張笑添, 張曉延, 黃賽亞, 王 倩, 劉 威
(1山西醫(yī)科大學(xué)汾陽(yáng)學(xué)院醫(yī)學(xué)檢驗(yàn)系, 山西 汾陽(yáng) 032200; 2山西醫(yī)科大學(xué)醫(yī)學(xué)影像學(xué)系, 山西 太原 030001)
miRNA-101-3p靶向調(diào)控EZH2蛋白抑制胃癌細(xì)胞增殖和遷移*
張笑添1△, 張曉延1, 黃賽亞1, 王 倩2, 劉 威1
(1山西醫(yī)科大學(xué)汾陽(yáng)學(xué)院醫(yī)學(xué)檢驗(yàn)系, 山西 汾陽(yáng) 032200;2山西醫(yī)科大學(xué)醫(yī)學(xué)影像學(xué)系, 山西 太原 030001)
目的研究微小RNA-101-3p (miRNA-101-3p) 對(duì)人胃癌細(xì)胞增殖、凋亡和遷移的影響及其可能的調(diào)控機(jī)制。方法Real-time PCR檢測(cè)2種人胃癌細(xì)胞和1種胃黏膜細(xì)胞中miRNA-101-3p和zeste增強(qiáng)子同源物2 (EZH2) 的表達(dá)水平;采用脂質(zhì)體瞬時(shí)轉(zhuǎn)染技術(shù)過(guò)表達(dá)miRNA-101-3p;流式細(xì)胞術(shù)檢測(cè)miRNA-101-3p對(duì)胃癌細(xì)胞周期和凋亡的影響;Transwell實(shí)驗(yàn)、CCK-8法和臺(tái)盼藍(lán)染色法檢測(cè)miRNA-101-3p對(duì)胃癌細(xì)胞遷移和增殖能力的影響;Western blot法檢測(cè)EZH2的表達(dá)。結(jié)果miRNA-101-3p在胃癌細(xì)胞的表達(dá)水平顯著低于胃黏膜細(xì)胞(P<0.05);過(guò)表達(dá)miRNA-101-3p后,流式細(xì)胞術(shù)結(jié)果顯示胃癌細(xì)胞的S期比例減少,G0/G1期比例增加,早期凋亡率增加(P<0.05);CCK-8法、臺(tái)盼藍(lán)染色法及Transwell實(shí)驗(yàn)結(jié)果顯示胃癌細(xì)胞的增殖和遷移能力顯著降低(P<0.05);Western blot結(jié)果顯示胃癌細(xì)胞中EZH2蛋白的表達(dá)明顯下降(P<0.05)。結(jié)論miRNA-101-3p可能通過(guò)靶向負(fù)調(diào)控EZH2蛋白的表達(dá)抑制胃癌細(xì)胞的增殖和遷移,進(jìn)而促進(jìn)胃癌細(xì)胞凋亡。
胃癌; 微小RNA; 細(xì)胞增殖; 細(xì)胞遷移; 細(xì)胞凋亡; Zeste增強(qiáng)子同源物2
胃癌是最常見(jiàn)的惡性腫瘤之一,是腫瘤死亡的第2常見(jiàn)的原因,其生存期和預(yù)后較差[1-2]。微小RNA (microRNA, miRNA) 作為一種高度保守的小分子內(nèi)源性的非編碼RNA,可以靶向結(jié)合mRNA 3’-UTR進(jìn)而調(diào)控靶基因的表達(dá)。已有研究發(fā)現(xiàn),miRNA-101-3p在結(jié)腸癌[3]、膀胱移行細(xì)胞癌[4]、肝癌[5]和乳腺癌[6]等發(fā)揮著抑癌的作用。近期研究發(fā)現(xiàn),miRNA-101-3p在胃癌中表達(dá)下降[7],但是其對(duì)胃癌細(xì)胞周期及凋亡的研究尚未見(jiàn)報(bào)道。
Zeste增強(qiáng)子同源物2(enhancer of zeste homolog 2,EZH2)是多梳家族(Polycomb group,PcG)的重要成員之一,在胚胎發(fā)育、細(xì)胞增殖和細(xì)胞周期調(diào)控中起著重要的作用。有研究發(fā)現(xiàn),EZH2與多種腫瘤的發(fā)生發(fā)展、轉(zhuǎn)移和侵襲都密切相關(guān)[8-9]。基于此,本研究將探討EZH2在胃癌中與miRNA-101-3p的相關(guān)性,以及miRNA-101-3p對(duì)胃癌細(xì)胞增殖、遷移以及凋亡的影響。
