miRNA-10a對(duì)小鼠肝纖維化細(xì)胞增殖及TGFβ1/Smads信號(hào)轉(zhuǎn)導(dǎo)通路表達(dá)的影響

鄧國(guó)孫，李鎮(zhèn)伽

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·基礎(chǔ)醫(yī)學(xué)· ·論著·

miRNA-10a對(duì)小鼠肝纖維化細(xì)胞增殖及TGFβ1/Smads信號(hào)轉(zhuǎn)導(dǎo)通路表達(dá)的影響

鄧國(guó)孫，李鎮(zhèn)伽

目的 通過(guò)觀察肝纖維化(hepatic fibrosis，HF)小鼠肝組織TGFβ1/Smads信號(hào)轉(zhuǎn)導(dǎo)通路的表達(dá)與HF進(jìn)展的關(guān)系，探討微小RNA(microRNA，miRNA)-10a對(duì)于HF的作用機(jī)制。方法 選用9周齡健康雄性小鼠(C57BL6/J)40只，按照數(shù)字表法隨機(jī)分為對(duì)照組和HF模型組(觀察組)，對(duì)照組生理鹽水5 μl/g腹腔注射，每周2次，注射8周；觀察組10% CCL4橄欖油5 μl/g腹腔注射，每周2次，注射8周，制作小鼠HF模型。RT-PCR法檢測(cè)HF細(xì)胞中miRNA-10a表達(dá)后，將觀察組HF細(xì)胞進(jìn)行培養(yǎng)及miRNA-10a模擬物轉(zhuǎn)染(轉(zhuǎn)染組)，CCK-8法檢測(cè)HF細(xì)胞增殖能力，Western blotting檢測(cè)HF細(xì)胞中TGFβ1、Smad7的表達(dá)水平。結(jié)果 與對(duì)照組比較，觀察組miRNA-10a表達(dá)水平明顯增加(P<0.05)；與對(duì)照組比較，轉(zhuǎn)染組肝細(xì)胞miRNA-10a表達(dá)水平明顯降低(P<0.05)；與對(duì)照組相比，低表達(dá)miRNA-10a明顯降低觀察組TGFβ1的表達(dá)水平，提高Smad7的表達(dá)水平(均P<0.05)。結(jié)論 小鼠HF細(xì)胞中低表達(dá)miRNA-10a，轉(zhuǎn)染miRNA-10a模擬物可明顯促進(jìn)小鼠HF細(xì)胞增殖，其作用機(jī)制是通過(guò)miRNA-10a調(diào)控TGFβ1/Smads信號(hào)轉(zhuǎn)導(dǎo)通路促進(jìn)小鼠HF。

