miR- 34a- 5p對(duì)胃腺癌細(xì)胞凋亡的影響及其作用機(jī)制

朱金峰，曾薇，王海江

(新疆醫(yī)科大學(xué)附屬腫瘤醫(yī)院，烏魯木齊830011)

miR- 34a- 5p對(duì)胃腺癌細(xì)胞凋亡的影響及其作用機(jī)制

朱金峰，曾薇，王海江

(新疆醫(yī)科大學(xué)附屬腫瘤醫(yī)院，烏魯木齊830011)

目的探討miR- 34a- 5p對(duì)胃腺癌細(xì)胞凋亡的影響及其作用機(jī)制。方法①采用生物信息學(xué)技術(shù)預(yù)測(cè)Bcl- 2是否為miR- 34a- 5p的靶基因。②體外培養(yǎng)人正常胃黏膜上皮細(xì)胞RGM- 1、人胃腺癌細(xì)胞SGC7901，采用qRT- PCR法檢測(cè)兩種細(xì)胞miR- 34a- 5p、Bcl- 2 mRNA表達(dá)。③將SGC7901細(xì)胞隨機(jī)分為觀察組和對(duì)照組，觀察組轉(zhuǎn)染miR- 34a- 5p mimic質(zhì)粒，對(duì)照組轉(zhuǎn)染scramble質(zhì)粒。轉(zhuǎn)染48 h，采用qRT- PCR法、Western blotting法檢測(cè)Bcl- 2 mRNA和蛋白表達(dá)。④將SGC7901細(xì)胞隨機(jī)分為陰性對(duì)照組、Bcl- 2 WT組、Bcl- 2 MT組，陰性對(duì)照組轉(zhuǎn)染miR- 34a- 5p mimic和pRL- TK，Bcl- 2 WT組轉(zhuǎn)染miR- 34a- 5p mimic、Bcl- 2野生型載體和pRL- TK，Bcl- 2 MT組轉(zhuǎn)染miR- 34a- 5p mimic、Bcl- 2突變型載體和pRL- TK。轉(zhuǎn)染48 h，采用雙熒光素酶報(bào)告基因?qū)嶒?yàn)檢測(cè)各組相對(duì)熒光素酶活性。⑤將SGC7901細(xì)胞隨機(jī)分為陰性對(duì)照組、miR- 34a- 5p mimic組、pcDNA3.1- Bcl- 2組、miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組，陰性對(duì)照組轉(zhuǎn)染scramble+pcDNA3.1- 空載體，miR- 34a- 5p mimic組轉(zhuǎn)染miR- 34a- 5p mimic，pcDNA3.1- Bcl- 2組轉(zhuǎn)染pcDNA3.1- Bcl- 2，miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組轉(zhuǎn)染miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2。轉(zhuǎn)染48 h，采用流式細(xì)胞儀檢測(cè)各組細(xì)胞凋亡率。結(jié)果① Bcl- 2為miR- 34a- 5p的靶基因。② SGC7901細(xì)胞miR- 34a- 5p mRNA相對(duì)表達(dá)量低于RGM- 1細(xì)胞(P<0.01)，Bcl- 2 mRNA相對(duì)表達(dá)量高于RGM- 1細(xì)胞(P<0.01)。③觀察組Bcl- 2 mRNA和蛋白相對(duì)表達(dá)量均低于對(duì)照組(P均<0.01)。④Bcl- 2 WT組相對(duì)熒光素酶活性低于陰性對(duì)照組和Bcl- 2 MT組(P均<0.01)。⑤miR- 34a- 5p mimic組細(xì)胞凋亡率高于陰性對(duì)照組，pcDNA3.1- Bcl- 2組、miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組低于陰性對(duì)照組及miR- 34a- 5p mimic組，組間比較P均<0.01。結(jié)論miR- 34a- 5p可通過(guò)靶向調(diào)控Bcl- 2抑制胃腺癌細(xì)胞凋亡，進(jìn)而參與胃腺癌的發(fā)生、發(fā)展。