miRNA反轉(zhuǎn)錄以及熒光定量試劑盒購(gòu)于北京天根生化有限公司;miRNA-101-3p mimics以及miRNA陰性對(duì)照(negative control, NC)購(gòu)于廣州銳博生物有限公司;Lipofectamine 2000購(gòu)自Invitrogen;抗EZH2單克隆抗體購(gòu)于Abcam;抗GAPDH多克隆抗體購(gòu)于Bioworld;Transwell小室購(gòu)自康寧公司;CCK-8以及細(xì)胞周期試劑盒購(gòu)自碧云天生物有限公司;Annexin V-FITC/PI凋亡試劑盒購(gòu)于聯(lián)科生物有限公司;RIPA裂解液以及BCA定量試劑盒購(gòu)自武漢博士德生物有限公司。
2.1細(xì)胞培養(yǎng)和轉(zhuǎn)染 胃癌細(xì)胞MGC-803和HGC-27的培養(yǎng)條件分別是RPMI-1640加10%胎牛血清(fetal bovine serum,F(xiàn)BS)和DMEM加10% FBS,胃黏膜細(xì)胞GES-1的培養(yǎng)條件是DMEM加10% FBS,于37 ℃、5% CO2條件下培養(yǎng),0.25%胰蛋白酶消化傳代。轉(zhuǎn)染前1 d取生長(zhǎng)狀態(tài)良好的細(xì)胞接種于6孔板中,每孔細(xì)胞數(shù)大約為3×105,次日細(xì)胞密度達(dá)到60%時(shí)進(jìn)行轉(zhuǎn)染實(shí)驗(yàn),使用Lipofectamine 2000將miRNA-101-3p mimics或miRNA mimics NC轉(zhuǎn)入胃癌細(xì)胞MGC-803中,相應(yīng)分為miRNA-101-3p mimics組、miRNA NC組及空白(blank)組,轉(zhuǎn)染8 h后,將無(wú)血清培養(yǎng)基更換為含10% FBS培養(yǎng)基繼續(xù)培養(yǎng),24 h后用流式細(xì)胞術(shù)觀察轉(zhuǎn)染效率,48 h后用熒光定量PCR檢測(cè)各組胃癌細(xì)胞中miRNA-101-3p和EZH2的表達(dá)水平。
2.2Real-time PCR 胰酶消化各組細(xì)胞后使用TRIzol試劑提取細(xì)胞中的RNA。應(yīng)用miRNA熒光定量PCR試劑盒檢測(cè)miRNA-101-3p的表達(dá),以U6為內(nèi)參照。擴(kuò)增條件是: 94 ℃ 2 min; 94 ℃ 20 s, 60 ℃ 34 s, 40個(gè)循環(huán)。使用SYBR Premix Ex Taq試劑盒檢測(cè)EZH2的表達(dá),以GAPDH為內(nèi)參照。EZH2上游引物序列為5’-AAAGAAAGCCGCCCACCT-3’,下游引物序列為5’-GGTCCATCTATGTTGGGGGTA-3’; GAPDH的上游引物序列為5’-GGATTTGGTGTCGTATTGGGC-3’,下游引物序列為5’-CGCTCCTGGAAGATGGTGAT-3’。擴(kuò)增條件是: 95 ℃ 3 min; 95 ℃ 30 s, 56 ℃ 30 s, 72 ℃ 30 s, 40個(gè)循環(huán)。擴(kuò)增結(jié)束后均進(jìn)行熔解曲線分析,miRNA-101-3p和EZH2的相對(duì)表達(dá)水平采用2-ΔΔCt法計(jì)算。
2.3CCK-8比色法和臺(tái)盼藍(lán)染色法檢測(cè)細(xì)胞增殖 將轉(zhuǎn)染了miRNA-101-3p mimics或miRNA NC的胃癌細(xì)胞經(jīng)胰酶消化后重新接種于96孔板中,每孔中的細(xì)胞約為3×105個(gè),于37 ℃、5% CO2培養(yǎng)箱中繼續(xù)培養(yǎng)24、48和72 h后,加入CCK-8試劑10 μL,混勻后繼續(xù)置于培養(yǎng)箱中1 h,使用酶聯(lián)免疫檢測(cè)儀測(cè)定450 nm處的吸光度(A)值,繪制生長(zhǎng)曲線,每組設(shè)定3個(gè)復(fù)孔。采用臺(tái)盼藍(lán)染色進(jìn)行活細(xì)胞計(jì)數(shù)時(shí),轉(zhuǎn)染前一天將細(xì)胞按3×108/L接種于6孔板中,轉(zhuǎn)染48 h后收集細(xì)胞,使用臺(tái)盼藍(lán)染色后直接計(jì)數(shù)活細(xì)胞。
