張婧婧， 吳 偉， 王 鵬， 楊景瑞， 段巨洪， 于海川△

(新鄉(xiāng)醫(yī)學(xué)院 1醫(yī)學(xué)檢驗(yàn)學(xué)院和分子診斷與醫(yī)學(xué)檢驗(yàn)技術(shù)河南省協(xié)同創(chuàng)新中心， 2第三附屬醫(yī)院，河南 新鄉(xiāng) 453003)

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長鏈非編碼RNA HOTAIR通過調(diào)控PIK3R3促進(jìn)肝癌HepG2細(xì)胞的轉(zhuǎn)移和侵襲*

張婧婧1， 吳 偉2， 王 鵬2， 楊景瑞1， 段巨洪1， 于海川1△

(新鄉(xiāng)醫(yī)學(xué)院1醫(yī)學(xué)檢驗(yàn)學(xué)院和分子診斷與醫(yī)學(xué)檢驗(yàn)技術(shù)河南省協(xié)同創(chuàng)新中心，2第三附屬醫(yī)院，河南 新鄉(xiāng) 453003)

目的： 探討長鏈非編碼RNA HOX轉(zhuǎn)錄反義RNA (HOTAIR)對(duì)肝癌HepG2細(xì)胞轉(zhuǎn)移和侵襲的影響。方法: 運(yùn)用免疫組化技術(shù)檢測磷脂酰肌醇3-激酶調(diào)節(jié)亞基3(phosphoinositide-3-kinase regulatory subunit 3，PIK3R3)在正常肝臟組織和肝癌組織的表達(dá)；運(yùn)用qPCR和Western blot檢測慢病毒LV3-shHOTAIR和LV3-shPIK3R3對(duì)HOTAIR和PIK3R3基因的沉默效率；Transwell侵襲實(shí)驗(yàn)檢測HOTAIR和PIK3R3的表達(dá)對(duì)肝癌細(xì)胞HepG2侵襲能力的影響；劃痕實(shí)驗(yàn)檢測HOTAIR和PIK3R3的表達(dá)對(duì)肝癌細(xì)胞HepG2遷移能力的影響；qPCR檢測沉默HOTAIR和PIK3R3后miR-214的表達(dá)；qPCR檢測轉(zhuǎn)染miR-214 mimics和miR-214 inhibitor后HOTAIR和PIK3R3的表達(dá)；雙螢光素酶報(bào)告基因系統(tǒng)檢測miR-214對(duì)HOTAIR和PIK3R3轉(zhuǎn)錄活性的影響。結(jié)果: 和正常肝組織比較，PIK3R3在肝癌組織中的表達(dá)明顯增加；沉默HOTAIR和PIK3R3基因后，肝癌細(xì)胞株HepG2的侵襲和轉(zhuǎn)移能力明顯降低；沉默HOTAIR和PIK3R3基因后，miR-214表達(dá)上調(diào)；轉(zhuǎn)染miR-214 mimics后，HOTAIR和PIK3R3的表達(dá)降低；轉(zhuǎn)染miR-214 inhibitor后，HOTAIR和PIK3R3的表達(dá)上調(diào)；雙螢光素酶報(bào)告基因系統(tǒng)檢測結(jié)果顯示miR-214可以直接調(diào)控HOTAIR和PIK3R3的轉(zhuǎn)錄活性。結(jié)論: HOTAIR可以通過miR-214調(diào)控PIK3R3的表達(dá)，從而促進(jìn)肝癌細(xì)胞的侵襲和遷移能力

HOX轉(zhuǎn)錄反義RNA； 肝癌； 磷脂酰肌醇3-激酶調(diào)節(jié)亞基3

肝細(xì)胞癌是我國常見的一種惡性腫瘤，其發(fā)病率較高，侵襲性極強(qiáng)，進(jìn)展非常迅速，預(yù)后極差。對(duì)患者生命造成嚴(yán)重威脅[1-2]。生長較為迅速，早期就出現(xiàn)淋巴管、血管以及遠(yuǎn)處器官轉(zhuǎn)移等惡性臨床病理特點(diǎn)。與肝癌患者預(yù)后較差有關(guān)。探索肝癌發(fā)生、發(fā)展的生物學(xué)機(jī)制，并闡述肝癌發(fā)生和進(jìn)展的分子生物學(xué)機(jī)制，可以為肝癌的診治提供新的重要途徑[3]。

近年來，盡管手術(shù)方式的改進(jìn)、化療和放療效果的不斷提高，但是肝癌患者尤其是晚期肝癌患者的5年生存率仍然很低。腫瘤轉(zhuǎn)移是肝癌患者死亡的最主要的原因[4-5]。腫瘤轉(zhuǎn)移第一步是降低腫瘤細(xì)胞黏附能力，增強(qiáng)細(xì)胞運(yùn)動(dòng)性，然后浸潤周圍臨近組織和遠(yuǎn)處轉(zhuǎn)移[6]。目前腫瘤轉(zhuǎn)移的分子生物學(xué)機(jī)制尚不明確，這對(duì)腫瘤治療造成很大的障礙。