肝纖維化；轉(zhuǎn)化生長(zhǎng)因子β1；Smad7蛋白；TGFβ1/Smads信號(hào)轉(zhuǎn)導(dǎo)通路

肝纖維化(hepatic fibrosis，HF)是機(jī)體對(duì)各種原因誘發(fā)的慢性肝細(xì)胞損傷后的一種自我修復(fù)反應(yīng)。有研究[1]顯示，HF是一個(gè)可逆的病理生理過(guò)程，消除相關(guān)致病因素或有效的早期干預(yù)治療可改善HF程度，反之則可進(jìn)展為終末失代償期肝硬化。微小RNA(microRNA，miRNA)是一類大小為18～22個(gè)核苷酸的內(nèi)源性非編碼單鏈小分子RNA，通過(guò)與靶mRNA完全或不完全互補(bǔ)配對(duì)，引起mRNA降解或翻譯抑制，從而能在轉(zhuǎn)錄后水平調(diào)控機(jī)體基因的表達(dá)[2]。有研究[3]顯示，一些miRNA在肝細(xì)胞內(nèi)表達(dá)相對(duì)豐富，并在疾病的進(jìn)展中表達(dá)有明顯差異，從而影響肝臟疾病的發(fā)生發(fā)展。也有研究[4-5]表明，miRNA-10a參與造血細(xì)胞的分化、腫瘤的發(fā)生發(fā)展、免疫功能的調(diào)節(jié)等多個(gè)病理生理過(guò)程。在博萊霉素所致的肺纖維化小鼠肺組織中，發(fā)現(xiàn)了161個(gè)差異表達(dá)的miRNA，其中miRNA-10a通過(guò)調(diào)節(jié)TGF-β信號(hào)通路參與調(diào)控纖維母細(xì)胞激活和膠原沉著[6]。有文獻(xiàn)[7]報(bào)道，肝星狀細(xì)胞(hepatic stellate cell，HSC)的激活或表型轉(zhuǎn)化為成纖維細(xì)胞是HF形成的中心環(huán)節(jié)，轉(zhuǎn)化生長(zhǎng)因子β1(transforming growth factor beta 1，TGFβ1)是公認(rèn)的最強(qiáng)致HF的細(xì)胞因子之一[8]，Smad蛋白是TGFβ1關(guān)鍵的作用底物[9]，可誘發(fā)HSC激活，啟動(dòng)膠原基因表達(dá)，從而導(dǎo)致HF的發(fā)生。TGFβ1/Smads信號(hào)轉(zhuǎn)導(dǎo)通路的激活及引起的相應(yīng)病理變化, 在HF的發(fā)生發(fā)展中具有非常重要的意義[10]。本研究探討miRNA-10a調(diào)控HF的分子機(jī)制，并將miRNA-10a作為HF藥物靶點(diǎn)，從而指導(dǎo)HF的診斷與治療。

1 材料與方法

1.1 主要材料與試劑 Pronase E購(gòu)于美國(guó)Merck公司，Collagenase II購(gòu)于美國(guó)Sigma公司，淋巴細(xì)胞分離液購(gòu)自中科院生工所，DMEM培養(yǎng)液及干粉購(gòu)于GIBCO公司，胎牛血清購(gòu)于杭州四季青公司，Interferin siRNA轉(zhuǎn)染劑購(gòu)自Polypus公司，miRNA分析系統(tǒng)購(gòu)自Applied 公司，PrimeScript RT-PCR反轉(zhuǎn)錄試劑盒購(gòu)自TOYOBO公司，miRNA-10購(gòu)于廣東銳博公司，各類單抗均購(gòu)于美國(guó)Abcam公司，細(xì)胞裂解液CLB購(gòu)自Cell Signaling T公司。

1.2 建立HF小鼠模型 選用9周齡健康雄性小鼠(C57BL6/J)40只，體質(zhì)量19～22 g，按照隨機(jī)數(shù)字表法分為對(duì)照組和HF模型組(觀察組)，對(duì)照組生理鹽水5 μl/g腹腔注射，每周2次，注射8周；觀察組10% CCL4橄欖油5 μl/g腹腔注射，每周2次，注射8周，制作小鼠HF模型。

1.3 制備標(biāo)本 實(shí)驗(yàn)小鼠禁食12 h，在乙醚麻醉下，斷頭取血，部分肝臟組織采用10%中性甲醛固定，備制病理切片，其余肝組織液氮快速冷凍，置于-80 ℃冰箱保存，備用。

1.4 HSC分離 75%酒精消毒腹部皮膚后，2%戊巴比妥鈉(40 mg/kg)小鼠腹腔注射，十字切口開腹，充分暴露小鼠心臟和下腔靜脈，18號(hào)套管針連接輸液瓶，從左心室進(jìn)針注入灌注液，下腔靜脈放血，10 ml/min流速肝臟灌流，直至肝臟變?yōu)橥咙S色，隨后采用酶灌注液繼續(xù)灌流14～18 min，直至肝臟呈現(xiàn)爛泥狀，取肝臟，生理鹽水沖洗后，剔除肝臟包膜及結(jié)締組織，剪碎后加入振蕩消化液，37 ℃振蕩20 min，200×g離心5 min，細(xì)胞懸液經(jīng)300目鋼網(wǎng)過(guò)濾后，收集于2個(gè)30 ml離心管中,1 400×g離心5 min，棄上清，D-Hanks液重懸沉淀，取上清進(jìn)行梯度分離。采用淋巴細(xì)胞分離液鋪梯度，上層加充分消化的單細(xì)胞懸液，25 ℃，1 500×g離心25 min，平拋離心后的分離細(xì)胞，小心吸出中層少量白色液體，即為HSC。DMEM制備，1 400 ×g離心10 min，洗滌2次，以1.0×106個(gè)細(xì)胞接種于100 ml塑料培養(yǎng)瓶中24 h，靜置培養(yǎng)72 h后換成含10%胎牛血清DMEM培養(yǎng)液，每3 d更換培養(yǎng)液1次。采用臺(tái)盼藍(lán)進(jìn)行染色，計(jì)算細(xì)胞成活率，以2組小鼠HSC成活率均達(dá)到90%以上表示分離成功。

