·論著·

微小RNA-132對胰腺癌SW1990細(xì)胞增殖及凋亡的影響

劉海斌華瑩金洲祥

【摘要】目的觀察微小RNA-132(miR-132)轉(zhuǎn)染人胰腺癌細(xì)胞株SW1990后對細(xì)胞增殖及凋亡的影響，并探討其作用機(jī)制。方法采用實時熒光定量PCR(RT-qPCR)法檢測28例胰腺癌及匹配的癌旁正常胰腺組織miR-132的表達(dá)。采用脂質(zhì)體將miR-132轉(zhuǎn)染SW1990細(xì)胞，以未轉(zhuǎn)染及轉(zhuǎn)染錯義miR-132的細(xì)胞分別作為空白對照和陰性對照。應(yīng)用CCK-8法、DAPI染色法檢測細(xì)胞的增殖及凋亡；將轉(zhuǎn)染細(xì)胞種植于裸鼠皮下成瘤，應(yīng)用TUNEL法檢測種植瘤細(xì)胞凋亡；免疫組化法檢測轉(zhuǎn)染細(xì)胞的mucin-4、HER-2、p-FAK蛋白表達(dá)及種植瘤組織mucin-4、Ki-67蛋白表達(dá)。結(jié)果28例胰腺癌及癌旁正常胰腺組織miR-132的相對表達(dá)量分別為0.46±0.11和1.24±0.36，差異有統(tǒng)計學(xué)意義(P<0.05)。轉(zhuǎn)染細(xì)胞的miR-132表達(dá)量為2.95±0.46，顯著高于陰性對照組的0.84±0.17(P<0.05)；轉(zhuǎn)染組細(xì)胞培養(yǎng)48、72、96 h時的存活率分別為陰性對照組56.5%、44.7%、37.4%(P值均<0.05)；細(xì)胞凋亡率為41.6%，顯著高于陰性對照組的5.7%(P<0.05)；轉(zhuǎn)染細(xì)胞mucin-4、HER-2、p-FAK蛋白的表達(dá)較陰性對照組顯著下調(diào)(0.76±0.14比2.94±0.42，0.34±0.04比1.75±0.33，0.27±0.03比2.74±0.24，P值均<0.01)。與陰性對照組比較，轉(zhuǎn)染組裸鼠皮下移植瘤生長明顯被抑制[(0.23±0.05)g比(0.59±0.13)g，P<0.05]，瘤內(nèi)腫瘤細(xì)胞凋亡明顯增加[(21.7±4.7)%比(5.2±0.7)%，P<0.05]，mucin-4和Ki-67蛋白表達(dá)顯著下調(diào)(64.35±7.16比281.34±36.62，30.75±4.61比148.05±21.34，P值均<0.01)。而陰性對照組與空白對照組間的差異均無統(tǒng)計學(xué)意義。結(jié)論轉(zhuǎn)染miR-132對胰腺癌SW1990細(xì)胞有顯著的增殖抑制和凋亡誘導(dǎo)作用，其機(jī)制可能與下調(diào)mucin-4、HER-2、p-FAK等蛋白的表達(dá)有關(guān)。

【關(guān)鍵詞】胰腺腫瘤；微RNAs；細(xì)胞增殖；細(xì)胞凋亡

DOI:10.3760/cma.j.issn.1674-1935.2015.01.010

基金項目：省衛(wèi)生廳資助項目(2012A122)

收稿日期：(2014-07-30)

Effects of microRNA-132 on the proliferation and apoptosis in human pancreatic cancer cells SW1990LiuHaibin,HuaYing,JinZhouxiang.DepartmentofHepaticSurgery,SecondAffiliatedHospitalofWenzhouMedicalUniversity,Wenzhou325000,China

Correspondingauthor:LiuHaibin,Email:wzykdx203109@126.com

Abstract【】ObjectiveTo observe the effect of miR-132 transfection on proliferation and apoptosis of pancreatic cancer SW1990, and to explore the underlying mechanism. MethodsThe expression of miR-132 in the pancreatic cancer tissue and the adjacent tissues in 28 pancreatic cancer patients were detected by real-time quantitative polymerase chain reaction (RT-qPCR). miR-132 was transfected into SW1990 cells by using liposome method, untransfected cells and cells with missense miR-132 transfection were used as black control and negative control. The proliferation and apoptosis was detected by CCK-8 assay and DAPI staining. The transfected cells were implanted in nude mice as xenograft tumor, and TUNEL was used to detect the apoptosis; immunohistochemistry was used to detect the expression of Ki-67 and mucin-4 protein in the xenograft tumors and mucin-4, HER-2, p-FAK protein in transfected cells. ResultsThe expression levels of miR-132 in pancreatic cancer tissue and adjacent tissues were 0.46±0.11 and 1.24±0.36, and the difference between

