hAFP及hTERT雙啟動(dòng)子調(diào)控的針對(duì)人IGF-II基因的siRNA特異性抑制人肝癌細(xì)胞生長(zhǎng)*

湯紹輝， 吳勝蘭， 王曠靖， 張良鵬， 羅羽宏， 曹明溶， 周鴻科

(暨南大學(xué)附屬第一醫(yī)院 1消化科， 2普通外科，廣東 廣州 510632)

hAFP及hTERT雙啟動(dòng)子調(diào)控的針對(duì)人IGF-II基因的siRNA特異性抑制人肝癌細(xì)胞生長(zhǎng)*

湯紹輝1， 吳勝蘭1， 王曠靖1， 張良鵬1， 羅羽宏2△， 曹明溶2， 周鴻科1

(暨南大學(xué)附屬第一醫(yī)院1消化科，2普通外科，廣東 廣州 510632)

目的設(shè)計(jì)并篩選高效沉默胰島素樣生長(zhǎng)因子II(IGF-II)基因的小干擾RNA(siRNA)序列，構(gòu)建由重組人甲胎蛋白(hAFP)和人端粒酶逆轉(zhuǎn)錄酶(hTERT)雙啟動(dòng)子調(diào)控的該siRNA表達(dá)載體，觀察其對(duì)肝癌細(xì)胞生長(zhǎng)的影響。方法根據(jù)siRNA設(shè)計(jì)原則，參照IGF-II mRNA序列設(shè)計(jì)3對(duì)siRNA序列及1對(duì)陰性對(duì)照序列，轉(zhuǎn)染人肝癌Huh7細(xì)胞，轉(zhuǎn)染24 h后采用實(shí)時(shí)熒光定量PCR檢測(cè)IGF-II mRNA表達(dá)量變化，篩選干擾效率最高的siRNA序列。采用PCR擴(kuò)增出hAFP及hTERT啟動(dòng)子的核心序列，應(yīng)用基因重組技術(shù)構(gòu)建重組hAFP和hTERT雙啟動(dòng)子調(diào)控的該siRNA表達(dá)載體。將上述siRNA表達(dá)載體轉(zhuǎn)染Huh7細(xì)胞及L-02人正常肝細(xì)胞，觀察IGF-II mRNA表達(dá)及細(xì)胞生長(zhǎng)情況變化。結(jié)果實(shí)時(shí)熒光定量PCR顯示，siRNA3在25 nmol/L濃度時(shí)抑制效率最高，約90%。成功擴(kuò)增hAFP及hTERT啟動(dòng)子核心序列，將其分別克隆入pGL3-Basic載體，構(gòu)建成重組pGL3-hAFP-hTERT載體；將siRNA3克隆至pGL3-hAFP-hTERT載體，構(gòu)建成重組pGL3-hAFP-hTERT-siRNA3表達(dá)載體。Huh7細(xì)胞IGF-II mRNA表達(dá)量顯著降低，抑制效率達(dá)86%，細(xì)胞增殖受到明顯抑制，G1期細(xì)胞比例顯著增加；L-02細(xì)胞上述指標(biāo)無(wú)明顯改變。結(jié)論成功構(gòu)建雙重RNA聚合酶II啟動(dòng)子(hAFP及hTERT雙啟動(dòng)子)調(diào)控的針對(duì)IGF-II基因的siRNA表達(dá)載體，即pGL3-hAFP-hTERT-siRNA3；該siRNA表達(dá)載體可特異性抑制IGF-II mRNA表達(dá)及肝癌細(xì)胞生長(zhǎng)。

