人microRNA-21真核過表達(dá)載體的構(gòu)建及其在HepG2.2.15細(xì)胞中上調(diào)c-myc基因的表達(dá)*

林永敏,任廣立,張衛(wèi)云,蔡啟茵,謝　聰,馬恒顥

(南方醫(yī)科大學(xué)附屬廣州軍區(qū)廣州總醫(yī)院：1.兒科；2.檢驗(yàn)科,廣州 510010)

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論著·基礎(chǔ)研究

人microRNA-21真核過表達(dá)載體的構(gòu)建及其在HepG2.2.15細(xì)胞中上調(diào)c-myc基因的表達(dá)*

林永敏1,任廣立1,張衛(wèi)云2,蔡啟茵1,謝聰1,馬恒顥1

(南方醫(yī)科大學(xué)附屬廣州軍區(qū)廣州總醫(yī)院：1.兒科；2.檢驗(yàn)科,廣州 510010)

目的構(gòu)建人微小RNA-21(microRNA-21,miRNA-21)真核過表達(dá)載體pmR-21,探討其在HepG2.2.15細(xì)胞中對(duì)c-myc基因表達(dá)的調(diào)控作用。方法PCR擴(kuò)增miRNA-21的前體基因片段(pre-miRNA-21),雙酶切后連接到pmR-mCherry載體上,通過雙酶切和測(cè)序驗(yàn)證重組載體的準(zhǔn)確性；將重組載體轉(zhuǎn)染到HepG2.2.15細(xì)胞中為實(shí)驗(yàn)組，另設(shè)空載體組(轉(zhuǎn)染pmR-mCherry空質(zhì)粒組)，空白組(未轉(zhuǎn)染組)，陽性對(duì)照組(HepG2細(xì)胞),24 h后觀察載體熒光蛋白表達(dá)情況,流式細(xì)胞術(shù)檢測(cè)轉(zhuǎn)染效率,實(shí)時(shí)熒光定量PCR評(píng)估m(xù)iRNA-21的表達(dá)情況；轉(zhuǎn)染72 h后,RT-PCR和Western blot檢測(cè)c-myc mRNA及蛋白表達(dá)水平；CCK-8法檢測(cè)各組細(xì)胞增殖情況。結(jié)果經(jīng)雙酶切和測(cè)序驗(yàn)證,目的基因片段插入載體中；實(shí)驗(yàn)組及空載體組轉(zhuǎn)染24 h后細(xì)胞內(nèi)可見強(qiáng)熒光,轉(zhuǎn)染效率大于50%；實(shí)驗(yàn)組細(xì)胞miRNA-21表達(dá)較空載體組、空白組水平升高；轉(zhuǎn)染72 h后實(shí)驗(yàn)組細(xì)胞c-myc mRNA表達(dá)較空載體組、空白組升高；實(shí)驗(yàn)組細(xì)胞增殖快于空載體組及空白組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論成功構(gòu)建miRNA-21真核過表達(dá)載體pmR-21,該重組載體可穩(wěn)定表達(dá)miRNA-21；miRNA-21可上調(diào)c-myc基因的表達(dá),c-myc基因是miRNA-21發(fā)揮促癌作用的靶點(diǎn)之一。

微RNA-21;遺傳載體;基因,c-myc;乙型肝炎相關(guān)性肝腫瘤;HepG2.2.15細(xì)胞

微小RNA(microRNA，miRNA)是一類長(zhǎng)度約22 nt的內(nèi)源性非編碼RNA,可以在轉(zhuǎn)錄后期作用于特定的mRNA,調(diào)控基因的表達(dá)。miRNA除了參與正常的生理過程外,還在感染、腫瘤發(fā)生發(fā)展、心腦血管疾病等方面發(fā)揮重要的作用[1],為相關(guān)疾病的診治提供新的突破點(diǎn)。miRNA-21是近年來發(fā)現(xiàn)的一種充當(dāng)促癌基因作用的miRNA,參與多種腫瘤細(xì)胞的增殖、凋亡和侵襲過程[2-3],尤為特殊的是miRNA-21在與HBV、EB等病毒感染相關(guān)的癌癥中起重要作用[4-5]。研究表明,原發(fā)性肝癌(hepatocelluar carcinoma,HCC)患者體內(nèi)的miRNA-21表達(dá)升高,miRNA-21與乙型肝炎病毒(HBV)HBx蛋白相互促進(jìn),在HCC發(fā)生和HBV感染中發(fā)揮重要作用[6-7]。c-myc基因是較早發(fā)現(xiàn)的癌基因之一,可與DNA結(jié)合,參與DNA修復(fù),在癌細(xì)胞的分化、增殖、凋亡過程起重要作用[8-10]。本研究通過構(gòu)建miRNA-21真核過表達(dá)載體,進(jìn)一步探索miRNA-21在HBV相關(guān)性HCC中的作用。