胃癌；微小RNA- 34a- 5p；B細(xì)胞淋巴瘤2；細(xì)胞凋亡

胃腺癌是消化系統(tǒng)最常見的惡性腫瘤之一，發(fā)病率及病死率均較高，但其發(fā)病機(jī)制尚未完全明確[1～3]。細(xì)胞凋亡是一種程序性細(xì)胞死亡[4]，各種腫瘤的發(fā)生均伴隨細(xì)胞凋亡受抑[5～7]。研究發(fā)現(xiàn)，微小RNA(miRNA)可通過(guò)直接靶向調(diào)控凋亡因子的表達(dá)，參與并調(diào)控與細(xì)胞凋亡相關(guān)的各種轉(zhuǎn)錄激活和信號(hào)傳導(dǎo)，影響細(xì)胞凋亡進(jìn)程，參與腫瘤的發(fā)生、發(fā)展[8,9]。目前已證實(shí)，miR- 34a- 5p與結(jié)直腸癌[10]、乳腺癌[11]等多種惡性腫瘤相關(guān)。但其與胃腺癌的關(guān)系尚不清楚。2016年1月～2017年3月，我們觀察了胃腺癌細(xì)胞miR- 34a- 5p表達(dá)。現(xiàn)分析結(jié)果并探討其臨床意義。

1 材料

人正常胃黏膜上皮細(xì)胞RGM- 1、人胃腺癌細(xì)胞SGC7901，購(gòu)自ATCC細(xì)胞庫(kù)。DMEM培養(yǎng)基、FBS、PBS、Trypsin Solution，購(gòu)自美國(guó)Gibco公司；Lipofectamine 2000，購(gòu)自美國(guó)Invitrogen公司；Bcl- 2兔抗人多克隆單抗，購(gòu)自美國(guó)Protein Tech公司；雙熒光素酶檢測(cè)試劑盒，購(gòu)自廣州市銳博生物科技有限公司；Annexin V- FITC/PI細(xì)胞凋亡檢測(cè)試劑盒，購(gòu)自美國(guó)Thermo Fisher Scientific公司；雙熒光素酶報(bào)告基因檢測(cè)試劑盒，購(gòu)自美國(guó)Promega公司；miR- 34a- 5p mimic、scramble、重組質(zhì)粒pcDNA3.1- Bcl- 2和空白質(zhì)粒pcDNA3.1，購(gòu)自美國(guó)Life Technologies公司。

2 方法與結(jié)果

2.1 miR- 34a- 5p的靶基因預(yù)測(cè) 采用miRNA靶標(biāo)預(yù)測(cè)軟件[miRNA數(shù)據(jù)庫(kù)miRBase(http://www.mirbase.org)、Targetscan human(http://www.targetscan.org/)]預(yù)測(cè)Bcl- 2是否為miR- 34a- 5p的靶基因。結(jié)果顯示，Bcl- 2為miR- 34a- 5p的靶基因。

2.2 細(xì)胞培養(yǎng) 將RGM- 1、SGC7901細(xì)胞分別置于含100 U/mL青霉素、100 μg/mL鏈霉素、10% FBS的DMEM培養(yǎng)基中，在37 ℃、5% CO2、飽和濕度條件下培養(yǎng)。倒置顯微鏡下觀察細(xì)胞生長(zhǎng)情況，隔日更換培養(yǎng)液。當(dāng)細(xì)胞融合達(dá)90%時(shí)，0.25%胰酶消化后傳代，取傳3代細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)。