作者單位：325000浙江溫州，浙江省溫州醫(yī)學(xué)院附屬第二醫(yī)院肝膽外科

通信作者：劉海斌，Email: wzykdx203109@126.com

the two groups was statistically significant (P<0.05). The expression level of miR-132 in transfected SW1990 cells was 2.95±0.46, which were significantly higher than those in negative control(0.84±0.17); the survival rate of transfected cells at 48, 72, 96 h was 56.5%, 44.7%, 37.4% of negative control cells. The apoptosis rate in transfected cells was 41.6%, and the corresponding value was 5.7% in negative control, and the difference was statistically significant(P<0.05). The expression levels of mucin-4, HER-2, p-FAK in nagative control were 2.94±0.42, 1.75±0.37 and 2.74±0.24, and the corresponding values in transfected cells were 0.76±0.14, 0.34±0.04 and 0.27±0.03, and the difference between the two groups was statistically significant (P<0.05). In vivo, the growth of xenograft tumors in transfected nude mice was significantly inhibited [(0.23±0.05)vs(0.59±0.13)g,P<0.05], the apoptosis of xenograft tumor cells was significant increased [(21.7±4.7)%vs(5.2±0.7)%,P<0.05]. The expressions of mucin-4 and Ki-67 protein in nagative control was 281.34±36.62 and 148.05±21.34, and the corresponding values in transfection group were 64.35±7.16 and 30.75±4.61, and the difference was statistically significant (P<0.05). ConclusionsmiR-132 transfection has an effect of inhibiting proliferation and promoting apoptosis on SW1990 cells, and the mechanism may be down-regulation of mucin-4, HER-2, p-FAK protein rxpression.

【Key words】Pancreatic neoplasms;MicroRNAs;Cell proliferation;Apoptosis

微小RNA(microRNA, miRNAs)是一種內(nèi)源性非編碼小分子RNA，在組織炎癥反應(yīng)、細(xì)胞增殖與凋亡、組織分化以及惡性腫瘤發(fā)生和發(fā)展等多種病理過程中發(fā)揮重要作用[1]。miR-132是當(dāng)前研究熱點之一，它不僅在結(jié)腸癌和肺癌等多種惡性腫瘤細(xì)胞中表達(dá)下調(diào)，同時與惡性腫瘤的轉(zhuǎn)移有著密切的關(guān)系[2-3]。本研究檢測miR-132在胰腺癌組織中的表達(dá)，并將miR-132轉(zhuǎn)染至胰腺癌細(xì)胞株SW1990，觀察其對SW1990細(xì)胞增殖和凋亡的影響，探討其作用機(jī)制。

材料與方法

一、材料

收集2004年8月至2013年7月間溫州醫(yī)學(xué)院附屬第二醫(yī)院肝膽外科行手術(shù)切除并經(jīng)病理證實的28例胰腺癌及匹配的癌旁正常胰腺組織標(biāo)本。28例患者中男性15例，女性13例，年齡46～72歲，中位年齡54歲。術(shù)前均未行放、化療。全部標(biāo)本取材后迅速置于-80℃冰箱保存?zhèn)溆?。人胰腺癌?xì)胞株SW1990購自中科院上海細(xì)胞庫，常規(guī)培養(yǎng)、傳代。4～6周齡雌性BALB/c-nu/nu品系裸鼠，體重18～20 g，購自中科院上海動物實驗中心，飼養(yǎng)于溫州醫(yī)學(xué)院動物實驗中心SPF級屏障系統(tǒng)的層流架內(nèi)，室溫(25±1)℃，相對濕度40%～60%。

二、方法

1．胰腺癌組織miR-132表達(dá)的檢測：用Trizol試劑提取胰腺癌組織及癌旁正常胰腺組織的總RNA。采用PrimeScript逆轉(zhuǎn)錄試劑盒(日本TakaRa公司)逆轉(zhuǎn)錄成cDNA，采用SYBR實時熒光定量試劑盒(Invitrogen公司)行實時定量PCR檢測miR-132表達(dá)，按說明書操作。PCR反應(yīng)條件：95℃ 30 s，95℃ 5 s，60℃ 34 s，40循環(huán)，72℃延伸7 min。以U6 snRNA作為內(nèi)參。通過儀器自帶軟件獲取Ct值，根據(jù)公式2-△△Ct計算miR-132的相對表達(dá)量。

