miR-130a-3p下調(diào)CYLD表達(dá)對(duì)乳腺癌MCF-7細(xì)胞功能的影響

李登峰++++魏傳奎++++宋洪明++++李曉宇++++羅頎楓++++陳磊++++黃毅祥++++菅偉++++房林*

摘 要 目的：探討miR-130a-3p對(duì)人乳腺癌MCF-7細(xì)胞功能的影響及其可能的靶向基因。方法：應(yīng)用脂質(zhì)體介導(dǎo)方法將miR-130a-3p模擬物（實(shí)驗(yàn)組）或?qū)φ漳M物（對(duì)照組）轉(zhuǎn)染MCF-7細(xì)胞，分別采用MTT方法和細(xì)胞劃痕實(shí)驗(yàn)檢測細(xì)胞增殖和遷移能力變化，印跡法檢測細(xì)胞中miR-130a-3p靶基因圓柱瘤基因（CYLD）的表達(dá)。結(jié)果：與對(duì)照組比較，實(shí)驗(yàn)組MCF-7細(xì)胞的增殖和遷移能力增強(qiáng)（P＜0.05），細(xì)胞中CYLD蛋白表達(dá)水平明顯下調(diào)（P＜0.05）。結(jié)論：miR-130a-3p可能通過靶向調(diào)節(jié)CYLD蛋白表達(dá)增強(qiáng)人乳腺癌MCF-7細(xì)胞的增殖和遷移。

關(guān)鍵詞乳腺腫瘤圓柱瘤基因miR-130a-3pMCF-7細(xì)胞

中圖分類號(hào)：R737.9文獻(xiàn)標(biāo)識(shí)碼：A文章編號(hào)：1006-1533（2014）12-0003-04

The effect of miR-130a-3p on function of human breast cancer cell line MCF-7 by down-regulating the expression of CYLD

LI Dengfeng, WEI Chuankui, SONG Hongming, LI Xiaoyu, LUO Qifeng, CHEN Lei，HUANG Yixiang, JIAN Wei, FANG Lin

（Department of Breast and Thyroid Surgery, Tenth Peoples Hospital, Tongji University, Shanghai 200072, China）

ABSTRACT Objective: To investigate the effect of miR-130a-3p on the function of human breast cancer cell line MCF-7, and to explore its possible target gene. Methods: miR-130a-3p mimics or its negative control (miR-130a-3p mimics-NC) was transfected into MCF-7 cells by lipofectamine. The capacities of proliferation and migration of MCF-7 cells were evaluated by MTT and Scratch assay, respectively. The expression of CYLD which was predicted as a target gene in MCF-7 cells was determined by Western blotting. Results: The capacities of proliferation and migration in MCF-7 cells transfected with miR-130a-3p mimics were significantly promoted and the expression level of CYLD was down-regulated when compared with those in MCF-7 cells transfected with miR-130a-3p mimics-NC（P＜0.05). Conclusion: The miR-130a-3p expression can promote the proliferation and migration of human breast cancer line MCF-7 by down-regulating the expression of CYLD.

KEY WORDSbreast neoplasms; cylindromatosis; mir-130a-3p;MCF-7 cell

乳腺癌是女性常見的惡性腫瘤，也是女性癌癥死亡的主要原因之一，全球每年死亡病例超過50萬[1]。雖然,近年來隨著乳腺癌篩查工作的開展，早期乳腺癌確診病例比例增加，但晚期死亡率并無明顯下降，因此，尋找乳腺癌特異標(biāo)志物對(duì)臨床診斷意義重大。已有研究提出，將微小RNA（miRNA）作為腫瘤分子標(biāo)志，可用于輔助癌癥的診斷和預(yù)后評(píng)估[2-3]。目前，已有研究顯示miR-130a-3p在肝胰島素敏感性和肝脂肪變中發(fā)揮作用[4]，并且在尿道膀胱癌中與腫瘤的發(fā)生和發(fā)展相關(guān)[5]。然而，miR-130a-3p在乳腺癌的發(fā)生、發(fā)展、侵襲和轉(zhuǎn)移中的作用及機(jī)制仍不明確。

圓柱瘤基因(CYLD)編碼蛋白是一種去泛素化酶，其缺陷或缺失主要導(dǎo)致頭部或面部皮膚腫瘤,如家族性圓柱瘤[6]，同時(shí)，發(fā)現(xiàn)CYLD 的表達(dá)在宮頸癌、結(jié)腸癌及黑色素肉瘤顯著下調(diào)[7]。最新研究發(fā)現(xiàn)， CYLD 在乳腺癌中表達(dá)也存在下調(diào),其異位表達(dá)顯著抑制乳腺癌相關(guān)細(xì)胞生長, 提示 CYLD 基因是腫瘤抑制性基因[8]。

