楊 婷 張衛(wèi)霞

miRNA-519d對卵巢癌的生長抑制作用及其對XIAP表達(dá)的影響

楊 婷 張衛(wèi)霞

目的探討MicroRNA-519d(miRNA-519d)對卵巢癌細(xì)胞生長抑制的作用及其對XIAP表達(dá)的影響。方法

對人卵巢癌細(xì)胞系OVCAR3瞬時(shí)轉(zhuǎn)染miR-519d mimic(實(shí)驗(yàn)組)和NC(陰性對照組)，不進(jìn)行任何轉(zhuǎn)染為空白對照組，轉(zhuǎn)染48 h后進(jìn)行MTT實(shí)驗(yàn)、流式細(xì)胞實(shí)驗(yàn)、Transwell侵襲實(shí)驗(yàn)，觀察卵巢癌細(xì)胞生長抑制狀況，WB實(shí)驗(yàn)測定XIAP的表達(dá)變化。結(jié)果定量Real-time PCR檢測顯示：miR-519d的表達(dá)水平均較對陰性對照組與空白對照組有明顯的增高(P<0.05)。MTT檢測顯示：上調(diào)OVCAR3細(xì)胞內(nèi)miR-519d水平后，細(xì)胞增殖抑制率為(29.81±8.24)%，而陰性照組與空白對照組分別為(2.49±0.28)%和(1.98±0.34)%，對比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。流式細(xì)胞術(shù)檢測顯示實(shí)驗(yàn)組OVCAR3細(xì)胞凋亡率為(8.33±1.39)%，而陰性對照組與空白對照組分別為(2.13±2.11)%和(1.89±1.08)%，對比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。Transwell侵襲實(shí)驗(yàn)顯示:轉(zhuǎn)染miR-519dmimics后實(shí)驗(yàn)組、陰性對照組、空白對照組的三組的細(xì)胞轉(zhuǎn)移能力對比無明顯差異(P>0.05)。Western實(shí)驗(yàn)結(jié)果顯示：處理后實(shí)驗(yàn)組的XIAP蛋白相對表達(dá)量為(0.98±0.34)，陰性對照組為(7.54±1.22)，空白對照組為(7.67±1.32)，對比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論增強(qiáng)miR-519d的表達(dá)能對卵巢癌細(xì)胞的增殖起抑制作用，并促進(jìn)其凋亡，而對卵巢癌細(xì)胞的轉(zhuǎn)移沒有影響，能抑制XIAP的表達(dá)，表明miR-519d在卵巢癌的生長中起到一定的抑制作用。

MicroRNA-519d；卵巢癌；細(xì)胞增殖；細(xì)胞侵襲；XIAP

(ThePracticalJournalofCancer,2017,32:1596～1599)

上皮性卵巢癌(epithelialovariancancer，EOC)是卵巢癌中最常見的類型，浸潤、轉(zhuǎn)移、擴(kuò)散是死亡的主要原因[1-2]。且卵巢癌發(fā)病隱匿，大于2/3的患者出現(xiàn)癥狀時(shí)已經(jīng)屬于晚期，為此對于治療的要求比較高[3]。在傳統(tǒng)治療中，腫瘤細(xì)胞減滅術(shù)和以鉑類化療藥物為主的聯(lián)合化療應(yīng)用比較多，但易復(fù)發(fā)，使得患者5年生存率大大下降[4]。miRNA通過堿基配對與靶mRNA序列的3’非翻譯區(qū)或編碼區(qū)結(jié)合，促進(jìn)目標(biāo)mRNA降解或抑制蛋白翻譯，調(diào)控靶基因的表達(dá)[5]。miRNA在腫瘤中的表達(dá)與其他腫瘤相似，卵巢癌也有其特異的miRNA表達(dá)譜，可調(diào)節(jié)卵巢癌的生長、侵襲和轉(zhuǎn)移[6-7]。MicroRNA-519d(miR-519d)是在人類組織或細(xì)胞中發(fā)現(xiàn)較早、廣泛存在的miRNAs之一[8-9]，但是在卵巢癌的應(yīng)用報(bào)道還比較少見。XIAP連鎖凋亡抑制蛋白作為凋亡抑制蛋白(IAPs)家族的一個(gè)重要成員，在多數(shù)腫瘤中呈現(xiàn)高表達(dá)狀況[10]。本文具體探討了miRNA-519d對卵巢癌的生長抑制作用及其對XIAP的表達(dá)影響，現(xiàn)報(bào)告如下。

