朱金峰，曾薇，王海江

(新疆醫(yī)科大學附屬腫瘤醫(yī)院，烏魯木齊830011)

miR- 34a- 5p對胃腺癌細胞凋亡的影響及其作用機制

朱金峰，曾薇，王海江

(新疆醫(yī)科大學附屬腫瘤醫(yī)院，烏魯木齊830011)

目的探討miR- 34a- 5p對胃腺癌細胞凋亡的影響及其作用機制。方法①采用生物信息學技術預測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胞RGM- 1、人胃腺癌細胞SGC7901，采用qRT- PCR法檢測兩種細胞miR- 34a- 5p、Bcl- 2 mRNA表達。③將SGC7901細胞隨機分為觀察組和對照組，觀察組轉染miR- 34a- 5p mimic質粒，對照組轉染scramble質粒。轉染48 h，采用qRT- PCR法、Western blotting法檢測Bcl- 2 mRNA和蛋白表達。④將SGC7901細胞隨機分為陰性對照組、Bcl- 2 WT組、Bcl- 2 MT組，陰性對照組轉染miR- 34a- 5p mimic和pRL- TK，Bcl- 2 WT組轉染miR- 34a- 5p mimic、Bcl- 2野生型載體和pRL- TK，Bcl- 2 MT組轉染miR- 34a- 5p mimic、Bcl- 2突變型載體和pRL- TK。轉染48 h，采用雙熒光素酶報告基因實驗檢測各組相對熒光素酶活性。⑤將SGC7901細胞隨機分為陰性對照組、miR- 34a- 5p mimic組、pcDNA3.1- Bcl- 2組、miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組，陰性對照組轉染scramble+pcDNA3.1- 空載體，miR- 34a- 5p mimic組轉染miR- 34a- 5p mimic，pcDNA3.1- Bcl- 2組轉染pcDNA3.1- Bcl- 2，miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組轉染miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2。轉染48 h，采用流式細胞儀檢測各組細胞凋亡率。結果① Bcl- 2為miR- 34a- 5p的靶基因。② SGC7901細胞miR- 34a- 5p mRNA相對表達量低于RGM- 1細胞(P<0.01)，Bcl- 2 mRNA相對表達量高于RGM- 1細胞(P<0.01)。③觀察組Bcl- 2 mRNA和蛋白相對表達量均低于對照組(P均<0.01)。④Bcl- 2 WT組相對熒光素酶活性低于陰性對照組和Bcl- 2 MT組(P均<0.01)。⑤miR- 34a- 5p mimic組細胞凋亡率高于陰性對照組，pcDNA3.1- Bcl- 2組、miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組低于陰性對照組及miR- 34a- 5p mimic組，組間比較P均<0.01。結論miR- 34a- 5p可通過靶向調控Bcl- 2抑制胃腺癌細胞凋亡，進而參與胃腺癌的發(fā)生、發(fā)展。

胃癌；微小RNA- 34a- 5p；B細胞淋巴瘤2；細胞凋亡

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)病機制尚未完全明確[1～3]。細胞凋亡是一種程序性細胞死亡[4]，各種腫瘤的發(fā)生均伴隨細胞凋亡受抑[5～7]。研究發(fā)現，微小RNA(miRNA)可通過直接靶向調控凋亡因子的表達，參與并調控與細胞凋亡相關的各種轉錄激活和信號傳導，影響細胞凋亡進程，參與腫瘤的發(fā)生、發(fā)展[8,9]。目前已證實，miR- 34a- 5p與結直腸癌[10]、乳腺癌[11]等多種惡性腫瘤相關。但其與胃腺癌的關系尚不清楚。2016年1月～2017年3月，我們觀察了胃腺癌細胞miR- 34a- 5p表達?，F分析結果并探討其臨床意義。

1 材料

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

2 方法與結果

2.1 miR- 34a- 5p的靶基因預測 采用miRNA靶標預測軟件[miRNA數據庫miRBase(http://www.mirbase.org)、Targetscan human(http://www.targetscan.org/)]預測Bcl- 2是否為miR- 34a- 5p的靶基因。結果顯示，Bcl- 2為miR- 34a- 5p的靶基因。