2.3 RGM- 1、SGC7901細(xì)胞miR- 34a- 5p、Bcl- 2 mRNA表達(dá)檢測(cè) 采用qRT- PCR法。取傳3代兩種細(xì)胞，TRIzol法提取細(xì)胞總RNA，以甲醛變性的瓊脂糖凝膠電泳鑒定總RNA完整，紫外分光光度計(jì)檢測(cè)總RNA濃度和純度合格，然后逆轉(zhuǎn)錄為cDNA；以cDNA為模板，進(jìn)行定量PCR擴(kuò)增。引物序列：miR- 34a- 5p上游引物：5′- TGGCAGTGTCTTAGCTGGTTGT- 3′，下游引物：5′- GCGAGCACAGAATTAATACGAC- 3′；U6 snRNA(miR- 34a- 5p內(nèi)參)上游引物：5′- CTCGCTTCGGCAGCACA- 3′，下游引物：5′- AACGCTTCACGA-AYYYGCGT- 3′。Bcl- 2上游引物：5′- GGTGCCACCT-GTGGTCCACCTG- 3′，下游引物： 5′- CTTCACTTGTGGCCCAGATAGG- 3′；β- actin(Bcl- 2內(nèi)參)上游引物：5′- CCTCACCCTGAAGTACCCCA- 3′，下游引物：5′- TCGTCCCAGTTGGTGACGAT- 3′。取擴(kuò)增產(chǎn)物進(jìn)行瓊脂糖凝膠電泳。采用2-ΔΔCt法計(jì)算miR- 34a- 5p、Bcl- 2 mRNA相對(duì)表達(dá)量。結(jié)果顯示，RGM- 1細(xì)胞miR- 34a- 5p mRNA相對(duì)表達(dá)量為0.94±0.04，SGC7901細(xì)胞為0.68±0.03，二者比較P<0.01；RGM- 1細(xì)胞Bcl- 2 mRNA相對(duì)表達(dá)量為0.93±0.03，SGC7901細(xì)胞為2.37±0.22，二者比較P<0.01。

2.4 轉(zhuǎn)染miR- 34a- 5p mimic質(zhì)粒的SGC7901細(xì)胞Bcl- 2 mRNA和蛋白表達(dá)檢測(cè) 取傳3代SGC7901細(xì)胞，胰酶消化后計(jì)數(shù)，細(xì)胞鋪板，待細(xì)胞融合50%～80%時(shí)，隨機(jī)分為觀察組和對(duì)照組。觀察組轉(zhuǎn)染miR- 34a- 5p mimic質(zhì)粒，對(duì)照組轉(zhuǎn)染scramble質(zhì)粒。轉(zhuǎn)染方法：用250 μL無(wú)血清DMEM培養(yǎng)基稀釋4.0 μg質(zhì)粒，250 μL DMEM培養(yǎng)基稀釋10 μL Lipofectamine 2000；混合質(zhì)粒與Lipofectamine 2000稀釋液室溫下孵育20 min；將混合液置于細(xì)胞培養(yǎng)板，輕輕混勻，37 ℃、5% CO2條件下培養(yǎng)4 h；更換為基礎(chǔ)培養(yǎng)基，繼續(xù)培養(yǎng)48 h，收集細(xì)胞進(jìn)行以下檢測(cè)。①Bcl- 2 mRNA表達(dá)：采用qRT- PCR法，具體方法同2.3。結(jié)果顯示，觀察組Bcl- 2 mRNA相對(duì)表達(dá)量為0.71±0.07，對(duì)照組為2.37±0.26，兩組比較P<0.01。②Bcl- 2蛋白表達(dá)：采用Western blotting法。取兩組細(xì)胞，分別加入RIPA細(xì)胞裂解液(含0.1%蛋白酶抑制劑)，冰上裂解30 min，超聲破碎2次、每次6 s；將細(xì)胞裂解液轉(zhuǎn)移至1.5 mL微量離心管中，4 ℃、12 000 r/min離心30 min，收集上清液，BCA法進(jìn)行蛋白定量。分別取50 μg蛋白進(jìn)行SDS- PAGE，恒壓100 V 70 min，冰浴轉(zhuǎn)膜至PVDF膜上；5% BSA封閉1 h；加入Bcl- 2一抗4 ℃搖床孵育過(guò)夜；TBST洗膜，加入辣根過(guò)氧化物酶標(biāo)記的二抗室溫孵育1 h；TBST洗滌，ECL化學(xué)發(fā)光顯像。以β- actin為內(nèi)參，計(jì)算Bcl- 2蛋白相對(duì)表達(dá)量。結(jié)果顯示，觀察組Bcl- 2蛋白相對(duì)表達(dá)量為0.94±0.04，對(duì)照組為1.47±0.21，兩組比較P<0.05。

