長(zhǎng)鏈非編碼RNA PANDAR促進(jìn)結(jié)直腸癌轉(zhuǎn)移的作用和機(jī)制研究

劉 寧，成冬冬，姜金波

1.肥城礦業(yè)中心醫(yī)院普外科，山東 泰安 271600；

2.山東大學(xué)齊魯醫(yī)院普外科，山東 濟(jì)南 250017

長(zhǎng)鏈非編碼RNA PANDAR促進(jìn)結(jié)直腸癌轉(zhuǎn)移的作用和機(jī)制研究

劉 寧1，成冬冬1，姜金波2

1.肥城礦業(yè)中心醫(yī)院普外科，山東 泰安 271600；

2.山東大學(xué)齊魯醫(yī)院普外科，山東 濟(jì)南 250017

背景與目的：越來(lái)越多的研究表明，長(zhǎng)鏈非編碼RNA(long non-coding RNA，lncRNA)在腫瘤發(fā)生、發(fā)展過(guò)程中有重要作用。LncRNA CDKN1A反義鏈啟動(dòng)子DNA損傷激動(dòng)RNA(promoter of CDKN1A antisense DNA damage activated RNA，PANDAR)與多種腫瘤的進(jìn)展及預(yù)后相關(guān)，在結(jié)直腸癌轉(zhuǎn)移中的作用尚未得到證實(shí)。該研究旨在探索lncRNA PANDAR在結(jié)直腸癌中的功能，并對(duì)其作用機(jī)制進(jìn)行初步探索。方法：采用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(real-time fluorescent quantitative polymerase chain reaction，RTFQ-PCR)檢測(cè)lncRNA PANDAR在結(jié)直腸癌細(xì)胞及組織中的表達(dá)，并分析其表達(dá)水平與結(jié)直腸癌臨床病理特征的關(guān)系。構(gòu)建lncRNA PANDAR沉默(HCT116-shPANDAR)和高表達(dá)(DLD1-PANDAR)及其對(duì)照(HCT116-shNC、DLD1-vector)穩(wěn)定轉(zhuǎn)染細(xì)胞系。通過(guò)Transwell和Matrigel實(shí)驗(yàn)檢測(cè)lncRNA PANDAR對(duì)細(xì)胞遷移和侵襲能力的影響。采用RTFQ-PCR檢測(cè)lncRNA PANDAR表達(dá)改變后介導(dǎo)細(xì)胞上皮-間質(zhì)轉(zhuǎn)化能力的主要調(diào)控基因表達(dá)情況，并對(duì)目的基因在介導(dǎo)lncRNA PANDAR調(diào)控結(jié)直腸癌細(xì)胞轉(zhuǎn)移中的作用進(jìn)行驗(yàn)證。結(jié)果：LncRNA PANDAR在結(jié)直腸癌細(xì)胞中的表達(dá)明顯高于結(jié)直腸正常上皮細(xì)胞；LncRNA PANDAR在結(jié)直腸癌組織中的表達(dá)明顯高于癌旁組織［(171.52±97.80)% vs (100.00±63.18)%，P＜0.05］，且其表達(dá)水平與結(jié)直腸癌的TNM分期、淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移相關(guān)(P＜0.05)。在Transwell及Matrigel實(shí)驗(yàn)中，lncRNA PANDAR沉默能夠明顯減弱結(jié)直腸癌細(xì)胞的遷移［100.00% vs (42.08±4.77)%，P＜0.05］和侵襲［100.00% vs (39.14±3.81)%，P＜0.05］能力，lncRNA PANDAR高表達(dá)能夠明顯促進(jìn)結(jié)直腸癌細(xì)胞的遷移［100.00% vs (194.12±9.33)%，P＜0.05］和侵襲［100.00% vs (204.08±12.27)%，P＜0.05］能力。采用RTFQ-PCR檢測(cè)lncRNA PANDAR表達(dá)改變后介導(dǎo)結(jié)直腸癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化的基因表達(dá)情況發(fā)現(xiàn)，鋅指E-盒結(jié)合同源異形盒(zinc-finger E-box binding homeobox 1，ZEB1)表達(dá)與lncRNA PANDAR表達(dá)呈顯著正相關(guān)；在lncRNA PANDAR高表達(dá)的細(xì)胞中干擾ZEB1表達(dá)能夠明顯逆轉(zhuǎn)lncRNA PANDAR高表達(dá)引起的細(xì)胞遷移和侵襲能力的增強(qiáng)。結(jié)論：LncRNA PANDAR能夠通過(guò)調(diào)控ZEB1表達(dá)進(jìn)而促進(jìn)結(jié)直腸癌轉(zhuǎn)移，lncRNA PANDAR可能成為結(jié)直腸癌新的診斷指標(biāo)及治療靶點(diǎn)。