hAFP/hTERT雙啟動(dòng)子； RNA干擾； 人胰島素樣生長(zhǎng)因子II； 癌,肝細(xì)胞

RNA干擾(RNA interference，RNAi)是一種高效、特異沉默靶基因的新技術(shù)。其作用機(jī)制是通過(guò)雙鏈RNA(double-stranded RNA, dsRNA)經(jīng)外界導(dǎo)入細(xì)胞或細(xì)胞內(nèi)合成后運(yùn)輸?shù)桨麧{中，被Dicer酶識(shí)別并加工成21～23 nt長(zhǎng)度的小干擾RNA(small interfering RNA，siRNA)，siRNA的反義鏈?zhǔn)雇磎RNA降解，從而抑制靶基因表達(dá)[1]。目前制備siRNA的方法主要分為兩類：體外合成法與表達(dá)載體法。構(gòu)建siRNA表達(dá)載體是通過(guò)將siRNA對(duì)應(yīng)的DNA雙鏈克隆到驅(qū)動(dòng)小發(fā)夾RNA表達(dá)所用的啟動(dòng)子下游而實(shí)現(xiàn)的。驅(qū)動(dòng)這些啟動(dòng)子轉(zhuǎn)錄的酶有RNA聚合酶II(RNA polymerase II，Pol II)和RNA聚合酶III(Pol III)，這些啟動(dòng)子分別被稱為Pol II和Pol III啟動(dòng)子[2]。Pol III啟動(dòng)子可在幾乎所有細(xì)胞中持續(xù)表達(dá)，無(wú)組織特異性，在腫瘤治療中可能導(dǎo)致人體正常組織損傷；Pol II啟動(dòng)子，如人甲胎蛋白(human alpha-fetoprotein，hAFP)啟動(dòng)子和人端粒酶逆轉(zhuǎn)錄酶(human telomerase reverse transcriptase，hTERT)啟動(dòng)子，則具有組織特異性[2]，可使靶基因在特定的腫瘤組織中沉默[3]。

人胰島素樣生長(zhǎng)因子II(insulin-like growth factor II，IGF-II)是一種由67個(gè)氨基酸殘基組成的多肽。研究表明，IGF-II的過(guò)表達(dá)是目前己知的肝細(xì)胞癌變過(guò)程中發(fā)生最早的基因改變事件，對(duì)肝癌的發(fā)生發(fā)展具有重要影響[4-6]。那么，抑制IGF-II的過(guò)表達(dá)對(duì)肝細(xì)胞癌是否具有較好的治療作用？目前國(guó)內(nèi)外尚未見(jiàn)深入的研究報(bào)道。鑒于此，本研究擬構(gòu)建雙重RNA聚合酶II啟動(dòng)子(hAFP及hTERT雙啟動(dòng)子)調(diào)控的針對(duì)IGF-II基因的siRNA表達(dá)載體，觀察其對(duì)肝癌細(xì)胞生長(zhǎng)的影響，為肝癌基因靶向治療開(kāi)辟新途徑奠定基礎(chǔ)。

材 料 和 方 法

1材料

pGL3-Basic 載體(圖1)、Lipofectamine? RNAiMAXTM轉(zhuǎn)染試劑和Wizard?基因組DNA 純化試劑盒均為Promega產(chǎn)品，Trizol試劑為Invitrogen產(chǎn)品，T4 DNA連接酶為TaKaRa產(chǎn)品，DNA凝膠回收試劑盒為廣州東盛生物科技有限公司產(chǎn)品，SYBR Green PCR Master Mix為Toyobo產(chǎn)品，人肝癌細(xì)胞株Huh7和人正常肝細(xì)胞株L-02購(gòu)自武漢大學(xué)中國(guó)典型培養(yǎng)物保藏中心。

Figure 1. Structure of pGL3-Basic vector.

