莊彪，閔志均，王延峰，張鵬，倪熊，瞿惠龍，丁育明(上海市浦東醫(yī)院，上海201399)

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miR-34a在結(jié)腸癌細(xì)胞增殖和遷移中的作用及其分子機(jī)制

莊彪，閔志均，王延峰，張鵬，倪熊，瞿惠龍，丁育明(上海市浦東醫(yī)院，上海201399)

摘要：目的探討miR-34a在結(jié)腸癌細(xì)胞增殖和遷移中的作用，并驗(yàn)證其靶點(diǎn)蛋白。方法傳代培養(yǎng)人結(jié)腸癌細(xì)胞株HCT-116，分別采用空病毒載體(pRI-CMV-GFP)轉(zhuǎn)染(陰性對(duì)照組)、慢病毒載體(pRI-CMV-GFP-miRNA-34a)轉(zhuǎn)染(慢病毒組)，另選未經(jīng)任何處理的HCT-116細(xì)胞作為空白對(duì)照組。采用Real-time PCR法檢測(cè)miR-34a的相對(duì)表達(dá)量，采用MTT實(shí)驗(yàn)、Transwell小室法檢測(cè)HCT-116細(xì)胞增殖和遷移能力，采用細(xì)胞免疫熒光試驗(yàn)和Western blotting法驗(yàn)證其靶點(diǎn)蛋白。結(jié)果以空白對(duì)照組miR-34a表達(dá)為1，陰性對(duì)照組為1.03±0.09，慢病毒組為6.41±1.56，慢病毒組高于陰性對(duì)照組及空白對(duì)照組(P均<0.01)，證實(shí)慢病毒轉(zhuǎn)染成功。與空白對(duì)照組、陰性對(duì)照組比較，慢病毒組細(xì)胞增殖和遷移能力顯著下降(P均<0.01)。細(xì)胞免疫熒光試驗(yàn)顯示，慢病毒組細(xì)胞c-Met的熒光強(qiáng)度顯著降低，而空白對(duì)照組與陰性對(duì)照組無(wú)明顯變化。Western blotting結(jié)果顯示，慢病毒組c-Met及磷酸化c-Met表達(dá)均低于陰性對(duì)照組、空白對(duì)照組(P均<0.01)。結(jié)論過(guò)表達(dá)miR-34a可抑制結(jié)腸癌細(xì)胞增殖及遷移，并下調(diào)靶點(diǎn)蛋白c-Met及磷酸化c-Met表達(dá)，提示miR-34a可作為結(jié)腸癌治療的分子靶點(diǎn)。

關(guān)鍵詞：結(jié)腸癌；miR-34a；細(xì)胞增殖；細(xì)胞遷移；c-Met

微小RNA(miRNA)是一種廣泛存在于真核生物細(xì)胞內(nèi)、長(zhǎng)約22個(gè)核苷酸、能與特定靶基因信使RNA的3′UTR配對(duì)結(jié)合來(lái)調(diào)控靶基因表達(dá)的非編碼RNA[1]。研究發(fā)現(xiàn)，miRNAs(如miR-21、miR-143、miR-145等)能通過(guò)調(diào)控不同蛋白的表達(dá)，影響結(jié)腸癌細(xì)胞的生物學(xué)行為[2]。miR-34a在肺癌、胃癌和肝癌等細(xì)胞中異常表達(dá)能夠誘導(dǎo)多種癌細(xì)胞凋亡并抑制其遷移[3, 4]；但其在結(jié)腸癌細(xì)胞中的作用及其機(jī)制鮮見(jiàn)報(bào)道。2014年5月，我們采用慢病毒miR-34a過(guò)表達(dá)載體轉(zhuǎn)染結(jié)腸癌細(xì)胞，觀察其對(duì)結(jié)腸癌細(xì)胞增殖和遷移的影響，并驗(yàn)證其靶點(diǎn)蛋白，旨在為結(jié)腸癌的臨床治療提供理論依據(jù)。

