miR-33a-5p對(duì)骨肉瘤細(xì)胞Saos-2增殖的影響及其機(jī)制

袁忠，胡俊，雷家維，付璐珩，俞曾強(qiáng)，汪海燕

(江西中醫(yī)藥大學(xué)附屬洪都中醫(yī)院，南昌330006)

miR-33a-5p對(duì)骨肉瘤細(xì)胞Saos-2增殖的影響及其機(jī)制

袁忠，胡俊，雷家維，付璐珩，俞曾強(qiáng)，汪海燕

(江西中醫(yī)藥大學(xué)附屬洪都中醫(yī)院，南昌330006)

目的 觀察miR-33a-5p對(duì)骨肉瘤細(xì)胞Saos-2增殖的影響，并探討其相關(guān)機(jī)制。方法 采用Real time-PCR法檢測(cè)骨肉瘤細(xì)胞Saos-2及正常人成骨細(xì)胞hFOB1.19中miR-33a-5p mRNA相對(duì)表達(dá)量。將對(duì)數(shù)生長(zhǎng)期的骨肉瘤細(xì)胞Saos-2分為兩組，分別轉(zhuǎn)染miR-33a-5p mimics(mimics組)及Scramble(對(duì)照組)。采用MTT法檢測(cè)兩組培養(yǎng)24、48、72、96及120 h細(xì)胞增殖能力，細(xì)胞克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞克隆形成能力(以克隆形成率表示)，流式細(xì)胞術(shù)比較細(xì)胞周期，Westen blotting法檢測(cè)細(xì)胞周期蛋白激酶2(CDK2)、CDK4、細(xì)胞周期蛋白D(CyclinD)及CyclinE蛋白相對(duì)表達(dá)量。結(jié)果 骨肉瘤細(xì)胞Saos-2及正常人成骨細(xì)胞hFOB1.19中miR-33a-5p mRNA相對(duì)表達(dá)量分別為2.65±0.43和24.32±3.21，兩者比較P<0.01。隨著培養(yǎng)時(shí)間延長(zhǎng)，兩組細(xì)胞增殖能力均呈升高趨勢(shì)；與對(duì)照組同時(shí)間點(diǎn)比較，mimics組細(xì)胞增殖能力均降低(P均<0.01)。mimics組和對(duì)照組克隆形成率分別為25.34%±3.46%、53.27%±5.82%,兩組比較P<0.01。mimics組和對(duì)照組分別有46.37%±4.28%、30.64%±3.52%的細(xì)胞處于G1期，分別有13.25%±1.25%、22.62%±2.01%的細(xì)胞處于G2期，兩組比較P均<0.01。mimics組CDK2、CDK4、CyclinD、CyclinE蛋白相對(duì)表達(dá)量均低于對(duì)照組(P均<0.01)。結(jié)論 骨肉瘤細(xì)胞Saos-2中miR-33a-5p表達(dá)降低，上調(diào)其表達(dá)可抑制細(xì)胞增殖；下調(diào) CDK2、CDK4、Cyclin D及Cyclin E等細(xì)胞周期相關(guān)蛋白的表達(dá)可能為miR-33a-5p的作用機(jī)制。

骨肉瘤；Saos-2細(xì)胞；miR-33a-5p；細(xì)胞增殖

骨肉瘤好發(fā)生于股骨遠(yuǎn)端、脛骨及肱骨，治療方法包括手術(shù)、化療、放療及綜合治療[1]。骨肉瘤患者的5年生存率較低，僅為63%[2]。微小RNA(miRNA)是一類非編碼小分子RNA，其長(zhǎng)度約為22個(gè)核苷酸，調(diào)節(jié)超過30%的人體基因，參與細(xì)胞分化、增殖、凋亡及細(xì)胞周期調(diào)控等各種生物學(xué)過程[3～6]。miR-33a-5p屬于新發(fā)現(xiàn)的miR-33a家族成員，目前對(duì)其具體功能所知甚少。2015年1月～2016年1月，我們觀察了骨肉瘤細(xì)胞Saos-2中miR-33a-5p表達(dá)變化，現(xiàn)分析結(jié)果，探討其對(duì)細(xì)胞增殖的影響及相關(guān)機(jī)制。