長鏈非編碼RNA(long noncoding RNA，lncRNA)是一類長度大于200個(gè)核苷酸、不編碼蛋白的RNA分子。其可在表觀遺傳水平、轉(zhuǎn)錄和轉(zhuǎn)錄后水平調(diào)控基因的表達(dá), 從而廣泛參與機(jī)體的生理與病理過程。 越來越多的證據(jù)表明lncRNA參與肝癌的發(fā)生與轉(zhuǎn)移過程。 研究表明在肝細(xì)胞癌中有一部分異常表達(dá)的lncRNA與腫瘤的發(fā)生、轉(zhuǎn)移、進(jìn)展或診斷有關(guān)[7-8]。 HOTAIR是一種反義lncRNA，同時(shí)也是第一個(gè)被發(fā)現(xiàn)具有反式轉(zhuǎn)錄作用的lncRNA。Bayram等[9]研究發(fā)現(xiàn)，人體11 種成纖維細(xì)胞中轉(zhuǎn)錄自HOX基因的非編碼RNA 中有231 個(gè)lncRNA，同時(shí)在位于第12號(hào)染色體的HOXC基因座發(fā)現(xiàn)了一種特殊的lncRNA，其作用方式和其它lncRNA不同，主要是通過反式作用沉默染色質(zhì)，并且編碼該lncRNA的DNA 僅一條鏈可以被轉(zhuǎn)錄，同時(shí)與HOX基因序列也不在同一條DNA 鏈上，因此將其命名為HOX轉(zhuǎn)錄反義RNA(HOX transcript antisense RNA，HOTAIR)。先前的研究發(fā)現(xiàn)，HOTAIR可以調(diào)控卵巢癌、乳腺癌、結(jié)腸癌等腫瘤的惡性生物學(xué)行為[9-10]。有文獻(xiàn)報(bào)道HOTAIR在肝癌中可以調(diào)控肝癌細(xì)胞的惡性生物學(xué)行為，但是在肝癌中其機(jī)制尚不明確。因此我們擬研究HOTAIR在肝癌細(xì)胞中的作用及其機(jī)制。

材 料 和 方 法

1 細(xì)胞株與主要試劑

人肝癌細(xì)胞株HepG2購自武漢大學(xué)中國典型培養(yǎng)物保藏中心。細(xì)胞在含10%胎牛血清的RPMI-1640，37 ℃、5% CO2條件下培養(yǎng)。胎牛血清和RPMI-1640培養(yǎng)基均購自Gibco；PIK3R3兔單克隆抗體均購自Abcam；Transwell小室購自Millipore；Matrigel購自BD；miR-214 mimics、miR-214 inhibitors、磷脂酰肌醇3-激酶調(diào)節(jié)亞基3(phosphoinositide-3-kinase regulatory subunit 3,PIK3R3)和HOTAIR沉默及對(duì)照慢病毒購自上海吉瑪制藥技術(shù)有限公司；PIK3R3和HOTAIR沉默慢病毒載體分別命名為LV3-shPIK3R3和LV3-shHOTAIR，其中LV3-NC為對(duì)照。miR-214、PIK3R3和HOTAIR的qPCR引物以及RNA提取試劑盒、逆轉(zhuǎn)錄試劑盒、PCR試劑盒均購自廣州復(fù)能基因有限公司。

2 實(shí)驗(yàn)方法

2.1 免疫組化實(shí)驗(yàn) 30例肝癌組織從手術(shù)中獲得；癌旁組織為距離腫瘤組織旁2 cm的組織。石蠟包埋組織，切片厚度4 μm。免疫組化實(shí)驗(yàn)步驟按免疫組化SP試劑盒(北京博奧森生物技術(shù)有限公司)說明書操作，經(jīng)組織脫蠟、水化，在枸櫞酸鹽緩沖液(pH 6.0)中使用微波爐抗原修復(fù)30 min，冷卻至室溫；然后用PBS洗3 min 3次，3% H2O237 ℃孵育15 min，PBS洗3 min 3次，5%驢血清(Abcam)37 ℃水浴30 min，兔抗人PIK3R3多克隆抗體(1∶100)37 ℃水浴2 h，PBS洗3 min 3次，加辣根過氧化物酶標(biāo)記的驢抗兔IgG(1∶200，北京博奧森生物技術(shù)有限公司)孵育，PBS洗3 min 3次，辣根酶標(biāo)記鏈酶卵白素工作液(北京博奧森生物技術(shù)有限公司)37 ℃水浴20 min，PBS洗3 min 3次，二氨基聯(lián)苯胺(DAB)顯色。陰性對(duì)照運(yùn)用PBS代替 I 抗。所有切片的閱片經(jīng)3名病理科醫(yī)生獨(dú)立完成，每個(gè)人選擇22個(gè)具有代表性的高倍視野(×400)，同時(shí)計(jì)數(shù)每個(gè)標(biāo)本陽性細(xì)胞數(shù)的比例，陽性結(jié)果的判定按照De Falco 等[11]描述的方法判定并評(píng)分：陽性細(xì)胞數(shù)不到1%為0分；陽性細(xì)胞數(shù)占1%到20%為1分；陽性細(xì)胞數(shù)占21%到40%為2分；陽性細(xì)胞數(shù)占41%到60%為3分；陽性細(xì)胞數(shù)超過61%為4分。