2.5 miR- 34a- 5p靶向調(diào)控Bcl- 2表達(dá)驗(yàn)證 采用雙熒光素酶報(bào)告基因?qū)嶒?yàn)。將SGC7901細(xì)胞以8×104個(gè)/孔密度接種至24孔板，以含10% FBS的RPMI 1640培養(yǎng)基培養(yǎng)至細(xì)胞80%融合。隨機(jī)分為陰性對(duì)照組、Bcl- 2 WT組、Bcl- 2 MT組，陰性對(duì)照組轉(zhuǎn)染miR- 34a- 5p mimic和pRL- TK；Bcl- 2 WT組轉(zhuǎn)染miR- 34a- 5p mimic、Bcl- 2 野生型載體和pRL- TK；Bcl- 2 MT組轉(zhuǎn)染miR- 34a- 5p mimic、Bcl- 2突變型載體和pRL- TK。轉(zhuǎn)染方法同2.4。轉(zhuǎn)染48 h，以被動(dòng)裂解液PBL裂解細(xì)胞，取20 μL細(xì)胞裂解液加入100 μL熒光素酶檢測(cè)試劑Ⅱ(LAR Ⅱ)，立即檢測(cè)螢火蟲熒光素酶活性。然后加入100 μL Stop & GloTM試劑，立即檢測(cè)海腎熒光素酶活性。以螢火蟲熒光素酶活性與海腎熒光素酶活性比值作為相對(duì)熒光素酶活性。結(jié)果顯示，陰性對(duì)照組相對(duì)熒光素酶活性為0.88±0.04，Bcl- 2 MT組為0.81±0.03，Bcl- 2 WT組為0.54±0.06。Bcl- 2 WT組相對(duì)熒光素酶活性明顯低于其他兩組(P均<0.01)。

2.6 細(xì)胞凋亡檢測(cè) 取SGC7901細(xì)胞隨機(jī)分為陰性對(duì)照組、miR- 34a- 5p mimic組、pcDNA3.1- Bcl- 2組、miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組，陰性對(duì)照組轉(zhuǎn)染scramble+pcDNA3.1- 空載體，miR- 34a- 5p mimic組轉(zhuǎn)染miR- 34a- 5p mimic，pcDNA3.1- Bcl- 2組轉(zhuǎn)染pcDNA3.1- Bcl- 2，miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組轉(zhuǎn)染miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2。各組轉(zhuǎn)染48 h，用不含EDTA的胰酶消化后收集細(xì)胞，預(yù)冷1×PBS洗滌，1×Binding Buffer懸浮，加入Annexin V- FITC，室溫避光孵育15 min，PI標(biāo)記后流式細(xì)胞儀檢測(cè)各組細(xì)胞凋亡率。結(jié)果顯示，陰性對(duì)照組細(xì)胞凋亡率為(39.90±3.21)%，miR- 34a- 5p mimic組為(70.73±2.45)%，pcDNA3.1- Bcl- 2組為(25.07±3.31)%，miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組為(30.97±2.73)%。miR- 34a- 5p mimic組細(xì)胞凋亡率明顯高于陰性對(duì)照組，pcDNA3.1- Bcl- 2組、miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組細(xì)胞凋亡率明顯低于陰性對(duì)照組及miR- 34a- 5p mimic組，組間比較P均<0.01。