1.5 Real-time PCR檢測(cè)miRNA-10a表達(dá) 采用TRIzol試劑，提取2組肝組織總RNA，利用SYBR Premix ExTaq熒光定量PCR試劑盒及LightCycler儀器，進(jìn)行操作及分析。miRNA-10a逆轉(zhuǎn)錄引物序列：5’-GTCGTATC-CAGCAGGGTCCGTATTCGCACTGGATACGACACA-3’；miRNA-10a定量引物上游序列：5’-ACGTACCTGTAGATCCG-3’，引物下游序列：5’-GTG-CAGGTCCGAGGT-3’。U6逆轉(zhuǎn)錄引物序列：5’-CGCTCACGAATTTGCGTGTAT-3’；U6定量引物上游序列：5’-CTCGCTTCGCAGCACA-3’，引物下游序列：5’-AACGCTTCACGAATTTCGT-3’。按3步法進(jìn)行DNA擴(kuò)增，反應(yīng)條件：95 ℃、20 s；94 ℃、25 s；55 ℃、15 s；75 ℃、10 s；40次循環(huán)條件：72 ℃、10 min。每次Real-timePCR被測(cè)標(biāo)本的Ct值減去對(duì)應(yīng)樣品中U6Ct值，即為ΔCt，HF細(xì)胞中miRNA-10a的表達(dá)量采用2-ΔΔCt計(jì)算，HF組織標(biāo)本中miRNA-10a表達(dá)量采用log22-ΔΔCt計(jì)算。

1.6 HF細(xì)胞培養(yǎng)和miRNA-10a模擬物轉(zhuǎn)染 小鼠HF細(xì)胞置于含10%胎牛血清的DMEM培養(yǎng)液中，37 ℃、5%CO2飽和濕度培養(yǎng)箱進(jìn)行傳代培養(yǎng)。將處于對(duì)數(shù)期生長(zhǎng)的HF細(xì)胞接種于12孔板，每孔細(xì)胞密度為3×105個(gè)，當(dāng)細(xì)胞生長(zhǎng)融合度至50%時(shí)，再將其分為2組，一組為miRNA-10a模擬物轉(zhuǎn)染組，一組為對(duì)照組，分別用含有miRNA-10a模擬物和對(duì)照miRNAOpti-MEM培養(yǎng)基轉(zhuǎn)染細(xì)胞，同時(shí)加入Interferin提高轉(zhuǎn)染率。轉(zhuǎn)染時(shí)每孔miRNA-10a模擬物或?qū)φ誱iRNAOpti-MEM的終濃度均為20 nmol/L，Interferin為4 μl，轉(zhuǎn)染72 h。本實(shí)驗(yàn)重復(fù)3次。

1.6 CCK-8法檢測(cè)HF細(xì)胞的增殖能力 根據(jù)CCK-8試劑盒說(shuō)明書操作，HF細(xì)胞轉(zhuǎn)染miRNA-10a模擬物或?qū)φ誱iRNA Opti-MEM 72 h后，將2組細(xì)胞根據(jù)每孔2×104個(gè)，接種于96孔培養(yǎng)板中，并各設(shè)置3個(gè)平行孔，3～5 h后細(xì)胞貼壁，加入100 μl 1640培養(yǎng)液、10 μl CCK-8，置于37 ℃、5%CO2培養(yǎng)箱培養(yǎng)2 h，酶標(biāo)儀測(cè)定光密度(D)值。本試驗(yàn)重復(fù)3次。