2．miR-132轉(zhuǎn)染細(xì)胞及分組：取對數(shù)生長期SW1990細(xì)胞，按每孔4×106個細(xì)胞密度接種于6孔板，每孔2 ml。細(xì)胞貼壁后分為空白對照組、陰性對照組及轉(zhuǎn)染組，在培養(yǎng)基中分別加入濃度為50 nmol/L的Lipofectamine 2000、CyTM3標(biāo)記miR-132錯義鏈、CyTM3標(biāo)記miR-132 mimic(均購自銳博生物科技有限公司)，培養(yǎng)24 h后收集細(xì)胞，在熒光顯微鏡下觀察細(xì)胞轉(zhuǎn)染效率。

3．轉(zhuǎn)染細(xì)胞miR-132表達(dá)檢測：取轉(zhuǎn)染48 h的SW1990細(xì)胞，用Trizol試劑提取細(xì)胞總RNA，采用Hairpin-itTMmiRNAs qPCR Quantitation Kit行實時定量PCR檢測miR-132表達(dá)，按說明書操作，以U6 snRNA作為內(nèi)參，根據(jù)公式2-△△Ct計算miR-132的相對表達(dá)量。

4．轉(zhuǎn)染細(xì)胞的增殖及凋亡檢測：取轉(zhuǎn)染24 h的SW1990細(xì)胞制成單細(xì)胞懸液，以每孔4×103個細(xì)胞密度接種于96孔板，每孔200 μl。細(xì)胞貼壁后分別培養(yǎng)24、48、72、96 h，每組設(shè)5個復(fù)孔，以僅加入等量PBS的孔調(diào)零。在各培養(yǎng)時間點結(jié)束前1 h加CCK-8溶液(南京凱基生物科技發(fā)展有限公司)0.01 ml，繼續(xù)培養(yǎng)1 h，上酶標(biāo)儀測各孔波長為490 nm的吸光度值(A490值)。細(xì)胞存活率=實驗組A490值/對照組A490值×100%。

取轉(zhuǎn)染24 h的SW1990細(xì)胞，調(diào)整細(xì)胞密度為1×105/ml。取1 ml細(xì)胞懸液滴于普通載玻片上培養(yǎng)過夜，用甲醛固定，再加DAPI溶液(南京凱基生物科技發(fā)展有限公司)染色5 min，置熒光顯微鏡下觀察。取20個低倍視野，每個視野計數(shù)200個細(xì)胞，計算凋亡細(xì)胞占總細(xì)胞數(shù)的百分率，取均值。

5．轉(zhuǎn)染細(xì)胞mucin-4、HER-2、p-FAK、FAK蛋白表達(dá)檢測：取轉(zhuǎn)染miR-132的SW1990細(xì)胞，用細(xì)胞裂解液裂解細(xì)胞，用Bradford法測量蛋白濃度。取50 μg蛋白常規(guī)行蛋白質(zhì)印跡法檢測mucin-4、HER-2、p-FAK、FAK蛋白的表達(dá)，以β-actin為內(nèi)參。兔抗人mucin-4、HER-2、p-FAK、FAK、β-actin一抗均購自Santa Cruz公司，工作濃度均為1∶1 000，HRP標(biāo)記的羊抗兔IgG工作濃度1∶2 500。最后ECL發(fā)光，X片曝光、顯影，用Quantity One 4.6.2(BIO, RAD)軟件分析各條帶灰度值，以目的條帶與內(nèi)參條帶的灰度值比表示蛋白的相對表達(dá)量。

6．裸鼠皮下胰腺癌種植瘤模型建立及分組：將15只裸鼠按完全隨機(jī)法分成空白對照組、陰性對照組和轉(zhuǎn)染組，分別在裸鼠右側(cè)大腿背部皮下注射對數(shù)生長期的空白對照組、陰性對照組和轉(zhuǎn)染組的SW1990細(xì)胞，細(xì)胞密度為2×107/ml，每只裸鼠注射0.2 ml。注射4周后處死裸鼠，剝離裸鼠皮下種植瘤標(biāo)本，稱重。

