雷莉妍,孫曉莉,朱利民,修志明,王麗萍,

(1.吉林大學(xué) 生命科學(xué)學(xué)院,長(zhǎng)春 130012；2.吉林大學(xué) 中日聯(lián)誼醫(yī)院急救醫(yī)學(xué)科,長(zhǎng)春 130033；3.博迅生物技術(shù)有限責(zé)任公司,長(zhǎng)春 130012)

過(guò)表達(dá)HER2/neu可促進(jìn)乳腺癌細(xì)胞的遷移[1-5].利用RNA干擾(RNA interference,RNAi)技術(shù)可從基因水平上抑制HER2/neu.RNA干擾是一種有效的沉默基因方法,是由21-23 mer雙鏈RNA介導(dǎo)后轉(zhuǎn)錄水平的基因沉默[6],如siRNA和shRNA.其中合成的siRNA介導(dǎo)干擾僅產(chǎn)生瞬時(shí)效果,shRNA可將干擾片段整合至真核表達(dá)載體上,并帶入細(xì)胞,一定時(shí)間內(nèi)可持續(xù)形成shRNA,被Dicer酶剪切為siRNA,從而達(dá)到長(zhǎng)期的RNA干擾效果[7-14],具有特異性、快速性、遺傳性和高效性等特點(diǎn).

本文構(gòu)建了針對(duì)人乳腺癌HER2/neu的shRNA干擾載體,轉(zhuǎn)染高表達(dá)HER2/neu的人乳腺癌細(xì)胞SK-BR-3,并觀察shRNA對(duì)HER2/neumRNA和蛋白的作用效果及對(duì)SK-BR-3細(xì)胞遷移能力的影響,以篩選有效的干擾靶點(diǎn),為進(jìn)一步研究HER2/neu蛋白功能提供理論依據(jù).

1 材料與方法

1.1 細(xì)胞株與主要試劑

熒光實(shí)時(shí)定量聚合酶鏈?zhǔn)椒磻?yīng)(PCR)試劑購(gòu)自大連寶生物有限公司；人乳腺癌細(xì)胞SK-BR-3購(gòu)自北京協(xié)和細(xì)胞庫(kù)；RPMI 1640和OPTI-MEM培養(yǎng)基購(gòu)自美國(guó)Invitrogen公司；胎牛血清購(gòu)自美國(guó)Thermo Scientific公司；pSUPERIOR.puro質(zhì)粒購(gòu)自美國(guó)Oligoengine公司；pEGFP-N1質(zhì)粒購(gòu)自美國(guó)BD Biosciences Clontech公司；LipofectamineTM2000購(gòu)自美國(guó)Invitrogen公司；BglⅡ和HindⅢ購(gòu)自大連寶生物工程有限公司；蛋白免疫印跡試劑均購(gòu)自武漢博士德公司;山羊抗人HER2/neu多克隆抗體(sc-31154)和小鼠抗人β-actin單克隆抗體(sc-8432)購(gòu)自美國(guó)Santa Cruz公司.

1.2 方 法

1.2.1 shRNA的設(shè)計(jì) 本文篩選出HER2/neumRNA (NM_001005862.1)中的3段核苷酸序列作為靶點(diǎn),根據(jù)siRNA的設(shè)計(jì)原則設(shè)計(jì)了shRNAs,分別命名為HER2/neu-shRNA-1,HER2/neu-shRNA-2,HER2/neu-shRNA-3和陰性對(duì)照HER2/neu-shRNA-C.由文獻(xiàn)[15]可知,該陰性對(duì)照不具靶向沉默功能.設(shè)計(jì)的序列由上海生物工程有限公司合成.

1.2.2HER2/neushRNA干擾載體的構(gòu)建 將pSUPERIOR.puro質(zhì)粒分別用限制性內(nèi)切酶BglⅡ和HindⅢ于37 ℃水浴中酶切2 h,與退火產(chǎn)物(dsDNA)在37 ℃水浴中連接過(guò)夜.連接產(chǎn)物轉(zhuǎn)化至感受態(tài)DH-5α大腸桿菌上,于固體選擇培養(yǎng)基上37 ℃選擇培養(yǎng),挑取陽(yáng)性克隆提取質(zhì)粒酶切,產(chǎn)物用質(zhì)量分?jǐn)?shù)為2%的葡聚糖凝膠分離鑒定,并由深圳華大基因研究院測(cè)序.

1.2.3 細(xì)胞培養(yǎng)及給藥處理 乳腺癌細(xì)胞SK-BR-3用RPMI 1640完全培養(yǎng)基(含體積分?jǐn)?shù)為10%的胎牛血清,1.667 μkat/mL青霉素和100 μg/mL鏈霉素)在37 ℃含體積分?jǐn)?shù)為5%的CO2恒溫培養(yǎng)箱內(nèi)培養(yǎng).將細(xì)胞接種于6孔板中用不含雙抗的培養(yǎng)基培養(yǎng).24 h后,用LipofectamineTM2000作為載體進(jìn)行轉(zhuǎn)染.轉(zhuǎn)染48 h后用熒光顯微鏡檢測(cè)綠色熒光蛋白(GFP)的表達(dá).轉(zhuǎn)染24 h后檢測(cè)HER2/neumRNA水平；轉(zhuǎn)染48 h后檢測(cè)HER2/neu蛋白水平.