1.7 Western blotting檢測(cè)HF細(xì)胞中TGFβ1、Smad 7蛋白的表達(dá) HF細(xì)胞轉(zhuǎn)染72 h后，收集轉(zhuǎn)染組和對(duì)照組待測(cè)細(xì)胞，細(xì)胞裂解液裂解，BCA法測(cè)定蛋白濃度，取50 μg蛋白經(jīng)8% SDS-PAGE分離后，轉(zhuǎn)至PVDF膜，25 ℃封閉1 h，分別加入TGFβ1抗體(1∶2 000)、Smad 7抗體(1∶2 000)、β-actin抗體(1∶1 000)，4 ℃孵育過(guò)夜，洗膜后加過(guò)氧化物酶標(biāo)記的二抗，25 ℃孵育1h，洗膜后ECL法顯影，Gene gnome采集圖片，利用Image J軟件進(jìn)行灰度分析。本試驗(yàn)重復(fù)3次。

1.8 統(tǒng)計(jì)學(xué)處理 采用SPSS 18.0統(tǒng)計(jì)學(xué)軟件，數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示，組間比較采用t檢驗(yàn)，P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 miRNA-10a在小鼠HF中高表達(dá) Real-time PCR檢測(cè)觀察與對(duì)照組中miRNA-10a相對(duì)表達(dá)量，結(jié)果顯示，觀察組HF組織中miRNA-10a表達(dá)量明顯高于對(duì)照組[(-7.84±1.38)vs(-9.97±1.59)，P<0.05]。

2.2 轉(zhuǎn)染miRNA-10a模擬物后HF細(xì)胞miRNA-10a的表達(dá) Real-time PCR檢測(cè)轉(zhuǎn)染組及對(duì)照組中miRNA-10a的相對(duì)表達(dá)量，結(jié)果(圖1)顯示，轉(zhuǎn)染組(miRNA-10a mimics)HF細(xì)胞miRNA-10a表達(dá)量明顯低于對(duì)照組(P<0.01)。

注：與對(duì)照組比較aP<0.01圖1 Real-time PCR檢測(cè)2組肝細(xì)胞miRNA-10a表達(dá)水平

2.3 低表達(dá)miRNA-10a促進(jìn)HF細(xì)胞增殖 為了研究低表達(dá)miRNA-10a對(duì)HF細(xì)胞增殖的影響，利用CCK-8法檢測(cè)轉(zhuǎn)染組及對(duì)照組HF細(xì)胞的增殖水平，結(jié)果(圖2)顯示，與對(duì)照組相比，低表達(dá)miRNA-10a的HF細(xì)胞增殖水平明顯提高(P<0.05)。

注：與對(duì)照組比較aP<0.05圖2 CCK-8法檢測(cè)轉(zhuǎn)染組及對(duì)照組HF細(xì)胞的增殖情況

2.4 miRNA-10a調(diào)控TGFβ1/Smads信號(hào)轉(zhuǎn)導(dǎo)通路 Western blotting檢測(cè)轉(zhuǎn)染miRNA-10a模擬物后HF細(xì)胞中TGFβ1、Smad 7蛋白的表達(dá)，結(jié)果(圖3)顯示，與對(duì)照組比較，轉(zhuǎn)染組TGFβ1蛋白的表達(dá)量明顯降低(P<0.05)，顯示miRNA-10a上調(diào)HF細(xì)胞中TGFβl表達(dá)，而轉(zhuǎn)染組Smad 7蛋白的表達(dá)量顯著增多(P<0.05)，顯示miRNA-10a下調(diào)Smad 7蛋白的表達(dá)。

注：與對(duì)照組比較aP<0.05圖3 Western blotting檢測(cè)miRNA-10a對(duì)TGFβ1/Smads表達(dá)的影響

3 討論

有研究顯示[11-12]，TGFβ1及其Smad信號(hào)轉(zhuǎn)導(dǎo)通路與HF的發(fā)生發(fā)展密切相關(guān)，其中TGFβ1為雙硫鍵結(jié)構(gòu)的二聚體堿性蛋白，是激活HF細(xì)胞的主要細(xì)胞因子，也是最強(qiáng)的HF促進(jìn)劑[13]；Smad蛋白是目前發(fā)現(xiàn)的TGFβ家族受體激酶的關(guān)鍵作用底物之一，根據(jù)其結(jié)構(gòu)和功能的不同分為3類，其中抑制型Smad蛋白中的Smad 7主要功能為抑制TGFβ的轉(zhuǎn)導(dǎo)通路。Smad 7可與活化的TGFβ1受體結(jié)合，抑制Smad蛋白磷酸化；進(jìn)入胞漿Smad 7，使Smad蛋白不能與其受體相結(jié)合，在TGFβ信號(hào)轉(zhuǎn)導(dǎo)中形成負(fù)反饋環(huán)路，從而發(fā)揮抗HF作用[14]。