長(zhǎng)鏈非編碼RNA；CDKN1A反意義鏈啟動(dòng)子DNA損傷激動(dòng)RNA；結(jié)直腸癌；轉(zhuǎn)移

結(jié)直腸癌是世界范圍內(nèi)常見(jiàn)的腫瘤之一。在中國(guó)，結(jié)直腸癌是常見(jiàn)的致死性腫瘤之一，并且其發(fā)病率呈不斷上升的趨勢(shì)。腫瘤轉(zhuǎn)移是造成結(jié)直腸癌患者死亡最為主要的原因［1］。在過(guò)去的幾十年中，研究者致力于探索結(jié)直腸癌的轉(zhuǎn)移機(jī)制，但目前其分子機(jī)制仍未得到明確的認(rèn)識(shí)。

長(zhǎng)鏈非編碼RNA(long non-coding RNA，lncRNA)是一種長(zhǎng)度超過(guò)200 nt，不具有蛋白質(zhì)編碼功能的轉(zhuǎn)錄RNA序列。有研究表明，lncRNA在細(xì)胞的多種生命活動(dòng)過(guò)程中發(fā)揮重要作用，包括細(xì)胞的生長(zhǎng)、分化及腫瘤的發(fā)生、發(fā)展。LncRNA表達(dá)異?？梢?jiàn)于乳腺癌、胃癌、肺癌、前列腺癌及肝癌等多種腫瘤中［2］。LncRNA CDKN1A反義鏈啟動(dòng)子DNA損傷激動(dòng)RNA(promoter of CDKN1A antisense DNA damage activated RNA，PANDAR)是一種新發(fā)現(xiàn)的lncRNA［3-6］。目前，由于其在腫瘤中的重要作用，lncRNA PANDAR被視為一種重要的治療潛在靶點(diǎn)［7］，但其在結(jié)直腸癌中的功能尚未見(jiàn)報(bào)道。

本研究通過(guò)檢測(cè)lncRNA PANDAR在結(jié)直腸癌細(xì)胞系及組織中的表達(dá)以證實(shí)lncRNA PANDAR在結(jié)直腸癌中高表達(dá)，且與結(jié)直腸癌的發(fā)生、發(fā)展明顯相關(guān)。體外實(shí)驗(yàn)進(jìn)一步驗(yàn)證lncRNA PANDAR在結(jié)直腸癌轉(zhuǎn)移中的作用，并對(duì)具體的機(jī)制進(jìn)行初步探索。該研究結(jié)果旨在進(jìn)一步揭示結(jié)直腸癌的發(fā)生、發(fā)展機(jī)制，并為將lncRNA PANDAR作為結(jié)直腸癌新的早期診斷指標(biāo)及治療靶點(diǎn)提供證據(jù)。

1 材料和方法

1.1 細(xì)胞培養(yǎng)

人結(jié)直腸癌細(xì)胞系DLD1、HCT116、LOVO、HCT8、HT29、SW620、SW480及人結(jié)直腸正常上皮細(xì)胞系NCM460均購(gòu)自中國(guó)科學(xué)院上海生命科學(xué)研究院生物化學(xué)與細(xì)胞生物學(xué)研究所細(xì)胞庫(kù)。所有細(xì)胞均按照細(xì)胞培養(yǎng)說(shuō)明使用RPMI-1640培養(yǎng)基或DMEM-H培養(yǎng)基(HYCLONE)添加胎牛血清、100 U/mL青霉素和

100 μg/mL鏈霉素進(jìn)行培養(yǎng)。細(xì)胞培養(yǎng)于37 ℃、CO2體積分?jǐn)?shù)為5%的飽和濕度的細(xì)胞培養(yǎng)箱中。