圖1pGL3-Basic載體結(jié)構(gòu)圖

2方法

2.1針對(duì)IGF-II基因的siRNA設(shè)計(jì)與篩選 根據(jù)siRNA設(shè)計(jì)原則，參照IGF-II mRNA序列(GenBank數(shù)據(jù)庫(kù)：NM_000612)設(shè)計(jì)并合成3對(duì)siRNA序列(siRNA1、siRNA2和siRNA3)及1對(duì)無(wú)任何靶基因的siRNA(NC siRNA)作為陰性對(duì)照。培養(yǎng)Huh7細(xì)胞至匯合度為30%～50%，采用Lipofectamine? RNAiMAXTM轉(zhuǎn)染試劑進(jìn)行細(xì)胞轉(zhuǎn)染siRNA。實(shí)驗(yàn)分為4個(gè)組，即空白對(duì)照組(NC siRNA)和3個(gè)實(shí)驗(yàn)組(siRNA1、siRNA2和siRNA3)，每組設(shè)置25 nmol/L、50 nmol/L和100 nmol/L siRNA濃度梯度。轉(zhuǎn)染24 h后，按Trizol試劑盒說(shuō)明抽提Huh7細(xì)胞總RNA，逆轉(zhuǎn)錄合成cDNA并進(jìn)行實(shí)時(shí)熒光定量PCR擴(kuò)增。IGF-II mRNA上游引物為5’-CTG GAG ACG TAC TGT GCT A-3’，下游為5’-GAC TGC TTC CAG GTG TCA T-3’，片段長(zhǎng)度為128 bp；以18S rRNA為內(nèi)參照。每個(gè)樣本同時(shí)擴(kuò)增3復(fù)管，并連續(xù)進(jìn)行2次實(shí)驗(yàn)。采用相對(duì)定量法(2-ΔΔCt) 計(jì)算目的基因表達(dá)量。

2.2重組hAFP和hTERT雙啟動(dòng)子調(diào)控的、針對(duì)IGF-II基因的siRNA表達(dá)載體構(gòu)建

2.2.1pGL3-hAFP-hTERT載體構(gòu)建 根據(jù)hAFP基因及hTERT基因啟動(dòng)子序列設(shè)計(jì)引物，從L-02細(xì)胞基因組中擴(kuò)增hAFP及hTERT啟動(dòng)子核心片段(分別為269 bp及456 bp)。hAFP啟動(dòng)子上游引物為5’-cgg ggt acc TGA GGA GAA TAT TTG TTA TAT-3’(含KpnI位點(diǎn))，下游引物為5’-cta gct agc TGT TAT TGG CAG TGG TGG AAG-3’(含NheI位點(diǎn))；hTERT啟動(dòng)子上游引物為5’-cta gct agc TGG CCC CTC CCT CGG GTT ACC C-3’(含NheI位點(diǎn))，下游引物為5’-ccg ctc gag CAT CGC GGG GGT GCC GGG G-3’(含XhoI位點(diǎn))。

取pGL3-Basic載體及hAFP啟動(dòng)子PCR產(chǎn)物分別行KpnI與NheI雙酶切，將hAFP啟動(dòng)子插入pGL3-Basic載體多克隆位點(diǎn)KpnI與NheI之間構(gòu)建成pGL3-hAFP載體。取pGL3-hAFP載體及hTERT啟動(dòng)子PCR產(chǎn)物分別行NheI與XhoI雙酶切，將hTERT啟動(dòng)子插入pGL3-hAFP載體hAFP啟動(dòng)子下游構(gòu)建成pGL3-hAFP-hTERT載體。上述重組載體送華大基因公司進(jìn)行測(cè)序分析。

2.3重組hAFP和hTERT雙啟動(dòng)子調(diào)控的siRNA對(duì)Huh7細(xì)胞IGF-II mRNA表達(dá)及細(xì)胞生長(zhǎng)的影響2.3.1細(xì)胞轉(zhuǎn)染及IGF-II mRNA表達(dá)分析 采用LipofectamineTM2000轉(zhuǎn)染試劑將pGL3-hAFP-hTERT-siRNA3表達(dá)載體及陰性對(duì)照載體pGL3-hAFP-hTERT分別轉(zhuǎn)染Huh7細(xì)胞及L-02細(xì)胞。實(shí)驗(yàn)中，每個(gè)共轉(zhuǎn)染實(shí)驗(yàn)同時(shí)做3復(fù)孔，并連續(xù)進(jìn)行2次實(shí)驗(yàn)。轉(zhuǎn)染48 h后收集細(xì)胞，按Trizol試劑盒說(shuō)明抽提細(xì)胞總RNA，檢測(cè)IGF-II mRNA 表達(dá)水平，方法同前。

