細(xì)胞組hTERT基因表達(dá)、端粒酶活性及細(xì)胞增殖變化. 結(jié)果： 在穩(wěn)定表達(dá)pGenesilshRNAhTERT的SMMC7721細(xì)胞株中，RNAi效力持續(xù)、穩(wěn)定存在，hTERT mRNA表達(dá)、端粒酶活性明顯降低，瘤細(xì)胞增殖被抑制. 結(jié)論： RNA干擾能持續(xù)、穩(wěn)定地抑制靶基因hTERT mRNA表達(dá)及腫瘤細(xì)胞增殖，是有潛力的基因治療腫瘤新方法.
　　【關(guān)鍵詞】 RNA干擾；肝腫瘤；端粒，末端轉(zhuǎn)移酶
　　0引言
　　端粒酶的異常表達(dá)與惡性腫瘤的發(fā)生發(fā)展和預(yù)后密切相關(guān)［1］， hTERT基因在肝癌中的表達(dá)陽性率為89.5%［2］. 研究表明hTERT的高表達(dá)能通過多分化刺激來抑制細(xì)胞凋亡，導(dǎo)致細(xì)胞永生化［3］. 我們依據(jù)RNA干擾（RNA interference, RNAi）原理，使用短發(fā)夾樣RNA（small hairpin RNA, shRNA）設(shè)計(jì)的RNAiDNA載體方法［4］，以hTERT基因?yàn)榘悬c(diǎn)，構(gòu)建穩(wěn)定表達(dá)小干擾RNA(small interfering RNA, siRNA) 的肝癌SMMC7721細(xì)胞株，從體外研究siRNA穩(wěn)定、特異誘導(dǎo)hTERT基因轉(zhuǎn)錄后沉默，逆轉(zhuǎn)癌細(xì)胞惡性表型的能力，探討RNAi基因治療惡性腫瘤的時(shí)效性.
　　1材料和方法
　　1.1材料
　　肝癌SMMC7721細(xì)胞株購自上海細(xì)胞生物研究所. 質(zhì)粒pGenesil1購自武漢晶賽生物工程技術(shù)有限公司. 限制性內(nèi)切酶BamHⅠ和HindⅢ購自Roche公司,總RNA提取試劑、轉(zhuǎn)染試劑LipofectamineTM2000等分別購自Takara和Invitrogen公司；端粒酶TRAPHyb Kit購自華美生物工程公司. Taqman探針和引物由Takara公司合成，hTERT及內(nèi)參PO的探針和引物序列參照文獻(xiàn)［4］. siRNAhTERT轉(zhuǎn)錄模板由上海博亞公司合成.
　　1.2方法
　　shRNA發(fā)夾結(jié)構(gòu)序列長(zhǎng)度為69 bp，兩端分別為BamHⅠ, HindⅢ酶切位點(diǎn)，中間為9 bp莖環(huán)序列分隔的反向重復(fù)靶序列，并以6個(gè)T作為RNA聚合酶Ⅲ的轉(zhuǎn)錄終止子. 質(zhì)粒構(gòu)建采用BamHⅠ, HindⅢ雙酶切空質(zhì)粒pGenesil1，將shRNAhTERT轉(zhuǎn)錄模板5′AAGTTCCTGCACTGGCTGATG3′（AF 015950 nucleotides 16821702）和陰性對(duì)照轉(zhuǎn)錄模板5′AAGCTTCATAAGGCGCATAGC3′ （與人基因無同源性）分別設(shè)計(jì)成發(fā)夾結(jié)構(gòu)，定向克隆至該載體上，經(jīng)篩選、酶切鑒定后，進(jìn)行測(cè)序確定，命名為pGenesilshRNAhTERT，陰性對(duì)照命名為pGenesilshRNAPK. 質(zhì)粒轉(zhuǎn)染與G418篩選采用LipofectamineTM2000脂質(zhì)體轉(zhuǎn)染法. 操作按說

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