1.2 臨床標(biāo)本收集

收集山東大學(xué)齊魯醫(yī)院和肥城礦業(yè)中心醫(yī)院2014年5月—2014年12月收治的76例結(jié)直腸癌患者的臨床標(biāo)本(其中50例患者有對(duì)應(yīng)癌旁組織標(biāo)本)，腫瘤及癌旁組織標(biāo)本手術(shù)切除后經(jīng)液氮凍存。所有患者術(shù)前均未行放療或化療等抗腫瘤治療，最終診斷由常規(guī)病理檢查確診。標(biāo)本使用經(jīng)患者簽字同意。該研究經(jīng)山東大學(xué)齊魯醫(yī)院倫理委員會(huì)審核批準(zhǔn)。

1.3 實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(real-time fluorescent quantitative polymerase chain reaction，RTFQ-PCR)

采用TRIzol(購(gòu)自美國(guó)Invitrogen公司)法提取細(xì)胞和組織總RNA。使用PrimeScriptTM反轉(zhuǎn)錄試劑盒［購(gòu)自寶生物工程(大連)有限公司］進(jìn)行反轉(zhuǎn)錄反應(yīng)合成cDNA。按照SYBR? Premix Ex Taq?試劑盒［購(gòu)自寶生物工程(大連)有限公司］使用說(shuō)明進(jìn)行RTFQ-PCR檢測(cè)。RTFQ-PCR及數(shù)據(jù)采集均使用ABI PRISM 7900HT序列檢測(cè)系統(tǒng)(購(gòu)自美國(guó)Applied Biosystems公司)。GAPDH作為內(nèi)參。LncRNA PANDAR引物序列順義鏈：5’-TGCACACATTTAACCCGAAG-3’，反義鏈：5’-CCCCAAAGCTACATCTATGACA-3’；GAPDH引物序列順義鏈：5’-CAAGGTCATCC ATGACAACTTTG-3’，反義鏈：5’-GTCCACCA CCCTGTTGCTGTAG-3’。

1.4 PANDAR沉默和高表達(dá)穩(wěn)定轉(zhuǎn)染細(xì)胞系及對(duì)照組細(xì)胞系構(gòu)建

LncRNA PANDAR序列按照NCBI提供的序列構(gòu)建，將lncRNA PANDAR序列克隆入pCDNA3.1質(zhì)粒(購(gòu)自美國(guó)Invitrogen公司)構(gòu)建lncRNA PANDAR高表達(dá)載體(pCDNA3.1-PANDAR)。pCDNA3.1空質(zhì)粒作為對(duì)照(pCDNA3.1-vector)。LncRNA PANDAR干擾序列：5’-TTTCGAACG GAACAGAGACTTATACAGATT-3’(購(gòu)自上海吉瑪制藥技術(shù)有限公司)，將其克隆入pGU6/Neo質(zhì)粒，構(gòu)建lncRNA PANDAR干擾質(zhì)粒(pGU6/ Neo-shPANDAR)。亂序的shRNA序列作為對(duì)照(pGU6/Neo-shNC)。慢病毒感染法構(gòu)建lncRNA PANDAR沉默穩(wěn)定轉(zhuǎn)染細(xì)胞系和對(duì)照組細(xì)胞系及高表達(dá)穩(wěn)定轉(zhuǎn)染細(xì)胞系和對(duì)照組細(xì)胞系。采用RTFQ-PCR檢測(cè)驗(yàn)證細(xì)胞系是否構(gòu)建成功。

1.5 Transwell遷移實(shí)驗(yàn)

細(xì)胞常規(guī)培養(yǎng)至對(duì)數(shù)生長(zhǎng)期，胰酶消化，沖洗懸浮液，按照5×104個(gè)/孔將細(xì)胞懸液加入Transwell小室上層，添加200 μL無(wú)血清基礎(chǔ)培養(yǎng)基，同時(shí)小室下層添加700 μL含10%胎牛血清培養(yǎng)基作為化學(xué)誘導(dǎo)。細(xì)胞培養(yǎng)箱中常規(guī)培養(yǎng)24 h。擦除濾膜上表面細(xì)胞，裁下Transwell小室濾膜，4%的甲醛溶液固定，吉姆薩染色。隨機(jī)選取5個(gè)200×高倍鏡視野記錄細(xì)胞數(shù)目并計(jì)算平均值。