研究[15-17]顯示，miRNA參與HF的發(fā)生發(fā)展，但關(guān)于miRNA調(diào)控HF的分子機(jī)制還不是很清楚。miRNA-10a作為miRNA家族中成員之一，本研究顯示，miRNA-10a在小鼠HF組織中高表達(dá)，并且低表達(dá)miRNA-10a能夠促進(jìn)HF細(xì)胞增殖，進(jìn)一步表明miRNA-10a在HF中發(fā)揮著促纖維化因子的作用。本研究同時(shí)發(fā)現(xiàn)，與對(duì)照組相比，轉(zhuǎn)染組TGFβ1蛋白表達(dá)明顯降低，表明miRNA-10a上調(diào)TGFβ1的表達(dá)；而轉(zhuǎn)染組Smad 7蛋白表達(dá)顯著增多，表明miRNA-10a能夠下調(diào)Smad 7表達(dá)，說(shuō)明miRNA-10a通過(guò)調(diào)控TGFβ1/Smads信號(hào)轉(zhuǎn)導(dǎo)通路發(fā)揮促HF作用，這為HF的治療提供了潛在的治療靶點(diǎn)。

[1] Lee YA, Friedman SL. Reversal, maintenance or progression: what happens to the liver after a virologic cure of hepatitis C[J]. Antiviral Res, 2014, 107(1): 23-30. DOI:10.1016/j.antiviral.2014.03.012.

[2] van der Ree MH, de Bruijne J, Kootstra NA, et al. MicroRNAs: role and therapeutic targets in viral hepatitis[J]. Antivir Ther (Lond), 2014, 19(6): 533-541. DOI:10.3851/IMP2766.

[3] Kim KM, Kim SG. Autophagy and microRNA dysregulation in liver diseases[J]. Arch Pharm Res, 2014, 37(9): 1097-1116. DOI:10.1007/s12272-014-0439-9.

[4] Havelange V, Ranganathan P, Geyer S, et al. Implications of the miR-10 family in chemotherapy response of NPM1-mutated AML[J]. Blood, 2014, 123(15): 2412-2415. DOI:10.1182/blood-2013-10-532374.

[5] Ohuchida K, Mizumoto K, Lin C, et al. MicroRNA-10a is overexpressed in human pancreatic cancer and involved in its invasiveness partially via suppression of the HOXA1 gene[J]. Ann Surg Oncol, 2012, 19(7): 2394-2402. DOI:10.1245/s10434-012-2252-3.

[6] Xie T, Liang J, Guo R, et al. Comprehensive microRNA analysis in bleomycin-induced pulmonary fibrosis identifies multiple sites of molecular regulation[J]. Physiol Genomics, 2011, 43(9): 479-487. DOI:10.1152/physiolgenomics.00222.2010.

[7] Uemura M, Swenson ES, Gamna MD, et al. Smad 2 and Smad 3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization[J]. Mol Biol Cell, 2005, 16(9): 4214-4224. DOI:10.1091/mbc.E05-02-0149.

[8] Tomita K, Tamiya G, Ando S, et al. Tumour necrosis factor alpha signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice[J]. Gut, 2006, 55(3): 415-424. DOI:10.1136/gut.2005.071118.

[9] Heldin CH, Miyazono K, Dijke P. TGF-beta signalling from cell membrane to nucleus through Smad proteins[J]. Nature, 1997, 390(6659): 465-471. DOI:10.1038/37284.

[10] Schnabl B, Kweon YO, Frederick JP, et al. The role of Smad3 in mediating mouse hepatic stellate cell activation[J]. Hepatology, 2001, 34(1): 89-100. DOI:10.1053/jhep.2001.25349.