2.2 qPCR 檢測miR-214、PIK3R3和HOTAIR的表達(dá) 用TRIzol (Gibco)抽提組織總RNA，逆轉(zhuǎn)錄，qPCR擴(kuò)增miR-214、PIK3R3和HOTAIR，PIK3R3和HOTAIR用GAPDH作為內(nèi)參照，miR-214使用U6作為內(nèi)參照。反應(yīng)條件為95 ℃ 10 min；95 ℃ 10 s，60 ℃ 20 s，72 ℃ 10 s，重復(fù)35個(gè)循環(huán)。每個(gè)樣品設(shè)3個(gè)平行復(fù)孔，取平均值。反應(yīng)結(jié)束后，由軟件自動(dòng)得出螢光反應(yīng)曲線以及每個(gè)標(biāo)本反應(yīng)體系的擴(kuò)增效率及Ct值，實(shí)驗(yàn)重復(fù)3次。

2.3 PIK3R3蛋白的檢測 裂解細(xì)胞以每孔20 μg上樣，經(jīng)聚丙烯酰胺凝膠電泳后移至PVDF膜，然后加入山羊抗人PIK3R3多克隆抗體(稀釋度1∶5 000)孵育2 h后洗膜，再加入辣根過氧化物酶標(biāo)記驢抗羊IgG，采用化學(xué)熒光發(fā)光法(ECL)顯影。運(yùn)用凝膠成像系統(tǒng)成像，計(jì)算PIK3R3相對(duì)表達(dá)量。

2.4 Transwell侵襲實(shí)驗(yàn)檢測肝癌細(xì)胞的遷移侵襲能力 所有試劑及器材均于冰上預(yù)冷，將Transwell小室置于24孔板內(nèi)，小室內(nèi)膜均勻涂抹Matrigel膠 50 μL(0.2 g/L)，37 ℃孵育15 min，使膠凝固；消化、離心、計(jì)數(shù)細(xì)胞后，按照2.5×107/L用無血清培養(yǎng)基稀釋細(xì)胞，制成細(xì)胞懸液；按照每孔200 μL，將細(xì)胞懸液加入Transwell上室，同時(shí)在Transwell下室加入10% 胎牛血清+培養(yǎng)基500 μL，放入37 ℃孵箱培養(yǎng)；甲醛固定，結(jié)晶紫染色10 min，然后用棉簽輕輕擦拭內(nèi)膜上的細(xì)胞。顯微鏡下計(jì)數(shù)4個(gè)高倍視野下穿過濾膜的細(xì)胞數(shù)。所有實(shí)驗(yàn)重復(fù)3次。

2.5 劃痕實(shí)驗(yàn)檢測肝癌細(xì)胞的遷移能力 將HepG2細(xì)胞接種于6孔板，待細(xì)胞融合度生長在90%時(shí)，用200 μL消毒槍頭從上而下劃線，并在顯微鏡下觀察，測量劃痕的初始距離(0 h)；在24 h、48 h和72 h后，分別測量劃痕的距離，并拍照，計(jì)算細(xì)胞的遷移率。所有實(shí)驗(yàn)重復(fù)3次。各組細(xì)胞任意3個(gè)部位的劃痕的寬度為D，則遷移率=[D(t=24 h、48 h或72 h)-D(t=0 h)]/D(t=0 h)。

2.6 雙螢光素酶報(bào)告基因檢測 于24孔板接種穩(wěn)定轉(zhuǎn)染的細(xì)胞，細(xì)胞濃度為2×104。放置于細(xì)胞培養(yǎng)箱培養(yǎng)過夜。觀察細(xì)胞狀態(tài)，細(xì)胞融合約70%時(shí)轉(zhuǎn)染質(zhì)粒。轉(zhuǎn)染試劑采用EndofectinTM-Plus。根據(jù)說明書步驟操作。將質(zhì)粒DNA(3.2 μg)與phRL-TK(0.8 μg)按1∶4混合后，加入12 μL EndoFectinTM試劑。一邊輕輕渦旋裝有質(zhì)粒溶液的試管，一邊將DMEM培養(yǎng)基稀釋的EndoFectinTM試劑滴加至試管中。充分混勻后，室溫靜置 10 min以形成 DNA-EndofectinTM復(fù)合物。將復(fù)合物加入24孔板中，輕輕搖細(xì)胞培養(yǎng)板，使均勻混合。24 h后，傾倒細(xì)胞培養(yǎng)液，每孔加入200 μL報(bào)告基因細(xì)胞裂解液。于室溫下裂解10 min，待細(xì)胞充分裂解后，12 000 r/min離心5 min，移液器吸取上清用于后續(xù)測定。融解海腎螢光素酶檢測緩沖液，待達(dá)到室溫。將海腎螢光素酶檢測底物(100×)放置于冰盒上備用。按照檢測每個(gè)孔的樣品需要100 μL檢測工作液的量，配置適量海腎螢光素酶檢測工作液。運(yùn)用多功能酶標(biāo)儀檢測。每個(gè)組3個(gè)復(fù)孔。并記錄數(shù)據(jù)。