本研究采用TargetScan等miRNA靶基因在線預(yù)測軟件預(yù)測miR-130a-3p與CYLD的可能結(jié)合，通過脂質(zhì)體介導(dǎo)將miRNA-130a-3p模擬物轉(zhuǎn)染人乳腺癌MCF-7細(xì)胞，檢測其對(duì)MCF-7細(xì)胞增殖、轉(zhuǎn)移的影響，以及CYLD蛋白的表達(dá)情況，探討其在人乳腺癌中可能參與的調(diào)節(jié)機(jī)制。

材料與方法

細(xì)胞培養(yǎng)

人乳腺癌MCF-7細(xì)胞購自中國科學(xué)院上海生命科學(xué)研究院細(xì)胞資源中心。MCF-7細(xì)胞加入含10% FBS、1%青-鏈霉素（10 000 U/ml青霉素加10 000μg/ml鏈霉素）的DMEM/高糖培養(yǎng)液，置于37 ℃、CO2體積分?jǐn)?shù)為5%的培養(yǎng)箱中培養(yǎng)。在倒置光學(xué)顯微鏡下觀察細(xì)胞生長情況，0.25%胰蛋白酶消化傳代，取對(duì)數(shù)生長期的細(xì)胞用于后續(xù)實(shí)驗(yàn)。

MiR-130a-3p 模擬物轉(zhuǎn)染乳腺癌細(xì)胞

miR-130a-3p模擬物（miR-130a-3p mimics）和陰性對(duì)照模擬物購自上海吉瑪制藥技術(shù)有限公司。

在培養(yǎng)瓶中常規(guī)培養(yǎng)MCF-7細(xì)胞，待細(xì)胞密度為80%～90%時(shí)，消化收集細(xì)胞；將細(xì)胞接種于6孔板中，2.5×105個(gè)細(xì)胞/孔，細(xì)胞培養(yǎng)至單層密度達(dá)40%～50%時(shí)，分別將終濃度為100 nmol/L的miR-130a-3p 模擬物（實(shí)驗(yàn)組）或陰性對(duì)照模擬物（對(duì)照組）轉(zhuǎn)染至MCF-7細(xì)胞。

MTT法檢測細(xì)胞增殖能力

將兩組細(xì)胞培養(yǎng)24 h，消化、收集細(xì)胞，以2 000/孔均勻接種于96孔板。按24、48、72、96 h時(shí)間段進(jìn)行如下操作：加入20 μl/孔MTT溶液（5 mg/L），繼續(xù)培養(yǎng)4 h，棄上清液，加入150 μl/孔DMSO，置于搖床上低速振蕩10 min，使結(jié)晶物充分溶解；在多功能酶聯(lián)免疫檢測儀上測量490 nm波長處各孔吸光度（A）值，繪制細(xì)胞增殖曲線。細(xì)胞增殖抑制率計(jì)算：抑制率＝（對(duì)照組A值－實(shí)驗(yàn)組A值）/對(duì)照組A值×100%。每組設(shè)置6個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次。

細(xì)胞劃痕法檢測細(xì)胞遷移能力

將兩組細(xì)胞培養(yǎng)24 h，消化、收集細(xì)胞，以4×105/孔均勻接種于12孔板（事先12孔板背側(cè)用刀片比直劃出橫線，4條/空直線橫穿過孔），待細(xì)胞培養(yǎng)至單層密度達(dá)80%～90%時(shí)，進(jìn)行劃痕，用200 μl槍頭在12孔中垂直背側(cè)橫線劃成“十”形，1×PBS液洗3次，然后加入含有2%的FBS的DMEM培養(yǎng)基，37 ℃、5%CO2 培養(yǎng)箱中培養(yǎng)。使用倒置相差顯微鏡分別在作用O和48 h時(shí)拍照，觀察兩組細(xì)胞遷移速度。

預(yù)測miR-130a-3p的靶基因

應(yīng)用在線生物信息預(yù)測軟件（TargetScan、Pictar、MicroRNA.org）對(duì)hsa-miR-130a-3p可能作用的靶基因進(jìn)行預(yù)測，選擇的原則是該基因具有物種保守性。預(yù)測結(jié)果顯示，hsa-miR-130a-3p靶向結(jié)合CYLD 3-UTR。

