長(zhǎng)鏈非編碼RNA CCAT2對(duì)肝癌細(xì)胞增殖和侵襲遷移能力的影響及其機(jī)制探討

紀(jì)易斐，劉金霞，江楓，劉肇修，管程齊，肖明兵，倪潤(rùn)洲

(南通大學(xué)附屬醫(yī)院，江蘇南通226000)

·論著·

長(zhǎng)鏈非編碼RNA CCAT2對(duì)肝癌細(xì)胞增殖和侵襲遷移能力的影響及其機(jī)制探討

紀(jì)易斐，劉金霞，江楓，劉肇修，管程齊，肖明兵，倪潤(rùn)洲

(南通大學(xué)附屬醫(yī)院，江蘇南通226000)

目的觀察長(zhǎng)鏈非編碼RNA CCAT2(lncRNA CCAT2)對(duì)肝細(xì)胞癌(HCC)細(xì)胞增殖和侵襲遷移能力的影響，并探討其機(jī)制。方法將對(duì)數(shù)生長(zhǎng)期的HCC細(xì)胞HepG2隨機(jī)分為A、B、C組，采用Lipofectamine?3000分別瞬時(shí)轉(zhuǎn)染對(duì)照質(zhì)粒、小干擾RNA質(zhì)粒(si-CCAT2)、si-CCAT2+多梳蛋白EZH2過(guò)表達(dá)質(zhì)粒。采用Real-time PCR法檢測(cè)細(xì)胞中的lncRNA CCAT2、EZH2 mRNA，Western blotting法檢測(cè)細(xì)胞中的EZH2蛋白，MTT法觀察培養(yǎng)24、48、72、96 h細(xì)胞增殖能力(OD490)，細(xì)胞劃痕及Transwell實(shí)驗(yàn)分別觀察細(xì)胞的遷移、侵襲能力。結(jié)果與A組比較，B組細(xì)胞中l(wèi)ncRNA CCAT2和EZH2的mRNA和蛋白表達(dá)水平均下降(P均<0.05)；與B組比較，C組胞中EZH2的mRNA和蛋白表達(dá)水平均增加(P均<0.05)。與A組同時(shí)點(diǎn)比較，B組細(xì)胞增殖的OD490值降低(P均<0.05)；與B組同時(shí)點(diǎn)比較，C組細(xì)胞增殖的OD490值升高(P均<0.05)。與A組比較，B組細(xì)胞劃痕愈合百分比、穿膜細(xì)胞數(shù)均下降(P均<0.05)；與B組比較，C組細(xì)胞劃痕愈合百分比、穿膜細(xì)胞數(shù)均增加(P均<0.05)。結(jié)論lncRNA CCAT2可通過(guò)上調(diào)EZH2的表達(dá)促進(jìn)HCC細(xì)胞的增殖、侵襲和遷移，進(jìn)而促進(jìn)HCC的發(fā)生發(fā)展。

肝細(xì)胞癌；長(zhǎng)鏈非編碼RNA CCAT2；EZH2基因；細(xì)胞增殖；細(xì)胞侵襲；細(xì)胞遷移

肝細(xì)胞癌(HCC)是發(fā)病率、病死率均較高的常見(jiàn)惡性腫瘤之一，具有惡性度高、易轉(zhuǎn)移、復(fù)發(fā)及預(yù)后差的特點(diǎn)。近幾十年來(lái)，盡管對(duì)HCC發(fā)生的認(rèn)識(shí)和治療取得了很大進(jìn)展和突破，但有關(guān)HCC增殖、侵襲轉(zhuǎn)移的機(jī)制至今尚未完全闡明。近年來(lái)，長(zhǎng)鏈非編碼RNA(lncRNA)在腫瘤中的重要作用逐漸引起人們廣泛關(guān)注，已經(jīng)成為目前腫瘤研究的熱點(diǎn)。lncRNA在多種腫瘤細(xì)胞中常表達(dá)異常，如HCC、乳腺癌、肺癌、前列腺癌、膀胱癌、腎癌等[1，2]。通過(guò)查閱文獻(xiàn)[3]發(fā)現(xiàn)，lncRNA CCAT2在HCC癌組織中的表達(dá)水平顯著高于癌旁組織，且lncRNA CCAT2高表達(dá)與HCC患者不良預(yù)后密切相關(guān)，但lncRNA CCAT2在HCC中發(fā)揮作用的具體機(jī)制卻不清楚。多梳蛋白EZH2在腫瘤中具有多重作用，具有作為腫瘤早期診斷及治療靶點(diǎn)的潛能[4]。2015年9月～2016年12月，我們利用RNA干擾技術(shù)研究降低lncRNA CCAT2表達(dá)對(duì)HCC細(xì)胞增殖和侵襲轉(zhuǎn)移的影響及其作用機(jī)制，以期為臨床HCC分子靶向治療提供新的潛在靶點(diǎn)和研究方向。