3 討論

miRNA是高度保守的內(nèi)源性非編碼小分子RNA，由20～23個(gè)核苷酸組成，在轉(zhuǎn)錄后水平調(diào)控基因表達(dá)[12]。miRNA可加速降解和(或)阻斷其靶基因的翻譯及誘導(dǎo)轉(zhuǎn)錄后基因表達(dá)，從而參與調(diào)節(jié)細(xì)胞增殖、分化、代謝和凋亡等各種生物學(xué)過(guò)程[13]，且與多種腫瘤的發(fā)生密切相關(guān)[14～17]。miR- 34a- 5p是miRNA中比較重要的一種。Sun等[18]研究發(fā)現(xiàn)，與癌旁正常肝組織相比，miR- 34a- 5p在肝癌組織中低表達(dá)；上調(diào)肝癌細(xì)胞中miR- 34a- 5p表達(dá)可抑制肝癌細(xì)胞增殖，促進(jìn)肝癌細(xì)胞凋亡；提示miR- 34a- 5p可能作為抑癌基因參與肝癌的發(fā)生、發(fā)展。Wu等[19]研究發(fā)現(xiàn)，在多發(fā)性骨髓瘤干細(xì)胞中過(guò)表達(dá)miR- 34a- 5p可抑制骨髓瘤細(xì)胞增殖和克隆形成，并促進(jìn)細(xì)胞凋亡，在多發(fā)性骨髓瘤的發(fā)生、發(fā)展過(guò)程中發(fā)揮負(fù)性調(diào)控作用。但是，關(guān)于miR- 34a- 5p與胃腺癌的關(guān)系鮮見報(bào)道。本研究結(jié)果顯示，與人正常胃黏膜上皮細(xì)胞RGM- 1相比，人胃腺癌細(xì)胞SGC7901 miR- 34a- 5p低表達(dá)，提示miR- 34a- 5p可能作為抑癌基因參與胃腺癌的發(fā)生、發(fā)展。進(jìn)一步對(duì)miR- 34a- 5p在胃腺癌細(xì)胞凋亡中的作用進(jìn)行研究，發(fā)現(xiàn)轉(zhuǎn)染miR- 34a- 5p mimic上調(diào)miR- 34a- 5p表達(dá)可促進(jìn)胃腺癌細(xì)胞凋亡，提示miR- 34a- 5p可能通過(guò)促進(jìn)胃腺癌細(xì)胞凋亡，在胃腺癌的發(fā)生、發(fā)展中發(fā)揮抑癌基因作用。

