人microRNA-21真核過表達載體的構建及其在HepG2.2.15細胞中上調c-myc基因的表達*

林永敏,任廣立,張衛云,蔡啟茵,謝　聰,馬恒顥

(南方醫科大學附屬廣州軍區廣州總醫院：1.兒科；2.檢驗科,廣州 510010)

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論著·基礎研究

人microRNA-21真核過表達載體的構建及其在HepG2.2.15細胞中上調c-myc基因的表達*

林永敏1,任廣立1,張衛云2,蔡啟茵1,謝聰1,馬恒顥1

(南方醫科大學附屬廣州軍區廣州總醫院：1.兒科；2.檢驗科,廣州 510010)

目的構建人微小RNA-21(microRNA-21,miRNA-21)真核過表達載體pmR-21,探討其在HepG2.2.15細胞中對c-myc基因表達的調控作用。方法PCR擴增miRNA-21的前體基因片段(pre-miRNA-21),雙酶切后連接到pmR-mCherry載體上,通過雙酶切和測序驗證重組載體的準確性；將重組載體轉染到HepG2.2.15細胞中為實驗組，另設空載體組(轉染pmR-mCherry空質粒組)，空白組(未轉染組)，陽性對照組(HepG2細胞),24 h后觀察載體熒光蛋白表達情況,流式細胞術檢測轉染效率,實時熒光定量PCR評估miRNA-21的表達情況；轉染72 h后,RT-PCR和Western blot檢測c-myc mRNA及蛋白表達水平；CCK-8法檢測各組細胞增殖情況。結果經雙酶切和測序驗證,目的基因片段插入載體中；實驗組及空載體組轉染24 h后細胞內可見強熒光,轉染效率大于50%；實驗組細胞miRNA-21表達較空載體組、空白組水平升高；轉染72 h后實驗組細胞c-myc mRNA表達較空載體組、空白組升高；實驗組細胞增殖快于空載體組及空白組，差異有統計學意義(P<0.05)。結論成功構建miRNA-21真核過表達載體pmR-21,該重組載體可穩定表達miRNA-21；miRNA-21可上調c-myc基因的表達,c-myc基因是miRNA-21發揮促癌作用的靶點之一。

微RNA-21;遺傳載體;基因,c-myc;乙型肝炎相關性肝腫瘤;HepG2.2.15細胞

微小RNA(microRNA，miRNA)是一類長度約22 nt的內源性非編碼RNA,可以在轉錄后期作用于特定的mRNA,調控基因的表達。miRNA除了參與正常的生理過程外,還在感染、腫瘤發生發展、心腦血管疾病等方面發揮重要的作用[1],為相關疾病的診治提供新的突破點。miRNA-21是近年來發現的一種充當促癌基因作用的miRNA,參與多種腫瘤細胞的增殖、凋亡和侵襲過程[2-3],尤為特殊的是miRNA-21在與HBV、EB等病毒感染相關的癌癥中起重要作用[4-5]。研究表明,原發性肝癌(hepatocelluar carcinoma,HCC)患者體內的miRNA-21表達升高,miRNA-21與乙型肝炎病毒(HBV)HBx蛋白相互促進,在HCC發生和HBV感染中發揮重要作用[6-7]。c-myc基因是較早發現的癌基因之一,可與DNA結合,參與DNA修復,在癌細胞的分化、增殖、凋亡過程起重要作用[8-10]。本研究通過構建miRNA-21真核過表達載體,進一步探索miRNA-21在HBV相關性HCC中的作用。

1　材料與方法

1.1材料HepG2.2.15細胞、HepG2細胞、大腸埃希菌DH5α由廣州軍區廣州總醫院醫學實驗科保存。DNA提取試劑盒、PCR產物純化試劑盒、CCK-8購于碧云天公司；DMEN高糖培養基購于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頸環引物由廣州銳博生物科技有限公司合成,PCR酶、限制性內切酶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,去內毒素質粒小提試劑盒購于Omega公司。轉染試劑Lipofectamine2000、 Trizol試劑、逆轉錄試劑盒、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。其他試劑均為國內分析純。熒光定量PCR儀購于Qiagen公司,流式細胞儀購于Beckman公司。

1.2方法

1.2.1pmR-21真核過表達載體的構建通過miRBase數據庫查找pre-miRNA-21序列及其上下游各100 bp側翼序列,用Oligo7軟件設計引物。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′(最前端序列護堿基,下劃線序列分別為EcoRⅠ和BamHⅠ酶切位點),擴增片段長度為328 bp。 提取HepG2.2.15細胞的DNA作為模板,用PCR擴增miRNA-21前體序列;反應條件為：94 ℃預變性2 min；94 ℃ 30 s,56 ℃ 30 s,72 ℃ 30 s,30個循環。純化后的pre-miRNA-21和pmR-mCherry質粒雙酶切后用T4連接酶于16 ℃過夜連接。將連接后的產物轉化感受態大腸埃希菌DH5α,進行卡那霉素抗性平板篩選。16 h后挑取陽性菌落搖菌擴大培養,提取質粒進行雙酶切,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行測序作鑒定。