1　材料與方法

1.1材料HepG2.2.15細(xì)胞、HepG2細(xì)胞、大腸埃希菌DH5α由廣州軍區(qū)廣州總醫(yī)院醫(yī)學(xué)實(shí)驗(yàn)科保存。DNA提取試劑盒、PCR產(chǎn)物純化試劑盒、CCK-8購于碧云天公司；DMEN高糖培養(yǎng)基購于Hycolne公司,胎牛血清(FBS)購于四季青公司。miRNA-21前體PCR引物和c-myc PCR引物由上海英濰捷公司基合成,miRNA-21 RT-PCR和qPCR頸環(huán)引物由廣州銳博生物科技有限公司合成,PCR酶、限制性內(nèi)切酶EcoRⅠ和BamHⅠ、T4連接酶購于Takara公司。瓊脂糖購于Biowest公司,瓊脂糖凝膠回收試劑盒購于天根生化科技有限公司,去內(nèi)毒素質(zhì)粒小提試劑盒購于Omega公司。轉(zhuǎn)染試劑Lipofectamine2000、 Trizol試劑、逆轉(zhuǎn)錄試劑盒、熒光定量PCR試劑盒qPCR SYBR select master mix 購于Invitrogen公司。c-myc和β-acitin蛋白一抗購于CST公司,二抗購于Asbio公司。其他試劑均為國內(nèi)分析純。熒光定量PCR儀購于Qiagen公司,流式細(xì)胞儀購于Beckman公司。

1.2方法

1.2.1pmR-21真核過表達(dá)載體的構(gòu)建通過miRBase數(shù)據(jù)庫查找pre-miRNA-21序列及其上下游各100 bp側(cè)翼序列,用Oligo7軟件設(shè)計(jì)引物。其中正向引物為：5′-TCG AGA ATT CAT TGG GGT TCG ATC TTA ACA G-3′,反向引物為：5′-TCG AGG ATC CCA CAA AAG ACT CTA AGT GCC-3′(最前端序列護(hù)堿基,下劃線序列分別為EcoRⅠ和BamHⅠ酶切位點(diǎn)),擴(kuò)增片段長(zhǎng)度為328 bp。 提取HepG2.2.15細(xì)胞的DNA作為模板,用PCR擴(kuò)增miRNA-21前體序列;反應(yīng)條件為：94 ℃預(yù)變性2 min；94 ℃ 30 s,56 ℃ 30 s,72 ℃ 30 s,30個(gè)循環(huán)。純化后的pre-miRNA-21和pmR-mCherry質(zhì)粒雙酶切后用T4連接酶于16 ℃過夜連接。將連接后的產(chǎn)物轉(zhuǎn)化感受態(tài)大腸埃希菌DH5α,進(jìn)行卡那霉素抗性平板篩選。16 h后挑取陽性菌落搖菌擴(kuò)大培養(yǎng),提取質(zhì)粒進(jìn)行雙酶切,以1%瓊脂糖凝膠電泳分離回收。最后選取電泳陽性組的菌株送檢進(jìn)行測(cè)序作鑒定。