1.6 Matrigel侵襲實(shí)驗(yàn)

Matrigel侵襲實(shí)驗(yàn)基本步驟同Transwell遷移實(shí)驗(yàn)，不同之處在于Matrigel侵襲實(shí)驗(yàn)中需先制備侵襲小室，方法如下：用不含血清的基礎(chǔ)培養(yǎng)基稀釋Matrigel基質(zhì)膠(體積比為1∶3)，均勻鋪在小室底部，將小室放置在37 ℃細(xì)胞培養(yǎng)箱中，待膠凝固即制成Matrigel侵襲小室。鋪細(xì)胞、培養(yǎng)、固定及染色方法同Transwell遷移實(shí)驗(yàn)。

1.7 統(tǒng)計(jì)學(xué)處理

2 結(jié) 果

2.1 LncRNA PANDAR在結(jié)直腸癌中高表達(dá)且能夠促進(jìn)結(jié)直腸癌轉(zhuǎn)移

采用RTFQ-PCR檢測(cè)人結(jié)直腸癌細(xì)胞系DLD1、HCT116、LOVO、HCT8、HT29、SW620、SW480及人結(jié)直腸正常上皮細(xì)胞系NCM460中l(wèi)ncRNA PANDAR表達(dá)發(fā)現(xiàn)，lncRNAPANDAR在人結(jié)直腸正常上皮細(xì)胞系NCM460中的表達(dá)量明顯低于人結(jié)直腸癌細(xì)胞系DLD1、HCT116、LOVO、HCT8、HT29、SW620和SW480(P＜0.05，圖1)。

為進(jìn)一步驗(yàn)證lncRNA PANDAR在結(jié)直腸癌中的作用，本研究采用RTFQ-PCR檢測(cè)了76例結(jié)直腸癌組織和50例結(jié)直腸癌癌旁組織中l(wèi)ncRNA PANDAR的表達(dá)水平，結(jié)果發(fā)現(xiàn)，結(jié)直腸癌組織中l(wèi)ncRNA PANDAR表達(dá)水平明顯高于結(jié)直腸癌癌旁組織［(171.52±97.80)% vs (100.00±63.18)%，P＜0.05，圖2］。

圖 1 采用RTFQ-PCR檢測(cè)結(jié)直腸正常上皮細(xì)胞和結(jié)直腸癌細(xì)胞中l(wèi)ncRNA PANDAR相對(duì)表達(dá)量Fig. 1 The relative expression level of lncRNA PANDAR in colorectal normal epithelial cells (NCM460) and colorectal cancer cells by RTFQ-PCR

圖 2 采用RTFQ-PCR檢測(cè)lncRNA PANDAR在結(jié)直腸癌組織和癌旁組織中的相對(duì)表達(dá)量Fig. 2 The relative expression level of lncRNA PANDAR in colorectal cancer tissues and tumor adjacent tissues by RTFQ-PCR

此外，以76例結(jié)直腸癌患者lncRNA PANDAR相對(duì)表達(dá)量均數(shù)(171.52)為截點(diǎn)，將患者分為lncRNA PANDAR低表達(dá)組(42例)和lncRNA PANDAR高表達(dá)組(34例)。統(tǒng)計(jì)分析發(fā)現(xiàn)，lncRNA PANDAR高表達(dá)與結(jié)直腸癌的TNM分期、淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移明顯相關(guān)(P＜0.05，表1)。

表 1 LncRNA PANDAR表達(dá)量與結(jié)直腸癌患者臨床病理特點(diǎn)分析Tab. 1 The correlation between lncRNA PANDAR expression and clinicopathological features of colorectal patients

2.2 體外實(shí)驗(yàn)證實(shí)lncRNA PANDAR能夠促進(jìn)結(jié)直腸癌轉(zhuǎn)移

為進(jìn)一步探索lncRNA PANDAR在結(jié)直腸癌轉(zhuǎn)移中的作用，本研究首先使用lncRNA PANDAR表達(dá)量較高的結(jié)直腸癌細(xì)胞系HCT116構(gòu)建lncRNA PANDAR穩(wěn)定沉默細(xì)胞系及其對(duì)照組細(xì)胞系(HCT116-shPANDAR及HCT116-shNC)，使用lncRNA PANDAR表達(dá)量較低的DLD1細(xì)胞系構(gòu)建lncRNA PANDAR穩(wěn)定高表達(dá)細(xì)胞系及其對(duì)照組細(xì)胞系(DLD1-PANDAR及DLD1-vector)，經(jīng)RTFQ-PCR檢測(cè)驗(yàn)證細(xì)胞系構(gòu)建成功(圖3)。