1 材料與方法

1.1 材料 骨肉瘤細(xì)胞Saos-2及正常人成骨細(xì)胞hFOB1.19均購自南昌大學(xué)實(shí)驗(yàn)醫(yī)學(xué)中心。實(shí)驗(yàn)所需一抗均購自美國(guó)Santa Cruz公司，HRP標(biāo)記的二抗均購自英國(guó)Abcam公司，MTT試劑盒和亞甲基藍(lán)均購自美國(guó)Sigma公司，miR-33a-5p mimics及Scramble均由上海吉馬制藥技術(shù)有限公司合成。

1.2 細(xì)胞miR-33a-5p mRNA相對(duì)表達(dá)量檢測(cè) 采用Real time-PCR法。取對(duì)數(shù)生長(zhǎng)期的骨肉瘤細(xì)胞Saos-2及正常人成骨細(xì)胞hFOB1.19，采用TRIzol試劑提取總RNA。以總RNA為模板，使用M-MLV反轉(zhuǎn)錄酶在25 μL體系中對(duì)cDNA進(jìn)行反轉(zhuǎn)錄。采用SYBR Green Ⅰ mix試劑盒于ABI VIIA7熒光定量PCR儀上進(jìn)行檢測(cè)。miR-33a-5p上游引物：5′- GGTGCATTGTAGTTGCATTGC-3′，下游引物5′- GTGCAGGGTCCGAGGTATTC -3′。以GAPDH為內(nèi)參，上游引物5′-GACTTCAACAGCAACTCCCAC-3′，下游引物5′-TCCACCACCCTGTTGCTGTA-3′。PCR反應(yīng)條件：95 ℃預(yù)變性30 s，95 ℃變性5 s，60 ℃退火34 s，40個(gè)循環(huán)。以2-ΔΔCt法計(jì)算miR-33a-5p mRNA相對(duì)表達(dá)量。

1.3 細(xì)胞分組處理 將對(duì)數(shù)生長(zhǎng)期的骨肉瘤細(xì)胞Saos-2分為mimics組及對(duì)照組，參照李振華等[7]的方法利用Lipofectamine 2000轉(zhuǎn)染試劑分別轉(zhuǎn)染miR-33a-5p mimics(可以增強(qiáng)細(xì)胞miR-33a-5p表達(dá))及Scramble(把RNA序列打亂，對(duì)目的基因表達(dá)無影響)。將兩組加入DMEM培養(yǎng)基，置于37 ℃、5% CO2的飽和濕度恒溫細(xì)胞培養(yǎng)箱中進(jìn)行培養(yǎng)，取對(duì)數(shù)生長(zhǎng)期細(xì)胞用于以下實(shí)驗(yàn)。

1.4 miR-33a-5p表達(dá)對(duì)骨肉瘤細(xì)胞Saos-2增殖的影響

1.4.1 細(xì)胞增殖能力 采用MTT法。取對(duì)數(shù)生長(zhǎng)期的兩組細(xì)胞，以1×104個(gè)/孔接種于96孔板中，分別于培養(yǎng)24、48、72、96及120 h每孔加入40 μL的MTT溶液(5 mg/mL)。孵育4 h后每孔加入200 μL 二甲基亞砜(DMSO)，置于搖床上充分震蕩。采用酶標(biāo)儀檢測(cè)兩組各時(shí)間點(diǎn)570 nm處的吸光度(OD)值，每組設(shè)置6個(gè)復(fù)孔，取平均值。