2.4流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期和凋亡 轉(zhuǎn)染48 h后,收集miRNA-101-3p mimics、miRNA NC及空白對(duì)照組的細(xì)胞,細(xì)胞數(shù)大約為1×106個(gè),使用預(yù)冷的PBS清洗2次,用70%的乙醇固定后置于4 ℃冰箱中過(guò)夜,再用預(yù)冷的PBS洗滌細(xì)胞2次,每管分別加入PI染色液(20×)和RNase A(50×),于37 ℃避光溫浴0.5 h后,使用流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期。
轉(zhuǎn)染48 h后使用不含EDTA的胰酶消化各組細(xì)胞,用上清培養(yǎng)液終止消化,1 000 r/min離心5 min收集細(xì)胞,PBS清洗細(xì)胞2次,收集大約(1~5)×105個(gè)細(xì)胞,使用500 μL 1×Binding Buffer重懸細(xì)胞,相繼加入5 μL Annexin V-FITC和10 μL PI,室溫避光5 min后使用流式細(xì)胞術(shù)檢測(cè)各組的凋亡率。
2.5Transwell細(xì)胞遷移實(shí)驗(yàn) 轉(zhuǎn)染24 h后,胰酶消化各組細(xì)胞并使用無(wú)血清培養(yǎng)基重懸細(xì)胞,調(diào)整細(xì)胞密度為2×108/L,取細(xì)胞懸液100 μL加在Transwell小室的上層,Transwell小室下層加入500 μL含10%血清培養(yǎng)基,置于37 ℃、5% CO2孵箱中繼續(xù)培養(yǎng)24 h后取出小室,PBS清洗后使用濕棉簽輕輕拭去上室中未遷移的細(xì)胞, 4%多聚甲醛固定20 min,PBS清洗3次后使用0.1%結(jié)晶紫染色10 min,PBS清洗后隨機(jī)選取5個(gè)視野,計(jì)數(shù)下室染色細(xì)胞。
2.6Western blot實(shí)驗(yàn) 收集細(xì)胞后,使用RIPA裂解液(含1% PMSF)提取細(xì)胞中的蛋白質(zhì),使用BCA法測(cè)定蛋白濃度。取大約30 μg蛋白行10% SDS-PAGE分離蛋白,使用濕轉(zhuǎn)法電轉(zhuǎn)至NC膜上,5%脫脂奶粉室溫封閉2 h,抗EZH2(1∶2 000)和GAPDH(1∶5 000)抗體4 ℃孵育過(guò)夜,TBST洗3次后,加入HRP標(biāo)記 II 抗(1∶8 000)室溫孵育1 h,TBST清洗3次后,ECL發(fā)光法顯影曝光,灰度值采用ImageJ軟件掃描并記錄,并分析目的蛋白與內(nèi)參照蛋白灰度的比值作為目的蛋白的相對(duì)表達(dá)量。
用SPSS 18.0統(tǒng)計(jì)軟件進(jìn)行分析。數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,多組間比較采用單因素方差分析(one-way ANOVA),組間兩兩比較采用Bonferroni校正的t檢驗(yàn)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
PCR結(jié)果顯示,miRNA-101-3p在胃癌細(xì)胞MGC-803和HGC-27中的表達(dá)量顯著低于胃黏膜細(xì)胞GES-1(P<0.05),其中胃癌細(xì)胞MGC-803中miRNA-101-3p的表達(dá)水平最低,故以胃癌細(xì)胞MGC-803作為后續(xù)的研究對(duì)象,見(jiàn)圖1。轉(zhuǎn)染48 h后,與miRNA NC組相比,miRNA-101-3p mimics組的miRNA-101-3p表達(dá)水平顯著上調(diào)(P<0.05),而miRNA NC組與空白組之間的差異沒(méi)有統(tǒng)計(jì)學(xué)顯著性,說(shuō)明轉(zhuǎn)染成功; EZH2在miRNA-101-3p mimics組的水平顯著低于miRNA NC組和空白組(P<0.05),見(jiàn)圖2。
Figure 1. The expression of miRNA-101-3p in gastric mucosal cells and gastric cancer cells detected by real-time PCR. Mean±SD.n=3.*P<0.05vsGES-1.