3 統(tǒng)計(jì)學(xué)分析

采用SPSS 20.0軟件，計(jì)量數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，2組間均數(shù)比較采用t檢驗(yàn)；3組

之間比較采用單因素方差分析，事后兩兩比較采用SNK-q檢驗(yàn)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 PIK3R3在肝癌組織中表達(dá)增加

免疫組化結(jié)果顯示，PIK3R3定位在細(xì)胞膜和細(xì)胞漿。PIK3R3在肝癌組織中呈強(qiáng)陽性表達(dá)，在癌旁組織中呈弱陽性表達(dá)。肝癌組織中PIK3R3的表達(dá)明顯高于癌旁組織，差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，見圖1。

Figure 1.The expression of PIK3R3 in the liver cancer detected by immunohistochemical staining (×20). Mean±SD.n=3.*P<0.05vsadjacent to carcinoma.

圖1 免疫組化檢測PIK3R3在肝癌組織和癌旁組織的表達(dá)情況

2 HOTAIR通過miR-214調(diào)控 PIK3R3的表達(dá)

通過生物信息學(xué)預(yù)測(TargetScan)表明，HOTAIR和PIK3R3互為內(nèi)源競爭性RNA。HOTAIR和PIK3R3可能通過miR-214相互調(diào)控。我們的結(jié)果發(fā)現(xiàn)，和對(duì)照組(LV3-NC)比較，肝癌細(xì)胞株HepG2感染慢病毒LV3-shHOTAIR后，PIK3R3的mRNA和蛋白水平明顯下調(diào)(P<0.05)；和對(duì)照組(LV3-NC)比較，肝癌細(xì)胞株HepG2感染慢病毒LV3-shPIK3R3后，HOTAIR的mRNA水平明顯下調(diào)(P<0.05)，提示HOTAIR和 PIK3R3可以相互調(diào)控。分別沉默HOTAIR和PIK3R3后，miR-214表達(dá)上調(diào)(P<0.05)，提示HOTAIR可能通過miR-214調(diào)控PIK3R3的表達(dá)，見圖2。

Figure 2.HOTAIR regulated PIK3R3 by miR-214. A: silencing ofHOTAIRregulated the mRNA and protein expression of PIK3R3; B: silencing ofPIK3R3 regulated the mRNA expression of HOTAIR; C: silencing ofHOTAIRandPIK3R3 regulated the expression of miR-214. Mean±SD.n=3.*P<0.05vsLV3-NC group.

圖2 HOTAIR通過miR-214調(diào)控 PIK3R3的表達(dá)

3 HOTAIR和PIK3R3是miR-214的直接靶點(diǎn)

為了進(jìn)一步證實(shí)HOTAIR和PIK3R3互為內(nèi)源競爭性RNA，運(yùn)用miR-214 mimics和miR-214 inhibitor轉(zhuǎn)染HepG2細(xì)胞。結(jié)果顯示，和對(duì)照組比較，轉(zhuǎn)染miR-214 mimics后，HOTAIR和 PIK3R3的表達(dá)降低；轉(zhuǎn)染miR-214 inhibitors后，HOTAIR和 PIK3R3的表達(dá)上調(diào)。同時(shí)雙螢光素酶報(bào)告系統(tǒng)檢測發(fā)現(xiàn)，HOTAIR和PIK3R3是miR-214的直接靶點(diǎn),可能通過miR-214相互調(diào)控，見圖3。

Figure 3.HOTAIR and PIK3R3 were direct targets of miR-214. A: the expression of HOTAIR and PIK3R3 was decreased when transfected with miR-214 mimics; B: the expression of HOTAIR and PIK3R3 was increased when transfected with miR-214 inhibitor; C: transfection of miR-214 mimics suppressed luciferase activity of miR-214 and HOTAIR binding sites; D: transfection of miR-214 mimics suppressed luciferase activity of miR-214 and PIK3R3 binding sites. Mean±SD.n=3.*P<0.05vsNC group.