Bcl- 2基因家族與細(xì)胞凋亡密切相關(guān)，根據(jù)其功能和結(jié)構(gòu)分為兩類：一類是具有抗凋亡作用的基因，如Bcl- 2、Bcl- xl、Bcl- w、MCL- 1等；另一類是具有促凋亡作用的基因，如Bax、Bak、Bad、Bid、Bim等[20]。越來(lái)越多的研究表明，Bcl- 2信號(hào)通路在細(xì)胞增殖與凋亡過(guò)程中發(fā)揮重要作用[21]。Chen等[22]報(bào)道，miR- 744可直接靶向調(diào)控Bcl- 2，在宮頸癌細(xì)胞中過(guò)表達(dá)miR- 744可下調(diào)Bcl- 2表達(dá)，并激活Caspase- 3表達(dá)，從而抑制宮頸癌細(xì)胞增殖，促進(jìn)宮頸癌細(xì)胞凋亡，對(duì)宮頸癌的發(fā)生、發(fā)展起負(fù)性調(diào)控作用。季濤等[23]研究發(fā)現(xiàn)，在胃黏膜腸上皮化生、胃黏膜不典型增生及胃癌組織中Bcl- 2陽(yáng)性表達(dá)率呈逐漸升高趨勢(shì)，同時(shí)細(xì)胞凋亡率呈逐漸降低趨勢(shì)，提示Bcl- 2可能作為癌基因，通過(guò)使細(xì)胞凋亡受抑，參與胃癌的發(fā)生、發(fā)展。本研究結(jié)果發(fā)現(xiàn)，與人正常胃黏膜上皮細(xì)胞RGM- 1相比，Bcl- 2在人胃腺癌細(xì)胞SGC7901中高表達(dá)，提示Bcl- 2可能作為癌基因參與胃腺癌的發(fā)生、發(fā)展。為進(jìn)一步驗(yàn)證Bcl- 2在胃腺癌中的表達(dá)調(diào)控機(jī)制，我們采用miRNA數(shù)據(jù)庫(kù)對(duì)miR- 34a- 5p的下游靶基因進(jìn)行預(yù)測(cè)，結(jié)果發(fā)現(xiàn)miR- 34a- 5p可與Bcl- 2的3′- UTR結(jié)合，提示miR- 34a- 5p可能靶向調(diào)控Bcl- 2表達(dá)。我們進(jìn)一步在人胃腺癌細(xì)胞SGC7901中轉(zhuǎn)染miR- 34a- 5p mimic以上調(diào)miR- 34a- 5p表達(dá)，結(jié)果發(fā)現(xiàn)，過(guò)表達(dá)miR- 34a- 5p可抑制Bcl- 2在轉(zhuǎn)錄和蛋白水平上的表達(dá)，進(jìn)一步驗(yàn)證了miR- 34a- 5p可對(duì)Bcl- 2表達(dá)具有調(diào)控作用。細(xì)胞凋亡檢測(cè)結(jié)果顯示，轉(zhuǎn)染pcDNA3.1- Bcl- 2以進(jìn)一步上調(diào)Bcl- 2表達(dá)，可顯著抑制人胃腺癌細(xì)胞SGC7901凋亡，提示Bcl- 2可能通過(guò)抑制胃腺癌細(xì)胞凋亡，在胃腺癌的發(fā)生、發(fā)展中發(fā)揮癌基因作用。為驗(yàn)證miR- 34a- 5p是否通過(guò)調(diào)控Bcl- 2的表達(dá)參與胃腺癌的凋亡過(guò)程，我們?cè)谌宋赶侔┘?xì)胞SGC7901中同時(shí)轉(zhuǎn)染miR- 34a- 5p mimic和pcDNA3.1- Bcl- 2，結(jié)果顯示，共同過(guò)表達(dá)miR- 34a- 5p和Bcl- 2可逆轉(zhuǎn)miR- 34a- 5p促進(jìn)胃腺癌細(xì)胞凋亡的作用，進(jìn)而使胃腺癌細(xì)胞凋亡受抑。提示miR- 34a- 5p靶向作用于Bcl- 2，通過(guò)下調(diào)Bcl- 2表達(dá)促進(jìn)胃腺癌細(xì)胞凋亡。但是，當(dāng)胃腺癌細(xì)胞共同轉(zhuǎn)染miR- 34a- 5p mimic和pcDNA3.1- Bcl- 2時(shí)，解除了miR- 34a- 5p對(duì)Bcl- 2表達(dá)的抑制作用，從而逆轉(zhuǎn)了miR- 34a- 5p促進(jìn)胃腺癌細(xì)胞凋亡的作用。

綜上所述，miR- 34a- 5p通過(guò)靶向調(diào)控Bcl- 2參與胃腺癌細(xì)胞凋亡，進(jìn)而參與胃腺癌的發(fā)生、發(fā)展。

[1] Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015[J]. CA Cancer J Clin, 2016,66(2):115- 132.

[2] 張?chǎng)踅鹑f(wàn).胃癌的流行病學(xué)及其分型[J].中國(guó)全科醫(yī)學(xué)，2010，13(11)：16- 17.

[3] 鄒文斌，李兆.中國(guó)胃癌發(fā)病率及死亡率研究進(jìn)展[J].中國(guó)實(shí)用內(nèi)科雜志，2014，34(4)：408- 415.

