張恒 彭輝勇 滿昌峰 徐娟 祁衛(wèi)東 蔣鵬程 范鈺

·論著·

下調miR-10a表達對人胰腺癌細胞AsPC-1遷移和侵襲的影響

張恒 彭輝勇 滿昌峰 徐娟 祁衛(wèi)東 蔣鵬程 范鈺

目的探討miR-10a表達對人胰腺癌AsPC-1細胞遷移和侵襲力的影響及其作用機制。方法構建針對miR-10a的小干擾RNA(miR-10a-siRNA)，轉染處理人胰腺癌AsPC-1細胞，同時設陰性對照siRNA(NC-siRNA)組和空白對照組。采用熒光實時定量PCR法檢測3組細胞中miR-10a的表達水平，劃痕實驗檢測細胞遷移，Transwell小室檢測細胞侵襲能力，ELISA法檢測各組癌細胞培養(yǎng)上清液中基質金屬蛋白質酶13(MMP-13)含量。結果對照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細胞miR-10a表達水平分別為1.05±0.08、1.03±0.06、0.02±0.01，穿膜細胞數(shù)分別為(150±2.6)、(145±2.2)、(62±1.8)個，培養(yǎng)上清中MMP-13表達量分別為(108.5±2.8)、(107.8±2.5)、(35.8±1.5)pg/ml，miR-10a-siRNA組均顯著低于對照組和NC-siRNA組，差異均有統(tǒng)計學意義(P值均<0.01)。 對照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細胞的遷移后間距分別為(385±15)、(395±13) 、(736±18) μm，miR-10a-siRNA組顯著大于對照組和NC-siRNA組，差異有統(tǒng)計學意義(P值均<0.01)。結論下調miR-10a表達可抑制胰腺癌AsPC-1細胞遷移和侵襲能力，其機制可能與MMP-13表達下調有關。

胰腺腫瘤； 微RNA； 細胞運動； 腫瘤浸潤； 基質金屬蛋白質酶-13； miR-10a

MicroRNAs(miRNAs)的異常表達與多種人類癌癥的發(fā)生密切相關[1-8]。近年的研究發(fā)現(xiàn)，miR-10a在人胃癌、甲狀腺癌中高表達[9]。Ohuchida等[10]報道，miR-10a在胰腺癌中高表達，并通過抑制HOXA1基因的表達參與細胞的浸潤。本研究采用小干擾RNA(small interfering RNA,siRNA)沉默人胰腺癌AsPC-1細胞miR-10a的表達，觀察其對細胞遷移、侵襲的影響，探討其機制。

材料與方法

一、細胞培養(yǎng)及siRNA轉染

人胰腺癌AsPC-1細胞系購自南京凱基生物科技發(fā)展有限公司，常規(guī)培養(yǎng)、傳代。靶向miR-10a的siRNA(miR-10a-siRNA)及陰性對照siRNA(NC-siRNA)購自廣州銳博公司。轉染前一天接種適量細胞至細胞培養(yǎng)板中，每孔加入不含抗生素的培養(yǎng)基。細胞密度達到30%～50%時用50 μl不含血清的培養(yǎng)基Opti-MEM分別稀釋1.25 μl 20 μmol/L的siRNA貯存液及1 μl脂質體，輕輕混勻，室溫孵育5 min后將稀釋的siRNA和脂質體輕輕混勻，室溫孵育20 min，加入到細胞的培養(yǎng)液中輕輕混勻。培養(yǎng)4～6 h，更換新鮮培養(yǎng)基，置37℃的CO2培養(yǎng)箱中繼續(xù)培養(yǎng)48 h。以未轉染的AsPC-1細胞作為對照。

二、轉染效果鑒定

采用Trizol方法抽提兩組轉染細胞及對照組細胞總RNA，先逆轉錄為cDNA，再采用熒光實時定量PCR方法檢測細胞miR-10a表達。參照文獻[11]方法設計miR-10a引物、PCR 反應條件及計算miR-10a表達量。

三、細胞遷移實驗

先用記號筆在6孔板背后用直尺均勻地劃橫線，大約每隔0.5～1 cm一道，橫穿過孔。取各組對數(shù)生長期細胞，調整細胞密度為8×105個/ml。每孔加入1 ml細胞懸液，常規(guī)培養(yǎng)至細胞鋪滿單層。用100 μl槍頭沿培養(yǎng)板底部呈“一”字形劃痕。棄去培養(yǎng)液，用PBS洗細胞3次以去除劃下的細胞后繼續(xù)培養(yǎng)24 h，拍照，計算細胞未爬入的間距。

四、細胞侵襲實驗

參照文獻[12]，應用Transwell小室檢測細胞侵襲力。最后在倒置顯微鏡(×100)下觀察穿膜的細胞數(shù)。每張膜中央部分和周圍部分各隨機取3個視野，計數(shù)每個視野內的穿膜細胞數(shù)，取均值。

五、細胞基質金屬蛋白酶13(MMP-13)表達量測定

收集各組細胞培養(yǎng)上清液，采用MMP-13 ELISA試劑盒測定MMP-13含量，按說明書操作。

六、統(tǒng)計學處理

結 果

一、細胞miR-10a表達水平的變化

對照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細胞miR-10a表達量分別為1.05±0.08、1.03±0.06、0.02±0.01(圖1)，miR-10a-siRNA組顯著低于對照組和NC-siRNA組，差異有統(tǒng)計學意義(F=983.280，P=0.000)。

圖1 實時PCR的擴增曲線(a)和溶解曲線(b)

