miR-155對(duì)HBV蛋白表達(dá)的抑制作用及機(jī)制

蔡啟茵，任廣立，張衛(wèi)云，馮宇鵬，馬恒顥

miR-155對(duì)HBV蛋白表達(dá)的抑制作用及機(jī)制

蔡啟茵，任廣立，張衛(wèi)云，馮宇鵬，馬恒顥

目的觀察miR-155對(duì)SOCS1 mRNA及蛋白水平的影響，探究miR-155對(duì)HBsAg、HBeAg表達(dá)的抑制作用。方法從HepG2.2.15細(xì)胞中提取基因組，經(jīng)PCR擴(kuò)增得到miR-155前體序列，純化并連接入pmR-mCherry載體，構(gòu)建重組質(zhì)粒pmiR-155，經(jīng)去內(nèi)毒素后轉(zhuǎn)染至HepG2.2.15細(xì)胞。將細(xì)胞分為重組組(轉(zhuǎn)染pmiR-155質(zhì)粒)、空載組(轉(zhuǎn)染pmR-mCherry質(zhì)粒)、轉(zhuǎn)染試劑組、空白組。采用熒光實(shí)時(shí)定量PCR檢測各組細(xì)胞內(nèi)miR-155的表達(dá)，RT-PCR檢測各組細(xì)胞SOCS1 mRNA的表達(dá)，Western blotting檢測各組細(xì)胞SOCS1 蛋白的表達(dá)，ELISA檢測各組細(xì)胞HBsAg、HBeAg的表達(dá)。結(jié)果熒光實(shí)時(shí)定量PCR結(jié)果顯示，以空白組細(xì)胞內(nèi)miR-155的表達(dá)量為基準(zhǔn)，重組組miR-155表達(dá)量(519.43±52.10)明顯高于空載組(24.24±16.70)及轉(zhuǎn)染試劑組(35.04±26.09，P＜0.05)；RT-PCR結(jié)果顯示，重組組SOCS1 mRNA表達(dá)量(0.63±0.91)顯著低于空白組(P＜0.05)；Western blotting結(jié)果顯示，重組組SOCS1蛋白表達(dá)量顯著低于空白組。以空白組的表達(dá)量為基準(zhǔn)，重組組HBsAg和HBeAg蛋白表達(dá)的抑制率分別為55.62%±3.77%和47.87%±2.46%(P＜0.01)。結(jié)論過表達(dá)的miR-155可抑制細(xì)胞內(nèi)SOCS1和HBV蛋白的表達(dá)。

乙型肝炎，慢性；微RNAs；乙型肝炎表面抗原；乙型肝炎核心抗原

乙型肝炎病毒(hepatitis B virus，HBV)呈全球性流行，每年約100萬人死于HBV相關(guān)的肝衰竭或原發(fā)性肝癌。干擾素和核苷類似物是目前抗HBV的主要藥物[1]，可在一定程度上抑制HBV的復(fù)制，減輕肝損害，但無法徹底清除體內(nèi)的病毒[2]。最近研究發(fā)現(xiàn)，miRNAs不僅可干擾病毒聚合酶或相關(guān)蛋白合成，而且可改變抗原提呈，從而達(dá)到抑制病毒復(fù)制的作用[3]。miR-155被認(rèn)為是新興的炎癥因子，可調(diào)控免疫系統(tǒng)發(fā)育和應(yīng)答、抗病毒感染等生物進(jìn)程[4]。研究指出，miR-155通過抑制細(xì)胞因子信號(hào)轉(zhuǎn)導(dǎo)抑制因子1(suppressor of cytokine signaling1，SOCS1)調(diào)節(jié)體內(nèi)的干擾素信號(hào)通路，增強(qiáng)抗病毒效應(yīng)[5]。然而，目前尚無證據(jù)證明特定的miRNA可通過調(diào)控信號(hào)通路來達(dá)到干擾HBV表達(dá)的作用。本研究通過構(gòu)建miR-155真核過表達(dá)載體，體外轉(zhuǎn)染HepG2.2.15細(xì)胞，從蛋白和基因水平評(píng)估m(xù)iR-155 對(duì)SO CS1表達(dá)的影響，同時(shí)觀察細(xì)胞所分泌HBsAg、HBeAg水平的改變，為進(jìn)一步研究miR-155 在HBV慢性感染中抗病毒作用提供線索。