1 材料與方法

1.1 材料 人HCC細(xì)胞系HepG2(美國(guó)ATCC公司)，胎牛血清、DMEM培養(yǎng)液(美國(guó)Gibico公司)，CO2無(wú)菌培養(yǎng)箱(美國(guó)Thermo公司)；lncRNA CCAT2的小干擾RNA質(zhì)粒(si-CCAT2)、對(duì)照質(zhì)粒(si-CON)及EZH2過(guò)表達(dá)質(zhì)粒(ex-EZH2)由上海捷瑞生物工程有限公司構(gòu)建，逆轉(zhuǎn)錄試劑盒、SYRB Green染料(大連寶生物工程有限公司)，定量引物(上海捷瑞生物工程有限公司)；RIPA裂解液(上海碧云天生物技術(shù)有限公司)，兔抗人EZH2多克隆抗體(美國(guó)Abcam公司)，鼠抗兔二抗(北京中杉金橋生物技術(shù)有限公司)，ECL顯影液(美國(guó)Millipore公司)，凝膠成像儀(美國(guó)UVP公司)；MTT溶液(上海翊圣生物科技有限公司)，酶標(biāo)儀(美國(guó)Biotek公司)；Transwell小室(美國(guó)Corning公司)，ECM膠(美國(guó)Sigma公司)。

1.2 實(shí)驗(yàn)方法

1.2.1 細(xì)胞分組與轉(zhuǎn)染處理 將對(duì)數(shù)生長(zhǎng)期的HepG2隨機(jī)分為A、B、C組，采用Lipofectamine?3000分別瞬時(shí)轉(zhuǎn)染si-CON、si-CCAT2、si-CCAT2+ex-EZH2。

1.2.2 細(xì)胞中l(wèi)ncRNA CCAT2、EZH2 mRNA檢測(cè) 采用Real-time PCR法。取各組細(xì)胞，用TRIzol法提取細(xì)胞總RNA；取1 μg RNA逆轉(zhuǎn)錄得到cDNA，用SYRB Green染料檢測(cè)lncRNA CCAT2或EZH2 mRNA的表達(dá)水平。lncRNA CCAT2上游引物為3′-CCCTGGTCAAATTGCTTAACCT-5′、下游引物為3′-TTATTCGTCCCTCTGTTTTATGGAT-5′，EZH2上游引物為3′-AATCAGAGTACATGCGACTGAGA-5′、下游引物為3′-GCTGTATCCTTCGCTGTTTCC-5′，內(nèi)參引物β-actin上游引物為3′-TCATGAAGTGTGACGTGGACAT-5′、下游引物為3′-CTCAGGAGGAGCAATGATCTTG-5′。lncRNA CCAT2或EZH2轉(zhuǎn)錄水平的相對(duì)表達(dá)量用2-ΔΔCt法分析。

1.2.3 細(xì)胞中EZH2蛋白檢測(cè) 采用Western blotting法。收取各組細(xì)胞，用冰浴的PBS洗滌2次；加入RIPA裂解液收取總蛋白，測(cè)定蛋白濃度；將30 μg蛋白經(jīng)聚丙烯酰胺凝膠電泳后，轉(zhuǎn)移至PVDF膜；5%脫脂奶粉室溫封閉1 h，4 ℃下結(jié)合EZH2或GAPDH(內(nèi)參)一抗過(guò)夜；TST洗3次，室溫結(jié)合二抗1 h；TST洗3次，ECL顯影，凝膠成像儀對(duì)蛋白條帶進(jìn)行灰度分析，計(jì)算目的蛋白與內(nèi)參蛋白條帶的灰度值比值。