圖1Real-timePCR檢測(cè)胃黏膜細(xì)胞和胃癌細(xì)胞中mi-RNA-101-3p的表達(dá)水平
Figure 2. The expression levels of miRNA-101-3p and EZH2 mRNA in MGC-803 cells after transfected with miRNA-101-3p mimics for 48 h were detected by real-time PCR. Mean±SD.n=3.*P<0.05vsmiRNA-101-3p mimics group.
圖2Real-timePCR檢測(cè)轉(zhuǎn)染48h后miRNA-101-3p和EZH2mRNA的表達(dá)水平
轉(zhuǎn)染24 h后,使用熒光顯微鏡觀察,結(jié)果顯示大約90%以上的細(xì)胞均呈紅色熒光蛋白Cy3陽(yáng)性,并進(jìn)一步使用流式細(xì)胞儀進(jìn)行分選,結(jié)果說(shuō)明轉(zhuǎn)染成功,見(jiàn)圖3。
轉(zhuǎn)染后使用CCK-8法測(cè)定各組細(xì)胞的吸光度(A)值,結(jié)果顯示,miRNA-101-3p mimics組在48和72 h時(shí)其吸光度值明顯低于miRNA NC組和空白對(duì)照組(P<0.05),而miRNA NC組和空白對(duì)照組之間的差異無(wú)統(tǒng)計(jì)學(xué)顯著性,見(jiàn)圖4A;同時(shí)采用臺(tái)盼藍(lán)染色法計(jì)數(shù)轉(zhuǎn)染后活細(xì)胞數(shù)量,結(jié)果顯示,miRNA-101-3p mimics組的活細(xì)胞數(shù)量顯著低于miRNA NC組和空白對(duì)照組(P<0.05),而miRNA NC組和空白對(duì)照組之間的活細(xì)胞數(shù)量沒(méi)有統(tǒng)計(jì)學(xué)顯著性,見(jiàn)圖4B。此結(jié)果說(shuō)明過(guò)表達(dá)miRNA-101-3p后胃癌細(xì)胞MGC-803的生長(zhǎng)受到抑制,細(xì)胞活力明顯降低。
Figure 3. The transfection efficiency after transfected with miRNA-101-3p mimics for 24 h (×200). A: the MGC-803 cells under phase-contrast microscope; B: the image of Cy3 positive staining of MGC-803 cells under fluorescence microscope; C: the intensity of red fluorescence in MGC-803 cells analyzed by flow cytometry.
圖3脂質(zhì)體轉(zhuǎn)染24h細(xì)胞轉(zhuǎn)染效率
Figure 4. The effect of miRNA-101-3p overexpression on the proliferation of gastric cancer cells. A: the viability of gastric cancer MGC-803 cells was dectected by CCK-8 assay; B: the number of gastric cancer cells was counted by trypan blue staining. Mean±SD.n=3.*P<0.05vsmiRNA-101-3p mimics.