印跡法檢測CYLD蛋白表達(dá)

將兩組細(xì)胞用預(yù)冷的PBS洗滌2次，RIPA裂解液裂解細(xì)胞，BCA法測定蛋白濃度。取30 μg/孔蛋白進(jìn)行10% SDS-PAGE，將電泳分離后的蛋白轉(zhuǎn)移至硝酸纖維素膜；5%脫脂奶粉溶液室溫下封閉1 h，加入1︰1 000稀釋的抗CYLD和β-actin（內(nèi)參照）抗體，4 ℃反應(yīng)過夜；PBST洗膜3次，加入1︰1 000稀釋的二抗鼠抗兔（820 nm）IgG，室溫反應(yīng)30 min；PBST洗膜3次，應(yīng)用Odyssey雙色紅外激光成像系統(tǒng)檢測蛋白條帶，結(jié)果用以目的蛋白條帶的灰度值與內(nèi)參照β-actin蛋白條帶灰度值的比值表示目的蛋白的相對(duì)表達(dá)水平。實(shí)驗(yàn)重復(fù)3次。

統(tǒng)計(jì)學(xué)方法

應(yīng)用SPSS 17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。計(jì)量數(shù)據(jù)以表示，樣本間均數(shù)比較采用獨(dú)立樣本t檢驗(yàn)，MTT檢測結(jié)果應(yīng)用兩因素方差分析。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

MiR-130a-3p抑制乳腺癌細(xì)胞增殖

與對(duì)照組比較，實(shí)驗(yàn)組MCF-7細(xì)胞的增殖促進(jìn)率上升，在一定程度上呈時(shí)間依賴性；在作用時(shí)間為96 h時(shí)，對(duì)MCF-7細(xì)胞的增殖促進(jìn)作用最強(qiáng)，促進(jìn)率為38.2%（圖1）。

MiR-130a-3p對(duì)乳腺癌MCF-7細(xì)胞遷移能力的影響

與對(duì)照組比較，實(shí)驗(yàn)組MCF-7細(xì)胞遷移率有明顯促進(jìn)（圖2）。

MiR-130a-3p抑制乳腺癌細(xì)胞CYLD蛋白的表達(dá)

與對(duì)照組比較，實(shí)驗(yàn)組MCF-7細(xì)胞中CYLD蛋白的表達(dá)水平明顯下調(diào)（t＝4.249，P＜0.05）（圖3）。

討論

近年來，中國乳腺癌的發(fā)病率呈逐年上升趨勢，嚴(yán)重危害女性的健康和生命，腫瘤晚期轉(zhuǎn)移是導(dǎo)致患者死亡的主要原因[9]。 隨著研究的深入，人們對(duì)miRNAs與惡性腫瘤的關(guān)系得到了進(jìn)一步的了解。腫瘤轉(zhuǎn)移的miRNA異常表達(dá)可能是由于腫瘤相關(guān)染色體區(qū)域miRNA增多或缺失[10]。研究表明，腫瘤相關(guān)的miRNA主要受一些癌基因和抑癌基因調(diào)節(jié)，其本身也可調(diào)節(jié)癌基因和抑癌基因[11]。對(duì)基因組中腫瘤相關(guān)基因區(qū)域的序列分析發(fā)現(xiàn)，約52.5%的miRNAs編碼基因位于其附近，提示miRNAs對(duì)腫瘤相關(guān)基因具有調(diào)控作用[12]?，F(xiàn)已證實(shí)，miRNAs可導(dǎo)致靶基因降解或抑制其翻譯，參與腫瘤的發(fā)生、發(fā)展、浸潤和轉(zhuǎn)移，甚至耐藥性的形成，對(duì)于miRNA靶基因的確定將為人類認(rèn)識(shí)和攻克腫瘤提供廣闊的前景[13]。