7．皮下種植瘤的細(xì)胞凋亡檢測：采用TUNEL法檢測種植瘤的細(xì)胞凋亡。按試劑盒(深圳晶美生物技術(shù)有限公司)說明書操作。TUNEL染色陽性細(xì)胞判定標(biāo)準(zhǔn)為胞質(zhì)不著色，胞核染成棕褐色，核固縮，染色質(zhì)凝集成塊或邊集。取5個低倍視野，每個視野計數(shù)200個細(xì)胞。凋亡指數(shù)=凋亡細(xì)胞數(shù)/細(xì)胞總數(shù)×100%，取均值。

8．種植瘤組織Ki-67、mucin-4蛋白表達(dá)檢測:取種植瘤組織，經(jīng)甲醛固定、石蠟包埋、切片，采用免疫組化法檢測瘤組織Ki-67、mucin-4蛋白表達(dá)。免疫組化SP試劑盒購自德國寶靈曼公司，按說明書操作。在低倍鏡下隨機(jī)選擇20個視野，用Image-Pro Plus6.0圖像處理軟件分析Ki-67、mucin-4的IOD值。

三、統(tǒng)計學(xué)處理

結(jié)果

一、胰腺癌及癌旁正常胰腺組織miR-132 表達(dá)

28例胰腺癌及癌旁正常胰腺組織miR-132的相對表達(dá)量分別為0.46±0.11和1.24±0.36，差異有統(tǒng)計學(xué)意義(t=6.509，P<0.05)。

二、各組SW1990細(xì)胞miR-132的表達(dá)量

空白對照組、陰性對照組和轉(zhuǎn)染組SW1990細(xì)胞的miR-132表達(dá)量分別為0.73±0.15、0.84±0.17和2.95±0.46，差異有統(tǒng)計學(xué)意義(t=52.181，P<0.05)，轉(zhuǎn)染效率達(dá)到實驗要求(圖1)。

圖1　轉(zhuǎn)染miR-132的SW1990細(xì)胞

三、各組SW1990細(xì)胞增殖及凋亡的變化

轉(zhuǎn)染組SW1990細(xì)胞培養(yǎng)48、72、96 h時的存活率分別為陰性對照組56.5%、44.7%、37.4%，差異有統(tǒng)計學(xué)意義(F=127.947，P<0.05)。

熒光顯微鏡下觀察到空白對照組和陰性對照組細(xì)胞的核完整，大小一致，而轉(zhuǎn)染組細(xì)胞核出現(xiàn)明顯固縮和凋亡小體等改變(圖2)。轉(zhuǎn)染組、陰性對照組、空白對照組的細(xì)胞凋亡率分別為41.6%、5.7%和6.4%，差異有統(tǒng)計學(xué)意義(F=137.634，P<0.05)。

圖2　空白對照組(2A)、陰性對照組(2B)、轉(zhuǎn)染組(2C)細(xì)胞的凋亡(熒光顯微鏡　×400)

四、各組SW1990細(xì)胞mucin-4、HER-2、p-FAK、FAK蛋白表達(dá)

轉(zhuǎn)染組細(xì)胞mucin-4、HER-2和p-FAK蛋白的表達(dá)較兩對照組明顯下調(diào)，而FAK的表達(dá)無明顯變化(表1，圖3)。

組 別mucin-4HER-2p-FAK空白對照組2.57±0.311.38±0.263.12±0.43陰性對照組2.94±0.421.75±0.332.74±0.24轉(zhuǎn)染組0.76±0.140.34±0.040.27±0.03F值18.12923.75342.889P值0.0030.001<0.001

圖3　空白對照組(1)、陰性對照組(2)、轉(zhuǎn)染組(3)SW1990細(xì)胞的mucin-4、HER-2、p-FAK、FAK蛋白表達(dá)

五、miR-132轉(zhuǎn)染對裸鼠胰腺癌皮下種植瘤生長的影響

各組裸鼠皮下種植瘤的成瘤率均為100%，實驗過程中裸鼠均無死亡?？瞻讓φ战M、陰性對照組和轉(zhuǎn)染組平均種植瘤重量分別為(0.48±0.16)、(0.59±0.13)、(0.23±0.05)g(圖4)，轉(zhuǎn)染組裸鼠種植瘤重量顯著低于空白對照組和陰性對照組，差異有統(tǒng)計學(xué)意義(F=25.107，P<0.05)。

圖4　各組裸鼠的皮下種植瘤大小

六、各組裸鼠胰腺癌皮下種植瘤的細(xì)胞凋亡

空白對照組、陰性對照組、轉(zhuǎn)染組種植瘤的細(xì)胞凋亡指數(shù)分別為(3.8±0.5)%、(5.2±0.7)%、(21.7±4.7)%(圖5)，差異有統(tǒng)計學(xué)意義(F=53.698，P<0.05)。