經(jīng)Transwell遷移實(shí)驗(yàn)證實(shí)，HCT116-shPANDAR細(xì)胞穿過(guò)濾膜數(shù)目明顯少于HCT116-shNC細(xì)胞［(42.08±4.77)% vs 100.00%，P＜0.05］；DLD1-PANDAR細(xì)胞穿過(guò)濾膜數(shù)目明顯多于DLD1-vector細(xì)胞［(194.12±9.33)% vs 100.00%，P＜0.05］。經(jīng)Matrigel侵襲實(shí)驗(yàn)證實(shí)，HCT116-shPANDAR細(xì)胞穿過(guò)濾膜數(shù)目明顯少于HCT116-shNC細(xì)胞［(39.14±3.81)% vs 100.00%，P＜0.05］；DLD1-PANDAR細(xì)胞穿過(guò)濾膜數(shù)目明顯多于DLD1-vector細(xì)胞［(204.08±12.27)% vs 100.00%，P＜0.05，圖4］。

2.3 鋅指E-盒結(jié)合同源異形盒(zinc-finger E-box binding homeobox 1，ZEB1)介導(dǎo)lncRNA PANDAR促進(jìn)結(jié)直腸癌轉(zhuǎn)移的作用

采用RTFQ-PCR檢測(cè)lncRNA PANDAR表達(dá)改變后影響結(jié)直腸癌上皮-間質(zhì)轉(zhuǎn)化的主要轉(zhuǎn)錄調(diào)控因子(Slug、Snail、ZEB1、ZEB2、Twist1和Twist2)表達(dá)改變發(fā)現(xiàn)，ZEB1變化最為明顯［100.00% vs (244.00±10.87)%，P＜0.05］，與lncRNA PANDAR表達(dá)呈明顯正相關(guān)(圖5)。

使用siRNA技術(shù)在lncRNA PANDAR高表達(dá)的細(xì)胞中干擾ZEB1表達(dá)能夠明顯逆轉(zhuǎn)lncRNA PANDAR高表達(dá)引起的細(xì)胞遷移［100.00%、(192.11±8.02)%和(119.80±5.02)%，P＜0.05)和侵襲［100.00%、(213.32±10.11)%和(112.71±6.38)%，P＜0.05］能力的增強(qiáng)(圖6)。

圖 3 RTFQ-PCR檢測(cè)lncRNA PANDAR沉默穩(wěn)定轉(zhuǎn)染細(xì)胞系HCT116細(xì)胞及其對(duì)照細(xì)胞系和lncRNA PANDAR穩(wěn)定高表達(dá)DLD1細(xì)胞及其對(duì)照組細(xì)胞中l(wèi)ncRNA PANDAR相對(duì)表達(dá)量Fig. 3 RTFQ-PCR was adopted to measure the expression levels of lncRNA PANDAR in lncRNA PANDAR stably overexpressed HCT116 cells, its control vector cells and lncRNA PANDAR stably silencing DLD1 cells and its control cells

圖 4 Transwell實(shí)驗(yàn)和Matrigel實(shí)驗(yàn)檢測(cè)lncRNA PANDAR表達(dá)沉默和高表達(dá)后細(xì)胞遷移和侵襲能力改變Fig. 4 Transwell assay and Matrigel assay were adopted to detect the migration and invasion capabilities in lncRNA PANDAR silencing and overexpressed cells

圖 5 LncRNA PANDAR高表達(dá)的細(xì)胞中上皮-間質(zhì)轉(zhuǎn)化轉(zhuǎn)錄調(diào)控因子mRNA表達(dá)變化Fig. 5 The mRNA levels of transcriptional factors regulating epithelial-mesenchymal transition in lncRNA PANDAR overexpressed cells

圖 6 Transwell實(shí)驗(yàn)和Matrigel實(shí)驗(yàn)檢測(cè)在lncRNA PANDAR高表達(dá)的細(xì)胞中干擾ZEB1對(duì)細(xì)胞遷移和侵襲能力的影響Fig. 6 The migration and invasion capabilities of lncRNA PANDAR overexpressed cells with ZEB1 silencing were investigated by Transwell assay and Matrigel assay