本研究證實(shí)miR-130a-3p對(duì)人乳腺癌細(xì)胞的增殖和轉(zhuǎn)移有促進(jìn)作用。應(yīng)用生物信息學(xué)軟件預(yù)測發(fā)現(xiàn)，miR-130a-3p有超過100個(gè)靶基因，在眾多靶基因中選擇與腫瘤細(xì)胞周期有明確相關(guān)性的CYLD進(jìn)行后續(xù)實(shí)驗(yàn)。CYLD作為一種去泛素酶，通過負(fù)向調(diào)控NF-κB活性，從而達(dá)到抑制腫瘤細(xì)胞增殖和遷移的作用[14]。已有研究報(bào)道，CYLD在乳腺癌中，通過負(fù)向調(diào)節(jié)RANKL-RANK介導(dǎo)的NF-κB活性，發(fā)揮抑癌基因的作用，通過相關(guān)樣本分析，CYLD與臨床乳腺癌無病存活率（disease-free survival, DFS)具有顯著相關(guān)性。該研究發(fā)現(xiàn)，在ER陰性、PgR陰性或三陰性的乳腺癌組織中，CYLD陰性表達(dá)比例更高， CYLD陰性表達(dá)與ki-67高表達(dá)和細(xì)胞多核情況存在顯著相關(guān)性。同時(shí)，該研究通過Kaplan–Meier 分析發(fā)現(xiàn)CYLD低表達(dá)通常導(dǎo)致不良DFS[8]。本實(shí)驗(yàn)結(jié)果表明，實(shí)驗(yàn)組乳腺癌細(xì)胞中CYLD蛋白的表達(dá)水平明顯下調(diào),提示了miR-130a-3p可能通過下調(diào)CYLD蛋白的表達(dá)水平，發(fā)揮促進(jìn)MCF-7細(xì)胞的增殖和遷移作用。本研究結(jié)果還需得到后續(xù)實(shí)驗(yàn)的進(jìn)一步證實(shí)。若明確miR-130a-3p在乳腺癌中的調(diào)控作用，可以為今后乳腺癌的治療提供新的思路。

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（收稿日期：2014-05-07）*作者簡介：房林，主任醫(yī)師，教授，甲狀腺乳腺科主任，大外科副主任，外科總論教研室主任，博士生導(dǎo)師，醫(yī)學(xué)博士。擅長乳腺癌、纖維瘤、乳腺增生等乳腺疾病及甲狀腺瘤、甲狀腺癌等疾病的診治。miR-130a-3p下調(diào)CYLD表達(dá)對(duì)乳腺癌MCF-7

細(xì)胞功能的影響

李登峰魏傳奎宋洪明李曉宇羅頎楓陳磊黃毅祥菅偉房林*

（同濟(jì)大學(xué)附屬第十人民醫(yī)院甲狀腺乳腺外科上海200072）

摘 要 目的：探討miR-130a-3p對(duì)人乳腺癌MCF-7細(xì)胞功能的影響及其可能的靶向基因。方法：應(yīng)用脂質(zhì)體介導(dǎo)方法將miR-130a-3p模擬物（實(shí)驗(yàn)組）或?qū)φ漳M物（對(duì)照組）轉(zhuǎn)染MCF-7細(xì)胞，分別采用MTT方法和細(xì)胞劃痕實(shí)驗(yàn)檢測細(xì)胞增殖和遷移能力變化，印跡法檢測細(xì)胞中miR-130a-3p靶基因圓柱瘤基因（CYLD）的表達(dá)。結(jié)果：與對(duì)照組比較，實(shí)驗(yàn)組MCF-7細(xì)胞的增殖和遷移能力增強(qiáng)（P＜0.05），細(xì)胞中CYLD蛋白表達(dá)水平明顯下調(diào)（P＜0.05）。結(jié)論：miR-130a-3p可能通過靶向調(diào)節(jié)CYLD蛋白表達(dá)增強(qiáng)人乳腺癌MCF-7細(xì)胞的增殖和遷移。

關(guān)鍵詞乳腺腫瘤圓柱瘤基因miR-130a-3pMCF-7細(xì)胞

中圖分類號(hào)：R737.9文獻(xiàn)標(biāo)識(shí)碼：A文章編號(hào)：1006-1533（2014）12-0003-04

The effect of miR-130a-3p on function of human breast cancer cell line MCF-7 by down-regulating the expression of CYLD

LI Dengfeng, WEI Chuankui, SONG Hongming, LI Xiaoyu, LUO Qifeng, CHEN Lei，HUANG Yixiang, JIAN Wei, FANG Lin

（Department of Breast and Thyroid Surgery, Tenth Peoples Hospital, Tongji University, Shanghai 200072, China）

ABSTRACT Objective: To investigate the effect of miR-130a-3p on the function of human breast cancer cell line MCF-7, and to explore its possible target gene. Methods: miR-130a-3p mimics or its negative control (miR-130a-3p mimics-NC) was transfected into MCF-7 cells by lipofectamine. The capacities of proliferation and migration of MCF-7 cells were evaluated by MTT and Scratch assay, respectively. The expression of CYLD which was predicted as a target gene in MCF-7 cells was determined by Western blotting. Results: The capacities of proliferation and migration in MCF-7 cells transfected with miR-130a-3p mimics were significantly promoted and the expression level of CYLD was down-regulated when compared with those in MCF-7 cells transfected with miR-130a-3p mimics-NC（P＜0.05). Conclusion: The miR-130a-3p expression can promote the proliferation and migration of human breast cancer line MCF-7 by down-regulating the expression of CYLD.