圖3 HOTAIR和PIK3R3是miR-214的直接靶點(diǎn)

4 沉默HOTAIR和PIK3R3后，肝癌細(xì)胞株HepG2侵襲能力降低

Transwell結(jié)果顯示，感染LV3-shHOTAIR或者LV3-shPIK3R3后，肝癌細(xì)胞HepG2通過Matrigel基質(zhì)膠的細(xì)胞數(shù)量分別為45.5±6.8和 50.7±4.7,明顯少于感染LV3-NC的HepG2細(xì)胞，差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05),說明沉默HOTAIR和PIK3R3可以抑制肝癌細(xì)胞HepG2的侵襲能力，見圖4。

Figure 4.The effects ofHOTAIRandPIK3R3 silencing on the invasion ability of HepG2 cells detected by Transwell Matrigel invasion assay. Mean±SD.n=3.*P<0.05vsLV3-NC group.

圖4 Transwell侵襲實(shí)驗(yàn)檢測沉默HOTAIR和PIK3R3對(duì)肝癌細(xì)胞HepG2侵襲能力的影響

5 沉默HOTAIR和PIK3R3后，肝癌細(xì)胞株HepG2的遷移能力降低

劃痕實(shí)驗(yàn)結(jié)果表明，與LV3-NC轉(zhuǎn)染細(xì)胞組比較，在48 h時(shí)，LV3-shHOTAIR或LV3-shPIK3R3感染細(xì)胞組的遷移率明顯降低，差異有統(tǒng)計(jì)學(xué)顯著性,表明沉默HOTAIR和PIK3R3后，肝癌細(xì)胞株HepG2遷移能力降低，見圖5。

討 論

HOTAIR由HOXC基因的反義鏈轉(zhuǎn)錄產(chǎn)生，可以通過招募PCR2蛋白復(fù)合體，從而促使組蛋白H3K27的甲基化修飾，進(jìn)而下調(diào)抑癌基因的表達(dá)，最后促進(jìn)腫瘤的生長和轉(zhuǎn)移[12-14]。Yang等[15]運(yùn)用檢測50 位肝癌患者肝癌組織和癌旁組織中HOTAIR 的表達(dá)發(fā)現(xiàn)，肝癌組織中HOTAIR 表達(dá)水平明顯高于癌旁組織，同時(shí)他們研究發(fā)現(xiàn)，肝癌復(fù)發(fā)患者中有65.7% 的患者HOTAIR 存在高表達(dá)狀態(tài)，但是未現(xiàn)復(fù)發(fā)患者HOTAIR低表達(dá)。因此推測HOTAIR可以作為肝癌患者預(yù)后的一個(gè)獨(dú)立標(biāo)志。Ishibashi等[16]研究發(fā)現(xiàn)，在64位肝癌患者肝癌組織檢測發(fā)現(xiàn)， 13例患者存在HOTAIR的高表達(dá)，和不表達(dá)HOTAIR的患者比， HOTAIR高表達(dá)患者的腫瘤體積更大，預(yù)后顯著更差，因此提出HOTAIR可以作為判斷肝癌患者預(yù)后的一個(gè)獨(dú)立因子。Li 等[17]等研究發(fā)現(xiàn)，HOTAIR通過下調(diào)SETD2調(diào)控肝癌干細(xì)胞的干性，從而促進(jìn)肝癌的惡性生物學(xué)行為。我們的研究發(fā)現(xiàn)，沉默HOTAIR后肝癌HepG2細(xì)胞侵襲和轉(zhuǎn)移能力降低。這說明HOTAIR在肝癌的發(fā)生和發(fā)展中發(fā)揮重要的作用。

Figure 5. The effects ofHOTAIRandPIK3R3 silencing on the migration ability of HepG2 cells detected by wound healing assay. Mean±SD.n=3.*P<0.05vsLV3-NC group.

圖5 劃痕實(shí)驗(yàn)檢測沉默HOTAIR和PIK3R3對(duì)肝癌細(xì)胞HepG2轉(zhuǎn)移能力的影響

目前關(guān)于lncRNA的研究包括生物信息學(xué)預(yù)測結(jié)合實(shí)驗(yàn)驗(yàn)證以及生物芯片篩選結(jié)合實(shí)驗(yàn)驗(yàn)證[18-20]。lncRNA的作用機(jī)制非常復(fù)雜，涉及調(diào)控蛋白的穩(wěn)定性或者定位[20-22]和DNA以及RNA相互作用調(diào)控基因表達(dá)[23-25]、lncRNA的“海綿作用”調(diào)控靶基因轉(zhuǎn)錄、通過影響轉(zhuǎn)錄因子和啟動(dòng)子的結(jié)合抑制附近基因的轉(zhuǎn)錄、形成miRNA或者Piwi調(diào)控基因的表達(dá)[26]、調(diào)控mRNA前體的可變剪接、作為內(nèi)源性競爭性RNA發(fā)揮調(diào)控作用、表觀遺傳學(xué)調(diào)控基因的調(diào)控[27]、與miRNA產(chǎn)生調(diào)控網(wǎng)絡(luò)的相互作用等多個(gè)方面。本研究通過生物信息學(xué)分析我們的研究證實(shí)，HOTAIR通過miR-214調(diào)控PIK3R3的表達(dá)，從而促進(jìn)肝癌的發(fā)生和發(fā)展。