1.2.2HepG2.2.15細(xì)胞和HepG2細(xì)胞培養(yǎng)及pmR-21重組載體轉(zhuǎn)染HepG2.2.15細(xì)胞培養(yǎng)于含15% FBS的高糖DMEM培養(yǎng)液中(同時(shí)添加200 mg/L的G418、10 g/L L-谷氨酰胺及100 000 IU/L青、鏈霉素),HepG2細(xì)胞培養(yǎng)于含10% FBS和100 000 IU/L青、鏈霉素的高糖DMEM培養(yǎng)液中,置于37 ℃含5% CO2的培養(yǎng)箱內(nèi)。細(xì)胞穩(wěn)定傳代3次后,接種到6孔板中,每孔細(xì)胞密度為4×105個(gè)。種板24 h后(匯合度約50%)取去經(jīng)內(nèi)毒素提取的pmR-21重組載體和pmR-mCherry空質(zhì)粒按試劑盒操作說明進(jìn)行轉(zhuǎn)染,分為實(shí)驗(yàn)組(轉(zhuǎn)染pmR21重組載體)，空載體組(轉(zhuǎn)染pmR-mCherry空質(zhì)粒)、空白組(未轉(zhuǎn)染質(zhì)粒)、陽性對(duì)照組(HepG2細(xì)胞)。每孔轉(zhuǎn)染試劑Lipofectamine2000 5 μL,質(zhì)粒2.5 μg，轉(zhuǎn)染24 h后熒光顯微鏡下觀察載體上熒光蛋白表達(dá)效果。

1.2.3miRNA-21 qPCR相對(duì)定量檢測(cè)轉(zhuǎn)染24 h后,Trizol法提取各組細(xì)胞總RNA,鑒定其完整性和純度后以總RNA為模板,設(shè)計(jì)針對(duì)miRNA-21成熟序列的特異性RT-PCR莖環(huán)引物,逆轉(zhuǎn)錄后進(jìn)行qPCR,以U6為內(nèi)參照,反應(yīng)條件為95 ℃預(yù)變性20 s；95 ℃ 10 s，60 ℃ 20 s，70 ℃ 10 s，共40個(gè)循環(huán)。采用2-ΔΔct相對(duì)定量分析方法分析各組細(xì)胞miRNA-21的相對(duì)表達(dá)量。

1.2.4c-myc mRNA水平的RT-PCR檢測(cè)以總RNA為模板,逆轉(zhuǎn)錄成cDNA,以此為模板進(jìn)行PCR擴(kuò)增。c-myc上游引物為5′-CGT CCT CGG ATT CTC TGC TC-3′,下游引物為5′-GCT GGT GCA TTT TCG GTT GT-3′，產(chǎn)物大小為380 bp。內(nèi)參為GAPDH,上游引物為5′-GCT CTC TGC TCC TCC TGT-3′,下游引物為5′-ATG AGT CCT TCC ACG ATA C-3′,產(chǎn)物大小為540 bp。反應(yīng)條件均為：94℃預(yù)變性2 min；94 ℃ 30 s,58 ℃ 30 s,72 ℃ 30 s，共30個(gè)循環(huán)。PCR產(chǎn)物進(jìn)行2%瓊脂糖凝膠電泳后進(jìn)行灰度值分析。

1.2.5Western blot檢測(cè)c-myc蛋白表達(dá)水平在轉(zhuǎn)染后72 h取各組蛋白樣品,BCA法測(cè)定蛋白濃度,變性后進(jìn)行十二烷基硫酸鈉-聚丙烯酰胺凝膠(SDS-PAGE)電泳,半干轉(zhuǎn)至聚偏氟乙烯(PVDF)膜,以濃度為1∶1 500的一抗4 ℃過夜封閉、1∶2 000的二抗37 ℃封閉1 h,TBST洗膜后滴加ECL發(fā)光液顯影,用灰度值分析條帶。內(nèi)參為β-actin。

1.2.6CCK-8法檢測(cè)細(xì)胞增殖取轉(zhuǎn)染24 h后各組細(xì)胞,胰酶消化后接種到96孔板中,每孔1 000個(gè)細(xì)胞,培養(yǎng)液200 μL,每組接種4孔,分別于1～4 d檢測(cè)細(xì)胞增殖情況。檢測(cè)時(shí)每孔加CCK-8試劑20 μL,培養(yǎng)1 h后用酶標(biāo)儀檢測(cè)各孔450 nm吸光度(A)值,繪制生長(zhǎng)曲線圖。每組設(shè)3個(gè)復(fù)孔,重復(fù)3次。