2.3.2MTS法檢測(cè)細(xì)胞增殖 取上述各組轉(zhuǎn)染細(xì)胞，計(jì)數(shù)，調(diào)整細(xì)胞濃度為1×108/L，分到96孔板，每孔100 μL。細(xì)胞貼壁后，收集各個(gè)時(shí)點(diǎn)的細(xì)胞(0 h、24 h、48 h和72 h)，加入MTS，比例為1/10。孵育4 h后，酶標(biāo)儀測(cè)定A490值。

2.3.3流式細(xì)胞儀檢測(cè)細(xì)胞周期 轉(zhuǎn)染48 h后每樣離心收集1×106細(xì)胞，用PBS洗細(xì)胞2次，加入預(yù)冷70%乙醇，于4 ℃固定過(guò)夜。離心收集細(xì)胞，用PBS洗細(xì)胞1次，加入含50 mg/L溴化丙啶和100 mg/L RNase A的PBS，4 ℃避光孵育30 min，以標(biāo)準(zhǔn)程序用流式細(xì)胞儀檢測(cè)。

3統(tǒng)計(jì)學(xué)處理

數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,組間均數(shù)比較采用One-way ANOVA，應(yīng)用SPSS 13.0統(tǒng)計(jì)軟件進(jìn)行分析，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1針對(duì)IGF-II基因的siRNA設(shè)計(jì)與篩選結(jié)果

根據(jù)IGF-II mRNA序列，設(shè)計(jì)并合成了3對(duì)siRNA，即siRNA1、siRNA2及siRNA3,見(jiàn)表1，分別轉(zhuǎn)染Huh7細(xì)胞。轉(zhuǎn)染24 h后，采用實(shí)時(shí)熒光定量PCR檢測(cè)Huh7細(xì)胞IGF-II mRNA表達(dá)量變化。結(jié)果顯示，轉(zhuǎn)染siRNA1、siRNA2和siRNA3的Huh7細(xì)胞中IGF-II mRNA表達(dá)量均有不同程度的降低，抑制效率約為65%～90%，其中siRNA3在25 nmol/L濃度時(shí)抑制效率最高，達(dá)90%,見(jiàn)圖2。

表1 siRNA序列

Figure 2. Effects of siRNAs targetingIGF-IIgene on IGF-II mRNA expression in Huh7 cells detected by real-time fluorescence quantitative PCR.Mean±SD.n=6.

圖2各siRNA轉(zhuǎn)染Huh7肝癌細(xì)胞24h后對(duì)IGF-IImRNA表達(dá)的影響

2hAFP和hTERT雙啟動(dòng)子調(diào)控的、針對(duì)IGF-II基因的siRNA表達(dá)載體構(gòu)建結(jié)果

圖3顯示pGL3-hAFP-hTERT重組載體2個(gè)陽(yáng)性克隆的雙酶切產(chǎn)物電泳結(jié)果。根據(jù)上述篩選出的siRNA3，設(shè)計(jì)表達(dá)siRNA3的序列，制備雙鏈siRNA3，克隆入pGL3-hAFP-hTERT載體雙啟動(dòng)子下游(XhoI與HindIII位點(diǎn)之間)構(gòu)建成pGL3-hAFP-hTERT-siRNA3表達(dá)載體。測(cè)序結(jié)果顯示，上述各重組載體插入片段序列正確，無(wú)突變。

Figure 3. Electrophoresis analysis of pGL3-hAFP-hTERTvectors (A) and their dual enzyme (KpnI andNheI) digestion products (B). M: DNA marker.Arrows indicate the positive target fragments of about 725 bp (hAFP-hTERTdual promoter fragment).

圖3pGL3-hAFP-hTERT載體及其雙酶(KpnI和NheI)切產(chǎn)物電泳分析

3重組hAFP和hTERT雙啟動(dòng)子調(diào)控的siRNA可特異性抑制Huh7細(xì)胞IGF-IImRNA表達(dá)及細(xì)胞生長(zhǎng)

圖4顯示，與轉(zhuǎn)染陰性對(duì)照載體pGL3-hAFP-hTERT(negative control)及空白對(duì)照(blank control)相比，轉(zhuǎn)染pGL3-hAFP-hTERT-siRNA3表達(dá)載體(siRNA)的Huh7細(xì)胞IGF-II mRNA表達(dá)量顯著降低，抑制效率達(dá)86%(P<0.05)。如此同時(shí)，在L-02細(xì)胞中，3組IGF-II mRNA表達(dá)量無(wú)明顯變化(P>0.05)。

Figure 4. Effects of siRNAs targetingIGF-IIgene driven byhAFP/hTERTdual promoters on IGF-II mRNA expression in Huh7 cells (A) or L-02 cells (B).Mean±SD.n=6.*P<0.05vsnegative control or blank control.