1材料與方法

1.1材料人結(jié)腸癌細(xì)胞株HCT-116，購(gòu)自中國(guó)科學(xué)院上海細(xì)胞庫(kù)；miRNA提取試劑盒，購(gòu)自德國(guó)QIAGEN公司；逆轉(zhuǎn)錄、熒光定量PCR試劑盒，購(gòu)自日本TaKaRa公司；miR-34a過(guò)表達(dá)的慢病毒載體(pRI-CMV-GFP-miRNA-34a，以下稱(chēng)慢病毒載體)和轉(zhuǎn)染增強(qiáng)劑(polybrene)，購(gòu)自上海吉瑪制藥技術(shù)有限公司；一抗(兔抗人anti-c-Met，anti-c-Met phosphorylation，GAPDH)和辣根過(guò)氧化物酶標(biāo)記的二抗(羊抗兔)，購(gòu)自美國(guó)Cell Signaling Technology公司；其他生化試劑購(gòu)自Sigma公司。

1.2實(shí)驗(yàn)方法

1.2.1細(xì)胞培養(yǎng)及轉(zhuǎn)染HCT-116細(xì)胞置于含10% FBS和100 U/mL青鏈霉素的DMEM培養(yǎng)基，37 ℃、5% CO2恒溫細(xì)胞培養(yǎng)箱內(nèi)培養(yǎng)，每2～3天更換培養(yǎng)液。待細(xì)胞融合>80%，胰蛋白酶消化并傳代。取對(duì)數(shù)生長(zhǎng)期細(xì)胞接種于6孔板，隨機(jī)分為空白對(duì)照組、陰性對(duì)照組、慢病毒組，37 ℃、5% CO2恒溫細(xì)胞培養(yǎng)箱培養(yǎng)。待細(xì)胞融合>30%時(shí)更換無(wú)血清培養(yǎng)基，陰性對(duì)照組轉(zhuǎn)染空病毒載體(pRI-CMV-GFP)、慢病毒組轉(zhuǎn)染慢病毒載體，空白對(duì)照組不予任何處理。按照1 μg載體∶500 μL DMEM混合均勻，加入polybrene(5 μg/mL)增強(qiáng)病毒轉(zhuǎn)染效率，室溫孵育30 min；將混合物加入6孔板中，混勻后繼續(xù)培養(yǎng)，轉(zhuǎn)染12 h更換DMEM完全培養(yǎng)液。采用濃度遞增的嘌呤霉素對(duì)轉(zhuǎn)染后的細(xì)胞進(jìn)行穩(wěn)定表達(dá)篩選，細(xì)胞出現(xiàn)GFP綠色熒光表明轉(zhuǎn)染成功。取各組細(xì)胞1×106個(gè)，采用miRNA提取試劑盒提取總miRNA，TaqMan microRNA逆轉(zhuǎn)錄試劑盒將miRNA逆轉(zhuǎn)錄成cDNA。以cDNA為模板進(jìn)行Real-time PCR反應(yīng)。以U6為內(nèi)參校正PCR模板的拷貝數(shù)。以2-ΔΔCt法[5]計(jì)算各組miR-34a的相對(duì)表達(dá)量。每組設(shè)3個(gè)復(fù)孔，取平均值。

1.2.2HCT-116細(xì)胞增殖能力檢測(cè)采用MTT法。細(xì)胞分組及轉(zhuǎn)染同上，待轉(zhuǎn)染成功后，采用胰蛋白酶消化制成單細(xì)胞懸液，按2×103個(gè)/孔接種于96孔板中。分別于0、24、48、72 h，加入10 μL MTT試劑，37 ℃孵育1 h，酶標(biāo)儀480 nm處讀取每孔的吸光度(OD)值。細(xì)胞增殖能力=(當(dāng)日OD值-前日OD值)/前日OD值。

1.2.3HCT-116細(xì)胞遷移能力檢測(cè)采用Transwell小室法。細(xì)胞分組及轉(zhuǎn)染同上，待轉(zhuǎn)染成功后，于Transwell小室下室加入1 mL完全培養(yǎng)基，遷移小室內(nèi)加入200 μL細(xì)胞懸液(細(xì)胞密度1×106/mL)，37 ℃孵育24 h；棉簽擦拭遷移小室內(nèi)未遷移至基底層細(xì)胞，向遷移小室內(nèi)加入200 μL染色液染色30 min，去除染色液，向每孔加入乙酸溶解染料，轉(zhuǎn)移至24孔板，置于酶標(biāo)儀570 nm處讀數(shù)。