1 材料與方法

1.1 實(shí)驗(yàn)材料

人卵巢癌細(xì)胞OVCAR3(上皮性卵巢癌)購于中國科學(xué)院典型培養(yǎng)物保藏委員會(huì)上海細(xì)胞庫，培養(yǎng)體系為RPMI-1640培養(yǎng)基+10%胎牛血清培養(yǎng)條件為37 ℃、5%CO2、飽和濕度培養(yǎng)。

1.2 細(xì)胞培養(yǎng)

待OVCAR3細(xì)胞密度至80%～90%時(shí)，用0.25%的胰酶與0.02%EDTA進(jìn)行消化傳代，繼續(xù)培養(yǎng)，取對數(shù)生長期的腫瘤細(xì)胞進(jìn)行實(shí)驗(yàn)。

1.3 瞬時(shí)轉(zhuǎn)染miR-519d

根據(jù)lipofectamine2000的轉(zhuǎn)染試劑盒的說明書，對人卵巢癌細(xì)胞系OVCAR3瞬時(shí)轉(zhuǎn)染miR-519d mimic(實(shí)驗(yàn)組)和NC(陰性對照組)，不進(jìn)行任何轉(zhuǎn)染為空白對照組，具體步驟如下：將5 μl的miR-519d mimic及陰性對照分別加入250 μl的無血清培養(yǎng)基液中。將5 μl的lipofectamine2000加入到250 μl的無血清培養(yǎng)基液中；將上述液體混勻，室溫放置20 min。各孔加入無血清培養(yǎng)基1 ml，再加入混懸液500 μl進(jìn)行細(xì)胞培養(yǎng)。在轉(zhuǎn)染細(xì)胞的鑒定中，分別從實(shí)驗(yàn)組、陰性對照組、空白對照組細(xì)胞中提取RNA，采用RT-PCR進(jìn)行miR-519d的表達(dá)量的測定。

1.4 MTT實(shí)驗(yàn)

取轉(zhuǎn)染后48 h的細(xì)胞，調(diào)整實(shí)驗(yàn)組、陰性對照組、空白對照組細(xì)胞密度為2×104個(gè)/ml，接種于96孔板，接種后48 h進(jìn)行MTT檢測。每孔加入20 μl的MTT，使其終濃度為0.5 mg/ml。37 ℃孵育4～6 h，570 nm處測定吸光度，計(jì)算細(xì)胞存活率與細(xì)胞增殖抑制率。

1.5 流式細(xì)胞實(shí)驗(yàn)

將細(xì)胞胰酶消化后接種于6孔板中，待細(xì)胞密度至30%時(shí)，轉(zhuǎn)染miR-519d mimics作用48 h。采用100 μl FITC-Annexin與binding buffer懸浮細(xì)胞，每組加Annexin V-FITC 5 μl和PI 5 μl，混勻避光。流式細(xì)胞儀上機(jī)檢測，檢測細(xì)胞凋亡情況。

1.6 Transwell侵襲實(shí)驗(yàn)

取轉(zhuǎn)染后48 h的細(xì)胞，調(diào)整實(shí)驗(yàn)組、陰性對照組、空白對照組細(xì)胞密度為3×105個(gè)/ml，取細(xì)胞懸液200 μl加入Transwell小室上室。24孔板下室加入550 μl含10%FBS的培養(yǎng)基進(jìn)行培養(yǎng)，37 ℃孵育36 h，PBS洗細(xì)胞3次，3.7%的甲醛固定10 min，結(jié)晶紫染色5 min，顯微鏡下計(jì)算每個(gè)視野的平均細(xì)胞數(shù)量。

1.7 XIAP的基因及蛋白表達(dá)

采用RIPA裂解液提取細(xì)胞總蛋白，BCA法定量。取100 μg總蛋白行10%SDS-PAGE電泳，將蛋白轉(zhuǎn)移到PVDF膜上，用5%脫脂奶封閉1h后，與XIAP一抗(1∶500，Abcam)4 ℃孵育過夜；常規(guī)進(jìn)行二抗雜交、洗膜、ECL顯影，實(shí)驗(yàn)以β-actin為內(nèi)參，計(jì)算XIAP蛋白的相對表達(dá)量。所有實(shí)驗(yàn)重復(fù)3次。

1.8 統(tǒng)計(jì)學(xué)方法

2 結(jié)果

2.1 轉(zhuǎn)染miR-519d mimics后細(xì)胞的miR-152水平過表達(dá)