二、細胞遷移力的變化

對照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細胞遷移后的間距分別為(385±15)、(395±13)、(736±18) μm(圖2)。miR-10a-siRNA組細胞遷移后的間距顯著大于對照組和NC-siRNA組，差異有統(tǒng)計學意義(F=883.118，P=0.000)。

圖2對照組(a)、NC-siRNA組(b)、miR-10a-siRNA組(c)AsPC-1細胞的遷移間距

三、細胞侵襲力的變化

對照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細胞的穿膜細胞數(shù)分別為(150±2.6)、(145±2.2)、(62±1.8)個(圖3)，miR-10a-siRNA組明顯少于對照組和NC-siRNA組，差異有統(tǒng)計學意義(F=523.500，P=0.000)。

圖3對照組(a)、NC-siRNA組(b)、miR-10a-siRNA組(c)的穿膜細胞

四、細胞MMP-13的表達量

對照組、NC-siRNA組和miR-10a-siRNA組AsPC-1細胞的培養(yǎng)上清中MMP-13表達量分別為(108.5±2.8)、(107.8±2.5)、(35.8±1.5)pg/ml，miR-10a-siRNA組顯著低于對照組和NC-siRNA組(F=463.853，P=0.000)。

討 論

大量證據表明，miRNAs的異常表達與多種人類癌癥的發(fā)生密切相關[1-8]。MiRNAs的發(fā)現(xiàn)和應用為胰腺癌等惡性腫瘤的診斷和治療提供了新的靶點。

胰腺癌細胞轉移是一個復雜的過程，遷移和侵襲是重要步驟，也是胰腺癌治療失敗的主要原因之一。積極尋找與胰腺癌侵襲有關的分子機制無疑會有助于胰腺癌的綜合診療水平。實驗研究表明，在許多實體瘤如胃癌、甲狀腺癌等瘤組織或細胞中均高表達miRNAs[10]，但目前尚不清楚miR-10a在胰腺癌細胞侵襲中的可能作用。

本研究結果顯示，與對照組和NC-siRNA組比較，miR-10a組AsPC-1細胞的遷移和侵襲能力均顯著減弱，表明miR-10a參與細胞的遷移及侵襲。

腫瘤的浸潤與轉移的關鍵是細胞外基質(extracellular matrix, ECM)成分的降解。MMPs能降解ECM。按照底物特異性，人類MMP主要分為明膠酶、間質膠原酶、基質溶解素、機制溶解因子、模型機制金屬蛋白質酶及其他等6類。MMP主要功能是可降解一種或多種ECM成分，調控血管形成，又可通過與整合素的相互活化而加強細胞間的粘附作用。MMP-13是MMPs重要成員之一，它在人許多惡性腫瘤包括人胰腺腺癌細胞中高表達[12-15]，且與人胰腺癌侵襲有關[16]。本研究結果顯示， miR-10a組細胞MMP-13表達量顯著低于對照組和NC-siRNA組，提示下調miR-10a表達后可能通過抑制MMP-13的表達而抑制AsPCA-1細胞的遷移和侵襲能力。

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EffectsofmiR-10adown-regulatedbysiRNAonmigrationandinvasionofhumanpancreaticcancercellAsPC-1

ZHANGHeng,PENGHui-yong,MANChang-feng,XUJuan,QIWei-dong,JIANGPeng-cheng,FANYu.

CancerInstitute,People′sHospitalofZhenjiang,JiangsuUniversity,Zhenjiang212002,China

FANYu,Email：yuf36@sina.com

ObjectiveTo investigate the effects of miR-10a expression on migration and invasion of human pancreatic cancer cells AsPC-1.MethodsSmall interfering RNA targeting at miR-10a(miR-10a-siRNA) was constructed, then it was transfected into pancreatic cancer AsPC-1 cells, and nonsense siRNA (Nc-siRNA) group and blank control group was established. Real time PCR assay was used to detect the expression of miR-10a in the 3 groups, and wound healing assay and Transwell assay were used to determine the migration and invasion abilities of cancer cells. The amount of matrix metalloproteinase-13 (MMP-13) in supernatant of cancer cell culture of each group was examined by ELISA assay.ResultsThe miR-10a levels in control group, NC-siRNA group and miR-10a-siRNA group were 1.05±0.08, 1.03±0.06, 0.02±0.01; and the number of transmembrane cell were (150±2.6), (145±2.2), (62±1.8), the levels of MMP-13 in the supernatant were (108.5±2.8), (107.8±2.5), (35.8±1.5)pg/ml. The values were significantly lower in miR-10a-siRNA group than those in control group and NC-siRNA group (P<0.01). The distance of cultured clone in miR-10a treated cancer cells (736±18 μm) was significantly longer than those in the controls (385±5 μm) and NC-siRNA group (395±13 μm,P<0.01).ConclusionsDown-regulation of miR-10a by siRNA may inhibit migration and invasion of pancreatic cancer AsPC-1 cells, and the down-regulated expression of MMP-13 may be one of the important mechanisms.

Pancreatic neoplasms; MicroRNAs; Cell movement; Neoplasm invasiveness; Matrix metalloproteinase-13; miR-10a

2013-09-29)

(本文編輯：呂芳萍)

10.3760/cma.j.issn.1674-1935.2013.06.004

江蘇省普通高校研究生科研創(chuàng)新計劃項目(CXLX12_0679)；江蘇大學臨床科技基金(JLY2010007)

212002 鎮(zhèn)江，江蘇大學附屬人民醫(yī)院腫瘤研究所

范鈺，Email：yuf36@sina.com