[4] Wlodkowic D, Telford W, Skommer J, et al. Apoptosis and beyond: cytometry in studies of programmed cell death[J]． Methods Cell Biol, 2011,10(3):55- 98．

[5] Burz C, Berindan NL, Balacescu O, et al. Apoptosis in cancer: key molecular signaling pathways and therapy targets[J]. Acta Oncol, 2009,48(6):811- 821．

[6] Wang RA, Li ZS, Yan QG, et al. Resistance to apoptosis should not be taken as a hallmark of cancer[J]. Chin J Cancer, 2014,33(2):47- 50．

[7] Strasser A, Cory S, Adams JM. Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases[J]. EMBO J, 2011,30(18):3667- 3683．

[8] Gregory RI, Yan KP, Amuthan G, et al. The Microprocessor complex mediates the genesis of microRNAs[J]. Nature, 2004,432(7014):235- 240.

[9] 李潔，秦性良，邵寧生.MicroRNA及其靶基因的時(shí)空特異性與動(dòng)態(tài)變化[J].生物化學(xué)與生物物理進(jìn)展，2013，40(7)：617- 626.

[10] Koelzer VH, Sokol L, Zahnd S, et al. Digital analysis and epigenetic regulation of the signature of rejection in colorectal cancer[J]. Oncoimmunology, 2017,6(4):e1288330.

[11] Imani S, Zhang X, Hosseinifard H, et al. The diagnostic role of microRNA- 34a in breast cancer: a systematic review and meta- analysis[J]. Oncotarget, 2017,8(14):23177- 23187.

[12] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004,116(2):281- 297.

[13] Bartel DP. MicroRNAs: target recognition and regulatory functions[J]. Cell, 2009,136(2):215- 233.

[14] Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias[J]. Proc Natl Acad Sci U S A, 2004,101(32):11755- 11760.

[15] Chen X, Wang X, Ruan A, et al. miR- 141 is a key regulator of renal cell carcinoma proliferation and metastasis by controlling EphA2 expression[J]. Clin Cancer Res, 2014,20(10):2617- 2630.

[16] Siu MK, Abou- Kheir W, Yin JJ, et al. Loss of EGFR signaling regulated miR- 203 promotes prostate cancer bone metastasis and tyrosine kinase inhibitors resistance[J]. Oncotarget, 2014,5(11):3770- 3784.

[17] Rokavec M, Oner MG, Li H, et al. IL- 6R/STAT3/miR- 34a feedback loop promotes EMT- mediated colorectal cancer invasion and metastasis[J]. J Clin Invest, 2014,124(4):1853- 1867.

[18] Sun TY, Xie HJ, Li Z, et al. miR- 34a regulates HDAC1 expression to affect the proliferation and apoptosis of hepatocellular carcinoma[J]. Am J Transl Res, 2017,9(1):103- 114.

[19] Wu S, He X, Li M, et al. MiRNA- 34a overexpression inhibits multiple myeloma cancer stem cell growth in mice by suppressing TGIF2[J]. Am J Transl Res, 2016,8(12):5433- 5443.

[20] Wensveen FM, Alves NL, Derks Ingrid AM, et al. Apoptosis induced by overall metabolic stress converges on the Bcl- 2 family proteins Noxa and Mcl- 1[J]. Apoptosis, 2011,16(7):708- 721.

[21] Anvekar RA, Asciolla JJ, Missert DJ, et al. Born to be alive: a role for the Bcl- 2 family in melanoma tumor cell survival, apoptosis, and treatment[J]. Front Oncol, 2011,1(34).pii:fonc.2011.00034.

[22] Chen XF, Liu Y. MicroRNA- 744 inhibited cervical cancer growth and progression through apoptosis induction by regulating Bcl- 2[J]. Biomed Pharmacother, 2016(81):379- 387.

