miRNA-519d對卵巢癌的生長抑制作用及其對XIAP表達的影響

楊 婷 張衛霞

miRNA-519d對卵巢癌的生長抑制作用及其對XIAP表達的影響

楊 婷 張衛霞

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

對人卵巢癌細胞系OVCAR3瞬時轉染miR-519d mimic(實驗組)和NC(陰性對照組)，不進行任何轉染為空白對照組，轉染48 h后進行MTT實驗、流式細胞實驗、Transwell侵襲實驗，觀察卵巢癌細胞生長抑制狀況，WB實驗測定XIAP的表達變化。結果定量Real-time PCR檢測顯示：miR-519d的表達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OVCAR3細胞內miR-519d水平后，細胞增殖抑制率為(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)%，對比差異有統計學意義(P<0.05)。流式細胞術檢測顯示實驗組OVCAR3細胞凋亡率為(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)%，對比差異有統計學意義(P<0.05)。Transwell侵襲實驗顯示:轉染miR-519dmimics后實驗組、陰性對照組、空白對照組的三組的細胞轉移能力對比無明顯差異(P>0.05)。Western實驗結果顯示：處理后實驗組的XIAP蛋白相對表達量為(0.98±0.34)，陰性對照組為(7.54±1.22)，空白對照組為(7.67±1.32)，對比差異有統計學意義(P<0.05)。結論增強miR-519d的表達能對卵巢癌細胞的增殖起抑制作用，并促進其凋亡，而對卵巢癌細胞的轉移沒有影響，能抑制XIAP的表達，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；細胞增殖；細胞侵襲；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、轉移、擴散是死亡的主要原因[1-2]。且卵巢癌發病隱匿，大于2/3的患者出現癥狀時已經屬于晚期，為此對于治療的要求比較高[3]。在傳統治療中，腫瘤細胞減滅術和以鉑類化療藥物為主的聯合化療應用比較多，但易復發，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’非翻譯區或編碼區結合，促進目標mRNA降解或抑制蛋白翻譯，調控靶基因的表達[5]。miRNA在腫瘤中的表達與其他腫瘤相似，卵巢癌也有其特異的miRNA表達譜，可調節卵巢癌的生長、侵襲和轉移[6-7]。MicroRNA-519d(miR-519d)是在人類組織或細胞中發現較早、廣泛存在的miRNAs之一[8-9]，但是在卵巢癌的應用報道還比較少見。XIAP連鎖凋亡抑制蛋白作為凋亡抑制蛋白(IAPs)家族的一個重要成員，在多數腫瘤中呈現高表達狀況[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的表達影響，現報告如下。

1 材料與方法

1.1 實驗材料

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

1.2 細胞培養

待OVCAR3細胞密度至80%～90%時，用0.25%的胰酶與0.02%EDTA進行消化傳代，繼續培養，取對數生長期的腫瘤細胞進行實驗。

1.3 瞬時轉染miR-519d

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

1.4 MTT實驗

取轉染后48 h的細胞，調整實驗組、陰性對照組、空白對照組細胞密度為2×104個/ml，接種于96孔板，接種后48 h進行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，計算細胞存活率與細胞增殖抑制率。

1.5 流式細胞實驗

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

1.6 Transwell侵襲實驗

取轉染后48 h的細胞，調整實驗組、陰性對照組、空白對照組細胞密度為3×105個/ml，取細胞懸液200 μl加入Transwell小室上室。24孔板下室加入550 μl含10%FBS的培養基進行培養，37 ℃孵育36 h，PBS洗細胞3次，3.7%的甲醛固定10 min，結晶紫染色5 min，顯微鏡下計算每個視野的平均細胞數量。

1.7 XIAP的基因及蛋白表達

采用RIPA裂解液提取細胞總蛋白，BCA法定量。取100 μg總蛋白行10%SDS-PAGE電泳，將蛋白轉移到PVDF膜上，用5%脫脂奶封閉1h后，與XIAP一抗(1∶500，Abcam)4 ℃孵育過夜；常規進行二抗雜交、洗膜、ECL顯影，實驗以β-actin為內參，計算XIAP蛋白的相對表達量。所有實驗重復3次。

1.8 統計學方法

2 結果

2.1 轉染miR-519d mimics后細胞的miR-152水平過表達

對人卵巢癌細胞系OVCAR3轉染miR-519d mimics 48 h后，定量Real-time PCR檢測，結果發現miR-519d的表達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 轉染miR-519d mimics的人卵巢癌細胞系中miR-519d的相對表達水平