圖4hAFP和hTERT雙啟動(dòng)子調(diào)控的siRNA對(duì)Huh7細(xì)胞及L-02細(xì)胞IGF-IImRNA表達(dá)的影響

圖5與圖6顯示，與轉(zhuǎn)染negative control及blank control相比，轉(zhuǎn)染siRNA的Huh7細(xì)胞增殖受到明顯抑制，轉(zhuǎn)染后第2天及第3天差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；細(xì)胞周期檢測(cè)顯示G1期細(xì)胞比例顯著增加(P<0.05)。而3組L-02細(xì)胞增殖及G1期細(xì)胞比例無(wú)明顯差異(P>0.05)。

討 論

人IGF-II基因是一個(gè)復(fù)雜的轉(zhuǎn)錄調(diào)控單位，具有9個(gè)外顯子和4個(gè)啟動(dòng)子(P1～P4)[7]。在個(gè)體不同組織及不同生長(zhǎng)發(fā)育階段，4個(gè)啟動(dòng)子呈現(xiàn)動(dòng)態(tài)活性，總共編碼5種5′端非翻譯區(qū)不同的IGF-II mRNA，但其成熟蛋白產(chǎn)物相同。例如，在胎兒和新生兒肝臟中，IGF-II mRNA高水平表達(dá)，其轉(zhuǎn)錄來(lái)源于P2～P4啟動(dòng)子激活，其中P3 活性最大，P1 失活；在出生2個(gè)月后，肝臟IGF-II mRNA表達(dá)量則顯著下調(diào)，約為新生兒峰值的1/10，一直至成人期都維持于此低水平狀態(tài)，其表達(dá)主要受控于P1 啟動(dòng)子，P2～P4啟動(dòng)子活性弱或呈關(guān)閉狀態(tài)[8]。

Figure 5. Effects of siRNAs targetingIGF-IIgene driven byhAFP/hTERTdual promoters on the proliferation of Huh7 cells (A) or L-02 cells (B).Mean±SD.n=6.*P<0.05vsnegative control or blank control.

圖5hAFP和hTERT雙啟動(dòng)子調(diào)控的siRNA對(duì)Huh7細(xì)胞及L-02細(xì)胞增殖的影響

Figure 6. Effects of siRNAs targetingIGF-IIgene driven byhAFP/hTERTdual promoters on the cell cycle distribution of Huh7 cells or L-02 cells.Mean±SD.n=6. The percentage of Huh7 cells in G1phase increased significantly in siRNA group compared with negative control or blank control group (P<0.05), and there were no differences of the cell cycle distribution of L-02 cells among the above three groups (P>0.05).

圖6hAFP和hTERT雙啟動(dòng)子調(diào)控的siRNA對(duì)Huh7細(xì)胞及L-02細(xì)胞細(xì)胞周期分布的影響

我們課題組和其他學(xué)者研究發(fā)現(xiàn)，由于P3、P4啟動(dòng)子再激活而導(dǎo)致的IGF-II過(guò)表達(dá)與肝細(xì)胞癌的發(fā)生發(fā)展密切相關(guān)[4-6,9-10]。Dong等[10]報(bào)道，肝細(xì)胞癌的癌灶、癌旁和遠(yuǎn)離癌灶的非癌組織中IGF-II mRNA的表達(dá)率分別為100%、53.3%和0%，提示IGF-II特異表達(dá)于癌變肝細(xì)胞及部分不典型增生的癌前肝細(xì)胞，正常肝細(xì)胞則無(wú)表達(dá)。將IGF-II反義RNA導(dǎo)入HepG2肝癌細(xì)胞后，細(xì)胞增殖率下降，凋亡率增加，AFP分泌水平降低，且惡性表型受到顯著抑制[11-12]。上述結(jié)果提示，IGF-II的過(guò)表達(dá)對(duì)肝細(xì)胞癌的發(fā)生發(fā)展具有重要影響，抑制IGF-II的過(guò)表達(dá)可能成為肝細(xì)胞癌治療的新靶點(diǎn)。