磷脂酰肌醇3-激酶是細(xì)胞內(nèi)脂質(zhì)底物的轉(zhuǎn)換器,可以被蛋白酪氨酸激酶受體富集和激活，進(jìn)而產(chǎn)生第二信使參與多條信號(hào)通路，調(diào)控細(xì)胞的增殖和轉(zhuǎn)移[28]。PIK3R3在結(jié)腸癌中可以促進(jìn)其生長和轉(zhuǎn)移，抑癌因子miR-152可以直接靶向PIK3R3抑制結(jié)腸癌的惡性生物學(xué)行為[29]。有研究發(fā)現(xiàn)，PIK3R3可以誘導(dǎo)EMT從而促進(jìn)結(jié)腸癌的轉(zhuǎn)移能力[30]。在肝癌當(dāng)中，miR-132和miR-511也可以通過靶向PIK3R3抑制肝癌細(xì)胞HepG2的增殖、侵襲和轉(zhuǎn)移能力[31-32]。在三陰性乳腺癌中，PIK3R3的高表達(dá)可以促進(jìn)其轉(zhuǎn)移能力[33]。本研究中，我們發(fā)現(xiàn)，PIK3R3在肝癌組織中表達(dá)增加，同時(shí)沉默PIK3R3后，肝癌HepG2細(xì)胞轉(zhuǎn)移和侵襲能力降低，這和前面的報(bào)道一致。在本研究中，我們發(fā)現(xiàn)PIK3R3在肝癌組織中表達(dá)明顯增加，同時(shí)體外實(shí)驗(yàn)發(fā)現(xiàn)沉默PIK3R3后肝癌細(xì)胞轉(zhuǎn)移和侵襲能力降低。這說明PIK3R3在體內(nèi)和體外均可以調(diào)控肝癌細(xì)胞的生長。

Chen 等[34]研究發(fā)現(xiàn)，在肝癌中，miR-214可以通過靶向成纖維細(xì)胞生長因子受體1(fibroblast growth factor receptor 1，F(xiàn)GFR1)，從而抑制肝癌細(xì)胞的轉(zhuǎn)移。另一項(xiàng)研究表明，順鉑通過促進(jìn)microRNA-214表達(dá)抑制肝癌細(xì)胞HepG2及Hep3b的增殖[35]。在肝癌細(xì)胞Hep3b中，過表達(dá)miR-214后，肝癌細(xì)胞增殖能力明顯被抑制。這說明miR-214參與了肝癌的發(fā)生和進(jìn)展。我們的研究發(fā)現(xiàn)，miR-214可以直接靶向調(diào)控HOTAIR和PIK3R3。這說明HOTAIR和PIK3R3促進(jìn)腫瘤的作用，并且也間接說明HOTAIR和PIK3R3之間的相互調(diào)控是通過miR-214來實(shí)現(xiàn)的。

綜上所述，本研究表明HOTAIR在肝癌細(xì)胞的侵襲遷移中起重要作用，并對(duì)其相關(guān)機(jī)制做進(jìn)一步探討，表明HOTAIR可能通過miR-214調(diào)控PIK3R3的表達(dá)，從而調(diào)控肝癌侵襲遷移的作用。這提示HOTAIR可能參與了肝癌的發(fā)展和轉(zhuǎn)移過程，有可能成為預(yù)測疾病發(fā)展、治療效果及預(yù)測預(yù)后的生物標(biāo)志物。本研究主要從體外實(shí)驗(yàn)研究，后續(xù)將深入研究HOTAIR對(duì)肝癌在體內(nèi)的作用。

[1] Nielsen SR, Quaranta V, Linford A, et al. Macrophage-secreted granulin supports pancreatic cancer metastasis by inducing liver fibrosis[J]. Nat Cell Biol, 2016, 18(5):549-560.

[2] Zhang Y, Xi Y, Fang J, et al. Identification and characterization of monoclonal antibodies against GP73 for use as a potential biomarker in liver cancer screening and diagnosis[J]. J Immunoassay Immunochem, 2016， 37(4):390-406.

[3] Wang Y, He H, Chang J, et al. Mueller matrix microscope: a quantitative tool to facilitate detections and fibrosis scorings of liver cirrhosis and cancer tissues[J]. J Biomed Opt, 2016, 21(8):71112.

[4] Hong SH, Eun JW, Choi SK, et al. Epigenetic reader BRD4 inhibition as a therapeutic strategy to suppress E2F2-cell cycle regulation circuit in liver cancer[J]. Oncotarget, 2016, 7(22)：32628-32640.

[5] Silberhumer GR, Paty PB, Denton B, et al. Long-term oncologic outcomes for simultaneous resection of synchronous metastatic liver and primary colorectal cancer[J]. Surgery, 2016, 160(1)：67-73.