注：與實驗組對比，△為P<0.05。

2.2 轉染miR-519d mimics對卵巢癌細胞增殖的影響

在OVCAR3細胞中轉染miR-519d mimics 48 h后，應用MTT檢測顯示上調OVCAR3細胞內miR-519d水平后，細胞增殖抑制率為(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)%，對比差異有統計學意義(P<0.05)。

2.3 轉染miR-519d mimics對卵巢癌細胞凋亡作用的影響

在卵巢癌細胞中轉染miR-519d mimics 48 h后，流式細胞術檢測顯示實驗組OVCAR3細胞凋亡率為(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)%，對比差異有統計學意義(P<0.05)。

2.4 轉染miR-519d mimic對卵巢癌細胞侵襲的影響

轉染miR-519d mimics后，觀察實驗組、陰性對照組、空白對照組在接種后第48 h細胞的轉移能力，發現細胞轉移能力沒有明顯下降(P>0.05)。

2.5 XIAP表達分析

Western實驗結果顯示：處理后實驗組的XIAP蛋白相對表達量為(0.98±0.34)，陰性對照組為(7.54±1.22)，空白對照組為(7.67±1.32)，對比差異有統計學意義(P<0.05)。

3 討論

現代研究表明腫瘤的發生是1個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胞的惡性轉化所必須[11-12]。miRNA是1類內源性的非編碼的小分子RNA，miRNA在細胞增殖、免疫反應、凋亡、腫瘤發生及侵襲、轉移、血管生成等各個過程中均發揮了重要的調控作用[13-14]。研究證實，microRNA在細胞增殖、分化、凋亡、基因調控及疾病的發生中扮演重要的角色，iR-222能抑制血管內皮細胞的增殖和遷移，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控[15-16]。

研究顯示卵巢癌組織中有35個miRNA的表達與正常對照標本相比有統計學差異，其中31個miRNA呈現低表達[17]。有研究發現了miR-10b參與乳腺癌的侵襲和轉移過程，其表達量的增加可以特異性的增強腫瘤細胞的侵襲轉移能力[18]。miR-519d具有調控細胞增殖的功能，敲除小鼠miR-519d基因簇會引起心臟、肺的發育缺陷，從而導致新生小鼠致死率，但是miR-519d在腫瘤的浸潤和轉移過程中的具體作用機制尚不清楚[19]。因此我們選取卵巢瘤細胞系OVCAR3，通過過表達miR-519d的功能研究其對卵巢癌細胞的增殖及轉移能力的影響。實時定量RT-PCR檢顯示過表達miR-519d后，miR-519d的表達水平隨之升高；MTT實驗結果證明，過表達miR-519d 48 h能影響細胞的活性，對細胞長期的增殖能力有抑制作用，且能促進細胞凋亡。但是miR-519d促進OVCAR3增殖及轉移的靶基因以及調控機制仍需進一步研究。

腫瘤的轉移過程是原發性腫瘤細胞浸潤鄰近組織、進入體循環，然后最終由微小轉移灶增殖成為繼發性腫瘤的過程。盡管已經證明一些miRNA具有癌基因或者抑癌基因的功能，但是miRNA在介導腫瘤轉移的研究還有待研究[20]。有研究發現miR-519d不僅具有癌基因的功能，還與腫瘤的侵襲和轉移功能密切相關[21]。有研究表明磷酸化的XIAP能發揮泛素連接酶的功能，通過直接結合Mdm2，最終導致細胞質中p53蛋白水平的升高，介導Mdm2的快速降解，以此來抑制細胞自噬[21-22]。本研究發現轉染miR-519d mimics后，細胞轉移能力沒有明顯下降(P>0.05)，表明miR-519d對于腫瘤細胞的生長調控作用可能與侵襲及轉移過程無關。Western實驗結果顯示處理后實驗組的XIAP蛋白相對表達量為(0.98±0.34)，陰性對照組為(7.54±1.22)，空白對照組為(7.67±1.32)，對比差異有統計學意義(P<0.05)，表明miR-519d的應用能抑制XIAP的表達。

總之，增加miR-519d的表達能對卵巢癌細胞的增殖起抑制作用，并促進其凋亡，而對卵巢癌細胞的轉移沒有影響，能抑制XIAP的表達，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 西安醫學院第二附屬醫院

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)