RNAi是一種經(jīng)濟(jì)、快捷、高效抑制特異基因表達(dá)的新技術(shù)[13]，可能成為惡性腫瘤基因治療新的選擇和希望。siRNA是RNAi過(guò)程中的關(guān)鍵中間產(chǎn)物，目前構(gòu)建siRNA表達(dá)載體采用的啟動(dòng)子有Pol II和Pol III啟動(dòng)子，前者包括survivin、hTERT、hAFP啟動(dòng)子等，后者包括H1、U6、tRNA啟動(dòng)子等[2]。Pol III啟動(dòng)子的優(yōu)點(diǎn)是能夠在精確的位置啟動(dòng)與終止轉(zhuǎn)錄，表達(dá)的siRNA可以在體內(nèi)實(shí)現(xiàn)高效而穩(wěn)定的基因沉默，但其不足是無(wú)組織特異性，在腫瘤基因治療時(shí)可能導(dǎo)致人體正常組織損傷[3]。Pol II啟動(dòng)子的優(yōu)點(diǎn)是具有組織特異性，可使靶基因在特定的腫瘤組織中沉默，但其不足是轉(zhuǎn)錄活性較低[14]。有研究顯示，串聯(lián)2個(gè)啟動(dòng)子的核心序列可以增強(qiáng)其轉(zhuǎn)錄活性[15-16]。

hAFP是目前特異性最強(qiáng)的肝細(xì)胞癌腫瘤標(biāo)記物，肝細(xì)胞癌中hAFP 的陽(yáng)性率為70%～90%。但是，生殖腺胚胎瘤及少數(shù)胃癌、肝炎、肝硬化可較低水平表達(dá)hAFP。hTERT是維持端粒酶活性所必需的催化亞單位，hTERT 在85%～90%的惡性腫瘤及86%肝細(xì)胞癌中高表達(dá)，而正常細(xì)胞則低表達(dá)或幾乎不表達(dá)[17]。因此，將hAFP啟動(dòng)子及hTERT啟動(dòng)子二者串聯(lián)構(gòu)建成雙啟動(dòng)子來(lái)調(diào)控siRNA表達(dá)，理論上可能只對(duì)表達(dá)hAFP和(或)hTERT的肝癌細(xì)胞靶基因產(chǎn)生特異性沉默作用，而對(duì)正常肝細(xì)胞幾乎不產(chǎn)生影響。

在本研究中，我們根據(jù)siRNA設(shè)計(jì)原則，參照IGF-II mRNA序列設(shè)計(jì)3對(duì)siRNA序列及1對(duì)陰性對(duì)照序列，分別以25 nmol/L、50 nmol/L和100 nmol/L濃度通過(guò)脂質(zhì)體法轉(zhuǎn)染肝癌細(xì)胞Huh7。結(jié)果顯示，轉(zhuǎn)染siRNA1、siRNA2和siRNA3的Huh7細(xì)胞IGF-II mRNA表達(dá)量均顯示不同程度的下降，其中siRNA3在25 nmol/L濃度時(shí)抑制效率最高，為90%。隨后，我們成功構(gòu)建成重組pGL3-hAFP-hTERT-siRNA3表達(dá)載體,并將其轉(zhuǎn)染Huh7細(xì)胞(表達(dá)IGF-II、hAFP 及hTERT)及L-02細(xì)胞(表達(dá)IGF-II，不表達(dá)hAFP 及hTERT)。結(jié)果顯示，Huh7細(xì)胞IGF-II mRNA表達(dá)量顯著下調(diào)，細(xì)胞生長(zhǎng)受到明顯抑制，而L-02細(xì)胞IGF-II mRNA表達(dá)量及細(xì)胞生長(zhǎng)無(wú)明顯改變，提示hAFP/hTERT雙啟動(dòng)子調(diào)控的siRNA可特異性抑制肝癌細(xì)胞IGF-II表達(dá)及細(xì)胞生長(zhǎng)，對(duì)正常肝細(xì)胞無(wú)明顯影響，為肝癌的基因靶向治療提供一種新的選擇奠定了基礎(chǔ)。本結(jié)果與Chen等[16]的研究報(bào)道相似，他們發(fā)現(xiàn)hAFP/hTERT雙啟動(dòng)子有較高的轉(zhuǎn)錄活性，并可靶向介導(dǎo)miR-26a在肝癌細(xì)胞中表達(dá)，特異性抑制其生長(zhǎng)。