對人卵巢癌細(xì)胞系OVCAR3轉(zhuǎn)染miR-519d mimics 48 h后，定量Real-time PCR檢測，結(jié)果發(fā)現(xiàn)miR-519d的表達(dá)水平均較陰性對照組與空白對照組有明顯的增高(P<0.05)，見表1。

表1 轉(zhuǎn)染miR-519d mimics的人卵巢癌細(xì)胞系中miR-519d的相對表達(dá)水平

注：與實(shí)驗(yàn)組對比，△為P<0.05。

2.2 轉(zhuǎn)染miR-519d mimics對卵巢癌細(xì)胞增殖的影響

在OVCAR3細(xì)胞中轉(zhuǎn)染miR-519d mimics 48 h后，應(yīng)用MTT檢測顯示上調(diào)OVCAR3細(xì)胞內(nèi)miR-519d水平后，細(xì)胞增殖抑制率為(29.81±0.24)%，而陰性對照組與空白對照組分別為(2.49±0.28)%和(1.98±0.34)%，對比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

2.3 轉(zhuǎn)染miR-519d mimics對卵巢癌細(xì)胞凋亡作用的影響

在卵巢癌細(xì)胞中轉(zhuǎn)染miR-519d mimics 48 h后，流式細(xì)胞術(shù)檢測顯示實(shí)驗(yàn)組OVCAR3細(xì)胞凋亡率為(8.33±1.39)%，而陰性對照組與空白對照組分別為(1.13±2.11)%和(1.89±1.08)%，對比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

2.4 轉(zhuǎn)染miR-519d mimic對卵巢癌細(xì)胞侵襲的影響

轉(zhuǎn)染miR-519d mimics后，觀察實(shí)驗(yàn)組、陰性對照組、空白對照組在接種后第48 h細(xì)胞的轉(zhuǎn)移能力，發(fā)現(xiàn)細(xì)胞轉(zhuǎn)移能力沒有明顯下降(P>0.05)。

2.5 XIAP表達(dá)分析

Western實(shí)驗(yàn)結(jié)果顯示：處理后實(shí)驗(yàn)組的XIAP蛋白相對表達(dá)量為(0.98±0.34)，陰性對照組為(7.54±1.22)，空白對照組為(7.67±1.32)，對比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。

3 討論

現(xiàn)代研究表明腫瘤的發(fā)生是1個(gè)涉及多種腫瘤基因功能改變的多步驟過程，腫瘤抑制基因的功能失活為細(xì)胞的惡性轉(zhuǎn)化所必須[11-12]。miRNA是1類內(nèi)源性的非編碼的小分子RNA，miRNA在細(xì)胞增殖、免疫反應(yīng)、凋亡、腫瘤發(fā)生及侵襲、轉(zhuǎn)移、血管生成等各個(gè)過程中均發(fā)揮了重要的調(diào)控作用[13-14]。研究證實(shí)，microRNA在細(xì)胞增殖、分化、凋亡、基因調(diào)控及疾病的發(fā)生中扮演重要的角色，iR-222能抑制血管內(nèi)皮細(xì)胞的增殖和遷移，miR-23、miR-27b和miR-130a能促血管生成，miR-155、miR-21和miR-126參與血管炎癥的調(diào)控[15-16]。

研究顯示卵巢癌組織中有35個(gè)miRNA的表達(dá)與正常對照標(biāo)本相比有統(tǒng)計(jì)學(xué)差異，其中31個(gè)miRNA呈現(xiàn)低表達(dá)[17]。有研究發(fā)現(xiàn)了miR-10b參與乳腺癌的侵襲和轉(zhuǎn)移過程，其表達(dá)量的增加可以特異性的增強(qiáng)腫瘤細(xì)胞的侵襲轉(zhuǎn)移能力[18]。miR-519d具有調(diào)控細(xì)胞增殖的功能，敲除小鼠miR-519d基因簇會(huì)引起心臟、肺的發(fā)育缺陷，從而導(dǎo)致新生小鼠致死率，但是miR-519d在腫瘤的浸潤和轉(zhuǎn)移過程中的具體作用機(jī)制尚不清楚[19]。因此我們選取卵巢瘤細(xì)胞系OVCAR3，通過過表達(dá)miR-519d的功能研究其對卵巢癌細(xì)胞的增殖及轉(zhuǎn)移能力的影響。實(shí)時(shí)定量RT-PCR檢顯示過表達(dá)miR-519d后，miR-519d的表達(dá)水平隨之升高；MTT實(shí)驗(yàn)結(jié)果證明，過表達(dá)miR-519d 48 h能影響細(xì)胞的活性，對細(xì)胞長期的增殖能力有抑制作用，且能促進(jìn)細(xì)胞凋亡。但是miR-519d促進(jìn)OVCAR3增殖及轉(zhuǎn)移的靶基因以及調(diào)控機(jī)制仍需進(jìn)一步研究。