1.4.2 細(xì)胞克隆形成能力 采用細(xì)胞克隆形成實(shí)驗(yàn)。取對(duì)數(shù)生長(zhǎng)期的兩組細(xì)胞，以1×104個(gè)/孔接種于96孔板中，培養(yǎng)24 h時(shí)接種于12孔板中，每孔接種600個(gè)細(xì)胞。培養(yǎng)8 d去除培養(yǎng)液，用PBS沖洗3次，甲醇固定20 min；1%亞甲基藍(lán)染色40 min,去離子水清洗2次，晾干。40倍顯微鏡下計(jì)算>50個(gè)細(xì)胞的克隆數(shù)量，計(jì)算克隆形成率?？寺⌒纬陕?(克隆數(shù)/接種細(xì)胞數(shù))×100%。每組設(shè)置3個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次，取平均值。

1.4.3 細(xì)胞周期 采用流式細(xì)胞術(shù)。取對(duì)數(shù)生長(zhǎng)期的兩組細(xì)胞，以1×104個(gè)/孔接種于96孔板中，培養(yǎng)24 h時(shí)PBS洗滌2次，70%乙醇固定，4 ℃保存過夜。PBS沖洗1次，將細(xì)胞密度調(diào)整為1×106個(gè)/mL。加入碘化丙啶染色液，至終濃度為0.05 mg/mL，4 ℃染色30 min。上流式細(xì)胞儀對(duì)兩組細(xì)胞周期進(jìn)行分析。每組設(shè)置3個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次，取平均值。

1.5 細(xì)胞周期相關(guān)蛋白相對(duì)表達(dá)量檢測(cè) 采用Westen blotting法。取對(duì)數(shù)生長(zhǎng)期的兩組細(xì)胞，采用RIPA裂解液裂解，PMSF處理后冰上孵育30 min。4 ℃條件下12 000 r/min離心30 min，取上清蛋白。采用BCA蛋白濃度測(cè)定試劑盒檢測(cè)蛋白濃度，進(jìn)行SDS-PAGE電泳，轉(zhuǎn)移至硝酸纖維素膜上。分別加入一抗anti-細(xì)胞周期蛋白激酶2(CDK2)、anti-CDK4、anti-細(xì)胞周期蛋白D(CyclinD)以及anti-Cyclin E(1∶2 000),加入anti-GAPDH、HRP標(biāo)記的二抗(1∶2 000)，通過ECL法進(jìn)行顯影。將膠片進(jìn)行掃描或拍照，用凝膠圖像處理系統(tǒng)分析目標(biāo)條帶的平均光密度值。以目的蛋白與GAPDH蛋白平均光密度值的比值計(jì)算細(xì)胞CDK2、CDK4以及CyclinD、CyclinE蛋白相對(duì)表達(dá)量。

2 結(jié)果

2.1 骨肉瘤細(xì)胞Saos-2及正常人成骨細(xì)胞miR-33a-5p相對(duì)表達(dá)量比較 miR-33a-5p在骨肉瘤細(xì)胞Saos-2及正常人成骨細(xì)胞hFOB1.19中的相對(duì)表達(dá)量分別為2.65±0.43和24.32±3.21，兩者比較P<0.01。

2.2 兩組細(xì)胞增殖相關(guān)指標(biāo)比較

2.2.1 細(xì)胞增殖能力 隨著培養(yǎng)時(shí)間的延長(zhǎng)，兩組細(xì)胞增殖能力均呈升高趨勢(shì)；與對(duì)照組同時(shí)間點(diǎn)比較，mimics組細(xì)胞增殖能力均降低(P均<0.01)。見表1。

表1 兩組細(xì)胞增殖能力比較±s)

注：與對(duì)照組同時(shí)間點(diǎn)比較，*P<0.01。

2.2.2 細(xì)胞克隆形成能力 mimics組和對(duì)照組克隆形成率為25.34%±3.46%、53.27%±5.82%，兩組比較P<0.01。

2.2.3 細(xì)胞周期 mimics組和對(duì)照組分別有46.37%±4.28%、30.64%±3.52%的細(xì)胞處于G1期，分別有13.25%±1.25%、22.62%±2.01%的細(xì)胞處于G2期，兩組比較P均<0.01。