圖4過(guò)表達(dá)miRNA-101-3p對(duì)胃癌細(xì)胞MGC-803增殖的影響
過(guò)表達(dá)miRNA-101-3p 48 h后,胃癌細(xì)胞的S期比例為(27.39±0.66)%,顯著低于NC組和空白對(duì)照組,而細(xì)胞分裂處于G0/G1期的胃癌細(xì)胞明顯增多(P<0.05),說(shuō)明過(guò)表達(dá)miRNA-101-3p可以導(dǎo)致胃癌細(xì)胞出現(xiàn)G1期阻滯,進(jìn)而抑制細(xì)胞的增殖,見(jiàn)圖5。
流式細(xì)胞儀檢測(cè)結(jié)果顯示,過(guò)表達(dá)miRNA-101-3p 48 h后,胃癌細(xì)胞早期凋亡率為(15.62±1.26)%,明顯高于NC組(6.20±1.07)%和空白對(duì)照組(4.54±1.32)%(P<0.05),說(shuō)明miRNA-101-3p過(guò)表達(dá)可以促進(jìn)胃癌細(xì)胞出現(xiàn)凋亡,見(jiàn)圖6。
Transwell細(xì)胞遷移實(shí)驗(yàn)的結(jié)果顯示,過(guò)表達(dá)miRNA-101-3p后,胃癌細(xì)胞的遷移能力顯著降低,遷移的細(xì)胞數(shù)明顯低于miRNA NC組和空白對(duì)照組(P<0.05),說(shuō)明過(guò)表達(dá)miRNA-101-3p可以抑制胃癌細(xì)胞遷移,見(jiàn)圖7。
利用在線軟件TargetScan進(jìn)行生物信息學(xué)分析,預(yù)測(cè)結(jié)果顯示,EZH2是miRNA-101-3p的潛在靶基因。為了進(jìn)一步確認(rèn)miRNA-101-3p對(duì)EZH2基因的調(diào)控,Western blot實(shí)驗(yàn)結(jié)果顯示,過(guò)表達(dá)miRNA-101-3p后,EZH2的表達(dá)明顯低于NC組和空白對(duì)照組(P<0.05),而NC組和空白對(duì)照組之間EZH2的表達(dá)沒(méi)有顯著變化,說(shuō)明miRNA-101-3p可以抑制胃癌細(xì)胞MGC803中EZH2蛋白的表達(dá);而胃癌細(xì)胞MGC-803和HGC-27中EZH2的水平顯著高于胃黏膜細(xì)胞GES-1(P<0.05),見(jiàn)圖8。
Figure 5. The effect of miRNA-101-3p overexpression on the cell cycle distribution of gastric cancer cells. Mean±SD.n=3.*P<0.05vsmiRNA-101-3p mimics.
圖5過(guò)表達(dá)miRNA-101-3p對(duì)胃癌細(xì)胞周期的影響
Figure 6. The effect of miRNA-101-3p overexpression on the apoptosis of gastric cancer cells. Mean±SD.n=3.*P<0.05vsmiRNA-101-3p mimics.
圖6過(guò)表達(dá)miRNA-101-3p對(duì)胃癌細(xì)胞凋亡的影響
Figure 7. The effect of miRNA-101-3p on the migration ability of gastric cancer cells detected by Transwell assay (×200). Mean±SD.n=3.*P<0.05vsmiRNA-101-3p mimics.
圖7Transwell遷移實(shí)驗(yàn)檢測(cè)miRNA-101-3p對(duì)胃癌細(xì)胞遷移能力的影響
Figure 8. The protein expression of EZH2 in gastric cancer cells and gastric mucosal cells was detected by Western blot. Mean±SD.n=3.*P<0.05vsmiRNA-101-3p mimics;#P<0.05vsGES-1.