1 材料與方法

1.1 主要材料及試劑 pmR-mCherry質(zhì)粒由美國Dr. Keith Peden惠贈(zèng)。大腸埃希菌菌株DH5α及肝癌細(xì)胞株HepG2.2.15細(xì)胞由本院醫(yī)學(xué)實(shí)驗(yàn)科保存。PCRTaq、QuickCutEcoR Ⅰ、QuickCutBamH Ⅰ、T4DNA連接酶及DNA切膠回收試劑盒均購自TaKaRa公司；去內(nèi)毒素質(zhì)粒小提中量試劑盒購自O(shè)mega公司；Trizol總RNA提取試劑盒、脂質(zhì)體Lipofectamine 2000及M-MLV 1st strand Kit購自Invitrogen公司；抗SOCS1多克隆抗體購自Cell Signaling公司；辣根過氧化物酶標(biāo)記的山羊抗兔IgG(二抗，1:4 000)購自北京中杉金橋科技有限公司；乙型肝炎病毒表面抗原診斷試劑盒、乙型肝炎病毒e抗原診斷試劑盒購自深圳華康生物醫(yī)學(xué)有限公司；用于熒光定量PCR檢測的miR-155及U6特異性引物均為廣州銳博生物科技有限公司產(chǎn)品。

1.2 載體構(gòu)建 根據(jù)miRBase及GenBank數(shù)據(jù)庫查找得到成熟miR-155莖環(huán)序列及miR-155前體側(cè)翼序列，利用Primer 5.0及Oligo 7設(shè)計(jì)引物(表1)。以HepG2.2.15細(xì)胞基因組為模板PCR擴(kuò)增得到miR-155前體序列，經(jīng)EcoRⅠ和BamHⅠ雙酶切，退火連接到pmR-mCherry載體，克隆轉(zhuǎn)化、搖菌、測序鑒定后，得到重構(gòu)質(zhì)粒pmiR-155。去內(nèi)毒素提取重組質(zhì)粒用于轉(zhuǎn)染實(shí)驗(yàn)[6]。

1.3 HepG2.2.15細(xì)胞培養(yǎng)及轉(zhuǎn)染 將HepG2.2.15細(xì)胞培養(yǎng)于含200mg/L G418、100g/L FBS、10萬U/L青鏈霉素、10g/L L-谷氨酰胺的高糖DMEM培養(yǎng)液中，于37℃、5%CO2培養(yǎng)箱中培養(yǎng)。細(xì)胞傳代培養(yǎng)3次后消化接種至6孔板，4.8×105個(gè)細(xì)胞/孔，培養(yǎng)24h后待細(xì)胞融合度達(dá)80%時(shí)轉(zhuǎn)染(轉(zhuǎn)染時(shí)不加雙抗)[7]。設(shè)重組組(轉(zhuǎn)染pmiR-155質(zhì)粒)、空載組(轉(zhuǎn)染pmR-mCherry質(zhì)粒)、轉(zhuǎn)染試劑組、空白組。轉(zhuǎn)染試劑Lipofectamine 2000(μl)與質(zhì)粒(μg)的比例為3:1。

表1 引物序列Tab.1 Primer sequence

1.4 熒光實(shí)時(shí)定量PCR檢測各組miR-155表達(dá) 應(yīng)用Trizol法提取各組細(xì)胞中總RNA，經(jīng)紫外分光光度法測定濃度及純度后，采用莖環(huán)法[8]反轉(zhuǎn)錄得到cDNA，進(jìn)行熒光實(shí)時(shí)定量PCR反應(yīng)。反應(yīng)程序?yàn)?5℃ 10s，60℃ 20s，70℃ 10s，循環(huán)40次。所得數(shù)據(jù)采用2-ΔΔCt表示，并評(píng)估樣本間差異。