[1] Melnyk CW, Molnar A, Baulcombe DC. Intercellular and systemic movement of RNA silencing signals [J]. EMBO J, 2011, 30(17):3553-3563.

[2] Giering JC, Grimm D, Storm TA, et al. Expression of shRNA from a tissue-specific pol II promoter is an effective and safe RNAi therapeutic [J]. Mol Ther, 2008, 16(9):1630-1636.

[3] Heng TL, Chang WT. Construction of simple and efficient DNA vector-based short hairpin RNA expression systems for specific gene silencing in mammalian cells [J]. Me-thods Mol Biol, 2007, 408:223-241.

[4] Breuhahn K, Schirmacher P. Reactivation of the insulin-like growth factor-II signaling pathway in human hepatocellular carcinoma [J]. World J Gastroenterol, 2008, 14(11):1690-1698.

[5] Tang SH, Yang DH, Huang W, et al. Differential promoter usage for insulin-like growth factor-II gene in Chinese hepatocellular carcinoma with hepatitis B virus infection [J]. Cancer Detect Prev, 2006, 30(2):192-203.

[6] Tang SH, Yang DH, Huang W, et al. Hypomethylated P4 promoter induces expression of the insulin-like growth factor-II gene in hepatocellular carcinoma in a Chinese population [J]. Clin Cancer Res, 2006, 12(14):4171-4177.

[7] 湯紹輝, 張良鵬, 吳小娟, 等. 乙型肝炎病毒X蛋白通過(guò)降低P4啟動(dòng)子甲基化水平上調(diào)人IGF-II基因的轉(zhuǎn)錄[J]. 中國(guó)病理生理雜志, 2012, 28(9):1633-1638.

[8] Li X，Cui H，Sandstedt B，et al．Expression levels of the insulin-like growth factor-II gene (IGF2) in the human liver: developmental relationships of the four promoters [J]． J Endocrinol, 1996, 149(1): 117-124.

[9] Qiu LW, Yao DF, Zong L,et al. Abnormal expression of insulin-like growth factor-II and its dynamic quantitative analysis at different stages of hepatocellular carcinoma development [J]. Hepatobiliary Pancreat Dis Int, 2008, 7(4):406-411.

[10] Dong ZZ, Yao DF, Yao DB, et al. Expression and alteration of insulin-like growth factor II-messenger RNA in hepatoma tissues and peripheral blood of patients with hepatocellular carcinoma [J]. World J Gastroenterol, 2005, 11(30):4655-4660.

[11] 楊冬華, 張鳴青, 杜 江, 等. 人胰島素樣生長(zhǎng)因子-Ⅱ反義RNA 對(duì)肝癌細(xì)胞惡性表型的抑制作用[J].中華肝臟病雜志, 1999, 7(1):39-41.

[12] 寧曉燕, 楊冬華, 杜 江, 等. 反義胰島素樣生長(zhǎng)因子Ⅱ?qū)θ烁伟┘?xì)胞生物學(xué)特性的影響[J]. 中華肝臟病雜志, 2001, 9(4):254.

[13] 鄭時(shí)玉, 王 麗, 劉澤兵, 等. 慢病毒介導(dǎo)的c-metRNA干擾對(duì)人乳頭狀甲狀腺癌K1細(xì)胞生物學(xué)行為的影響[J]. 中國(guó)病理生理雜志, 2012, 28(10):1819-1824.