圖8Westernblot實(shí)驗(yàn)檢測(cè)胃癌細(xì)胞和胃黏膜細(xì)胞中EZH2的蛋白表達(dá)
miRNA做為一種非編碼RNA,可以調(diào)控機(jī)體許多生理和病理過(guò)程,大約30%的編碼蛋白基因被miRNA所調(diào)控[10]。miRNA-101-3p在多種腫瘤中表達(dá)下調(diào),研究發(fā)現(xiàn),在肝癌中下調(diào)的miRNA-101-3p可以靶向調(diào)控髓細(xì)胞白血病基因1 (myeloid cell leukemia-1,Mcl-1)促進(jìn)肝癌細(xì)胞凋亡,抑制肝癌細(xì)胞增殖[5];在乳腺癌中,miRNA-101-3p與生存期及淋巴轉(zhuǎn)移密切相關(guān),并且可能通過(guò)調(diào)控CXC趨化因子受體7(CXC chemokine receptor, CXCR7)影響體內(nèi)細(xì)胞周期蛋白D1(cyclin D1)、B細(xì)胞白血病/淋巴瘤2(B-cell leukemia/lymphoma-2,Bcl-2)、基質(zhì)金屬蛋白酶2(matrixmetalloprotein 2,MMP2)、MMP-9和上皮鈣黏蛋白(E-cadherin)等蛋白的表達(dá),進(jìn)而抑制乳腺癌細(xì)胞的增殖[6]。在本研究中,miRNA-101-3p在胃癌細(xì)胞中的表達(dá)下調(diào),為了進(jìn)一步研究miRNA-101-3p對(duì)胃癌細(xì)胞增殖、凋亡以及遷移的影響,采用脂質(zhì)體轉(zhuǎn)染技術(shù)過(guò)表達(dá)胃癌細(xì)胞中miRNA-101-3p的水平,結(jié)果顯示上調(diào)miRNA-101-3p的水平后,胃癌細(xì)胞的生長(zhǎng)和遷移能力明顯受到抑制,細(xì)胞S期明顯減少,G0/G1期延長(zhǎng),細(xì)胞阻滯于G0/G1期,而細(xì)胞的早期凋亡率明顯增加;以上結(jié)果說(shuō)明,miRNA-101-3p在胃癌的發(fā)生和發(fā)展中可能起到了抑癌基因的作用。
EZH2是表觀遺傳調(diào)控因子PcG蛋白的核心組件,在多種腫瘤[11-12]中表達(dá)增高,是腫瘤治療的潛在靶點(diǎn)。已有研究發(fā)現(xiàn)[13-14],胃癌組織中EZH2的表達(dá)明顯增高,而且與生存期密切相關(guān)。在本研究中我們也發(fā)現(xiàn)了胃癌細(xì)胞中EZH2的表達(dá)高于胃黏膜細(xì)胞;有學(xué)者發(fā)現(xiàn)[15],沉默胃癌細(xì)胞中EZH2的表達(dá)后可以誘導(dǎo)P53和組蛋白脫乙酰酶1(histone deacetylase 1, HDAC1)的表達(dá),同時(shí)抑制cyclin D1和cyclin E的表達(dá)進(jìn)而調(diào)控細(xì)胞的增殖; Bai等[16]也發(fā)現(xiàn),抑制EZH2的表達(dá)后可以抑制胃癌細(xì)胞SGC-7901的生長(zhǎng),激活p21和p16的表達(dá),進(jìn)而調(diào)控細(xì)胞周期;吳雪雷等[17]發(fā)現(xiàn) EZH2可以通過(guò)核因子κB(nuclear factor kappa B, NF-κB) 而抑制胃癌細(xì)胞的生長(zhǎng);還有的學(xué)者發(fā)現(xiàn),長(zhǎng)鏈非編碼RNA 00152可通過(guò)結(jié)合EZH2而抑制p15和p21的表達(dá),進(jìn)而促進(jìn)胃癌的進(jìn)展[18],可見(jiàn)EZH2基因可能成為治療胃癌的重要靶點(diǎn)。在本研究中,通過(guò)生物學(xué)信息預(yù)測(cè)EZH2基因可能是miRNA-101-3p的潛在靶基因,也有學(xué)者[19]研究發(fā)現(xiàn)在前列腺癌和乳腺癌中EZH2基因受到miRNA-101-3p的靶向調(diào)控;而在其它腫瘤如膀胱癌[20]和肝癌[21]中通過(guò)雙熒光素報(bào)告基因也驗(yàn)證了miRNA-101-3p對(duì)EZH2基因的靶向調(diào)控;本研究中,Western blot實(shí)驗(yàn)顯示胃癌細(xì)胞中EZH2的表達(dá)顯著高于胃黏膜細(xì)胞,同時(shí)miRNA-101-3p的表達(dá)則低于胃黏膜細(xì)胞,而在上調(diào)胃癌細(xì)胞中的miRNA-101-3p后,EZH2的表達(dá)則明顯下調(diào)。因此我們推斷,在胃癌中EZH2與miRNA-101-3p的水平有可能呈負(fù)相關(guān),并由于miRNA-101-3p下調(diào),使得其對(duì)EZH2基因的負(fù)調(diào)控作用減弱, EZH2的表達(dá)增加,而增高的EZH2蛋白又會(huì)影響E-cadherin[22]、整合素2[23]和印跡基因CDKN1C(p57KP12)[24]等的表達(dá),從而最終促進(jìn)了腫瘤的演進(jìn)。
綜上所述,miRNA-101-3p可能通過(guò)靶向負(fù)調(diào)控EZH2基因的表達(dá),進(jìn)而抑制胃癌細(xì)胞的生長(zhǎng)和遷移,促進(jìn)胃癌細(xì)胞的凋亡。miRNA-101-3p有望成為治療胃癌的重要靶點(diǎn)。
[1] Crew KD, Neugut AI. Epidemiology of gastric cancer[J]. World J Gastroenterol, 2006, 12(3):354-362.