1.2.2HepG2.2.15細胞和HepG2細胞培養及pmR-21重組載體轉染HepG2.2.15細胞培養于含15% FBS的高糖DMEM培養液中(同時添加200 mg/L的G418、10 g/L L-谷氨酰胺及100 000 IU/L青、鏈霉素),HepG2細胞培養于含10% FBS和100 000 IU/L青、鏈霉素的高糖DMEM培養液中,置于37 ℃含5% CO2的培養箱內。細胞穩定傳代3次后,接種到6孔板中,每孔細胞密度為4×105個。種板24 h后(匯合度約50%)取去經內毒素提取的pmR-21重組載體和pmR-mCherry空質粒按試劑盒操作說明進行轉染,分為實驗組(轉染pmR21重組載體)，空載體組(轉染pmR-mCherry空質粒)、空白組(未轉染質粒)、陽性對照組(HepG2細胞)。每孔轉染試劑Lipofectamine2000 5 μL,質粒2.5 μg，轉染24 h后熒光顯微鏡下觀察載體上熒光蛋白表達效果。

1.2.3miRNA-21 qPCR相對定量檢測轉染24 h后,Trizol法提取各組細胞總RNA,鑒定其完整性和純度后以總RNA為模板,設計針對miRNA-21成熟序列的特異性RT-PCR莖環引物,逆轉錄后進行qPCR,以U6為內參照,反應條件為95 ℃預變性20 s；95 ℃ 10 s，60 ℃ 20 s，70 ℃ 10 s，共40個循環。采用2-ΔΔct相對定量分析方法分析各組細胞miRNA-21的相對表達量。

1.2.4c-myc mRNA水平的RT-PCR檢測以總RNA為模板,逆轉錄成cDNA,以此為模板進行PCR擴增。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′，產物大小為380 bp。內參為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′,產物大小為540 bp。反應條件均為：94℃預變性2 min；94 ℃ 30 s,58 ℃ 30 s,72 ℃ 30 s，共30個循環。PCR產物進行2%瓊脂糖凝膠電泳后進行灰度值分析。

1.2.5Western blot檢測c-myc蛋白表達水平在轉染后72 h取各組蛋白樣品,BCA法測定蛋白濃度,變性后進行十二烷基硫酸鈉-聚丙烯酰胺凝膠(SDS-PAGE)電泳,半干轉至聚偏氟乙烯(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發光液顯影,用灰度值分析條帶。內參為β-actin。

1.2.6CCK-8法檢測細胞增殖取轉染24 h后各組細胞,胰酶消化后接種到96孔板中,每孔1 000個細胞,培養液200 μL,每組接種4孔,分別于1～4 d檢測細胞增殖情況。檢測時每孔加CCK-8試劑20 μL,培養1 h后用酶標儀檢測各孔450 nm吸光度(A)值,繪制生長曲線圖。每組設3個復孔,重復3次。

2　結　　果

2.1重組質粒雙酶切及測序驗證重組質粒經EcoRⅠ和BamHⅠ雙酶切后,可見質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產物大小一致。最終測序證實pre-miRNA-21基因片段已經插入pmR-mCherry質粒中,pmR-21重組載體構建成功。

M：DNA分子標記物；A：未酶切重組質粒；B：酶切后重組質粒；C：未酶切空質粒；D：酶切后空質粒。

圖1重組載體雙酶切鑒定

2.2轉染24 h后熒光顯微鏡下觀察載體熒光蛋白表達效果各組細胞形態正常,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。流式細胞術檢測轉染效率為57.14%。

圖2　　轉染24 h后細胞熒光表達情況(×50)

圖3　　各組細胞miRNA-21相對表達量

A：c-myc mRNA凝膠電泳圖；1：為實驗組；2：為空載體組；3:為空白組；4:D為陽性對照組；M：為蛋白分子標記物；B：為c-myc mRNA表達分析圖。

圖4各組細胞c-myc mRNA表達量

2.3qPCR檢測miRNA-21表達量以空白組miRNA-21校正后,空載體組與空白組表達量差異無統計學意義(P>0.05),實驗組miRNA-21表達量高于空白組及空載體組，差異有統計學意義(P<0.05)。見圖3。

2.4c-myc mRNA表達水平檢測實驗組在轉染24 h后c-myc mRNA表達量高于空白組及空載體組，差異有統計學意義(P<0.05)。見圖4。

2.5c-myc蛋白表達水平檢測Western blot顯示,與空載體組和空白組比較，實驗組在轉染72 h后表達升高(P<0.05),與該基因mRNA檢測結果一致，見圖5。

2.6Hepg2.2.15細胞生長曲線鋪板48 h后,實驗組細胞生長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為實驗組；2為空載體組；3為空白組；4為陽性對照組；B：c-myc蛋白表達分析圖。

圖5各組細胞c-myc蛋白表達情況

圖6　　各組細胞生長曲線圖

3　討　　論

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

c-myc是重要的癌基因之一,其異常表達在包括HCC等多種癌細胞中發現,參與細胞癌變、增殖、抑制凋亡等過程[8,17-19]。本實驗通過構建miRNA-21真核過表達載體證實,Hepg2.2.15細胞在轉染該載體后miRNA-21的表達顯著升高,c-myc基因的表達隨之升高，細胞增殖增快。因此,在HBV感染細胞模型中,miRNA-21可上調c-myc基因的表達,促進癌細胞增殖。本實驗構建的miRNA-21真核過表達載體可為今后進一步實驗奠定基礎。

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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

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

R512.62

A

1671-8348(2016)12-1601-04

2015-11-08

2015-12-16)