1.5 RT-PCR檢測各組SOCS1 mRNA表達(dá) 應(yīng)用Trizol法提取各組總RNA，反轉(zhuǎn)錄得到cDNA，行RT-PCR。反應(yīng)程序：94℃ 30s，57℃ 30s, 72℃30s，循環(huán)30次。GAPDH序列的PCR反應(yīng)程序除退火溫度改為56℃外，其余與SOCS1相同。所得產(chǎn)物經(jīng)凝膠電泳及測序鑒定。應(yīng)用Gel-pro Analyzer分析各組mRNA的相對(duì)表達(dá)量。

1.6 Western blotting檢測各組SOCS1蛋白表達(dá)轉(zhuǎn)染72h后收集各組細(xì)胞，用預(yù)冷PBS沖洗3遍，每孔加入100μl裂解液，冰上放置30min后移入離心管，4℃、12 000r/min離心30min，收集上清液。BCA法測定蛋白濃度。加入50μl 2×上樣緩沖液混勻，99℃煮10min，取50μg樣品行12% SDS-PAGE凝膠電泳。電泳結(jié)束后用半轉(zhuǎn)移儀將蛋白從凝膠轉(zhuǎn)移到PVF膜，封閉液封閉，先后與抗SOCS1多克隆抗體(1:1 000稀釋)和辣根過氧化物酶標(biāo)記的山羊抗兔IgG(1:4 000稀釋)孵育，洗膜，加入化學(xué)發(fā)光劑ECL，在Sage Creation凝膠成像儀中曝光顯影。

1.7 HBsAg和HBeAg蛋白的檢測 收集細(xì)胞轉(zhuǎn)染24、48h和72h的培養(yǎng)上清液，1000r/min離心5min，吸取上清，應(yīng)用ELISA法檢測各組細(xì)胞HBsAg和HBeAg的分泌量。嚴(yán)格按試劑盒說明操作，結(jié)果用吸光度(A)值表示。

1.8 統(tǒng)計(jì)學(xué)處理 采用SPSS 17.0軟件進(jìn)行統(tǒng)計(jì)分析，所有數(shù)據(jù)均以表示，組間均數(shù)比較采用One-Way ANOVA法，P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 miR-155真核過表達(dá)載體的構(gòu)建及鑒定 經(jīng)測序鑒定，PCR擴(kuò)增的人miR-155前體序列連入pmR-mCherry真核過表達(dá)載體，序列正確，沒有堿基丟失、替換等，重組質(zhì)粒構(gòu)建成功(圖1)。

2.2 miR-155在轉(zhuǎn)染細(xì)胞中的表達(dá) 經(jīng)熒光實(shí)時(shí)定量PCR檢測，以空白組細(xì)胞內(nèi)miR-155的表達(dá)量為基準(zhǔn)，重組組miR-155表達(dá)量(519.43±52.10)明顯高于空載組(24.24±16.7)及轉(zhuǎn)染試劑組(35.04±26.09，P＜0.05)。

2.3 miR-155對(duì)SOCS1 mRNA表達(dá)的抑制作用RT-PCR檢測結(jié)果表明，以GAPDH表達(dá)為參照，空載組(0.95±0.79)、轉(zhuǎn)染試劑組(0.94±0.52)與空白組SOCS1 mRNA的表達(dá)量無明顯差異，重組組SOCS1 mRNA的表達(dá)量(0.63±0.91)顯著低于空白組(P＜0.05，圖2)。

2.4 miR-155對(duì)SOCS1蛋白表達(dá)的抑制作用Western blotting檢測表明，以β-actin表達(dá)為參照，轉(zhuǎn)染72h后，空載組、轉(zhuǎn)染試劑組與空白組SOCS1蛋白表達(dá)量均無明顯差異，而重組組SOCS1蛋白表達(dá)量顯著低于空白組(P＜0.05，圖3)。