1.2.4 細(xì)胞增殖情況觀察 采用MTT法。將對(duì)數(shù)生長(zhǎng)期的HepG2，以1×103/孔接種于96孔板，分組處理同1.2.1，每小組6個(gè)復(fù)孔。分別于培養(yǎng)24、48、72、96 h每孔加10 μL的MTT溶液(5 mg/mL)，孵育4 h棄掉上清；每孔加150 μL的DMSO，振蕩、融解，于酶標(biāo)儀上測(cè)量490 nm波長(zhǎng)時(shí)的光密度值(OD490)，取6個(gè)復(fù)孔的均數(shù)。

1.2.5 細(xì)胞遷移能力觀察 采用細(xì)胞劃痕實(shí)驗(yàn)。將細(xì)胞接種于12孔板，分組處理同1.2.1。用100 μL槍頭垂直于孔板制作細(xì)胞劃痕，盡量保證各個(gè)劃痕寬度一致。吸去細(xì)胞培養(yǎng)液，用PBS沖洗孔板3次，洗去劃痕產(chǎn)生的細(xì)胞碎片。加入無(wú)血清培養(yǎng)基，拍照記錄。將培養(yǎng)板放入培養(yǎng)箱培養(yǎng)，48 h后再取出拍照，計(jì)算劃痕愈合百分比。

1.2.6 細(xì)胞侵襲能力觀察 采用Transwell實(shí)驗(yàn)。在每個(gè)8 μm孔徑的Transwell小室內(nèi)加入50 μg ECM膠進(jìn)行包被，37 ℃無(wú)菌條件下放置6 h；ECM膠充分凝固后，上室中接種細(xì)胞2×105/孔。上室加入無(wú)抗生素、無(wú)血清培養(yǎng)基，下室加入含10%血清的無(wú)抗生素培養(yǎng)基，繼續(xù)培養(yǎng)24 h；將小室取出，用無(wú)菌棉簽擦拭去除上室內(nèi)的細(xì)胞，以1%結(jié)晶紫染液將小室多孔膜下表面的細(xì)胞染色，拍照。計(jì)數(shù)紫色染色的穿膜細(xì)胞，細(xì)胞數(shù)越多，即細(xì)胞侵襲能力越強(qiáng)。

2 結(jié)果

2.1 各組細(xì)胞中l(wèi)ncRNA CCAT2、EZH2表達(dá)情況比較 與A組比較，B組細(xì)胞中l(wèi)ncRNA CCAT2和EZH2的mRNA和蛋白表達(dá)水平下降(P均<0.05)；與B組比較，C組胞中EZH2的mRNA和蛋白表達(dá)水平均增加(P均<0.05)。見(jiàn)表1。

表1 各組細(xì)胞中l(wèi)ncRNA CCAT2、EZH2表達(dá)情況比較

注：與A組比較，*P<0.05；與B組比較，#P<0.05。

2.2 各組細(xì)胞增殖情況比較 與A組同時(shí)點(diǎn)比較，B組細(xì)胞增殖的OD490值降低(P均<0.05)；與B組同時(shí)點(diǎn)比較，C組細(xì)胞增殖的OD490值升高(P均<0.05)。見(jiàn)表2。

表2 各組細(xì)胞增殖情況比較

注：與A組同時(shí)點(diǎn)比較，*P<0.05；與B組同時(shí)點(diǎn)比較，#P<0.05。

2.3 各組細(xì)胞遷移、侵襲能力比較 與A組比較，B組細(xì)胞劃痕愈合百分比、穿膜細(xì)胞數(shù)均下降(P均<0.05)；與B組比較，C組細(xì)胞劃痕愈合百分比、穿膜細(xì)胞數(shù)均增加(P均<0.05)。見(jiàn)表3。