1.2.4 實(shí)時(shí)定量PCR檢測(cè)HER2/neumRNA的變化 本文用實(shí)時(shí)定量PCR檢測(cè)HER2/neumRNA的變化，以β-actin作為內(nèi)參[11].實(shí)時(shí)定量PCR條件：1) 94 ℃變性30 s；2) 58 ℃退火30 s；3) 72 ℃延伸30 s,循環(huán)45次,最后72 ℃延伸10 min.HER2/neumRNA相對(duì)表達(dá)量=2-Δ CT,其中:ΔCT=(CT目的-CT參比)實(shí)驗(yàn)-(CT目的-CT參比)對(duì)照;CT表示反應(yīng)過(guò)程中管內(nèi)熒光信號(hào)達(dá)到閾值時(shí)的擴(kuò)增循環(huán)數(shù).

1.2.5 蛋白免疫印跡(Western blot)檢測(cè)HER2/neu蛋白量的表達(dá) 細(xì)胞總蛋白樣品經(jīng)SDS-PAGE電泳分離后,轉(zhuǎn)至硝酸纖維素膜上,封閉液封閉2 h后,將膜置于山羊抗人HER2/neu或小鼠抗人β-actin一抗溶液中,4 ℃孵育過(guò)夜.洗脫液洗膜3次,用辣根過(guò)氧化酶(HRP)標(biāo)記的二抗溶液在室溫下孵育膜1 h,用化學(xué)發(fā)光法顯色.β-actin作為內(nèi)參.

1.2.6 細(xì)胞劃痕實(shí)驗(yàn) 當(dāng)生長(zhǎng)在12孔板中的SK-BR-3細(xì)胞在70%～80%匯合時(shí),用移液槍頭劃痕,磷酸鹽緩沖液(PBS)洗3次,去除脫壁的細(xì)胞,將劃痕拍照.細(xì)胞轉(zhuǎn)染48 h后再次拍照.根據(jù)劃痕寬度變化判斷SK-BR-3細(xì)胞的遷移情況[12].

1.3 數(shù)據(jù)統(tǒng)計(jì)

采用統(tǒng)計(jì)學(xué)軟件SPSS 10.0進(jìn)行數(shù)據(jù)統(tǒng)計(jì)分析,結(jié)果以平均值±標(biāo)準(zhǔn)差表示,組間比較采用Student’st-test進(jìn)行顯著性檢驗(yàn),P<0.05表示具有顯著的統(tǒng)計(jì)學(xué)差異.

2 結(jié)果與分析

2.1 重組質(zhì)粒構(gòu)建

構(gòu)建的重組質(zhì)粒經(jīng)測(cè)序比對(duì),結(jié)果與設(shè)計(jì)序列完全吻合,表明HER2/neu基因shRNA表達(dá)質(zhì)粒構(gòu)建成功.干擾序列列于表1.

表1 HER2/neu基因的特異性干擾片段Table 1 Target sequences of HER2/neu gene for RNAi

2.2 轉(zhuǎn)染48 h后報(bào)告基因GFP的表達(dá)

本文用pSUPERIOR.puro和pEGFP-N1質(zhì)粒共轉(zhuǎn)染,用熒光顯微鏡觀察細(xì)胞中GFP的表達(dá)檢測(cè)轉(zhuǎn)染效率,轉(zhuǎn)染成功的細(xì)胞存在GFP表達(dá),結(jié)果如圖1所示.由圖1可見(jiàn),共轉(zhuǎn)染組細(xì)胞中存在顯著的GFP表達(dá),轉(zhuǎn)染效率約為45%.

(A) 空載體轉(zhuǎn)染組明場(chǎng)；(B) 空載體轉(zhuǎn)染組暗場(chǎng)；(C) pSUPERIOR.puro和pEGFP-N1質(zhì)粒共轉(zhuǎn)染組明場(chǎng)；(D) pSUPERIOR.puro和pEGFP-N1質(zhì)粒共轉(zhuǎn)染組暗場(chǎng).圖1 pSUPER-HER2/neu-shRNA的轉(zhuǎn)染效率Fig.1 Transfection efficiency of pSUPER-HER2/neu-shRNA

2.3 HER2/neu-shRNA重組質(zhì)粒對(duì)HER2/neu mRNA表達(dá)的影響

各組質(zhì)粒轉(zhuǎn)染24 h后,熒光實(shí)時(shí)定量PCR檢測(cè)HER2/neumRNA表達(dá)情況,引物序列列于表2.結(jié)果顯示,轉(zhuǎn)染陰性對(duì)照質(zhì)粒組HER2/neumRNA表達(dá)量約為空載體轉(zhuǎn)染組的(90.58±5.26)%,t檢驗(yàn)顯示差異較小.轉(zhuǎn)染HER2/neu-shRNA-1～HER2/neu-shRNA-3質(zhì)粒組分別為空載體轉(zhuǎn)染組的(15.35±3.02)%,(53.8±15.06)%和(76.86±3.11)%,t檢驗(yàn)顯示差異較大.其中HER2/neu-shRNA-1對(duì)HER2/neu基因表達(dá)水平抑制作用最大,如圖2所示.

