KDR為靶的siRNA抑制乳腺癌細(xì)胞增殖的體內(nèi)外研究

1、KDR為靶的siRNA抑制乳腺癌細(xì)胞增殖的體內(nèi)外研究         11-01-31 14:44:00     編輯：studa20                  作者：張曉靜, 葛銀林*, 侯琳, 李泉【摘要】  目的: 采用化學(xué)修飾的小干擾RNA(siRNA)在體內(nèi)外抑制含激酶插入?yún)^(qū)受體(

2、KDR)基因表達(dá), 探討化學(xué)修飾的siRNA介導(dǎo)的RNA干擾（RNAi）技術(shù)在乳腺癌基因治療的可行性和特異性。方法: 采用陽離子脂質(zhì)體Lipofectamine2000TM作為轉(zhuǎn)染試劑將針對(duì)人KDR基因的siRNA轉(zhuǎn)染人類乳腺細(xì)胞株MCF7, 誘導(dǎo)RNAi, 采用四甲基偶氮唑藍(lán)（MTT）法, RTPCR, Western blot試驗(yàn)等檢測KDR基因和蛋白表達(dá)及細(xì)胞增殖變化。采用陽離子聚合物納米粒In vivo jetPEITM為轉(zhuǎn)染試劑將siRNA直接注射進(jìn)裸鼠移植瘤, 監(jiān)測腫瘤生長變化, RTPCR, 免疫組化方法等監(jiān)測KDR基因和蛋白表達(dá)變化。結(jié)果: 靶向KDR基因siRNA轉(zhuǎn)染MCF7

3、后, 細(xì)胞增殖被抑制, KDR mRNA和蛋白的表達(dá)明顯降低; 裸鼠體內(nèi)實(shí)驗(yàn)顯示siRNA治療組瘤組織的增長受到明顯抑制; RTPCR, 免疫組化結(jié)果同時(shí)表明治療組KDR表達(dá)下調(diào)。各對(duì)照組指標(biāo)無明顯變化。結(jié)論: 化學(xué)修飾的siRNA介導(dǎo)的RNAi在體內(nèi)外均能成功抑制靶基因的表達(dá)和MCF7細(xì)胞增殖, 是潛在的腫瘤治療新方法, 而KDR亦可作為腫瘤治療的新靶點(diǎn)。 【關(guān)鍵詞】  RNA干擾 siRNA MCF 7細(xì)胞 血管內(nèi)皮生長因子受體2 基因治療Abstract  AIM: To inhibit kinase insert domaincontaining receptor

4、(KDR) expression chemically modified siRNA in vitro and in vivo and to investigate the feasibility and specificity of gene therapy for breast cancer. METHODS: In vitro, siRNA was transfected into MCF7 cells to induce RNAi by using cationic liposome Lipofectamine2000TM. The changes of KDR mRNA and pr

5、otein expression in both siRNA treatment group and control group were measured by MTT assay and RTPCR, In vivo, the siRNA was transfected into transplanted tumor in nude mice by using cationic polymer nanoparticle In vivo jetPEITM. Tumor growth was observed. The mRNA and protein expression of KDR wa

6、s measured by RTPCR and immunohistochemical staining. RESULTS: Experiments in vitro showed that siRNA directed against KDR effectively inhibited the proliferation of MCF7 cells and downregulated KDR mRNA expression. In vivo, the growth of tumor was visibly suppressed. Furthermore, RTPCR and immunohi

7、stochemical results indicated that KDR mRNA and protein expression was reduced in excised tumors. CONCLUSION: RNAi mediated by chemically modified siRNA markedly decreased KDR gene expression and inhibited cellular proliferation. It may have the potential as a therapeutic method to treat human cance

8、r.KeywordsRNAi; siRNA; MCF7; VEGFR2; gene therapy惡性腫瘤生長過程中, 腫瘤組織內(nèi)細(xì)胞急劇、 持續(xù)性增殖的病理生理學(xué)基礎(chǔ)是腫瘤組織內(nèi)血管的再生。有研究認(rèn)為, 含激酶插入?yún)^(qū)受體(kinase insert domaincontaining receptor, KDR), 即血管內(nèi)皮生長因子受體2（vascular endothelial growth factor receptor2, VEGFR2）在血管內(nèi)皮和部分腫瘤細(xì)胞, 包括乳腺癌細(xì)胞上特異性表達(dá), 對(duì)于VEGF的信號(hào)轉(zhuǎn)導(dǎo)及血管內(nèi)皮生成起主導(dǎo)作用, 在腫瘤發(fā)生、 發(fā)展中起重要的調(diào)節(jié)作用1 。

9、腫瘤細(xì)胞分泌VEGF, 除了以旁分泌的形式作用于血管內(nèi)皮上的KDR, 促進(jìn)血管形成外, 還以自分泌的方式作用于自身細(xì)胞上的KDR, 直接促進(jìn)腫瘤細(xì)胞生長2, 3。所以, 抑制KDR 的表達(dá), 可以在兩個(gè)水平上抑制腫瘤生長。RNA干擾(RNA interference, RNAi)是一項(xiàng)新興的基因治療技術(shù)4, 是將雙鏈RNA（dsRNA）導(dǎo)入細(xì)胞引起特異基因mRNA降解的一種細(xì)胞反應(yīng)過程。siRNA(small interference RNA, siRNA)是RNAi的效應(yīng)分子。我們依據(jù)RNAi原理, 設(shè)計(jì)合成以KDR為靶的siRNA, 并對(duì)其進(jìn)行化學(xué)修飾, 增強(qiáng)其在體內(nèi)外的穩(wěn)定性, 并以人類