表3 各組細(xì)胞遷移、侵襲能力比較

注：與A組比較，*P<0.05；與B組比較，#P<0.05。

3 討論

HCC在腫瘤相關(guān)死亡中僅次于肺癌，位居第二[5]，嚴(yán)重威脅我國(guó)人民的健康和生命。HCC發(fā)病是一個(gè)綜合病因所致的復(fù)雜過(guò)程，目前尚未研究透徹。已有研究[6～8]發(fā)現(xiàn)，lncRNA的表達(dá)異常與HCC的發(fā)生發(fā)展密切相關(guān)。lncRNA CCAT2可促進(jìn)乳腺癌和胃癌細(xì)胞的增殖、轉(zhuǎn)移，促進(jìn)腫瘤細(xì)胞的惡性進(jìn)展[9～12]。HCC癌細(xì)胞中l(wèi)ncRNA CCAT2是否可以通過(guò)促進(jìn)細(xì)胞增殖和侵襲來(lái)促進(jìn)HCC進(jìn)展影響患者的預(yù)后，lncRNA CCAT2通過(guò)何種機(jī)制調(diào)節(jié)細(xì)胞增殖和侵襲，均尚未研究清楚。

多梳蛋白EZH2位于人類(lèi)染色體7q35，包含20個(gè)外顯子，全長(zhǎng)近40 kb，編碼746個(gè)氨基酸的多肽。EZH2編碼的蛋白含有高度保守的SET結(jié)構(gòu)域,該結(jié)構(gòu)域具有甲基轉(zhuǎn)移酶的活性,通過(guò)甲基化作用抑制下游基因的表達(dá)。EZH2是PRC2復(fù)合物中的一員，而PRC2是一個(gè)作用于組蛋白H3賴氨酸位點(diǎn)K27(H3K27)的高度保守的組蛋白甲基轉(zhuǎn)移酶；EZH2是PRC2核心組成部分，構(gòu)成PRC2蛋白復(fù)合物中的一個(gè)催化亞基[13]。近年研究[14]表明,EZH2在人類(lèi)多種癌組織過(guò)表達(dá)，如HCC、乳腺癌、前列腺癌、肺癌、胃癌、宮頸癌、淋巴瘤等。研究發(fā)現(xiàn)，EZH2與腫瘤的增殖、轉(zhuǎn)移和不良預(yù)后密切相關(guān)；EZH2表達(dá)水平越高，腫瘤惡性程度越高，預(yù)后越差。例如，宮頸癌組織中EZH2高表達(dá)與腫瘤增殖、不良預(yù)后密切相關(guān)[15]；乳腺癌組織中EZH2的降解可以削弱腫瘤的侵襲、轉(zhuǎn)移能力[16]。本研究發(fā)現(xiàn)，lncRNA CCAT2可以通過(guò)上調(diào)EZH2發(fā)揮其促進(jìn)HCC增殖、侵襲和遷移的作用，即EZH2可被異常表達(dá)的lncRNA CCAT2激活。除了本研究發(fā)現(xiàn)的lncRNA CCAT2外，ANCR、SPRY4-IT1、XIST等也可參與對(duì)EZH2的表達(dá)調(diào)控[17～19]。本研究中l(wèi)ncRNA CCAT2如何上調(diào)EZH2的表達(dá)并不十分清楚，需要進(jìn)一步的研究。

本研究發(fā)現(xiàn)，EZH2可促進(jìn)HCC的增殖、侵襲和遷移，但沒(méi)有深入探討EZH2促進(jìn)HCC細(xì)胞進(jìn)展的具體機(jī)制。EZH2可通過(guò)介導(dǎo)抑制性組蛋白修飾 H3K27me3下調(diào)一系列基因的表達(dá)，從而促進(jìn)腫瘤細(xì)胞的增殖、遷移、侵襲或抑制凋亡。表觀遺傳的改變是腫瘤的重要標(biāo)志之一，異常的組蛋白修飾可導(dǎo)致抑癌基因的沉默。例如，EZH2可通過(guò)抑制靶基因E-cadherin表達(dá)，調(diào)控腫瘤轉(zhuǎn)移能力[20]；EZH2可通過(guò)抑制促凋亡基因BIK、NOXA的表達(dá)來(lái)減弱乳腺癌細(xì)胞的凋亡反應(yīng)能力，從而促進(jìn)化療耐藥[21]；EZH2還可沉默IGTA2表達(dá)，從而促進(jìn)結(jié)直腸癌的上皮-間質(zhì)轉(zhuǎn)換[22]。