表2 實(shí)時(shí)PCR引物Table 2 Primers for real-time PCR

2.4 HER2/neu-shRNA對(duì)HER2/neu蛋白的影響

HER2/neu蛋白分子量為185 000,用Western blot法檢測(cè)RNAi對(duì)蛋白的影響,結(jié)果如圖3所示.由圖3可見(jiàn),以空載體轉(zhuǎn)染組作為空白對(duì)照,陰性對(duì)照組蛋白相對(duì)表達(dá)量約為(93.94±4.27)%,差異較小.HER2/neu-shRNA-1,HER2/neu-shRNA-2和HER2/neu-shRNA-3質(zhì)粒轉(zhuǎn)染組HER2/neu蛋白相對(duì)表達(dá)量分別為空載體轉(zhuǎn)染組的(26.02±3.62)%,(69.31±2.03)%和(78.09±5.29)%,均遠(yuǎn)低于空白和陰性對(duì)照組(P<0.05),其中HER2/neu-shRNA-1質(zhì)粒轉(zhuǎn)染組目的蛋白的表達(dá)水平最低.

1.空載體轉(zhuǎn)染組；2.HER2/neu-shRNA-C陰性對(duì)照轉(zhuǎn)染組；3.HER2/neu-shRNA-1轉(zhuǎn)染組；4.HER2/neu-shRNA-2轉(zhuǎn)染組；5.HER2/neu-shRNA-3轉(zhuǎn)染組.圖2 轉(zhuǎn)染HER2/neu-shRNA 24 h后HER2/neu mRNA的表達(dá)Fig.2 Expression of HER2/neu mRNA after transfection with HER2/neu-shRNA for 24 h

1.空載體轉(zhuǎn)染組；2.HER2/neu-shRNA-C陰性對(duì)照轉(zhuǎn)染組；3.HER2/neu-shRNA-1轉(zhuǎn)染組；4.HER2/neu-shRNA-2轉(zhuǎn)染組；5.HER2/neu-shRNA-3轉(zhuǎn)染組.圖3 轉(zhuǎn)染HER2/neu-shRNA 48 h后HER2/neu蛋白的表達(dá)Fig.3 Expression of HER2/neu protein after transfection with HER2/neu-shRNA for 48 h

2.5 HER2/neu-shRNA重組質(zhì)粒對(duì)SK-BR-3細(xì)胞遷移的影響

通過(guò)劃痕實(shí)驗(yàn)研究HER2/neu-shRNA重組質(zhì)粒對(duì)SK-BR-3細(xì)胞遷移的影響,結(jié)果如圖4所示.由圖4可見(jiàn),空載體轉(zhuǎn)染組和陰性重組質(zhì)粒轉(zhuǎn)染組細(xì)胞遷移明顯.各重組質(zhì)粒轉(zhuǎn)染組細(xì)胞遷移能力均不同程度地受到抑制,其中HER2/neu-shRNA-1轉(zhuǎn)染組對(duì)SK-BR-3細(xì)胞遷移抑制最大.這是由于本文構(gòu)建的干擾質(zhì)?？捎行б种艸ER2/neu蛋白,進(jìn)而抑制SK-BR-3細(xì)胞遷移所致.

1.空載體轉(zhuǎn)染組；2.HER2/neu-shRNA-C陰性對(duì)照轉(zhuǎn)染組；3.HER2/neu-shRNA-1轉(zhuǎn)染組；4.HER2/neu-shRNA-2轉(zhuǎn)染組；5.HER2/neu-shRNA-3轉(zhuǎn)染組.圖4 轉(zhuǎn)染HER2/neu-shRNA 48 h后SK-BR-3細(xì)胞的遷移Fig.4 Migration of SK-BR-3 after transfection with HER2/neu-shRNA for 48 h

綜上,本文以HER2/neumRNA序列中的3段核苷酸為靶點(diǎn),設(shè)計(jì)了3個(gè)shRNA干擾序列,先將其克隆至pSUPERIOR.puro質(zhì)粒上,再將重組的真核表達(dá)質(zhì)粒轉(zhuǎn)染至人乳腺癌細(xì)胞SK-BR-3中,通過(guò)熒光實(shí)時(shí)定量PCR、蛋白免疫印跡和劃痕實(shí)驗(yàn)檢測(cè)干擾效果.結(jié)果表明,本文設(shè)計(jì)的shRNAs均可不同程度下調(diào)HER2/neumRNA和蛋白水平,從而抑制SK-BR-3遷移,達(dá)到基因治療的目的.其中以質(zhì)粒HER2/neu-shRNA-1的作用效果最強(qiáng).

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