1.2.4miR-34a靶點(diǎn)蛋白驗(yàn)證①采用細(xì)胞免疫熒光試驗(yàn)。細(xì)胞分組及轉(zhuǎn)染同上，待轉(zhuǎn)染成功后，于細(xì)胞培養(yǎng)板中將已爬好細(xì)胞的玻片用PBS浸洗，4%多聚甲醛常溫下固定15 min，0.5% Triton X-100室溫通透，PBS浸洗；玻片上滴加正常山羊血清，室溫封閉30 min；每張玻片滴加一抗(anti-c-Met)并放入濕盒，4 ℃孵育過(guò)夜；次日去除一抗，PBS清洗后加入熒光二抗，濕盒37 ℃孵育1 h；使用含抗熒光淬滅劑的封片液封片，熒光顯微鏡下觀察。②采用Western blotting法。細(xì)胞分組及轉(zhuǎn)染同上，48 h后取1×106個(gè)細(xì)胞，加入10 μL RIPA裂解液，10 min后提取總蛋白，并測(cè)定蛋白濃度，各組取50 μg蛋白進(jìn)行10% SDS-PAGE電泳。電泳結(jié)束后轉(zhuǎn)膜至PVDF膜，用5% milk-TBS將膜封閉1 h，加入相應(yīng)的第一抗體，靜置，4 ℃過(guò)夜。次日使用TBST漂洗，辣根過(guò)氧化物酶(HRP)標(biāo)記的二抗室溫孵育1 h，顯色拍照。

2結(jié)果

2.1各組miR-34a相對(duì)表達(dá)量比較轉(zhuǎn)染48 h，慢病毒組和陰性對(duì)照組可見(jiàn)GFP綠色熒光，而空白對(duì)照組未見(jiàn)GFP綠色熒光，表明慢病毒轉(zhuǎn)染成功。見(jiàn)插頁(yè)Ⅰ圖1。以空白對(duì)照組miR-34a表達(dá)為1，陰性對(duì)照組為1.03±0.09，慢病毒組為6.41±1.56。慢病毒組miR-34a表達(dá)高于陰性對(duì)照組、空白對(duì)照組(P均<0.01)，而陰性對(duì)照組、空白對(duì)照組比較均無(wú)統(tǒng)計(jì)學(xué)差異(P>0.05)。

2.2各組細(xì)胞增殖能力及遷移能力比較見(jiàn)表1。

2.3miR-34a靶點(diǎn)蛋白驗(yàn)證結(jié)果細(xì)胞免疫熒光試驗(yàn)顯示，慢病毒組c-Met的熒光強(qiáng)度顯著降低，而空白對(duì)照組與陰性對(duì)照組無(wú)明顯變化。見(jiàn)插頁(yè)Ⅰ圖2。Western blotting結(jié)果顯示，以空白對(duì)照組c-Met及磷酸化c-Met表達(dá)為1，陰性對(duì)照組分別為1.10±0.22、1.14±0.31，慢病毒組分別為0.41±0.30、0.19±0.42。慢病毒組c-Met及磷酸化c-Met表達(dá)均低于陰性對(duì)照組、空白對(duì)照組(P均<0.01)，而陰性對(duì)照組、空白對(duì)照組比較均無(wú)統(tǒng)計(jì)學(xué)差異(P均<0.01)。

表1　各組細(xì)胞增殖能力及遷移能力比較±s)

注：與空白對(duì)照組、陰性對(duì)照組比較，*P<0.01。

3討論

miRNAs是人體細(xì)胞內(nèi)種類(lèi)最多的表達(dá)調(diào)控因子，具有多種生物學(xué)功能[6～8]。目前，miRNAs在癌細(xì)胞惡性生物學(xué)行為的調(diào)控已成為腫瘤研究熱點(diǎn)。有研究表明，腫瘤患者體內(nèi)miRNAs表達(dá)譜已經(jīng)出現(xiàn)顯著差異，通過(guò)對(duì)其中差異化表達(dá)明顯的miRNAs進(jìn)行初步篩選，證實(shí)部分miRNAs表達(dá)與腫瘤預(yù)后相關(guān)[9～13]。miR-34a作為一種最新鑒定的、與多種癌癥病理機(jī)制密切相關(guān)的miRNA，在前列腺癌、乳腺癌及胃癌中高表達(dá)，并與腫瘤的侵襲及轉(zhuǎn)移密切相關(guān)。