2.5 miR-155對(duì)HBsAg和HBeAg蛋白表達(dá)的抑制作用 ELISA檢測結(jié)果表明，轉(zhuǎn)染后重組組培養(yǎng)上清液的HBsAg、HBeAg表達(dá)量明顯受到抑制，尤其是轉(zhuǎn)染后48h。空載組、轉(zhuǎn)染試劑組對(duì)蛋白的表達(dá)幾乎沒有影響。轉(zhuǎn)染48h時(shí)，重組組HBsAg和HBeAg蛋白表達(dá)的抑制率分別為55.62%±3.77%和47.87%±2.46%，與其他3組相比差異均有統(tǒng)計(jì)學(xué)意義(P<0.05，圖4)。

圖1 重組質(zhì)粒pmiR-155部分核酸序列測序結(jié)果Fig.1 Sequencing results of partial nucleic acid of recombinant plasmid pmiR-155

圖2 RT-PCR檢測轉(zhuǎn)染后各組細(xì)胞內(nèi)SOCS1 mRNA表達(dá)Fig.2 Expression of SOCS1 mRNA in each group (RT-PCR) M. DL2000; 1. pmiR-155 plasmid group; 2. pmR-mCherry plasmid group; 3. Reagent group; 4. Blank group

圖3 Western blotting檢測轉(zhuǎn)染后各組細(xì)胞SOCS1蛋白表達(dá)Fig.3 Expression of SOCS1 proteins in each group (Western blotting)1. pmiR-155 plasmid group; 2. pmR-mCherry plasmid group; 3. Reagent group; 4. Blank group

圖4 ELISA檢測轉(zhuǎn)染后各組細(xì)胞HBsAg(A)和HBeAg(B)蛋白表達(dá)Fig.4 Expression of HBsAg (A) and HBeAg (B) proteins in each group (ELISA)

3 討 論

miRNA可通過降解靶細(xì)胞mRNAs或抑制蛋白翻譯來調(diào)控轉(zhuǎn)錄后期的基因表達(dá)[9-10]。miR-155是21nt的小分子RNA，在抗病毒感染、抗腫瘤和免疫調(diào)節(jié)中發(fā)揮重要作用[11-13]。Banerjee等[14]研究發(fā)現(xiàn)，miR-155通過激活I(lǐng)FN-γ信號(hào)通路誘導(dǎo)Th1細(xì)胞分化，活化CD4+T細(xì)胞來調(diào)節(jié)免疫系統(tǒng)穩(wěn)定。Rodriguez等[15]證實(shí)miR-155可作用于IL-2信號(hào)途徑，抑制SOCS1的表達(dá)，從而影響調(diào)節(jié)性T細(xì)胞的分化能力。此外，研究已證實(shí)了SOCS1是miR-155的靶位點(diǎn)，可負(fù)反饋性阻斷細(xì)胞因子信號(hào)轉(zhuǎn)導(dǎo)過程(如干擾素、IL-10、IL-12等信號(hào)通路)[16]。miR-155抑制SOCS1表達(dá)后可削弱其對(duì)相關(guān)細(xì)胞因子的負(fù)反饋調(diào)節(jié)，促進(jìn)細(xì)胞因子的分泌，增強(qiáng)抗病毒作用。然而，在HBV慢性感染中，miR-155對(duì)SOCS1的抑制及干擾HBV蛋白分泌的作用及機(jī)制尚未明確。