3 討 論

盡管過(guò)去幾十年在結(jié)直腸癌發(fā)生、發(fā)展的機(jī)制研究中取得了諸多進(jìn)展，且目前其根治性治療手段包括手術(shù)治療、化療及分子靶向治療等多種方法，但是由于結(jié)直腸癌篩查手段較少，且難以廣泛進(jìn)行，因此，很多患者就診時(shí)已出現(xiàn)淋巴結(jié)轉(zhuǎn)移或遠(yuǎn)處轉(zhuǎn)移，明顯影響患者預(yù)后。此外，結(jié)直腸癌具有較高的異質(zhì)性，不同患者間或同一例患者在腫瘤進(jìn)展的不同時(shí)期治療方案差異較大，分子靶向治療耐藥的發(fā)生率高。因此，進(jìn)一步揭示結(jié)直腸癌發(fā)生、發(fā)展的分子機(jī)制，尋找早期的診斷指標(biāo)及治療靶點(diǎn)，進(jìn)而根據(jù)患者不同基因表達(dá)譜和病理特點(diǎn)采取個(gè)體化的治療手段，對(duì)于改善患者預(yù)后有十分重要的意義［8-11］。

LncRNA是近年新發(fā)現(xiàn)一種非編碼RNA，自發(fā)現(xiàn)后便引起了極為廣泛的關(guān)注，其功能也不斷被揭示。LncRNA能夠通過(guò)調(diào)控基因及miRNA表達(dá)進(jìn)而影響腫瘤生物學(xué)行為，并參與染色體重塑、轉(zhuǎn)錄調(diào)控及RNA降解等多種過(guò)程。此外，由于其具有組織特異性，在疾病診斷中敏感性明顯高于DNA、蛋白編碼RNA及蛋白質(zhì)標(biāo)志物［12］。已有多項(xiàng)研究報(bào)道了lncRNA在結(jié)直腸癌中的功能，如lncRNA H19、MALAT1、CCAT1及HOTAIR等在促進(jìn)結(jié)直腸癌發(fā)生、發(fā)展中的作用已經(jīng)得到了較為廣泛的認(rèn)可。因此，進(jìn)一步探索lncRNA在結(jié)直腸癌中的功能不僅對(duì)于揭示結(jié)直腸癌發(fā)生、發(fā)展機(jī)制具有重要意義，同時(shí)可能為尋找結(jié)直腸癌新的診斷指標(biāo)及治療靶點(diǎn)提供證據(jù)［12-15］。

在DNA損傷反應(yīng)中，lncRNA PANDAR能夠由p53激活，lncRNA PANDAR的表達(dá)缺失能夠明顯促進(jìn)DNA損傷所誘導(dǎo)的損傷反應(yīng)。近期有研究顯示，lncRNA PANDAR與膀胱癌、肝癌、乳腺癌、胃癌及非小細(xì)胞肺癌的發(fā)生、發(fā)展和預(yù)后相關(guān)［16-19］，但其在結(jié)直腸癌中的功能尚未見(jiàn)報(bào)道。本研究發(fā)現(xiàn)，lncRNA PANDAR在結(jié)直腸正常上皮細(xì)胞和癌旁組織中的表達(dá)明顯低于結(jié)直腸癌細(xì)胞和組織，并且與結(jié)直腸癌的TNM分期、淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移相關(guān)，提示lncRNA PANDAR在結(jié)直腸癌中也是一種促癌基因，能夠促進(jìn)結(jié)直腸癌轉(zhuǎn)移。體外細(xì)胞實(shí)驗(yàn)進(jìn)一步證實(shí)，lncRNA PANDAR高表達(dá)能夠促進(jìn)結(jié)直腸癌細(xì)胞的轉(zhuǎn)移；相反，干擾lncRNA PANDAR表達(dá)能夠明顯抑制結(jié)直腸癌細(xì)胞的轉(zhuǎn)移。上述實(shí)驗(yàn)證實(shí)，lncRNA PANDAR能夠促進(jìn)結(jié)直腸癌細(xì)胞的轉(zhuǎn)移。上皮-間質(zhì)轉(zhuǎn)化在腫瘤轉(zhuǎn)移過(guò)程中發(fā)揮極為重要的作用，因此，本研究檢測(cè)了lncRNA PANDAR高表達(dá)的細(xì)胞中主要的上皮-間質(zhì)轉(zhuǎn)化轉(zhuǎn)錄調(diào)控因子mRNA的表達(dá)，結(jié)果發(fā)現(xiàn)，ZEB1表達(dá)變化明顯，且在lncRNA PANDAR高表達(dá)的細(xì)胞中干擾ZEB1表達(dá)會(huì)明顯逆轉(zhuǎn)lncRNA PANDAR高表達(dá)引起的結(jié)直腸癌細(xì)胞侵襲和遷移能力的增強(qiáng)，提示lncRNA PANDAR可能通過(guò)調(diào)控ZEB1表達(dá)進(jìn)而促進(jìn)結(jié)直腸癌上皮-間質(zhì)轉(zhuǎn)化，從而增強(qiáng)結(jié)直腸癌轉(zhuǎn)移能力。