KEY WORDSbreast neoplasms; cylindromatosis; mir-130a-3p;MCF-7 cell

乳腺癌是女性常見的惡性腫瘤，也是女性癌癥死亡的主要原因之一，全球每年死亡病例超過50萬[1]。雖然,近年來隨著乳腺癌篩查工作的開展，早期乳腺癌確診病例比例增加，但晚期死亡率并無明顯下降，因此，尋找乳腺癌特異標(biāo)志物對(duì)臨床診斷意義重大。已有研究提出，將微小RNA（miRNA）作為腫瘤分子標(biāo)志，可用于輔助癌癥的診斷和預(yù)后評(píng)估[2-3]。目前，已有研究顯示miR-130a-3p在肝胰島素敏感性和肝脂肪變中發(fā)揮作用[4]，并且在尿道膀胱癌中與腫瘤的發(fā)生和發(fā)展相關(guān)[5]。然而，miR-130a-3p在乳腺癌的發(fā)生、發(fā)展、侵襲和轉(zhuǎn)移中的作用及機(jī)制仍不明確。

圓柱瘤基因(CYLD)編碼蛋白是一種去泛素化酶，其缺陷或缺失主要導(dǎo)致頭部或面部皮膚腫瘤,如家族性圓柱瘤[6]，同時(shí)，發(fā)現(xiàn)CYLD 的表達(dá)在宮頸癌、結(jié)腸癌及黑色素肉瘤顯著下調(diào)[7]。最新研究發(fā)現(xiàn)， CYLD 在乳腺癌中表達(dá)也存在下調(diào),其異位表達(dá)顯著抑制乳腺癌相關(guān)細(xì)胞生長, 提示 CYLD 基因是腫瘤抑制性基因[8]。

本研究采用TargetScan等miRNA靶基因在線預(yù)測軟件預(yù)測miR-130a-3p與CYLD的可能結(jié)合，通過脂質(zhì)體介導(dǎo)將miRNA-130a-3p模擬物轉(zhuǎn)染人乳腺癌MCF-7細(xì)胞，檢測其對(duì)MCF-7細(xì)胞增殖、轉(zhuǎn)移的影響，以及CYLD蛋白的表達(dá)情況，探討其在人乳腺癌中可能參與的調(diào)節(jié)機(jī)制。

材料與方法

細(xì)胞培養(yǎng)

人乳腺癌MCF-7細(xì)胞購自中國科學(xué)院上海生命科學(xué)研究院細(xì)胞資源中心。MCF-7細(xì)胞加入含10% FBS、1%青-鏈霉素（10 000 U/ml青霉素加10 000μg/ml鏈霉素）的DMEM/高糖培養(yǎng)液，置于37 ℃、CO2體積分?jǐn)?shù)為5%的培養(yǎng)箱中培養(yǎng)。在倒置光學(xué)顯微鏡下觀察細(xì)胞生長情況，0.25%胰蛋白酶消化傳代，取對(duì)數(shù)生長期的細(xì)胞用于后續(xù)實(shí)驗(yàn)。

MiR-130a-3p 模擬物轉(zhuǎn)染乳腺癌細(xì)胞

miR-130a-3p模擬物（miR-130a-3p mimics）和陰性對(duì)照模擬物購自上海吉瑪制藥技術(shù)有限公司。

在培養(yǎng)瓶中常規(guī)培養(yǎng)MCF-7細(xì)胞，待細(xì)胞密度為80%～90%時(shí)，消化收集細(xì)胞；將細(xì)胞接種于6孔板中，2.5×105個(gè)細(xì)胞/孔，細(xì)胞培養(yǎng)至單層密度達(dá)40%～50%時(shí)，分別將終濃度為100 nmol/L的miR-130a-3p 模擬物（實(shí)驗(yàn)組）或陰性對(duì)照模擬物（對(duì)照組）轉(zhuǎn)染至MCF-7細(xì)胞。