[2] Baba H, Kuwabara K, Ishiguro T, et al. Prognostic factors for stage IV gastric cancer[J]. Int Surg, 2011, 98(2):181-187.
[3] Chandramouli A, Onyeagucha BC, Mercado-Pimentel ME, et al. MicroRNA-101 (miR-101) post-transcriptio-nally regulates the expression of EP4 receptor in colon can-cers[J]. Cancer Biol Ther, 2012, 13(3):175-183.
[4] Zhang H, Qi F, Cao Y, et al. Down-regulated microRNA-101 in bladder transitional cell carcinoma is associated with poor prognosis[J]. Med Sci Monit, 2014, 20(2):812-817.
[5] Su H, Yang JR, Xu T, et al. MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity[J]. Cancer Res, 2009, 69(3):1139-1142.
[6] Li JT, Jia LT, Liu NN, et al. MiRNA-101 inhibits breast cancer growth and metastasis by targeting CX chemokine receptor 7[J]. Oncotarget, 2015, 6(31):30818-30830.
[7] Wang HJ, Ruan HJ, He XJ, et al. MicroRNA-101 is down-regulated in gastric cancer and involved in cell migration and invasion[J]. Eur J Cancer, 2010, 46(12):2295-2303.
[8] Sudo T, Utsunomiya T, Mimori K, et al. Clinicopatholo-gical significance of EZH2 mRNA expression in patients with hepatocellular carcinoma[J]. Br J Cancer, 2005, 92(9):1754-1758.
[9] Mu Z, Li H, Fernandez SV,et al.EZH2 knockdown suppresses the growth and invasion of human inflammatory breast cancer cells[J]. J Exp Clin Cancer Res, 2013, 32:70.
[10] Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets[J]. Cell, 2005, 120(1):15-20.
[11] Varambally S, Dhanasekaran SM, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer[J]. Nature, 2002, 419(6907):624-629.
[12] Chang CJ, Yang JY, Xia W, et al. EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-β-catenin signaling[J]. Cancer Cell, 2011, 19(1):86-100.
[13] Matsukama Y, Semba S, Kato H,et al. Expression of the enhancer of zeste homolog 2 is correlated with poor prognosis in human gastric cancer[J]. Cancer Sci, 2006, 97(6):484-491.
[14] He LJ, Cai MY, Xu GL,et al. Prognostic significance of overexpression of EZH2 and H3k27me3 proteins in gastric cancer[J]. Asian Pac J Cancer Prev, 2012, 13(7):3173-3178.
[15] Choi JH, Song YS, Yoon JS, et al. Enhancer of zeste homolog 2 expression is associate with tumor cell proliferation and metastasis in gastric cancer[J]. APMIS, 2010, 118(3):196-202.
[16] Bai J, Chen J, Ma M, et al. Inhibiting enhancer of zeste homolog 2 promotes cellular senescence in gastric cancer cells SGC-7901 by activation of p21 and p16[J]. DNA Cell Biol, 2014, 33(6):337-344.
[17] 吳雪雷, 蔡耀武, 莊志忠, 等.EZH2對(duì)胃癌細(xì)胞核因子κB靶基因的調(diào)節(jié)作用[J]. 中國(guó)病理生理雜志,2015,31(12) :2169-2175.