EZH2還可以通過(guò)與多種lncRNA相互作用，被lncRNA帶至靶基因區(qū)域，發(fā)揮其調(diào)控功能。例如，Luo等[20]研究發(fā)現(xiàn)，EZH2可通過(guò)與lncRNA H19相互作用，促進(jìn)膀胱癌的轉(zhuǎn)移；在乳腺癌中，EZH2與H19相互作用促進(jìn)乳腺癌細(xì)胞對(duì)化療藥物耐受[21]；EZH2還可通過(guò)與lncRNA hotair相互作用促進(jìn)膠質(zhì)瘤的進(jìn)展[23]；EZH2可與lncRNA NEAT1作用調(diào)節(jié)乳腺癌細(xì)胞生長(zhǎng)[24]。本研究發(fā)現(xiàn)，CCAT2促進(jìn)HCC的作用之一是通過(guò)直接上調(diào)EZH2表達(dá)，lncRNA CCAT2是否也可以通過(guò)與EZH2相互作用被帶至下游特定靶基因處，需后續(xù)實(shí)驗(yàn)驗(yàn)證。

綜上所述，本研究結(jié)果證明，HCC細(xì)胞中高表達(dá)lncRNA CCAT2可促進(jìn)HCC細(xì)胞的增殖、侵襲和遷移，并且是通過(guò)直接上調(diào)組蛋白甲基轉(zhuǎn)移酶EZH2表達(dá)的方式來(lái)實(shí)現(xiàn)。這表明lncRNA CCAT2可行使癌基因的功能，有望成為HCC早期診斷和靶向治療的有效靶點(diǎn)。

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Effects of lncRNA CCAT2 on proliferation, invasion, and migration of hepatocellular carcinoma cells

JIYifei,LIUJinxia,JIANGFeng,LIUZhaoxiu,GUANChengqi,XIAOMingbing,NIRunzhou

(AffiliatedHospitalofNantongUniversity,Jiangsu226000,China)

ObjectiveTo observe the role and mechanism of lncRNA CCAT2 in proliferation, invasion and migration of hepatocellular carcinoma (HCC) cells.MethodsHepG2 cells in logarithmic phase were randomly divided into groups A, B, and C, which were transiently transfected with control plasmids, small interfering RNA (si-CCAT2), and si-CCAT2+EZH2 overexpression plasmid by using Lipofectamine?3000. The real-time PCR was used to detect the expression of lncRNA CCAT2 and EZH2 mRNA. Western blotting was used to detect the protein level of EZH2. MTT method was used to observe the proliferation of cells at 24, 48, 72 and 96 h (OD490). Scratch test and Transwell assay were used to detect cell migration and invasion.ResultsCompared with group A, the mRNA and protein expression of lncRNA CCAT2 and EZH2 decreased in the group B (bothP<0.05). Compared with group B, the mRNA and protien levels of EZH2 increased in the group C (bothP<0.05). The OD490value representing cell proliferation was lower in the group B than that of the group A, and the OD490value in the group C was higher than that of the group B (allP<0.05). The percentage of wound healing and the number of penetrating cells in the group B were lower than those of the group A (bothP<0.05); compared with group B, the percentage of wound healing and the number of penetrating cells were higher in the group C than in the group B (bothP<0.05).ConclusionsLncRNA CCAT2 can promote the proliferation, invasion, and migration of HCC cells by up-regulating the expression of EZH2, and thus promote the occurrence and development of HCC.

hepatocellular carcinoma; long non-coding RNA CCAT2; EZH2 gene; cell proliferation; cell invasion; cell migration

10.3969/j.issn.1002-266X.2017.34.001

R735.7

A

1002-266X(2017)34-0001-04

2017-07-05)

國(guó)家自然科學(xué)基金資助項(xiàng)目(81502055)。

紀(jì)易斐(1982-)，男，碩士，主治醫(yī)師，主要研究方向?yàn)橄到y(tǒng)腫瘤。E-mail: li863268624@foxmail.com

肖明兵(1972-)，男，碩士，副主任醫(yī)師，主要研究方向?yàn)橄到y(tǒng)疾病。E-mail: li863268@126.com