綜上所述，lncRNA PANDAR可促進(jìn)結(jié)直腸癌轉(zhuǎn)移能力，且ZEB1可能介導(dǎo)該過(guò)程。LncRNA PANDAR可能成為結(jié)直腸癌新的診斷指標(biāo)及治療靶點(diǎn)。

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The functional role of long non-coding RNA PANDAR in promoting colorectal cancer metastasis and its mechanism

LIU Ning1, CHENG Dongdong1, JIANG Jinbo2(1. Department of General Surgery, Feicheng Kuangye Center Hospital, Tai’an 271600, Shandong Province, China; 2. Department of General Surgery, Qilu Hospital of Shandong University, Jinan 250017, Shandong Province, China)

JIANG Jinbo E-mail: sddoctor8611@126.com

Background and purpose:Accumulating evidence has revealed that long non-coding RNA (lncRNA) is correlated with carcinogenesis and tumor development. Recent literature suggested that lncRNA promoter of CDKN1A antisense DNA damage activated RNA (PANDAR) was involved in the development of various cancers. However, the functional role of PANDAR in colorectal cancer (CRC) has not been elucidated yet. The present study aimed to explore the functional role of lncRNA PANDAR in promoting CRC metastasis and its mechanism.Methods:The expression of lncRNA PANDAR in CRC cell lines and tissues was detected by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR), and the correlation between lncRNA PANDAR expression and CRC clinicopathological characteristics was statistically analyzed. Then, lncRNA PANDAR stably silencing CRC cells (HCT116-shPANDAR), overexpression cells (DLD1-PANDAR) and control vector cells (HCT116-shNC and DLD1-vector) were established using lentiviral vectors. Moreover, Transwell assay and Matrigel assay were performed to investigate the function of lncRNA PANDAR in CRC migration and invasion. Furthermore, the expression of transcriptional factors mediating epithelial-mesenchymal transition of lncRNA PANDAR overexpression cells were monitored by RTFQ-PCR assay, and the function of the target gene in modulating lncRNA PANDAR mediated CRC metastasis was also explored.Results:The expression levels of lncRNA PANDAR in normal colorectal epithelial cells were much lower than in CRC cell. The levels of lncRNA PANDAR in tumor-adjacent tissues were verified to be much lower than in CRC tissues ［(171.52±97.80)% vs (100.00±63.18)%, P＜0.05］. Moreover, the expression of lncRNA PANDAR was detected to be significantly correlated with CRC TNM stage, lymph node metastasis and distant metastasis (P＜0.05). Besides, lncRNA PANDAR deficiency significantly reduced the migration ［100.00% vs (42.08±4.77)%, P＜0.05］ and invasion ［100.00% vs (39.14±3.81)%, P＜0.05］ capabilities in CRC cells, in contrast, the migration ［100.00% vs (194.12±9.33)%, P＜0.05］ and invasion ［100.00% vs (204.08±12.27)%, P＜0.05］ capabilities of CRC cells were obviously increased with lncRNA PANDAR overexpression. Furthermore, zinc-finger E-box binding homeobox 1 (ZEB1) expression was detected to be positively correlated with lncRNA PANDAR expression, and ZEB1 silencing could significantly reverse the increased migration and invasion capabilities induced by lncRNA PANDAR in CRC cells.Conclusion:LncRNA PANDAR could promote CRC metastasis by potentially targeting ZEB1. LncRNA PANDAR might be a promising diagnostic marker and therapeutic target for CRC patients.

Long non-coding RNA; Promoter of CDKN1A antisense DNA damage activated RNA; Colorectal cancer; Metastasis

10.19401/j.cnki.1007-3639.2017.04.005

R735.3

A

1007-3639(2017)04-0268-08

2016-09-20

2016-11-30）

姜金波 E-mail: sddoctor8611@126.com