圖5　空白對照組(5A)、陰性對照組(5B)、轉(zhuǎn)染組(5C)裸鼠胰腺癌皮下種植瘤的凋亡細(xì)胞(TUNEL法　×400)

七、各組裸鼠胰腺癌皮下種植瘤組織Ki-67、mucin-4表達(dá)

轉(zhuǎn)染組種植瘤組織的Ki-67和mucin-4的陽性表達(dá)強(qiáng)度均明顯低于空白對照組和陰性對照組，差異有統(tǒng)計學(xué)意義(圖6、表2)。

圖6　空白對照組(6A)、陰性對照組(6B)、轉(zhuǎn)染組(6C)裸鼠胰腺癌皮下種植瘤Ki-67(左)、mucin-4(右)的表達(dá)(免疫組化　×400)

近年來研究表明，miRNAs與惡性腫瘤的發(fā)生和發(fā)展關(guān)系密切，其中miR-132 是目前比較公認(rèn)的抑癌性miRNA。目前已發(fā)現(xiàn)幾十種miR-132的靶基因，其中大多數(shù)已被證實可直接調(diào)控腫瘤細(xì)胞的增殖、侵襲和轉(zhuǎn)移等[3-4]。但miR-132對胰腺癌生長和凋亡的作用尚未見報道。本研究結(jié)果表明，miR-132在胰腺癌組織中的表達(dá)明顯下調(diào)，表明miR-132參與胰腺癌的發(fā)展過程。

表2　各組裸鼠種植瘤組織Ki-67和mucin-4的表達(dá) ± s)

癌癥的發(fā)生是多基因和多因素共同參與的細(xì)胞增殖、分化以及凋亡異常的過程。既往的研究報道，miR-132能抑制惡性腫瘤生長，并誘導(dǎo)細(xì)胞凋亡[5-6]，但尚無體內(nèi)實驗的研究報道。本研究將miR-132轉(zhuǎn)染胰腺癌SW1990細(xì)胞，結(jié)果顯示轉(zhuǎn)染細(xì)胞的miR-132表達(dá)顯著上調(diào)，細(xì)胞的增殖被抑制，凋亡增加；轉(zhuǎn)染細(xì)胞種植于裸鼠皮下形成的胰腺癌皮下種植瘤的體積明顯縮小，重量明顯降低，且Ki-67陽性表達(dá)強(qiáng)度明顯下降，種植瘤內(nèi)細(xì)胞凋亡明顯增加，進(jìn)一步證實miR-132對胰腺癌細(xì)胞增殖和凋亡的調(diào)控作用。

研究發(fā)現(xiàn)，mucins家族蛋白在惡性腫瘤的發(fā)生和發(fā)展以及逃避機(jī)體免疫監(jiān)視等多種病理生理過程中發(fā)揮重要作用，在惡性腫瘤的診斷中亦具有一定價值[7-8]。mucin-4在正常組織細(xì)胞中低表達(dá)或無表達(dá)，但在腫瘤組織細(xì)胞中高表達(dá)[9]。此外，mucin-4與惡性腫瘤細(xì)胞對化療藥物耐藥性關(guān)系密切[10]。膜蛋白HER-2/ErbB-2作為與mucin-4共存蛋白，在穩(wěn)定mucin-4蛋白中起關(guān)鍵作用，與多種惡性腫瘤的發(fā)生和發(fā)展密切相關(guān)[11-12]。本研究結(jié)果顯示，SW1990細(xì)胞轉(zhuǎn)染miR-132后，mucin-4和HER-2蛋白的表達(dá)顯著下調(diào)，表明mucin-4可能作為miR-132的靶蛋白，在調(diào)控胰腺癌細(xì)胞增殖和凋亡過程中發(fā)揮一定的作用。文獻(xiàn)報道，F(xiàn)AK作為HER-2的下游蛋白[13]，其磷酸化(p-FAK)與惡性腫瘤的發(fā)生、發(fā)展、侵襲和轉(zhuǎn)移等行為密切相關(guān)[14]。本研究結(jié)果顯示，轉(zhuǎn)染miR-132后可顯著抑制SW1990細(xì)胞p-FAK的表達(dá)，與mucin-4和HER-2表達(dá)下調(diào)結(jié)果相一致。

參考文獻(xiàn)

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(本文編輯：屠振興)