[23]季濤,徐向明,譚洪武.胃癌變過(guò)程中凋亡基因生存素及Bcl- 2的表達(dá)與幽門螺桿菌感染相關(guān)性研究[J].中華醫(yī)院感染學(xué)雜志，2015，25(8):1707- 1709.

EffectofmiR- 34a- 5ponapoptosisofgastricadenocarcinoma

ZHUJinfeng,ZENGWei,WANGHaijiang

(AffiliatedCancerHospitalofXinjiangMedicalUniversity,Urumqi830011,China)

ObjectiveTo investigate the effect and mechanism of miR- 34a- 5p on the apoptosis of gastric adenocarcinoma.Methods①Bioinformatics tools were used to predict the potential target gene of miR- 34a- 5p. ②Normal gastric mucosa epithelial cells RGM- 1 and gastric adenocarcinoma cells SGC7901 were cultured, and qRT- PCR was performed to detect the expression of miR- 34a- 5p and Bcl- 2 mRNA in these two cell lines. ③SGC7901 cells were assigned into two groups: the observation group was transfected with miR- 34a- 5p mimic plasmid and the control group with scramble plamid for 48 h, and then qRT- PCR was performed to detect Bcl- 2 mRNA, and Western blotting was used to detect the Bcl- 2 protein. ④ SGC7901 cells were assigned into three groups: the negative control group was co- transfected with miR- 34a- 5p mimic and pRL- TK; the Bcl- 2 WT group was co- transfected with miR- 34a- 5p mimic, Bcl- 2 WT plasmid, and pRL- TK; the Bcl- 2 MT group was co- transfected with miR- 34a- 5p mimic, Bcl- 2 MT plasmid, and pRL- TK. The relative luciferase activity was measured by dual luciferase reporter assay at 48 h. ⑤SGC7901 cells were assigned into four groups: the negative control group was co- transfected with miRNA scramble and pcDNA3.1 empty vector, the miR- 34a- 5p mimic group was transfected with miR- 34a- 5p mimic, the pcDNA3.1- Bcl- 2 group was transfected with pcDNA3.1- Bcl- 2, and the miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2 group was co- transfected with miR- 34a- 5p mimic and pcDNA3.1- Bcl- 2. The apoptotic rate of the four groups was measured by flow cytometry after 48 h of transfection.Results①Bcl- 2 was predicted to be the potential target gene of miR- 34a- 5p. ② Compared with RGM- 1 cells, SGC7901 cells had relatively low expression of miR- 34a- 5p and high expression of Bcl- 2 mRNA (bothP<0.01). ③ The relative expression of Bcl- 2 mRNA and protein in the observation group was lower than that in the control group (P<0.01). ④The relative luciferase activity of the Bcl- 2 WT group was lower than that of the negative control group and Bcl- 2 MT group (P<0.01). ⑤The apoptotic rate of the miR- 34a- 5p mimic group was higher than that of the negative control group, and the apoptotic rates of the pcDNA3.1- Bcl- 2 group and miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2 group were lower than those of the negative control group and miR- 34a- 5p mimic (P<0.01).ConclusionThe miR- 34a- 5p can inhibit the apoptosis of gastric adenocarcinoma by up- regulating the expression of Bcl- 2 and thus be involved in the occurrence and development of gastric adenocarcinoma.

gastric carcinoma; miR- 34a- 5p; Bcl- 2; apoptosis

新疆維吾爾自治區(qū)自然科學(xué)基金面上項(xiàng)目(2016D01C347)。

朱金峰(1980- )，男，主治醫(yī)師，主要研究方向?yàn)槲改c腫瘤的診治。E- mail: zjf122@hotmail.com

王海江(1963- )，男，主任醫(yī)師，主要研究方向?yàn)槲改c腫瘤的診治。E- mail： wanghaijiang@medmail.com.cn

10.3969/j.issn.1002- 266X.2017.36.006

R735.2

A

1002- 266X(2017)36- 0021- 04

2017- 04- 12)