腫瘤的轉(zhuǎn)移過程是原發(fā)性腫瘤細(xì)胞浸潤?quán)徑M織、進(jìn)入體循環(huán)，然后最終由微小轉(zhuǎn)移灶增殖成為繼發(fā)性腫瘤的過程。盡管已經(jīng)證明一些miRNA具有癌基因或者抑癌基因的功能，但是miRNA在介導(dǎo)腫瘤轉(zhuǎn)移的研究還有待研究[20]。有研究發(fā)現(xiàn)miR-519d不僅具有癌基因的功能，還與腫瘤的侵襲和轉(zhuǎn)移功能密切相關(guān)[21]。有研究表明磷酸化的XIAP能發(fā)揮泛素連接酶的功能，通過直接結(jié)合Mdm2，最終導(dǎo)致細(xì)胞質(zhì)中p53蛋白水平的升高，介導(dǎo)Mdm2的快速降解，以此來抑制細(xì)胞自噬[21-22]。本研究發(fā)現(xiàn)轉(zhuǎn)染miR-519d mimics后，細(xì)胞轉(zhuǎn)移能力沒有明顯下降(P>0.05)，表明miR-519d對于腫瘤細(xì)胞的生長調(diào)控作用可能與侵襲及轉(zhuǎn)移過程無關(guān)。Western實(shí)驗(yàn)結(jié)果顯示處理后實(shí)驗(yàn)組的XIAP蛋白相對表達(dá)量為(0.98±0.34)，陰性對照組為(7.54±1.22)，空白對照組為(7.67±1.32)，對比差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，表明miR-519d的應(yīng)用能抑制XIAP的表達(dá)。

總之，增加miR-519d的表達(dá)能對卵巢癌細(xì)胞的增殖起抑制作用，并促進(jìn)其凋亡，而對卵巢癌細(xì)胞的轉(zhuǎn)移沒有影響，能抑制XIAP的表達(dá)，表明miR-519d在卵巢癌的生長中起到一定的抑制作用。

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EffectsofMicroRNA-519dontheGrowthandExpressionofXIAPofOvarianCancerCellLine

YANGTing,ZHANGWeixia.

TheSecondAffiliatedHospitalofXi’anMedicalUniversity,Xi’an,710038

ObjectiveTo study the effects of MicroRNA-519d (miR-519d) on the the growth and expression of XIAP of ovarian cancer cell line.MethodsThe human ovarian cancer cell lineOVCAR3 were transfected to miR-519d mimic (experimental group) and NC (negative control group),no transfection were the blank control group,after transfection of 48 h were given MTT assay,flow cytometry assay and Transwell invasion experiments,observed the growth of ovarian cancer cell.The expression of XIAP was determined by WB assay.Resultsquantitative PCR Real-time test showed that the expression level of miR-519 dwere significantly higher than that of negative group and blank control group (P<0.05).MTT showed that the cell proliferation inhibition rate in the experimental group was (29.81±8.24)%,and the negative control group and blank control group were (2.49±0.28)% and (1.98±0.34)%,compared were statistically significant difference (P<0.05).Flow cytometry showed that the apoptosis rate of experimental group was (8.33±1.39)%,while the negative group and the blank control group were (2.13±2.11)% and (1.89±1.08)% respectively.The Transwell invasion experiment showed that there were no significant difference compared among the three groups(P>0.05).The results of Western showed that the relative expression of XIAP protein in the experimental group was (0.98±0.34),and the negative control group was (7.52±1.22),with a blank control group of (7.67±1.32),and the difference was statistically significant (P<0.05).ConclusionTransient transfection of mimics miR-519d in the ovarian cancer cell line can inhibit the cell growth and promote its apoptosis but has no effect on the metastasis,it can inhibit the expression of XIAP,which indicates that miR-519d might inhibit the proliferation of ovarian cancer cells at the post transcriptional level.

MicroRNA-519d;Ovarian cancer;Cell proliferation;Cell invasion;XIAP

710038 西安醫(yī)學(xué)院第二附屬醫(yī)院

10.3969/j.issn.1001-5930.2017.10.009

R737.31

A

1001-5930(2017)10-1596-04

2017-04-06

2017-06-19)

(編輯吳小紅)