[11] Martinez FE, Lopez CL, Regordan C, et al. Meningitis by Cryptococcus neoformans in patients with HIV infection[J]. Neurologia, 1999,14(5):218-223.

[12] Paradis V, Dargere D, Bonvoust F, et al. Effects and regulation of connective tissue growth factor on hepatic stellate cells[J]. Lab Invest, 2002, 82(6): 767-774. DOI:10.1097/01.lab.0000017365.18894.d3.

[13] Long J, Wang G, Matsuura I, et al. Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3)[J]. Proc Natl Acad Sci USA, 2004, 101(1): 99-104. DOI:10.1073/pnas.0307598100.

[14] Kavsak P, Rasmussen RK, Causing CG, et al. Smad7 binds to Smurf 2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation[J]. Mol Cell, 2000, 6(6): 1365-1375. DOI:10.1016/s1097-2765(00)00134-9.

[15] Roderburg C, Luedde M, Vargas CD, et al. miR-133a mediates TGF dependent derepression of collagen synthesis in hepatic stellate cells during liver fibrosis[J]. J Hepatol, 2013, 58(4): 736-742. DOI:10.1016/j.jhep.2012.11.022.

[16] Kumar V, Mahato RI. Delivery and targeting of miRNAs for treating liver fibrosis[J]. Pharm Res, 2015, 32(2): 341-361. DOI:10.1007/s11095-014-1497-x.

[17] Chen SL, Zheng MH, Shi KQ, et al. A new strategy for treatment of liver fibrosis: letting microRNA do the job[J]. Bio Drugs, 2013, 27(1): 25-34. DOI:10.1007/s40259-012-0005-2.

(本文編輯：王映紅)

Effects of miRNA-10a on the proliferation of hepatic fibrosis cells and the expression of TGF beta 1/Smads signal transduction pathway in mice

DengGuosun,LiZhen′ga

(DepartmentofGastroenterology,ChongmingBranch,XinhuaHospitalAffiliatedtoShanghaiJiaotongUniversity,Shanghai202150,China)

Objective To investigate the possible mechanism involved in the effect of micro RNA-10a(miRNA)on hepatic fibrosis (HF), through the observation on the expression of TGFβ1/Smads signal transduction pathway in mice with HF and its correlation with the development of hepatic fibrosis.Methods Forty healthy male mice with an age of 9 weeks (C57BL6/J) were randomly divided into the control group and the HF model group (or the observation group). The animals in the control group were given normal saline abdominally at a dosage of 5 μl/g, twice a week, for a succession of 8 weeks, while the animals in the observation group received 10% CCL4 olive oil also at a dosage of 5 μl/g with the same frequency and for the same length of time to develop the model of hepatic fibrosis. The expression of miRNA-10a was detected by RT-PCR. HF cells were cultured in the animals of the observation group and miRNA-10a mimics were transfected (the transfection group). CCK-8 method was used to detect the proliferation of HF cells, and the expression levels of TGFβ1 and Smad 7 in the HF cells were detected by Western-blotting. Results As compared with that of the control group, the expression level of miRNA-10a in the observation group was increased significantly (P<0.05); and as compared with that of the control group, the expression level of miRNA-10a in the transfection group were significantly decreased (P<0.05); and furthermore, as compared with that of the control group, the lower expression of miRNA-10a significantly decreased the expression level of TGFβ1 and increased the expression level of Smad 7(P<0.05).Conclusion The expression of miRNA-10a in the HF cells was lower, and the transfected miRNA-10a mimics could significantly promote the proliferation of HF cells, the possible mechanism of which might be associated with the regulation of TGFβ1/Smads signal transduction pathway in HF promotion.

Hepatic fibrosis; Transforming growth factor β1; Smad 7 protein; TGFβ1/Smads signal pathway

202150 上海，上海交通大學(xué)附屬新華醫(yī)院崇明分院消化內(nèi)科(鄧國(guó)孫)；南昌大學(xué)醫(yī)學(xué)院第二附屬醫(yī)院普外科(李鎮(zhèn)伽)

R575.3

A

10.3969/j.issn.1009-0754.2017.03.013

2016-10-27)