MTT法檢測細(xì)胞增殖能力

將兩組細(xì)胞培養(yǎng)24 h，消化、收集細(xì)胞，以2 000/孔均勻接種于96孔板。按24、48、72、96 h時(shí)間段進(jìn)行如下操作：加入20 μl/孔MTT溶液（5 mg/L），繼續(xù)培養(yǎng)4 h，棄上清液，加入150 μl/孔DMSO，置于搖床上低速振蕩10 min，使結(jié)晶物充分溶解；在多功能酶聯(lián)免疫檢測儀上測量490 nm波長處各孔吸光度（A）值，繪制細(xì)胞增殖曲線。細(xì)胞增殖抑制率計(jì)算：抑制率＝（對(duì)照組A值－實(shí)驗(yàn)組A值）/對(duì)照組A值×100%。每組設(shè)置6個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次。

細(xì)胞劃痕法檢測細(xì)胞遷移能力

將兩組細(xì)胞培養(yǎng)24 h，消化、收集細(xì)胞，以4×105/孔均勻接種于12孔板（事先12孔板背側(cè)用刀片比直劃出橫線，4條/空直線橫穿過孔），待細(xì)胞培養(yǎng)至單層密度達(dá)80%～90%時(shí)，進(jìn)行劃痕，用200 μl槍頭在12孔中垂直背側(cè)橫線劃成“十”形，1×PBS液洗3次，然后加入含有2%的FBS的DMEM培養(yǎng)基，37 ℃、5%CO2 培養(yǎng)箱中培養(yǎng)。使用倒置相差顯微鏡分別在作用O和48 h時(shí)拍照，觀察兩組細(xì)胞遷移速度。

預(yù)測miR-130a-3p的靶基因

應(yīng)用在線生物信息預(yù)測軟件（TargetScan、Pictar、MicroRNA.org）對(duì)hsa-miR-130a-3p可能作用的靶基因進(jìn)行預(yù)測，選擇的原則是該基因具有物種保守性。預(yù)測結(jié)果顯示，hsa-miR-130a-3p靶向結(jié)合CYLD 3-UTR。

印跡法檢測CYLD蛋白表達(dá)

將兩組細(xì)胞用預(yù)冷的PBS洗滌2次，RIPA裂解液裂解細(xì)胞，BCA法測定蛋白濃度。取30 μg/孔蛋白進(jìn)行10% SDS-PAGE，將電泳分離后的蛋白轉(zhuǎn)移至硝酸纖維素膜；5%脫脂奶粉溶液室溫下封閉1 h，加入1︰1 000稀釋的抗CYLD和β-actin（內(nèi)參照）抗體，4 ℃反應(yīng)過夜；PBST洗膜3次，加入1︰1 000稀釋的二抗鼠抗兔（820 nm）IgG，室溫反應(yīng)30 min；PBST洗膜3次，應(yīng)用Odyssey雙色紅外激光成像系統(tǒng)檢測蛋白條帶，結(jié)果用以目的蛋白條帶的灰度值與內(nèi)參照β-actin蛋白條帶灰度值的比值表示目的蛋白的相對(duì)表達(dá)水平。實(shí)驗(yàn)重復(fù)3次。

統(tǒng)計(jì)學(xué)方法

應(yīng)用SPSS 17.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。計(jì)量數(shù)據(jù)以表示，樣本間均數(shù)比較采用獨(dú)立樣本t檢驗(yàn)，MTT檢測結(jié)果應(yīng)用兩因素方差分析。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

MiR-130a-3p抑制乳腺癌細(xì)胞增殖

與對(duì)照組比較，實(shí)驗(yàn)組MCF-7細(xì)胞的增殖促進(jìn)率上升，在一定程度上呈時(shí)間依賴性；在作用時(shí)間為96 h時(shí)，對(duì)MCF-7細(xì)胞的增殖促進(jìn)作用最強(qiáng)，促進(jìn)率為38.2%（圖1）。

MiR-130a-3p對(duì)乳腺癌MCF-7細(xì)胞遷移能力的影響

與對(duì)照組比較，實(shí)驗(yàn)組MCF-7細(xì)胞遷移率有明顯促進(jìn)（圖2）。

MiR-130a-3p抑制乳腺癌細(xì)胞CYLD蛋白的表達(dá)

與對(duì)照組比較，實(shí)驗(yàn)組MCF-7細(xì)胞中CYLD蛋白的表達(dá)水平明顯下調(diào)（t＝4.249，P＜0.05）（圖3）。