2.3 兩組細(xì)胞周期相關(guān)蛋白相對(duì)表達(dá)量比較 mimics組CDK2、CDK4、CyclinD、CyclinE蛋白相對(duì)表達(dá)量均低于對(duì)照組(P均<0.01)。見表2。

表2 兩組細(xì)胞周期相關(guān)蛋白相對(duì)表達(dá)量比較±s)

注：與對(duì)照組比較，*P<0.01。

3 討論

骨肉瘤主要累及骨髓腔及骨皮質(zhì)，波及骨外膜時(shí)可形成特征性的三角形隆起，患者生存率較低[8]。近年來，一系列的醫(yī)學(xué)研究指出miRNA表達(dá)失調(diào)與多種腫瘤的發(fā)病相關(guān)，包括結(jié)腸癌、乳腺癌、骨肉瘤等[9～11]。Kobayashi等[3]通過7個(gè)骨肉瘤樣本的miRNA表達(dá)譜證實(shí)多個(gè)miRNA表達(dá)失調(diào)，包括miR-34、miR-140、miR-92a、miR-99b、miR-193a-5p等。miR-33a-5p屬于miR-33a家族成員，是近期發(fā)現(xiàn)的一種miRNA，目前對(duì)其具體生理功能所知甚少。文獻(xiàn)報(bào)道,miR-33a-5p參與調(diào)節(jié)T細(xì)胞功能[12]，并在肝癌細(xì)胞中參與調(diào)節(jié)β-catenin信號(hào)通路[13]。

miRNA mimics是一種化學(xué)方法人工合成的成熟miRNA片段,能模擬生物體內(nèi)源性miRNAs[14]。通過轉(zhuǎn)染miRNA mimics能人為增加目標(biāo)miRNA的表達(dá)，從而凸顯出目標(biāo)miRNA的功能。miRNA Scramble是采用化學(xué)方法人工合成的隨機(jī)miRNA片段，無靶向性基因調(diào)控效應(yīng)，多用作陰性對(duì)照。本研究結(jié)果顯示，miR-33a-5p在骨肉瘤細(xì)胞Saos-2中的相對(duì)表達(dá)量明顯低于正常人成骨細(xì)胞hFOB1.19，說明骨肉瘤的發(fā)病與miR-33a-5p表達(dá)降低有關(guān)。由于miR-33a-5p在骨肉瘤細(xì)胞內(nèi)低表達(dá)，若采用常規(guī)基因沉默方法，將骨肉瘤細(xì)胞中miR-33a-5p表達(dá)進(jìn)一步降低，則其表達(dá)變化幅度較小，難以體現(xiàn)其調(diào)控功能。因此本研究通過轉(zhuǎn)染miR-33a-5p mimics，人為增加miR-33a-5p表達(dá)，能顯著體現(xiàn)其對(duì)骨肉瘤細(xì)胞增殖的調(diào)控作用。本研究結(jié)果顯示，隨著培養(yǎng)時(shí)間的延長(zhǎng)，兩組細(xì)胞增殖能力均呈升高趨勢(shì)；與對(duì)照組同時(shí)間點(diǎn)比較，mimics組細(xì)胞增殖能力均降低；mimics組克隆形成率也明顯低于對(duì)照組。證實(shí)miR-33a-5p對(duì)骨肉瘤細(xì)胞具有增殖抑制作用。細(xì)胞周期分為分裂間期和分裂期，分裂間期又分為G1期、S期及G2期。若細(xì)胞停留在G1期，則該細(xì)胞被稱為休止細(xì)胞或G0期細(xì)胞[15]。本研究結(jié)果表明，mimics組處于G1期的細(xì)胞比例明顯高于對(duì)照組，處于G2期的細(xì)胞比例明顯低于對(duì)照組。證實(shí)miR-33a-5p可誘導(dǎo)骨肉瘤細(xì)胞Saos-2停滯于G1期，從而抑制細(xì)胞增殖。