2　結(jié)　　果

2.1重組質(zhì)粒雙酶切及測(cè)序驗(yàn)證重組質(zhì)粒經(jīng)EcoRⅠ和BamHⅠ雙酶切后,可見質(zhì)粒大片段和插入的基因小片段兩條條帶(圖1),其中小片段位于300 bp與500 bp之間,與pre-miRNA-21的PCR產(chǎn)物大小一致。最終測(cè)序證實(shí)pre-miRNA-21基因片段已經(jīng)插入pmR-mCherry質(zhì)粒中,pmR-21重組載體構(gòu)建成功。

M：DNA分子標(biāo)記物；A：未酶切重組質(zhì)粒；B：酶切后重組質(zhì)粒；C：未酶切空質(zhì)粒；D：酶切后空質(zhì)粒。

圖1重組載體雙酶切鑒定

2.2轉(zhuǎn)染24 h后熒光顯微鏡下觀察載體熒光蛋白表達(dá)效果各組細(xì)胞形態(tài)正常,pmR-21重組載體組和空載體組觀察到紅色熒光，空白組無熒光，見圖2。流式細(xì)胞術(shù)檢測(cè)轉(zhuǎn)染效率為57.14%。

圖2　　轉(zhuǎn)染24 h后細(xì)胞熒光表達(dá)情況(×50)

圖3　　各組細(xì)胞miRNA-21相對(duì)表達(dá)量

A：c-myc mRNA凝膠電泳圖；1：為實(shí)驗(yàn)組；2：為空載體組；3:為空白組；4:D為陽性對(duì)照組；M：為蛋白分子標(biāo)記物；B：為c-myc mRNA表達(dá)分析圖。

圖4各組細(xì)胞c-myc mRNA表達(dá)量

2.3qPCR檢測(cè)miRNA-21表達(dá)量以空白組miRNA-21校正后,空載體組與空白組表達(dá)量差異無統(tǒng)計(jì)學(xué)意義(P>0.05),實(shí)驗(yàn)組miRNA-21表達(dá)量高于空白組及空載體組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見圖3。

2.4c-myc mRNA表達(dá)水平檢測(cè)實(shí)驗(yàn)組在轉(zhuǎn)染24 h后c-myc mRNA表達(dá)量高于空白組及空載體組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見圖4。

2.5c-myc蛋白表達(dá)水平檢測(cè)Western blot顯示,與空載體組和空白組比較，實(shí)驗(yàn)組在轉(zhuǎn)染72 h后表達(dá)升高(P<0.05),與該基因mRNA檢測(cè)結(jié)果一致，見圖5。

2.6Hepg2.2.15細(xì)胞生長(zhǎng)曲線鋪板48 h后,實(shí)驗(yàn)組細(xì)胞生長(zhǎng)速度快于空載體組和空白組(P<0.05),見圖6。

A:c-myc蛋白Wester blot圖；1為實(shí)驗(yàn)組；2為空載體組；3為空白組；4為陽性對(duì)照組；B：c-myc蛋白表達(dá)分析圖。

圖5各組細(xì)胞c-myc蛋白表達(dá)情況

圖6　　各組細(xì)胞生長(zhǎng)曲線圖

3　討　　論

miRNA-21在肝臟生理和病理過程起到廣泛而復(fù)雜的作用。研究發(fā)現(xiàn),miRNA-21參與肝臟損傷的修復(fù)過程，在急性肝衰竭、部分肝切除后miRNA-21表達(dá)升高,促進(jìn)正常肝細(xì)胞再生[11]；miRNA-21在酒精性肝損傷中起保護(hù)作用,抑制肝細(xì)胞凋亡[12]。同時(shí)miRNA-21也參與肝臟疾病的致病過程。目前研究發(fā)現(xiàn)的miRNA-21作用靶點(diǎn)多是腫瘤抑制基因。高表達(dá)的miRNA-21抑制促凋亡蛋白PTEN、Fas-L的表達(dá)[5-6],抑制HCC細(xì)胞凋亡；降低PDCD4的表達(dá),通過 miRNA-21-PDCD4-AP-1反饋回路促進(jìn)HCC細(xì)胞的遷移和侵襲；增強(qiáng)PI3K信號(hào)通路的傳導(dǎo),促進(jìn)癌細(xì)胞增殖等[13]。在HBV相關(guān)性HCC中,HBx蛋白被認(rèn)為是HBV持續(xù)感染和肝細(xì)胞癌變的重要因素[14-16]。研究發(fā)現(xiàn),HBx使miRNA-21表達(dá)升高,而miRNA-21通過IL-6途徑上調(diào)HBx的表達(dá),二者相互促進(jìn),促進(jìn)癌細(xì)胞增殖[6,8]。