討論

近年來，中國乳腺癌的發(fā)病率呈逐年上升趨勢，嚴(yán)重危害女性的健康和生命，腫瘤晚期轉(zhuǎn)移是導(dǎo)致患者死亡的主要原因[9]。 隨著研究的深入，人們對(duì)miRNAs與惡性腫瘤的關(guān)系得到了進(jìn)一步的了解。腫瘤轉(zhuǎn)移的miRNA異常表達(dá)可能是由于腫瘤相關(guān)染色體區(qū)域miRNA增多或缺失[10]。研究表明，腫瘤相關(guān)的miRNA主要受一些癌基因和抑癌基因調(diào)節(jié)，其本身也可調(diào)節(jié)癌基因和抑癌基因[11]。對(duì)基因組中腫瘤相關(guān)基因區(qū)域的序列分析發(fā)現(xiàn)，約52.5%的miRNAs編碼基因位于其附近，提示miRNAs對(duì)腫瘤相關(guān)基因具有調(diào)控作用[12]。現(xiàn)已證實(shí)，miRNAs可導(dǎo)致靶基因降解或抑制其翻譯，參與腫瘤的發(fā)生、發(fā)展、浸潤和轉(zhuǎn)移，甚至耐藥性的形成，對(duì)于miRNA靶基因的確定將為人類認(rèn)識(shí)和攻克腫瘤提供廣闊的前景[13]。

本研究證實(shí)miR-130a-3p對(duì)人乳腺癌細(xì)胞的增殖和轉(zhuǎn)移有促進(jìn)作用。應(yīng)用生物信息學(xué)軟件預(yù)測發(fā)現(xiàn)，miR-130a-3p有超過100個(gè)靶基因，在眾多靶基因中選擇與腫瘤細(xì)胞周期有明確相關(guān)性的CYLD進(jìn)行后續(xù)實(shí)驗(yàn)。CYLD作為一種去泛素酶，通過負(fù)向調(diào)控NF-κB活性，從而達(dá)到抑制腫瘤細(xì)胞增殖和遷移的作用[14]。已有研究報(bào)道，CYLD在乳腺癌中，通過負(fù)向調(diào)節(jié)RANKL-RANK介導(dǎo)的NF-κB活性，發(fā)揮抑癌基因的作用，通過相關(guān)樣本分析，CYLD與臨床乳腺癌無病存活率（disease-free survival, DFS)具有顯著相關(guān)性。該研究發(fā)現(xiàn)，在ER陰性、PgR陰性或三陰性的乳腺癌組織中，CYLD陰性表達(dá)比例更高， CYLD陰性表達(dá)與ki-67高表達(dá)和細(xì)胞多核情況存在顯著相關(guān)性。同時(shí)，該研究通過Kaplan–Meier 分析發(fā)現(xiàn)CYLD低表達(dá)通常導(dǎo)致不良DFS[8]。本實(shí)驗(yàn)結(jié)果表明，實(shí)驗(yàn)組乳腺癌細(xì)胞中CYLD蛋白的表達(dá)水平明顯下調(diào),提示了miR-130a-3p可能通過下調(diào)CYLD蛋白的表達(dá)水平，發(fā)揮促進(jìn)MCF-7細(xì)胞的增殖和遷移作用。本研究結(jié)果還需得到后續(xù)實(shí)驗(yàn)的進(jìn)一步證實(shí)。若明確miR-130a-3p在乳腺癌中的調(diào)控作用，可以為今后乳腺癌的治療提供新的思路。

參考文獻(xiàn)

DeSantis C, Ma J, Bryan L, et al. Breast cancer statistics, 2013[J]. CA Cancer J Clin, 2014, 64(1): 52-62.

Li X, Wang Q, Zheng Y, et al. Prioritizing human cancer microRNAs based on genes functional consistency between microRNA and cancer[J]. Nucleic Acids Res, 2011, 39(22): e153.

Liu J, Zheng M, Tang YL, et al. MicroRNAs, an active and versatile group in cancers[J]. Int J Oral Sci, 2011, 3(4): 165-175.

Xiao F, Yu J, Liu B, et al. A novel function of microRNA 130a-3p in hepatic insulin sensitivity and liver steatosis[J]. Diabetes, 2014, Mar 27. [Epub ahead of print]

Segersten U, Spector Y, Goren Y, et al. The role of microRNA profiling in prognosticating progression in Ta and T1 urinary bladder cancer[J]. Urol Oncol, 2014, Jan 14. pii: S1078-1439(13)00463-8. doi: 10.1016/j.urolonc.2013.11.001. [Epub ahead of print].

van den Ouweland AM, Elfferich P, Lamping R， et al. Identification of a large rearrangement in CYLD as a cause of familiaI cylindromatosis[J]. Fam Cancer, 2011, 10(1): 127-132.