[18] Chen WM, Huang MD, Sun DP, et al. Long intergenic non-coding RNA 00152 promotes tumor cell cycle progression by binding to EZH2 and repressing p15 and p21 in gastric cancer[J]. Oncotarget, 2016, 7(9):9773-9787.
[19] Varambally S, Cao Q, Mani RS, et al. Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer[J]. Science, 2008, 322(5908):1695-1699.
[20] Friedman JM, Liang G, Liu CC, et al. The putative tumor suppressor microRNA-101 modulates the cancer epigenome by repressing the polycomb group protein EZH2[J]. Cancer Res, 2009, 69(6):2623-2629.
[21] Xu L, Beckebaum S, Lacob S, et al. MicroRNA-101 inhibits human hepatocellular carcinoma progression through EZH2 downregulation and increased cytostatic drug sensitivity[J]. J Hepatol, 2014, 60(3):590-598.
[22] Han T, Jiao F, Hu H, et al. EZH2 promotes cell migrarion and invasion but not alters cell proliferation by suppressing E-cadherin, partly through association with MALAT-1 in pancreatic cancer[J]. Oncotarget, 2016, 7(10):11194-11207.
[23] Ferraro A, Boni T, Pintzas A. EZH2 regulates cofilin activity and colon cancer cell migration by targeting ITGA2 gene[J]. PLoS One, 2014, 9(12):e115276.
[24] Yang X, Karuturi RK, Sun F, et al.CDKN1C(p57KP12) is a direct target of EZH2 and suppressed by multiple epigenetic mechanisms in breast cancer cells[J]. PLoS One, 2009, 4(4):e5011.
miRNA-101-3p inhibits proliferation and migration of gastric cancer cells by targeting EZH2
ZHANG Xiao-tian1, ZHANG Xiao-yan1, HUANG Sai-ya1, WANG Qian2, LIU Wei1
(1DepartmentofMedicalLaboratoryScience,FenyangCollegeofShanxiMedicalUniversity,Fenyang032200,China;2DepartmentofMedicalImaging,ShanxiMedicalUniversity,Taiyuan030001,China.E-mail: 116048164@qq.com)
AIM: To investigate the role of microRNA-101-3p (miRNA-101-3p) on the proliferation, apoptosis and invasion of gastric cancer cells and the possible regulatory mechanisms.METHODSThe expression of miRNA-101-3p in two kinds of gastric cancer cells and a gastric mucosal cell line was detected by real-time PCR. The miRNA-101-3p was overexpressed by Lipofectamine 2000 transfection with miRNA-101-3p mimics. The effects of miRNA-101-3p on cell cycle distribution and apoptosis were analyzed by flow cytometry. The effects of miRNA-101-3p on cell proliferation and migration abilities were detected by CCK-8 assay, trypan blue exclusion test and Transwell assay. The protein expression of enhancer of zeste homolog 2 (EZH2) was determined by Western blot.RESULTSThe expression of miRNA-101-3p in gastric cancer cells was lower than that in gastric mucosal cells (P<0.05). The gastric cancer cell MGC-803 had the lowest expression level of miRNA-101-3p. The result of flow cytometry showed that the population of S phase was reduced, and the population of G0/G1phase and the early stage apoptotic rate were increased after the expression of miRNA-101-3p was overexpressed (P<0.05). The results of CCK-8 assay, trypan blue exclusion test and Transwell assay showed that overexpression of miRNA-101-3p significantly reduced the proliferation and migration abilities of gastric cancer cells (P<0.05). Overexpression of miRNA-101-3p decreased the protein level of EZH2 (P<0.05).CONCLUSIONmiRNA-101-3p may suppresses the gastric cancer cell proliferation and migration, and promotes the gastric cancer cell apotosis by down-regulation of EZH2.
Gastric cancer; MicroRNA; Cell proliferation; Cell migration; Apotosis; Enhancer of zeste homolog 2
1000- 4718(2017)12- 2143- 08
2017- 07- 17
2017- 11- 07
國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 81301426);山西醫(yī)科大學(xué)汾陽(yáng)學(xué)院科研項(xiàng)目(No. 2017B05)
△通訊作者 Tel: 0358-2100372; E-mail: 116048164@qq.com
R735.2; R363.2
A
10.3969/j.issn.1000- 4718.2017.12.006
(責(zé)任編輯: 林白霜, 羅 森)