[14] Ren GL, Fang Y, Ma HH, et al. The short hairpin RNA driven by polymerase II suppresses both wild-type and lamivudine-resistant hepatitis B virus strains [J]. Antivir Ther, 2007, 12(6):865-876.

[15] Tomizawa M, Saisho H, Tagawa M. Regulatory regions of growth-related genes can activate an exogenous gene of the alpha-fetoprotein promoter to a comparable degree in human hepatocellular carcinoma cells [J]. Anticancer Res, 2003, 23(4): 3273-3277.

[16] Chen L, Zheng J, Zhang Y, et al. Tumor-specific expression of microRNA-26a suppresses human hepatocellular carcinoma growth via cyclin-dependent and-independent pathways [J]. Mol Ther, 2011, 19(8):1521-1528.

[17] Hu Y, Shen Y, Ji B, et al. Combinational RNAi gene therapy of hepatocellular carcinoma by targeting human EGFR and TERT [J]. Eur J Pharm Sci, 2011, 42(4): 387-391.

HumanIGF-IIsiRNAdrivenbyhAFP/hTERTdualpromotersspecificallysuppressesgrowthofhumanhepatocellularcarcinomacells

TANG Shao-hui1, WU Sheng-lan1, WANG Kuang-jing1, ZHANG Liang-peng1, LUO Yu-hong2, CAO Ming-rong2, ZHOU Hong-ke1

(1DepartmentofGastroenterology,2DepartmentofGeneralSurgery,theFirstAffiliatedHospital,JinanUniversity,Guangzhou510632,China.E-mail:yuhongluo123@163.com)

AIM: To design and screen the most effective siRNA targeting human insulin-like growth factor II (IGF-II) gene, to construct an siRNA expression vector driven byhAFP(human alpha-fetoprotein)/hTERT(human telomerase reverse transcriptase) dual promoters, and to observe the effect of the siRNA on the growth of human hepatocellular carcinoma cells.METHODSThree siRNAs targetingIGF-IIgene and one negative control siRNA were designed and synthesized. They were transfected into human hepatocellular carcinoma Huh7 cells. The expression IGF-II mRNA in Huh7 cells was detected by real-time fluorescence quantitative PCR 24 h after transfection to screen the most effective siRNA. The core sequences ofhAFPandhTERTpromoters and the most effective siRNA were cloned into pGL3-basic vector to construct the siRNA expression vector driven byhAFPandhTERTpromoters. The siRNA expression vector was transfected into Huh7 cells and human normal liver L-02 cells, and then the IGF-II mRNA expression in these cells and the cell growth were evaluated.RESULTSThe inhibitory effect of siRNA3 on IGF-II mRNA expression was the strongest at the concentration of 25 nmol/L, with an inhibitory rate of approximately 90%. The core sequence fragments ofhAFPandhTERTpromoters and siRNA3 were successfully cloned into pGL3-basic vector. The expression of IGF-II mRNA in the transfected Huh7 cells was reduced by approximately 86%, and proliferation inhibition and G1-phase arrest of the cells were also observed. However, there were no differences of the above parameters in L-02 cells before and after transfection.CONCLUSIONThe siRNA expression vector targeting humanIGF-IIgene driven byhAFP/hTERTdual promoters was successfully constructed. The siRNA3 expression vector may specifically suppress the expression of IGF-II mRNA and the growth of hepatocellular carcinoma cells.

hTERT/hAFPdual promoters; RNA interference; Insulin-like growth factor II; Carcinoma,hepatocellular

R735

A

1000- 4718(2013)08- 1422- 06

2013- 03- 18

2013- 07- 02

廣東省科技計(jì)劃(No.2012B031800399)；廣州市科技計(jì)劃(No.2011J4100077)；暨南大學(xué)第一臨床醫(yī)學(xué)院科研培育專項(xiàng)基金資助項(xiàng)目(No.2013107)

△通訊作者Tel: 020-38688039; E-mail: yuhongluo123@163.com

10.3969/j.issn.1000- 4718.2013.08.014