Massoumi R, Kuphal S, Hellerbrand C, et al. Down-regulation of CYLD expression by SnaiI promotes tumor progression in maIignant melanoma[J]. J Exp Med, 2009, 206(1): 221-232.

Hayashi M, Jono H, Shinriki S, et al. Clinical significance of CYLD downregulation in breast cancer[J]. Breast Cancer Res Treat, 2014, 143(3): 447-457.

Lubinski J. Breast cancer genetics: 20 years later[J]. Clin Genet, 2014, 85(1): 5-6.

Harquail J, Benzina S, Robichaud GA. MicroRNAs and breast cancer m alignancy:an overview of miRNA-regulated cancer processes leading to metastasis[J]. Cancer Biomark, 2012, 11(6): 269-280.

Wee EJ, Peters K, Nair SS, et al. Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer[J]. Oncogene, 2012, 31(38): 4182-4195.

Chen W, Cai F, Zhang B, et al. The level of circulating miRNA-10b and miRNA-373 in detecting lymph node metastasis of breast cancer: potential biomarkers[J]. Tumour Biol, 2013, 34(1): 455-462.

Zhong XY, Yu JH, Zhang WG, et al. MicroRNA-421 functions as an oncogenic miRNA in biliary tract cancer through down-regulating farnesoid X receptor expression[J]. Gene, 2012, 493(1): 44-51.

Iliopoulos D, Jaeger SA, Hirsch HA, et al. STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer[J]. Mol Cell, 2010, 39(4): 493-506.

（收稿日期：2014-05-07）

參考文獻(xiàn)

DeSantis C, Ma J, Bryan L, et al. Breast cancer statistics, 2013[J]. CA Cancer J Clin, 2014, 64(1): 52-62.

Li X, Wang Q, Zheng Y, et al. Prioritizing human cancer microRNAs based on genes functional consistency between microRNA and cancer[J]. Nucleic Acids Res, 2011, 39(22): e153.

Liu J, Zheng M, Tang YL, et al. MicroRNAs, an active and versatile group in cancers[J]. Int J Oral Sci, 2011, 3(4): 165-175.

Xiao F, Yu J, Liu B, et al. A novel function of microRNA 130a-3p in hepatic insulin sensitivity and liver steatosis[J]. Diabetes, 2014, Mar 27. [Epub ahead of print]

Segersten U, Spector Y, Goren Y, et al. The role of microRNA profiling in prognosticating progression in Ta and T1 urinary bladder cancer[J]. Urol Oncol, 2014, Jan 14. pii: S1078-1439(13)00463-8. doi: 10.1016/j.urolonc.2013.11.001. [Epub ahead of print].

van den Ouweland AM, Elfferich P, Lamping R， et al. Identification of a large rearrangement in CYLD as a cause of familiaI cylindromatosis[J]. Fam Cancer, 2011, 10(1): 127-132.

Massoumi R, Kuphal S, Hellerbrand C, et al. Down-regulation of CYLD expression by SnaiI promotes tumor progression in maIignant melanoma[J]. J Exp Med, 2009, 206(1): 221-232.

Hayashi M, Jono H, Shinriki S, et al. Clinical significance of CYLD downregulation in breast cancer[J]. Breast Cancer Res Treat, 2014, 143(3): 447-457.

Lubinski J. Breast cancer genetics: 20 years later[J]. Clin Genet, 2014, 85(1): 5-6.

Harquail J, Benzina S, Robichaud GA. MicroRNAs and breast cancer m alignancy:an overview of miRNA-regulated cancer processes leading to metastasis[J]. Cancer Biomark, 2012, 11(6): 269-280.

Wee EJ, Peters K, Nair SS, et al. Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer[J]. Oncogene, 2012, 31(38): 4182-4195.

Chen W, Cai F, Zhang B, et al. The level of circulating miRNA-10b and miRNA-373 in detecting lymph node metastasis of breast cancer: potential biomarkers[J]. Tumour Biol, 2013, 34(1): 455-462.

Zhong XY, Yu JH, Zhang WG, et al. MicroRNA-421 functions as an oncogenic miRNA in biliary tract cancer through down-regulating farnesoid X receptor expression[J]. Gene, 2012, 493(1): 44-51.

Iliopoulos D, Jaeger SA, Hirsch HA, et al. STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer[J]. Mol Cell, 2010, 39(4): 493-506.

（收稿日期：2014-05-07）

參考文獻(xiàn)

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（收稿日期：2014-05-07）