10、乳腺癌細(xì)胞株MCF7為研究對(duì)象, 在體外細(xì)胞水平及裸鼠動(dòng)物水平, 探討siRNA對(duì)KDR基因特異性沉默及對(duì)細(xì)胞和瘤體增殖的抑制作用。1  材料和方法1.1  材料  乳腺癌MCF7細(xì)胞株購自中國協(xié)和基礎(chǔ)學(xué)院細(xì)胞中心, 常規(guī)培養(yǎng)于含100 mL/L胎牛血清的高糖DMEM(Gibco公司產(chǎn)品)培養(yǎng)液中, 50 mL/L CO2、 37飽和濕度的孵箱中培養(yǎng)傳代。BALB/c nu/nu裸小鼠（雌性, 4周齡, 體質(zhì)量1214 g）, 購自上海斯萊克動(dòng)物有限公司, 按規(guī)定飼養(yǎng)于無菌動(dòng)物試驗(yàn)室, 經(jīng)高壓滅菌的標(biāo)準(zhǔn)飼料和水供動(dòng)物自由飲食。LipofectamineTM2000

11、及TRIzol為Invitrogen公司產(chǎn)品; In vivo jetPEITM為Poly Plus公司產(chǎn)品; AMV 逆轉(zhuǎn)錄試劑盒購自Promega公司; 鼠抗flk1多克隆抗體（sc6251）為santa cruz公司產(chǎn)品; 免疫組化檢測試劑盒PV6002購自北京中杉公司; ECL發(fā)光試劑購自北京普利萊公司。1.2  方法2  結(jié)果2.1  KDR siRNA的設(shè)計(jì)  根據(jù)人KDR mRNA的序列, 利用siRNA在線設(shè)計(jì)軟件及RNA二級(jí)結(jié)構(gòu)分析軟件structure 4.4, 將隨機(jī)設(shè)計(jì)的siRNA與預(yù)測的mRNA二級(jí)結(jié)構(gòu)對(duì)比, 選擇出一條與預(yù)測的

12、KDR mRNA容易結(jié)合的siRNA,  其KDR siRNA序列為: 5GCCACCAUGUUCUCUAAUATT3(正義鏈), 5UAUUAGAGAACAUGGUGGCAT3(反義鏈)。2.2  MTT比色法檢測siRNA對(duì)腫瘤細(xì)胞生長抑制作用  轉(zhuǎn)染KDR siRNA 48 h后, MCF7細(xì)胞增長明顯減慢, MTT數(shù)據(jù)統(tǒng)計(jì)處理結(jié)果顯示: KDR siRNA 4個(gè)濃度組A值與脂質(zhì)體組比較有統(tǒng)計(jì)學(xué)意義（P<0.01）; 錯(cuò)義序列組（即siRNASCR組）與脂質(zhì)體組比較差異不大（P>0.05）。100 nmol/L組和200 nmol/L組比較無統(tǒng)計(jì)

13、學(xué)意義。這表明:   KDR siRNA對(duì)細(xì)胞增殖的抑制作用, 在一定范圍內(nèi)抑制率隨著濃度增加而增加, 達(dá)到一定濃度后, 再增加siRNA濃度并不提高沉默作用。各組A值及抑制率數(shù)值（表1）。表1  MTT比色法檢測MCF7轉(zhuǎn)染siRNA 48 h后增殖情況（略）Tab 1  Detection of proliferation of MCF7 cells 48 h after siRNA transfection by MTT colorimetrybP<0.01 vs blank control group.2.3RTPCR檢測KDR mRNA表

14、達(dá)的變化與未轉(zhuǎn)染組比較, 脂質(zhì)體組與錯(cuò)義序列組無統(tǒng)計(jì)學(xué)意義; KDR siRNA 50nmol/L、 100nmol/L和200nmol/L 3組數(shù)據(jù)均有統(tǒng)計(jì)學(xué)意義（P<0.05）, 50 nmol/L與100   nmol/L和200 nmol/L組相比有統(tǒng)計(jì)學(xué)意義（P<0.05）, 而100 nmol/L和200 nmol/L這兩組之間比較無統(tǒng)計(jì)學(xué)意義（圖1）, 結(jié)果表明, 達(dá)到一定濃度的KDR siRNA轉(zhuǎn)染細(xì)胞24 h后明顯下調(diào)了KDR mRNA的表達(dá), 與MTT法檢測結(jié)果相符。圖1  RTPCR檢測轉(zhuǎn)染siRNA 后MCF7細(xì)胞KDR mRNA表達(dá)水平（略）Fig 1  Expression of KDR mRNA in MCF7 cells transfected with siRNA measured by RTPCRRnasefree water was used as negative PCR control; GAPDH was used as a control. M: DNA markers; 1: