王顯艷，　高　峰，　趙春明，　孫玉榮，　溫秋婷，　于秀文，　張曉杰

(齊齊哈爾醫(yī)學(xué)院 1病理學(xué)院， 2第三附屬醫(yī)院麻醉科，黑龍江 齊齊哈爾 161006)

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miR-140在人胃癌組織中的表達(dá)及對(duì)SGC-7901胃癌細(xì)胞功能的影響*

王顯艷1△，高峰2，趙春明1，孫玉榮1，溫秋婷1，于秀文1，張曉杰1

(齊齊哈爾醫(yī)學(xué)院1病理學(xué)院，2第三附屬醫(yī)院麻醉科，黑龍江 齊齊哈爾 161006)

目的： 研究microRNA-140(miR-140)在人胃癌和正常胃組織中的表達(dá)水平，以及調(diào)控miR-140表達(dá)后對(duì)SGC-7901胃癌細(xì)胞功能的影響。方法： 采用實(shí)時(shí)熒光定量PCR檢測(cè)miR-140在人胃癌和正常胃組織中的表達(dá)水平；將miR-140 mimics(miR-140上調(diào)表達(dá))和miR-140 inhibitors(miR-140下調(diào)表達(dá))分別通過(guò)脂質(zhì)體轉(zhuǎn)染至人胃癌SGC-7901細(xì)胞中，同時(shí)設(shè)置未轉(zhuǎn)染對(duì)照組(control組)和miRNA無(wú)義序列轉(zhuǎn)染對(duì)照組(NC組)。實(shí)時(shí)熒光定量PCR檢測(cè)轉(zhuǎn)染后各組細(xì)胞中miR-140的表達(dá)變化；MTT方法檢測(cè)各組細(xì)胞的生長(zhǎng)活力和順鉑(DDP)作用下的生長(zhǎng)抑制率；流式細(xì)胞術(shù)檢測(cè)各組的細(xì)胞周期和凋亡率；Transwell實(shí)驗(yàn)檢測(cè)各組細(xì)胞侵襲能力；Western blot檢測(cè)各組細(xì)胞中組蛋白脫乙酰酶4(HDAC4)蛋白表達(dá)。結(jié)果：miR-140在人胃癌組織中表達(dá)水平顯著低于正常胃組織(P<0.05)。與control和NC組相比，miR-140 mimics組中SGC-7901細(xì)胞活力和侵襲能力下降，細(xì)胞周期被阻滯，DDP作用下細(xì)胞生長(zhǎng)抑制率和凋亡率上升，且HDAC4蛋白表達(dá)下調(diào)，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；而miR-140 inhibitors 組中SGC-7901細(xì)胞活力和侵襲能力上升，細(xì)胞周期被促進(jìn)，DDP作用下細(xì)胞生長(zhǎng)抑制率和凋亡率下降，且HDAC4蛋白表達(dá)上調(diào)，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論：miR-140在胃癌組織中低表達(dá)，可作為抑癌因子調(diào)控胃癌細(xì)胞活力、細(xì)胞周期變化、凋亡、侵襲并通過(guò)下調(diào)HDAC4發(fā)揮作用。miR-140可能作為胃癌診斷和治療的新靶點(diǎn)。

胃癌； MicroRNA-140； SGC-7901細(xì)胞； 細(xì)胞活力； 細(xì)胞凋亡； 侵襲； 組蛋白脫乙酰酶4

胃癌是我國(guó)最常見(jiàn)的惡性腫瘤之一，在我國(guó)消化道惡性腫瘤中居首位，而胃癌細(xì)胞的增殖、侵襲和轉(zhuǎn)移是導(dǎo)致患者死亡的主要原因，已成為臨床胃癌治療的主要障礙[1]。同時(shí)胃癌細(xì)胞的增殖侵襲等細(xì)胞活動(dòng)是一個(gè)極其復(fù)雜的過(guò)程，研究表明有多種基因、酶類(lèi)及細(xì)胞因子等參與調(diào)控促進(jìn)或抑制胃癌的細(xì)胞活動(dòng)，但至今胃癌細(xì)胞發(fā)生發(fā)展的確切分子機(jī)制仍未充分闡明[2]。越來(lái)越多的研究表明 microRNAs (miRNA)與腫瘤關(guān)系密切，它不僅調(diào)控腫瘤細(xì)胞增殖、凋亡，而且參與調(diào)控腫瘤細(xì)胞侵襲轉(zhuǎn)移、耐藥等[3]。在多種腫瘤中均已發(fā)現(xiàn)相關(guān)miRNA表達(dá)量的改變，并發(fā)揮促癌或抑癌作用，提示miRNA參與到腫瘤的病理機(jī)制中[4]。miR-140是一種在軟骨增生和發(fā)育及骨關(guān)節(jié)炎發(fā)生過(guò)程中起重要作用的miRNA，但其在腫瘤發(fā)生與進(jìn)展中發(fā)揮作用的研究尚少且無(wú)定論[5]。有研究發(fā)現(xiàn)miR-140在非小細(xì)胞肺癌(non-small-cell lung cancer，NSCLC)中低表達(dá)，其通過(guò)下調(diào)IGF1R抑制NSCLC的增殖和轉(zhuǎn)移[6]。另一項(xiàng)在腦膠質(zhì)瘤中的研究則發(fā)現(xiàn)miR-140在由II級(jí)到IV級(jí)的進(jìn)展過(guò)程中表達(dá)增高[7]。目前miR-140在胃癌中表達(dá)情況及其在胃癌細(xì)胞活動(dòng)中的作用機(jī)制研究未見(jiàn)。本研究檢測(cè)miR-140在臨床胃癌標(biāo)本中表達(dá)情況，通過(guò)上調(diào)和下調(diào)miR-140表達(dá)揭示其對(duì)胃癌SGC-7901細(xì)胞增殖、凋亡、侵襲等細(xì)胞功能的影響并闡明可能的作用機(jī)制，以探討miR-140能否作為臨床胃癌治療的靶點(diǎn)。

材　料　和　方　法

1標(biāo)本來(lái)源

選取齊齊哈爾醫(yī)學(xué)院第三附屬醫(yī)院腫瘤外科2012年6月～2014年6月收治的30例經(jīng)病理科證實(shí)為胃癌的病例，進(jìn)行病理學(xué)檢查手術(shù)切除胃癌組織，并以患者遠(yuǎn)離胃癌腫瘤大于10 cm的正常胃組織作為對(duì)照，標(biāo)本取樣后用生理鹽水沖洗，去除黏膜組織放入液氮中凍存。腫瘤TNM分期以UICC/AJCC TNM病理分期第7版為準(zhǔn)，具體患者臨床資料見(jiàn)表1。標(biāo)本采集均通過(guò)齊齊哈爾醫(yī)學(xué)院醫(yī)學(xué)倫理委員會(huì)批準(zhǔn)和患者知情同意。

表1miR-140的相對(duì)表達(dá)水平與臨床病理因素的關(guān)系

Table 1.Relationships of miR-140 expression and clinicopathological features of gastric carcinoma

FeaturesnmiR-140relativeexpressionPAverageexpression<0.05 Up-expression61.47±0.24 Down-expression240.38±0.21Gender>0.05 Male170.51±0.18 Female130.67±0.13Age(year)>0.05 ≤5591.01±0.11 >55210.65±0.21Tumorsize(cm)<0.05 ≤3.5120.83±0.14 >3.5180.44±0.12Histologicaldifferentiation>0.05 High140.74±0.13 Low160.61±0.15Depthofinvasion(Tstage)<0.05 T1-390.89±0.11 T4210.42±0.16Lymphnodemetastasis(Nstage)<0.05 N061.13±0.17 N1-3240.43±0.21

2細(xì)胞及試劑

人胃癌細(xì)胞株SGC-7901購(gòu)自中國(guó)科學(xué)院上海生命科學(xué)研究院細(xì)胞資源中心；RPMI-1640細(xì)胞培養(yǎng)基、0.25%的EDTA胰酶購(gòu)自HyClone；胎牛血清購(gòu)自Gibco；miR-140 mimics和miR-140 inhibitors及其無(wú)義序列購(gòu)自Invitrogen；LipofectamineTM2000轉(zhuǎn)染試劑購(gòu)自上海生工公司；real-time PCR試劑盒購(gòu)自TaKaRa；四甲基偶氮唑鹽(MTT)、二甲基亞砜(DMSO)和順鉑(diamminedichloroplatinum，DDP)購(gòu)自Sigma；Annexin V/PI細(xì)胞凋亡試劑盒購(gòu)自北京鼎國(guó)公司；Transwell板購(gòu)自Corning；多克隆兔抗人組蛋白脫乙酰酶4(histone deacetylase 4, HDAC4)和β-actin蛋白抗體購(gòu)自Abcam。

3主要方法

3.1Real-time PCR檢測(cè)miR-140在人胃癌和正常胃組織中表達(dá)利用TRIzol提取試劑盒提取胃癌和正常胃組織樣品中總RNA，后用miR-140特異性逆轉(zhuǎn)錄引物(表2)和NCodeTMmiRNA First-Strand cDNA Synthesis Kit轉(zhuǎn)錄合成cDNA。由Invitrogen設(shè)計(jì)合成miR-140和內(nèi)參照U6 real-time PCR引物。根據(jù)OpenArray MicroRNA Real-time PCR Master Mix 試劑盒分別加入miR-140或U6引物、cDNA模板置于ABI7300實(shí)時(shí)熒光定量PCR儀上經(jīng)行擴(kuò)增檢測(cè)。miR-140和內(nèi)參照U6均設(shè)置3個(gè)復(fù)孔，每個(gè)樣品經(jīng)3次重復(fù)檢測(cè)，采用2-ΔΔCt方法對(duì)人胃癌和正常胃組織中miR-140 相對(duì)表達(dá)量進(jìn)行比較。

表2　逆轉(zhuǎn)錄和實(shí)時(shí)熒光定量PCR引物

3.2miR-140 mimics和miR-140 inhibitors 轉(zhuǎn)染SGC-7901細(xì)胞將人胃癌細(xì)胞株SGC-7901復(fù)蘇后在含10%胎牛血清的RPMI-1640細(xì)胞培養(yǎng)基中培養(yǎng)，置于37 ℃、5% CO2細(xì)胞培養(yǎng)箱中。利用脂質(zhì)體Lipofectamine 2000分別將miR-140 mimics和miR-140 inhibitors轉(zhuǎn)染至SGC-7901細(xì)胞中以使miR-140上調(diào)和下調(diào)。細(xì)胞分組為：空白對(duì)照(control)組：僅加入脂質(zhì)體Lipofectamine 2000；陰性對(duì)照組(negative control， NC)：加入miRNA無(wú)義序列和Lipofectamine 2000；miR-140上調(diào)轉(zhuǎn)染組：加入miR-140 mimics和Lipofectamine 2000；miR-140下調(diào)轉(zhuǎn)染組：加入miR-140 inhibitors和Lipofectamine 2000。按照上述方法用real-time PCR檢測(cè)各組轉(zhuǎn)染后SGC-7901細(xì)胞中miR-140表達(dá)變化。

3.3MTT檢測(cè)細(xì)胞活力變化和生長(zhǎng)抑制率將各組轉(zhuǎn)染后的SGC-7901細(xì)胞以5×107/L接種于96孔板，分別培養(yǎng)12、24、36、48、60、72 h，后在每孔中加入20 μL的MTT溶液(5 g/L)，繼續(xù)培養(yǎng)4 h后棄上清培養(yǎng)基，每孔加入150 μL DMSO，振蕩混勻10 min后置于酶標(biāo)儀上測(cè)定570 nm波長(zhǎng)時(shí)每孔的吸光度A值以反映細(xì)胞生長(zhǎng)活力。同時(shí)將各組轉(zhuǎn)染后的SGC-7901細(xì)胞接種96孔板貼壁生長(zhǎng)后分別更換含不同濃度(0、5、10、15、20 μmol/L)DDP的培養(yǎng)基繼續(xù)培養(yǎng)24 h，按照MTT方法測(cè)定各孔細(xì)胞的A值，并計(jì)算各組細(xì)胞生長(zhǎng)抑制率(%)=(1-加藥組A值/未加藥對(duì)照組A值)×100%以反映各組細(xì)胞對(duì)順鉑藥物敏感性。

3.4流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期和凋亡率收集各組轉(zhuǎn)染24 h后的SGC-7901細(xì)胞，用預(yù)冷的75%乙醇混勻固定后，離心收集細(xì)胞，用200 μL的PBS重懸細(xì)胞并避光孵育PI 20 min后直接經(jīng)BDcantoⅡ流式細(xì)胞儀檢測(cè)各組細(xì)胞周期變化。同時(shí)根據(jù)上述實(shí)驗(yàn)選擇10 μmol/L的DDP作用于各組轉(zhuǎn)染后SGC-7901細(xì)胞以測(cè)定細(xì)胞凋亡變化。將各組轉(zhuǎn)染后細(xì)胞接種于6孔板中，待細(xì)胞貼壁生長(zhǎng)后更換含10 μmol/L DDP的培養(yǎng)基繼續(xù)培養(yǎng)12 h，后用胰酶消化細(xì)胞并收集。分別在各組細(xì)胞中加入5 μL的FITC標(biāo)記Annexin V和10 μL的PI細(xì)胞凋亡檢測(cè)試劑，37 ℃下避光孵育20 min后，PBS洗滌1次重懸細(xì)胞，經(jīng)BDcantoⅡ流式細(xì)胞儀測(cè)定各組細(xì)胞凋亡率變化。

3.5Transwell檢測(cè)細(xì)胞侵襲能力將轉(zhuǎn)染后的各組SGC-7901細(xì)胞調(diào)整細(xì)胞濃度為1×109/L，以每孔200 μL種植于預(yù)先用1 g/L的Matrigel膠包被的Transwell板上室中，下室中加入20%胎牛血清的RPMI-1640完全細(xì)胞培養(yǎng)基600 μL，每組設(shè)置6個(gè)復(fù)孔，置于細(xì)胞培養(yǎng)箱中繼續(xù)培養(yǎng)48 h。培養(yǎng)結(jié)束后用4%多聚甲醛固定Transwell小室的濾膜，擦去上表面的細(xì)胞，用結(jié)晶紫染色液染色10 min后，PBS洗滌3次，在顯微鏡下觀察各組侵襲穿過(guò)濾膜的細(xì)胞，并隨機(jī)拍照計(jì)算細(xì)胞侵襲率=(各組侵襲細(xì)胞數(shù)/未處理對(duì)照組侵襲細(xì)胞數(shù))×100%。

3.6Western blot檢測(cè)HDAC4表達(dá)變化收集各組轉(zhuǎn)染后SGC-7901細(xì)胞，用RAPI細(xì)胞裂解液提取各組細(xì)胞中總蛋白，BCA試劑盒測(cè)定細(xì)胞中總蛋白濃度。各組上樣30 μg總蛋白經(jīng)行10%的SDS-PAGE凝膠電泳2 h，后轉(zhuǎn)PVDF膜并封閉，4 ℃孵育多克隆兔抗人HDAC4和β-actin I 抗(1∶1 000)過(guò)夜；TBST洗滌后室溫孵育HRP標(biāo)記的山羊抗兔IgG II 抗(1∶5 000)2 h，洗滌后ECL化學(xué)發(fā)光顯影，曝光掃描拍照。用Quantity One軟件分析各組轉(zhuǎn)染后細(xì)胞中HDAC4蛋白相對(duì)表達(dá)情況。

4統(tǒng)計(jì)學(xué)處理

所有數(shù)據(jù)采用SPSS 13.0軟件經(jīng)行統(tǒng)計(jì)學(xué)分析，定量數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。多組間數(shù)據(jù)比較采用單因素方差(one-way ANOVA)分析，用SNK-q進(jìn)行均數(shù)之間的兩兩比較。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

對(duì)鋼鐵行業(yè)的用鋼標(biāo)準(zhǔn)和建筑工程設(shè)計(jì)規(guī)范應(yīng)進(jìn)行修訂和完善，將400 MPa級(jí)、500 MPa級(jí)和600 MPa級(jí)鋼筋納入相關(guān)的結(jié)構(gòu)設(shè)計(jì)和施工驗(yàn)收規(guī)范中，使相關(guān)條文具有強(qiáng)制性。同時(shí)，要加強(qiáng)與交通、鐵路、水利等行業(yè)的協(xié)調(diào)，使各行業(yè)的設(shè)計(jì)規(guī)范有效銜接，納入高強(qiáng)鋼筋生產(chǎn)和應(yīng)用的新工藝、新技術(shù)，進(jìn)行配套修訂，從而推進(jìn)高強(qiáng)鋼筋在工程建設(shè)中的應(yīng)用。

結(jié)　　果

1miR-140在胃癌中表達(dá)情況及與臨床病理參數(shù)之間的關(guān)系

利用real-time PCR檢測(cè)發(fā)現(xiàn)miR-140在人胃癌組織中表達(dá)水平顯著降低，miR-140低表達(dá)率為80%，見(jiàn)表1。對(duì)胃癌組織中miR-140的表達(dá)水平與臨床病理參數(shù)之間進(jìn)行統(tǒng)計(jì)，全組30例胃癌患者，男17例，女13例，年齡在38～67歲，中位年齡58.9歲，miR-140總平均表達(dá)量為正常胃組織的(0.63±0.13)倍(P<0.05)。同時(shí)檢測(cè)發(fā)現(xiàn)，人胃癌組織中miR-140表達(dá)水平與腫瘤大小、胃癌侵襲度和淋巴轉(zhuǎn)移相關(guān)。

2miR-140 mimics和miR-140 inhibitors 轉(zhuǎn)染SGC-7901細(xì)胞后miR-140表達(dá)的變化

Real-time PCR檢測(cè)發(fā)現(xiàn)miR-140 mimics轉(zhuǎn)染SGC-7901細(xì)胞后能夠使miR-140表達(dá)上調(diào)，而轉(zhuǎn)染miR-140 inhibitors后能夠使miR-140表達(dá)下調(diào)，見(jiàn)圖1。與control組相比，NC轉(zhuǎn)染組中miR-140表達(dá)水平無(wú)明顯變化；與control組和NC組相比，miR-140 mimics組中miR-140表達(dá)水平上升至原來(lái)的(3.85±0.17)倍(P<0.05)；而miR-140 inhibitors組中miR-140表達(dá)水平下降至(15±9)%(P<0.05)，說(shuō)明miR-140 mimics和miR-140 inhibitors 轉(zhuǎn)染SGC-7901細(xì)胞成功，能夠上調(diào)和下調(diào)細(xì)胞中miR-140表達(dá)。

Figure 1.The expression of miR-140 in SGC-7901 cells transfected with miR-140 mimics and miR-140 inhibitors. Mean±SD.n=6.*P<0.05vscontrol group and NC group.

圖1miR-140在miR-140 mimics和miR-140 inhibitors轉(zhuǎn)染SGC-7901細(xì)胞后的表達(dá)差異

3miR-140調(diào)控表達(dá)對(duì)SGC-7901細(xì)胞活力和順鉑作用下生長(zhǎng)抑制率的影響

MTT實(shí)驗(yàn)發(fā)現(xiàn)miR-140 mimics轉(zhuǎn)染SGC-7901細(xì)胞后能夠降低細(xì)胞生長(zhǎng)活力，而miR-140 inhibitors轉(zhuǎn)染后SGC-7901細(xì)胞活力有所提升。與control和NC組相比，在24～72 h時(shí)段內(nèi)，miR-140 mimics轉(zhuǎn)染組SGC-7901的細(xì)胞活力均顯著降低，差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05)；而在36～72 h時(shí)段內(nèi)，miR-140 inhibitors轉(zhuǎn)染組中SGC-7901細(xì)胞活力均顯著提升，差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，見(jiàn)圖2。同時(shí)用MTT實(shí)驗(yàn)檢測(cè)各組細(xì)胞對(duì)DDP的敏感性，發(fā)現(xiàn)在不同濃度DDP作用下miR-140 mimics轉(zhuǎn)染組SGC-7901細(xì)胞生長(zhǎng)抑制率顯著高于miR-140 inhibitors轉(zhuǎn)染組。與control和NC組相比，在DDP濃度大于10 μmol/L時(shí)，miR-140 mimics轉(zhuǎn)染組SGC-7901細(xì)胞生長(zhǎng)抑制率顯著上升，而miR-140 inhibitors轉(zhuǎn)染組細(xì)胞生長(zhǎng)抑制率顯著下降，差異均具有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，見(jiàn)圖3。

Figure 2.The changes of SGC-7901 cell activity curve were detected by MTT assay. Mean±SD.n=6.*P<0.05vscontrol group and NC group.

圖2MTT檢測(cè)miR-140調(diào)控表達(dá)后各組SGC-7901細(xì)胞生長(zhǎng)活力曲線的變化

Figure 3.The sensitivity changes of SGC-7901 cell growth inhibition rate to DDP were detected by MTT assay. Mean±SD.n=6.*P<0.05vscontrol and NC group.

圖3miR-140調(diào)控表達(dá)后各組SGC-7901細(xì)胞在DDP作用下生長(zhǎng)抑制率的變化

4miR-140調(diào)控表達(dá)對(duì)SGC-7901細(xì)胞周期的影響

流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)miR-140 mimics轉(zhuǎn)染SGC-7901細(xì)胞后能夠阻滯細(xì)胞周期變化，而miR-140 inhibitors轉(zhuǎn)染后能夠促進(jìn)細(xì)胞周期。與control和NC組相比，miR-140 mimics轉(zhuǎn)染組SGC-7901細(xì)胞G0/G1期比例顯著上升，而G2/M期細(xì)胞比例顯著下降(P<0.05)；而miR-140 inhibitors轉(zhuǎn)染組SGC-7901細(xì)胞G0/G1期比例顯著下降，但S期和G2/M期細(xì)胞比例顯著上升(P<0.05)，見(jiàn)圖4。

Figure 4.The changes of SGC-7901 cell cycle detected by flow cytometry. Mean±SD.n=6.*P<0.05vscontrol group and NC group.

圖4miR-140調(diào)控表達(dá)對(duì)各組SGC-7901細(xì)胞周期的影響

5miR-140調(diào)控表達(dá)對(duì)SGC-7901細(xì)胞凋亡的影響

流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)在10 μmol/L順鉑藥物的誘導(dǎo)下，miR-140 mimics轉(zhuǎn)染SGC-7901細(xì)胞能夠促進(jìn)細(xì)胞凋亡，而miR-140 inhibitors轉(zhuǎn)染能夠抑制細(xì)胞凋亡。經(jīng)過(guò)統(tǒng)計(jì)，與control和NC組相比，miR-140 mimics轉(zhuǎn)染組SGC-7901細(xì)胞凋亡率顯著上升，而miR-140 inhibitors轉(zhuǎn)染組細(xì)胞凋亡率顯著下降，差異均有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，見(jiàn)圖5。

6miR-140調(diào)控表達(dá)對(duì)SGC-7901細(xì)胞侵襲能力的影響

Transwell實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn)miR-140 mimics轉(zhuǎn)染SGC-7901細(xì)胞能夠抑制細(xì)胞侵襲能力，而miR-140 inhibitors轉(zhuǎn)染能夠促進(jìn)細(xì)胞侵襲能力。與control和NC組相比，miR-140 mimics轉(zhuǎn)染組SGC-7901細(xì)胞侵襲率顯著下降，而miR-140 inhibitors轉(zhuǎn)染組細(xì)胞侵襲率顯著上升，差異均有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，見(jiàn)圖6。

7miR-140調(diào)控表達(dá)對(duì)SGC-7901細(xì)胞中HDAC4蛋白表達(dá)的影響

Western blot檢測(cè)發(fā)現(xiàn)miR-140調(diào)控表達(dá)能夠調(diào)節(jié)SGC-7901細(xì)胞中HDAC4蛋白表達(dá)。 與control和NC組相比，miR-140 mimics轉(zhuǎn)染組SGC-7901細(xì)胞中HDAC4蛋白相對(duì)表達(dá)量顯著下降，而miR-140 inhibitors轉(zhuǎn)染組細(xì)胞HDAC4蛋白相對(duì)表達(dá)量顯著上升，差異均有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，見(jiàn)圖7。

討　　論

目前，miRNA和腫瘤的相關(guān)性受到廣泛的關(guān)注，如miR-15a、miR-16-1、miR-34a等多種miRNA可能成為腫瘤治療的新靶點(diǎn)[8-9]。miR-140是一種在軟骨增生和發(fā)育中發(fā)揮重要作用的微小RNA，與腫瘤細(xì)胞增殖、侵襲等活動(dòng)也相關(guān)[10]。Iorio等[11]應(yīng)用microRNA芯片技術(shù)篩選上皮源性卵巢癌與正常卵巢組織之間的差異表達(dá)miRNA，其中發(fā)現(xiàn)miR-140在卵巢癌中的表達(dá)水平顯著降低。但另一項(xiàng)研究應(yīng)用實(shí)時(shí)熒光定量PCR比較了4例原發(fā)性II級(jí)神經(jīng)膠質(zhì)瘤與自發(fā)進(jìn)展的繼發(fā)性IV級(jí)惡性膠質(zhì)瘤之間的microRNA表達(dá)譜，發(fā)現(xiàn)miR-140在神經(jīng)膠質(zhì)瘤由II級(jí)到IV級(jí)的進(jìn)展過(guò)程中表達(dá)增高[7]。因此，miR-140在不同腫瘤病理發(fā)生或不同進(jìn)展階段中可能發(fā)揮不同的調(diào)節(jié)作用。本研究取30例臨床胃癌和正常胃組織標(biāo)本檢測(cè)發(fā)現(xiàn)總體上miR-140在胃癌組織中表達(dá)水平低于正常胃組織，并且與腫瘤大小、侵襲度、淋巴轉(zhuǎn)移分期相關(guān)，胃癌腫瘤生長(zhǎng)越大，侵襲度、淋巴轉(zhuǎn)移分期越高，miR-140相對(duì)表達(dá)水平越低。因此，本研究結(jié)果與上述Iorio等在其它腫瘤組織中的發(fā)現(xiàn)一致，并提示miR-140可能作為抑癌因子在人胃癌侵襲和轉(zhuǎn)移等細(xì)胞功能中發(fā)揮作用。

Figure 5.The changes of SGC-7901 cell apoptotic rate detected by flow cytometry. Mean±SD.n=6.*P<0.05vscontrol group and NC group.