[6] Araújo OC, Rosa AS, Fernandes A, et al. RASSF1A and DOK1 promoter methylation levels in hepatocellular carcinoma, cirrhotic and non-cirrhotic liver, and correlation with liver cancer in brazilian patients[J]. PLoS One, 2016,11(4):e0153796.

[7] Ma Y, Huang D, Yang F, et al. Long noncoding RNA highly upregulated in liver cancer regulates the tumor necrosis factor-α-induced apoptosis in human vascular endothelial cells[J].DNA Cell Biol, 2016, 35(6):296-300.

[8] Dickson I. Hepatocellular carcinoma: a role for lncRNA in liver cancer[J]. Nat Rev Gastroenterol Hepatol, 2016,13(3):122-123.

[9] Bayram S, Sümbül AT, DadaE. A functionalHOTAIRrs12826786 C>T polymorphism is associated with breast cancer susceptibility and poor clinicopathological characteristics in a Turkish population: a hospital-based case-control study[J]. Tumour Biol, 2016, 37(4):5577-5584.

[11]De Falco M, Fedele V, Cobellis L, et al. Pattern of expression of cyclin D1/CDK4 complex in human placenta during gestation[J]. Cell Tissue Res, 2004, 317(2):187-194.

[12]Liu Y, Wang B, Liu X, et al. Epigenetic silencing of p21 by long non-coding RNA HOTAIR is involved in the cell cycle disorder induced by cigarette smoke extract[J]. Toxicol Lett, 2016, 240(1):60-67.

[13]Betancur JG, Tomari Y. Cryptic RNA-binding by PRC2 components EZH2 and SUZ12[J]. RNA Biol, 2015,12(9):959-965.

[14]Sharma S, Mandal P, Sadhukhan T, et al. Bridging links between long noncoding RNA HOTAIR and HPV oncoprotein E7 in cervical cancer pathogenesis[J]. Sci Rep, 2015, 5:11724.

[15]Yang Z, Zhou L, Wu LM, et al. Overexpression of long non-coding RNA HOTAIR predicts tumor recurrence in hepatocellular carcinoma patients following liver transplantation[J]. Ann Surg Oncol, 2011,18(5):1243-1250.

[16]Ishibashi M, Kogo R, Shibata K, et al. Clinical significance of the expression of long non-coding RNA HOTAIR in primary hepatocellular carcinoma[J]. Oncol Rep, 2013, 29(3):946-950.

[17]Li H, An J, Wu M, et al. LncRNA HOTAIR promotes human liver cancer stem cell malignant growth through downregulation of SETD2[J]. Oncotarget, 2015, 6(29): 27847-27864.

[18]Zhou M, Wang X, Shi H, et al. Characterization of long non-coding RNA-associated ceRNA network to reveal potential prognostic lncRNA biomarkers in human ovarian cancer[J]. Oncotarget, 2016, 7(11):12598-12611.

[19]Guo Q, Cheng Y, Liang T, et al. Comprehensive analysis of lncRNA-mRNA co-expression patterns identifies immune-associated lncRNA biomarkers in ovarian cancer malignant progression[J]. Sci Rep, 2015, 5:17683.

[20]Liu E, Liu Z, Zhou Y. Carboplatin-docetaxel-induced activity against ovarian cancer is dependent on up-regulated lncRNA PVT1[J]. Int J Clin Exp Pathol, 2015, 8(4):3803-3810.

[21]Tian T, Li C, Xiao J, et al. Quantitative assessment of the polymorphisms in theHOTAIRlncRNA and cancer risk: A meta-analysis of 8 case-control studies[J]. PLoS One, 2016,11(3):e0152296.

[22]Gao JZ, Li J, DU JL, et al. Long non-coding RNA HOTAIR is a marker for hepatocellular carcinoma progression and tumor recurrence[J]. Oncol Lett, 2016, 11(3):1791-1798.

[23]Liu S, Cui B, Dai ZX, et al. Long non-coding RNA HOTAIR promotes Parkinson’s disease induced by MPTP through up-regulating the expression of LRRK2[J]. Curr Neurovasc Res, 2016,13(2):115-120.

[24]Zhou Q, Chen F, Fei Z, et al. Genetic variants of lncRNA HOTAIR contribute to the risk of osteosarcoma[J]. Oncotarget, 2016,DOI: 10.18632/oncotarget.7957.

[25]Liu FT, Qui C, Zhang Y, et al. The association of HOTAIR expression with clinicopathological features and prognosis in gastric cancer patients[J]. Panminerva Med, 2016, 58(2):167-174.

[26]Zhang L, Liu Z, Li X, et al. Low long non-coding RNA HOTAIR expression is associated with down-regulation of Nrf2 in the spermatozoa of patients with asthenozoospermia or oligoasthenozoospermia[J]. Int J Clin Exp Pathol, 2015, 8(11):14198-14205.