細(xì)胞周期蛋白(Cyclins)是一類對(duì)細(xì)胞周期進(jìn)行調(diào)節(jié)的蛋白，其中CyclinD和CyclinE分別在G1期和S期進(jìn)行表達(dá)，并對(duì)細(xì)胞分裂增殖起促進(jìn)作用[16]。細(xì)胞周期蛋白激酶(CDKs)是一類調(diào)節(jié)細(xì)胞周期，具有促進(jìn)細(xì)胞增殖作用的激酶[17]。CDK2與CDK4的功能在于促進(jìn)細(xì)胞從G1期進(jìn)入S期，進(jìn)而促進(jìn)細(xì)胞增殖。本研究結(jié)果顯示，mimics組CDK2、CDK4、CyclinD、CyclinE蛋白相對(duì)表達(dá)量均低于對(duì)照組，表明上述蛋白表達(dá)降低可能是miR-33a-5p抑制細(xì)胞增殖的機(jī)制之一。

綜上所述，骨肉瘤細(xì)胞Saos-2中miR-33a-5p表達(dá)降低，上調(diào)其表達(dá)后可抑制細(xì)胞增殖；下調(diào)CDK2、CDK4、CyclinD及CyclinE等細(xì)胞周期相關(guān)蛋白的表達(dá)可能為miR-33a-5p的作用機(jī)制。miR-33a-5p在體外實(shí)驗(yàn)中表現(xiàn)出抑癌基因的功能，其作為一種新發(fā)現(xiàn)的miRNA，對(duì)抑制骨肉瘤細(xì)胞增殖具有重要作用，可能成為未來治療骨肉瘤新的作用靶點(diǎn)。

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Effect and mechanism of miR-33a-5p on the proliferation of osterosarcoma cells Saos-2

YUANZhong,HUJun,LEIJiawei,FULuheng,YUZengqiang,WANGHaiyan

(HongduHospitalAffiliatedtoJiangxiUniversityofTraditionalChineseMedicine,Nanchang330006,China)

Objective To investigate the effect and mechanism of miR-33a-5p on the proliferation of osteosarcoma cancer cells Saos-2. Methods RT-PCR was conducted to investigate the expression level of miR-33a-5p in Saos-2 cells and hFOB1.19 cells. Saos-2 cell line was divided into two groups, the mimics group was transfected with miR-33a-5p mimics and the control group was transfected with Scramble. The proliferation ability was analyzed by MTT cell viability assay at 24, 48, 72, 96 and 120 h of cultivation, the cloning ability was detected by clone-formation assay, the cell cycle was detected by flow cytometry, and the expression levels of CDK2, CDK4, CyclinD and CyclinE were measured by Western blotting. Results The expression levels of miR-33a-5p were 2.65±0.43 in the miR-33a-5p mimics group and 24.32±3.21 in the control group, respectively,P<0.01. The OD value of the two groups was increased as culturing. The OD value of mimics group was lower than that of the control group (allP<0.01). The clone-formation rate was 25.34%±3.46% in mimics group, which was significantly lower than that of the control group (53.27%±5.82%),P<0.01. There was 46.37%±4.28%% of the total mimics group of Saos-2 cells in the G1 phase, much higher than 30.64% ±3.52% in the control group; the percentage of G2phase in the mimics group was 13.25%±1.25%, which was much lower than 22.62%±2.01% in the control group (allP<0.01). The expression level of CDK2, CDK4, Cyclin D and Cyclin E in the mimics group was significantly lower than that of the control group (allP<0.01). Conclusion The miR-33a-5p expression was reduced, and up-regulation of miR-33a-5p may inhibit the proliferation of osterosarcoma cells Saos-2, whose mechanism may be down-regulating the cell cycles of CDK2, CDK4, Cyclin D and Cyclin E.

osteosarcoma; Saos-2 cells; miR-33a-5p; cell proliferation

袁忠(1969-)，男，副主任醫(yī)師，研究方向?yàn)楣悄[瘤及骨質(zhì)疏松。E-mail： yuanzhong122@sina.com

10.3969/j.issn.1002-266X.2016.32.002

R738.1

A

1002-266X(2016)32-0005-04

2016-02-27)