圖5miR-140調(diào)控表達(dá)對(duì)各組SGC-7901細(xì)胞凋亡率的影響

Figure 6.The changes of SGC-7901 cell invasion rate detected by Transwell assay (×200). Mean±SD.n=6.*P<0.05vscontrol group and NC group.

圖6miR-140調(diào)控表達(dá)對(duì)各組SGC-7901細(xì)胞侵襲率的影響

Figure 7.The changes of HDAC4 protein expression in SGC-7901 cells detected by Western blot. Mean±SD.n=6.*P<0.05vscontrol and NC group.

圖7miR-140調(diào)控表達(dá)對(duì)各組SGC-7901細(xì)胞中HDAC4蛋白表達(dá)的影響

研究表明，miRNA的異常表達(dá)與腫瘤增殖活性、藥物敏感性和侵襲轉(zhuǎn)移行為等細(xì)胞功能密切相關(guān)，如miR-21過(guò)表達(dá)能夠促進(jìn)大腸癌、胃癌、肝癌和胰腺癌等腫瘤侵襲和轉(zhuǎn)移，而miR-34a過(guò)表達(dá)則抑制膠質(zhì)瘤、乳腺癌等腫瘤增殖、侵襲，并增強(qiáng)藥物敏感性[12-13]。為進(jìn)一步驗(yàn)證miR-140在胃癌細(xì)胞功能中發(fā)揮的作用，選擇人胃癌細(xì)胞株SGC-7901為研究對(duì)象將miR-140模擬物(miR-140 mimics)和miR-140抑制物(miR-140 inhibitor)分別轉(zhuǎn)染SGC-7901細(xì)胞，real-time PCR檢測(cè)發(fā)現(xiàn)分別可以顯著上調(diào)和下調(diào)miR-140表達(dá)。同時(shí)，本研究也發(fā)現(xiàn)miR-140 mimics轉(zhuǎn)染組促進(jìn)miR-140表達(dá)水平后，SGC-7901細(xì)胞活力下降，對(duì)順鉑敏感性增強(qiáng)，細(xì)胞周期被阻滯在G0/G1期，細(xì)胞凋亡率顯著上升，而侵襲能力下降；但miR-140 inhibitor轉(zhuǎn)染組下調(diào)miR-140表達(dá)水平后SGC-7901各細(xì)胞作用與之相反，證明了miR-140在人胃癌細(xì)胞功能中發(fā)揮抑癌作用。本研究結(jié)果與Yang等[14]報(bào)道m(xù)iR-140過(guò)表達(dá)后抑制肝癌、非小細(xì)胞肺癌的增殖和侵襲轉(zhuǎn)移水平結(jié)果相一致。

Tuddenham等[10]在小鼠3T3細(xì)胞中，應(yīng)用螢光素酶報(bào)告系統(tǒng)證實(shí)了HDAC4是miR-140的靶基因，miR-140通過(guò)抑制HDAC4表達(dá)來(lái)促進(jìn)軟骨細(xì)胞的發(fā)育。有研究表明，HDAC4在乳腺癌和子宮內(nèi)膜癌中高表達(dá)，能夠作為促癌基因調(diào)控相關(guān)腫瘤增殖凋亡和順鉑耐藥性[15]。為揭示miR-140在人胃癌細(xì)胞中發(fā)揮作用是否通過(guò)調(diào)控HDAC4靶基因及其調(diào)控關(guān)系，本研究利用Western blot直接檢測(cè)miR-140轉(zhuǎn)染各組中HDAC4蛋白的表達(dá)，發(fā)現(xiàn)對(duì)照組和NC組中HDAC4蛋白呈高表達(dá)水平，而miR-140 mimics轉(zhuǎn)染組中HDAC4蛋白表達(dá)顯著降低，相應(yīng)miR-140 inhibitor轉(zhuǎn)染組中HDAC4蛋白表達(dá)顯著上升。結(jié)合上述研究說(shuō)明miR-140可通過(guò)負(fù)調(diào)控HDAC4發(fā)揮抑制胃癌細(xì)胞增殖、侵襲并促進(jìn)凋亡的作用。

綜上所述，本研究結(jié)果首先證明miR-140是調(diào)控胃癌細(xì)胞增殖、凋亡、侵襲等功能的關(guān)鍵因子之一，為其作為臨床胃癌治療的新靶點(diǎn)提供了依據(jù)。

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[5]Liang ZJ, Zhuang H, Wang GX, et al. MiRNA-140 is a negative feedback regulator of MMP-13 in IL-1β-stimulated human articular chondrocyte C28/I2 cells[J]. Inflamm Res, 2012, 61(5):503-509.