c-myc是重要的癌基因之一,其異常表達(dá)在包括HCC等多種癌細(xì)胞中發(fā)現(xiàn),參與細(xì)胞癌變、增殖、抑制凋亡等過程[8,17-19]。本實(shí)驗(yàn)通過構(gòu)建miRNA-21真核過表達(dá)載體證實(shí),Hepg2.2.15細(xì)胞在轉(zhuǎn)染該載體后miRNA-21的表達(dá)顯著升高,c-myc基因的表達(dá)隨之升高，細(xì)胞增殖增快。因此,在HBV感染細(xì)胞模型中,miRNA-21可上調(diào)c-myc基因的表達(dá),促進(jìn)癌細(xì)胞增殖。本實(shí)驗(yàn)構(gòu)建的miRNA-21真核過表達(dá)載體可為今后進(jìn)一步實(shí)驗(yàn)奠定基礎(chǔ)。

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Construction of human microRNA-21 eukaryotic overexpression vector and its up-regulation of c-myc gene expression in HepG2.2.15 cells*

LinYongmin1,RenGuangli1,ZhangWeiyun2,CaiQiyin1,XieCong1,MaHenghao1

(1.DepartmentofPediatric;2.DepartmentofClinicalLaboratory,AffiliatedGeneralHospitalofGuangzhouMilitaryCommand,SouthernMedicalUniversity,Guangzhou,Guangdong510010,China)

ObjectiveTo construct the miRNA-21 eukaryotic overexpression vector pmR-21 and to explore its regulation effect on the expression of c-myc gene in HepG2.2.15 cells.MethodsThe miRNA-21 precursor gene fragment pre-miRNA-21 was amplified by PCR,then connected to the pmR-mCherry plasmid vector after double enzyme digestion，the accuracy of the recombinant vector was verified by double enzyme digestion and sequencing;then the recombinant vector was transfected into HepG2.2.15 cells,the fluorescent protein expression was observed under the fluorescence microscopy at 24 h and the transfection efficiency was detected by flow cytometry；the expression of miRNA-21 was evaluated by real-time quantitative PCR；at 72 h after transfection,the expression levels of c-myc gene were detected by RT-PCR and Western blot;CCK-8 was used to detect the cell proliferation in each group.ResultsThe double enzyme digestion and Western blot verified that the target gene fragment was inserted into the pmR-mCherry vector；at 24 h after transfection,intracellular strong fluorescence was seen,the transfection efficiency was higher than 50%;miRNA-21 expression level of the pmR-21 recombinant vector group was significantly increased；c-myc gene expression was increased in the pmR-21 recombinant vector group at 72 h after transfection,the cell proliferation in the pmR-21 recombinant group was faster than that in the control group(P<0.05).ConclusionThe pmR-21 eukaryotic overexpression vector is successfully constructed,this recombinant vector can express miRNA-21 stably;miRNA-21 can up-regulate c-myc gene expression，c-myc gene is one of miR-21′s targets for playing a cancer-promoting action.

microRNAs-21;genetic vectors;genes,c-myc;HBV related liver neoplasms;HepG2.2.15 cells

10.3969/j.issn.1671-8348.2016.12.006

廣州市科技計(jì)劃基金資助項(xiàng)目(2013J4100116)。作者簡(jiǎn)介：林永敏(1988-)，在讀碩士，主要從事兒科病毒感染相關(guān)疾病的研究。

R512.62

A

1671-8348(2016)12-1601-04

2015-11-08

2015-12-16)