本研究所選用的H BV慢性感染細(xì)胞模型HepG2.2.15細(xì)胞能持續(xù)分泌HBV蛋白及表達(dá)HBV DNA，是探討miR-155在HBV慢性感染中所發(fā)揮作用的較佳細(xì)胞模型。目前尚未見在HepG2.2.15細(xì)胞中研究miR-155對(duì)SOCS1表達(dá)影響的報(bào)道。本研究成功構(gòu)建了人miR-155真核過表達(dá)載體，并轉(zhuǎn)染至HepG2.2.15細(xì)胞，結(jié)果顯示，SOCS1的mRNA和蛋白表達(dá)量隨著細(xì)胞內(nèi)的miR-155表達(dá)上調(diào)而下降，且轉(zhuǎn)染了人miR-155過表達(dá)載體的HepG2.2.15細(xì)胞HBsAg、HBeAg分泌量也明顯降低。

綜上，本研究證實(shí)了在HBV慢性感染中，過表達(dá)miR-155可抑制SOCS1及HBV蛋白的表達(dá)，其作用機(jī)制很有可能是由于過表達(dá)的miR-155下調(diào)了SOCS1的表達(dá)，削弱了其負(fù)反饋調(diào)節(jié)，增強(qiáng)了機(jī)體的抗病毒作用，從而抑制HBsAg、HBeAg的表達(dá)。本研究結(jié)果為進(jìn)一步探討miR-155在HBV慢性感染中的免疫調(diào)節(jié)及抗病毒作用奠定了基礎(chǔ)，并為miRNAs治療HBV提供了實(shí)驗(yàn)證據(jù)。

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Inhibitory effect of miR-155 on expression of hepatitis B virus and its mechanism

CAI Qi-yin, REN Guang-li*, ZHANG Wei-yun, FENG Yu-peng, MA Heng-hao
Department of Pediatrics, General Hospital of Guangzhou Command, Guangzhou 510010, China
*< class="emphasis_italic">Corresponding author, E-mail: guangliren@hotmail.com

, E-mail: guangliren@hotmail.com
This work was supported by the Science and Technology Foundation of Guangzhou (2013J4100116)

ObjectiveTo investigate the mechanism of miR-155 regulating SOCS1 in the inhibition of HBsAg and HBeAg.MethodsThe pre-miR-155 was amplified from total DNA of HepG2.2.15 by PCR. The target gene fragment was digested and cloned into the pmR-mCherry plasmid. PmiR-155 was transfected into HepG2.2.15 cells (recombinant group) by liposome-mediated method. The empty plasmid, the reagent group and untreated cells were set as control. Firstly the expression of miR-155 was detected by the real-time quantitative PCR. Secondly, the expression of SOCS1 mRNA was detected by RT-PCR, and then the expression of SOCS1 protein was determined by Western blotting. Finally, the expression of HBsAg and HBeAg was determined by ELISA.ResultsThe pmiR-155 eukaryotic over-expression vector was successfully constructed. MiR-155 level of HepG2.2.15 cells which was transfected with the recombinant plasmid (519.43±52.10) was obviously higher than those of the empty plasmid (24.24±16.70) and reagent groups (35.04±26.09,P<0.05). RT-PCR showed the expression of SOCS1 mRNA was lower in recombinant group than in untreated group. The expression of SOCS1 protein markedly decreased as shown with Western blotting. Over-expression of miR-155 could inhibit the expressions of HBsAg and HBeAg (55.62±3.77)% and (47.87±2.46)% (P<0.01) respectively.ConclusionsOver-expression of miR-155 can down regulate the expression of SOCS1, and inhibit the expressions of HBsAg and HBeAg.

hepatitis B, chronic; microRNAs; hepatitis B surface antigens; hepatitis B core antigens

R512.6

A

0577-7402(2015)11-0902-04

10.11855/j.issn.0577-7402.2015.11.09

2015-05-18；

2015-07-29)

(責(zé)任編輯：熊曉然)

廣州市科技計(jì)劃項(xiàng)目(2013J4100116)

蔡啟茵，醫(yī)學(xué)碩士。主要從事兒科病毒感染性疾病的研究

510010 廣州 廣州軍區(qū)廣州總醫(yī)院兒科(蔡啟茵、任廣立、張衛(wèi)云、馮宇鵬、馬恒顥)

任廣立，E-mail: guangliren@hotmail.com