[6]Yuan Y, Shen Y, Xue L, et al. miR-140 suppresses tumor growth and metastasis of non-small cell lung cancer by targeting insulin-like growth factor 1 receptor[J]. PLoS One, 2013, 8(9):e73604.

[7]Malzkorn B, Wolter M, Liesenberg F, et al. Identification and functional characterization of microRNAs involved in the malignant progression of gliomas[J]. Brain Pathol, 2010, 20(3):539-550.

[8]邱瑜，黃建平，周勤仙，等. Hsa-miR-218 靶向調(diào)控LASP1 對(duì)宮頸癌 HeLa 細(xì)胞生長(zhǎng)的影響[J]. 中國(guó)病理生理雜志, 2015, 31(9):1572-1577.

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(責(zé)任編輯： 林白霜， 羅森)

Expression of miR-140 in human gastric cancer and its effect on function of SGC-7901 cells

WANG Xian-yan1, GAO Feng2, ZHAO Chun-ming1, SUN Yu-rong1, WEN Qiu-ting1, YU Xiu-wen1, ZHANG Xiao-jie1

(1SchoolofPathology,2DepartmentofAnesthesiology,TheThirdAffiliatedHospital,QiqiharMedicalUniversity,Qiqihar161006,China.E-mail:wxygf1976@sina.com)

AIM: To explore the expression level of microRNA-140 (miR-140) in human gastric cancer and normal gastric tissues, and the regulatory effect of miR-140 expression on the function of SGC-7901 cells. METHODS: The expression levels of miR-140 in human gastric cancer and normal gastric tissues were detected by real-time PCR. miR-140 mimics (miR-140 up-regulated expression) and miR-140 inhibitors (miR-140 down-regulated expression) were transfected into human gastric cancer SGC-7901 cells by liposome method. At the same time, the untransfected control group (control group) and miRNA nonsense sequence transfection group (NC group) were set up . The expression of miR-140 in the cells after transfection was detected by real-time PCR. The cell viability and growth inhibition rate with DDP were measured by MTT assay. The cell cycle and apoptotic rate of SGC-7901 cells were analyzed by flow cytometry. The invasion ability of SGC-7901 cells was measured by Transwell assay. The protein expression of histone deacetylase 4(HDAC4) in the cells was determined by Western blot. RESULTS: The expression level of miR-140 in human gastric cancer tissues was significantly lower than that in normal gastric tissues (P<0.05). Compared with control group and NC group, the viability and invasion ability of the SGC-7901 cells were decreased, the cell cycle was arrested, the cell growth inhibition rate and apoptotic rate with DDP treatment were increased, and the protein expression of HDAC4 was down-regulated (P<0.05) in miR-140 mimics group. However, in miR-140 inhibitors group, the viability and invasion ability of the SGC-7901 cells were increased, the cell cycle was promoted, the cell growth inhibition rate and apoptotic rate with DDP treatment were decreased, and the protein expression of HDAC4 was up-regulated (P<0.05). CONCLUSION: The expression level of miR-140 in the gastric cancer tissues is low. miR-140 serves as a tumor suppressor to regulate the viability, apoptosis and invasion ability of gastric cancer cells, and to play a role by down-regulating HDAC4 protein. miR-140 may serve as a new target for diagnosis and treatment of gastric cancer.

Gastric cancer; MicroRNA-140; SGC-7901 cells; Cell viability; Apoptosis; Invasion; Histone deacetylase 4

1000- 4718(2016)04- 0651- 07

2015- 12- 07

2016- 02- 04

黑龍江省教育廳科學(xué)技術(shù)研究面上項(xiàng)目(No. 12531804)

Tel: 0452-2663483; E-mail: wxygf1976@sina.com

R730.23

A

10.3969/j.issn.1000- 4718.2016.04.012

雜志網(wǎng)址： http://www.cjpp.net