[27]Berrondo C, Flax J, Kucherov V, et al. Expression of the long non-coding RNA HOTAIR correlates with disease progression in bladder cancer and is contained in bladder cancer patient urinary exosomes[J]. PLoS One, 2016, 11(1):e0147236.

[28]Wang G, Yang X, Li C, et al. PIK3R3 induces epithelial-to-mesenchymal transition and promotes metastasis in colorectal cancer[J]. Mol Cancer Ther, 2014, 13(7): 1837-1847.

[29]Li B, Xie Z, Li B. miR-152 functions as a tumor suppressor in colorectal cancer by targeting PIK3R3[J]. Tumour Biol, 2016, 37(8):10075-10084.

[30]Wang G, Yang X, Li C, et al. PIK3R3 induces epithelial-to-mesenchymal transition and promotes metastasis in colorectal cancer[J]. Mol Cancer Ther, 2014, 13(7):1837-1847.

[31]Liu K, Li X, Cao Y, et al. MiR-132 inhibits cell proliferation, invasion and migration of hepatocellular carcinoma by targeting PIK3R3[J]. Int J Oncol, 2015, 47(4):1585-1593.

[32]Cao G, Dong W, Meng X, et al. MiR-511 inhibits growth and metastasis of human hepatocellular carcinoma cells by targeting PIK3R3[J]. Tumour Biol, 2015, 36(6):4453-4459.

[33]Klahan S, Wu MS, Hsi E, et al. Computational analysis of mRNA expression profiles identifies the ITG family and PIK3R3 as crucial genes for regulating triple negative breast cancer cell migration[J]. Biomed Res Int, 2014, 2014:536591.

[34]Chen DL, Wang ZQ, Zeng ZL, et al. Identification of microRNA-214 as a negative regulator of colorectal cancer liver metastasis by way of regulation of fibroblast growth factor receptor 1 expression[J]. Hepatology, 2014, 60(2):598-609.

[35]劉子文, 杜永星, 由 磊, 等. 順鉑通過促進(jìn)microRNA-214表達(dá)抑制肝癌細(xì)胞增殖[J].基礎(chǔ)醫(yī)學(xué)與臨床,2014, 34(9):1199-1203.

(責(zé)任編輯： 盧 萍， 羅 森)

Long noncoding RNA HOTAIR promotes liver cancer HepG2 cell migration and invasion by regulating PIK3R3

ZHANG Jing-jing1, WU Wei2, WANG Peng2, YANG Jing-rui1, DUAN Ju-hong1, YU Hai-chuan1

(1CollaborativeInnovationCenterofHenanProvince,InstituteofMedicalLaboratoryScienceandMolecularDiagnosticsandMedicalLaboratoryTechnology,2TheThirdAffiliatedHospital,XinxiangMedicalUniversity,Xinxiang453003,China.E-mail:fjingsc@163.com)

AIM: To investigate the effect of HOX transcript antisense RNA (HOTAIR) on the migration and invasion abilities of liver carcinoma HepG2 cells.METHODS: The expression of phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) in the liver cancer and normal liver tissues was detected by immunohistochemistry. The efficiency of gene silencing ofHOTAIRorPIK3R3 by LV3-shHOTAIR or LV3-shPIK3R3 was determined by qPCR and Western blot. The migration and invasion abilities of HepG2 cells after silencing ofHOTAIRandPIK3R3 were measured by wound healing assay and Transwell Matrigel invasion assay. The expression of miR-214 after silencing ofHOTAIRandPIK3R3 was analyzed by qPCR. The expression of HOTAIR and PIK3R3 in the HepG2 cells was also evaluated by qPCR after transfected with miR-214 mimics or miR-214 inhibitor. Dual-luciferase reporter assay system was used to determine the regulatory effect of miR-214 on HOTAIR andPIK3R3 expression.RESULTS: PIK3R3 expression increased significantly in the liver cancer tissues compared with normal liver tissues. The abilities of invasion and metastasis of hepatocellular carcinoma were reduced after silencing ofHOTAIRandPIK3R3. miR-214 expression was increased when silencing ofHOTAIRandPIK3R3 was performed. HOTAIR and PIK3R3 expression was reduced after transfection with miR-214 mimics. HOTAIR and PIK3R3 expression was increased after transfection with miR-214 inhibitor. The results of dual-luciferase reporter assay test showed that miR-214 directly regulatedHOTAIRandPIK3R3 transcription activity. CONCLUSION: HOTAIR regulates the expression of PIK3R3 through miR-214, thus promoting the migration and invasion abilities in the liver cancer cells.

HOX transcript antisense RNA; Liver cancer; Phosphoinositide-3-kinase regulatory subunit 3

1000- 4718(2016)10- 1775- 07

2016- 05- 18

2016- 06- 20

國家自然科學(xué)基金資助項(xiàng)目(No.31301135)

△通訊作者 Tel: 13938715600； E-mail: fjingsc@163.com

R730.23

A

10.3969/j.issn.1000- 4718.2016.10.008

雜志網(wǎng)址： http://www.cjpp.net