為闡明miR-34a在結(jié)腸癌發(fā)生中的作用，本研究構(gòu)建了miR-34a過(guò)表達(dá)的慢病毒載體，并穩(wěn)定轉(zhuǎn)染HCT-116細(xì)胞，通過(guò)嘌呤霉素和Real-time PCR法證實(shí)轉(zhuǎn)染成功，并通過(guò)細(xì)胞功能試驗(yàn)證實(shí)過(guò)表達(dá)miR-34a可顯著抑制HCT-116細(xì)胞增殖和遷移。

c-Met作為原癌基因，在多種癌細(xì)胞增殖、遷移和分化等過(guò)程中具有重要調(diào)節(jié)作用。c-Met在惡性腫瘤中過(guò)表達(dá)，可能與腫瘤的進(jìn)展密切相關(guān)。Landskron等[14]研究發(fā)現(xiàn)，潰瘍性結(jié)腸炎癌變黏膜c-Met表達(dá)明顯增高。本研究結(jié)果顯示，miR-34a可下調(diào)HCT-116細(xì)胞中靶點(diǎn)蛋白c-Met及磷酸化c-Met的表達(dá)。證實(shí)結(jié)腸癌細(xì)胞c-Met表達(dá)受miR-34a的調(diào)控，過(guò)表達(dá)miR-34a能夠顯著下調(diào)c-Met的表達(dá)，繼而抑制結(jié)腸癌細(xì)胞的增殖和遷移。但在癌細(xì)胞的增殖、分化和遷移中，往往存在多種基因網(wǎng)絡(luò)或信號(hào)通路的共同參與，如TREM2、SIRT1等，也受miR-34a的抑制或增強(qiáng)[15]。因此，要進(jìn)一步明確miR-34a的作用及其機(jī)制，仍需對(duì)結(jié)腸癌相關(guān)基因的調(diào)控網(wǎng)絡(luò)進(jìn)行深入研究。

綜上所述，過(guò)表達(dá)miR-34a可抑制結(jié)腸癌細(xì)胞增殖及遷移，并下調(diào)靶點(diǎn)蛋白c-Met及磷酸化c-Met表達(dá)，提示miR-34a可作為結(jié)腸癌治療的分子靶點(diǎn)。

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Function and molecular mechanism of miR-34a in proliferation and migration of colon cancer

ZHUANGBiao,MINZhijun,WANGYanfeng,ZHANGPeng,NIXiong,QUHuilong,DINGYuming

(ShanghaiPudongHospital,Shanghai201399,China)

Abstract:ObjectiveTo explore the role of miR-34a in the proliferation and migration of colon cancer cells, and to validate the target protein. MethodsThe empty vector (pRI-CMV-GFP) and lentiviral vector (pRI-CMV-GFP-miRNA-34a vector) were transfected into human colon cancer cell line (HCT-116) as the negative control group and lentiviral group, while HCT116 cells without any processing were regarded as the blank control group. The expression of miR-34a was detected by real-time PCR. MTT and Transwell assays were used to detect HCT-116 cell proliferation and migration. Immunofluorescence and Western blotting were applied to verify the target gene of miR-34a in HCT-116. ResultsThe expression of miR-34a in the blank control group was taken as 1, the expression of miR-34a in the negative control group and lentiviral group was 1.03±0.09 and 6.41±1.56, respectively. The lentiviral group was higher than the negative control group and blank control group (all P<0.01), which confirmed lentiviral transfection was successful. Compared with the blank control group and negative control group, the proliferation and migration abilities of HCT-116 cells in the lentiviral group were significantly decreased (all P<0.01). Moreover, the fluorescence intensity of c-Met in the lentiviral group was significantly decreased while no change was found in the other two groups. Western blotting showed that the expression of c-Met and phosphorylated c-Met in the lentiviral group was lower than that of the negative control and blank control group (all P<0.01). ConclusionOver-expression of miR-34a inhibits the proliferation and migration of colon cancer cells and down-regulates the target protein c-Met and phosphorylated c-Met expression, which suggests miR-34a could be a effective molecular target in treatment of colon cancer.

Key words:colon cancer; miR-34a; cell proliferation; cell migration; c-Met

(收稿日期：2015-11-21)

中圖分類(lèi)號(hào)：R735.3

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1002-266X(2016)08-0014-03

doi:10.3969/j.issn.1002-266X.2016.08.005

作者簡(jiǎn)介：第一莊彪(1969-)，男，副主任醫(yī)師，主要研究方向?yàn)槲改c道腫瘤綜合治療。E-mail： zhuangbiao2000@163.com

基金項(xiàng)目：上海市浦東新區(qū)衛(wèi)生和計(jì)劃生育委員會(huì)衛(wèi)生科技項(xiàng)目(PW2014A-28)。