2.2 細胞培養(yǎng) 將RGM- 1、SGC7901細胞分別置于含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胞生長情況，隔日更換培養(yǎng)液。當細胞融合達90%時，0.25%胰酶消化后傳代，取傳3代細胞進行后續(xù)實驗。

2.3 RGM- 1、SGC7901細胞miR- 34a- 5p、Bcl- 2 mRNA表達檢測 采用qRT- PCR法。取傳3代兩種細胞，TRIzol法提取細胞總RNA，以甲醛變性的瓊脂糖凝膠電泳鑒定總RNA完整，紫外分光光度計檢測總RNA濃度和純度合格，然后逆轉錄為cDNA；以cDNA為模板，進行定量PCR擴增。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內參)上游引物：5′- CCTCACCCTGAAGTACCCCA- 3′，下游引物：5′- TCGTCCCAGTTGGTGACGAT- 3′。取擴增產物進行瓊脂糖凝膠電泳。采用2-ΔΔCt法計算miR- 34a- 5p、Bcl- 2 mRNA相對表達量。結果顯示，RGM- 1細胞miR- 34a- 5p mRNA相對表達量為0.94±0.04，SGC7901細胞為0.68±0.03，二者比較P<0.01；RGM- 1細胞Bcl- 2 mRNA相對表達量為0.93±0.03，SGC7901細胞為2.37±0.22，二者比較P<0.01。

2.4 轉染miR- 34a- 5p mimic質粒的SGC7901細胞Bcl- 2 mRNA和蛋白表達檢測 取傳3代SGC7901細胞，胰酶消化后計數，細胞鋪板，待細胞融合50%～80%時，隨機分為觀察組和對照組。觀察組轉染miR- 34a- 5p mimic質粒，對照組轉染scramble質粒。轉染方法：用250 μL無血清DMEM培養(yǎng)基稀釋4.0 μg質粒，250 μL DMEM培養(yǎng)基稀釋10 μL Lipofectamine 2000；混合質粒與Lipofectamine 2000稀釋液室溫下孵育20 min；將混合液置于細胞培養(yǎng)板，輕輕混勻，37 ℃、5% CO2條件下培養(yǎng)4 h；更換為基礎培養(yǎng)基，繼續(xù)培養(yǎng)48 h，收集細胞進行以下檢測。①Bcl- 2 mRNA表達：采用qRT- PCR法，具體方法同2.3。結果顯示，觀察組Bcl- 2 mRNA相對表達量為0.71±0.07，對照組為2.37±0.26，兩組比較P<0.01。②Bcl- 2蛋白表達：采用Western blotting法。取兩組細胞，分別加入RIPA細胞裂解液(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；將細胞裂解液轉移至1.5 mL微量離心管中，4 ℃、12 000 r/min離心30 min，收集上清液，BCA法進行蛋白定量。分別取50 μg蛋白進行SDS- PAGE，恒壓100 V 70 min，冰浴轉膜至PVDF膜上；5% BSA封閉1 h；加入Bcl- 2一抗4 ℃搖床孵育過夜；TBST洗膜，加入辣根過氧化物酶標記的二抗室溫孵育1 h；TBST洗滌，ECL化學發(fā)光顯像。以β- actin為內參，計算Bcl- 2蛋白相對表達量。結果顯示，觀察組Bcl- 2蛋白相對表達量為0.94±0.04，對照組為1.47±0.21，兩組比較P<0.05。

2.5 miR- 34a- 5p靶向調控Bcl- 2表達驗證 采用雙熒光素酶報告基因實驗。將SGC7901細胞以8×104個/孔密度接種至24孔板，以含10% FBS的RPMI 1640培養(yǎng)基培養(yǎng)至細胞80%融合。隨機分為陰性對照組、Bcl- 2 WT組、Bcl- 2 MT組，陰性對照組轉染miR- 34a- 5p mimic和pRL- TK；Bcl- 2 WT組轉染miR- 34a- 5p mimic、Bcl- 2 野生型載體和pRL- TK；Bcl- 2 MT組轉染miR- 34a- 5p mimic、Bcl- 2突變型載體和pRL- TK。轉染方法同2.4。轉染48 h，以被動裂解液PBL裂解細胞，取20 μL細胞裂解液加入100 μL熒光素酶檢測試劑Ⅱ(LAR Ⅱ)，立即檢測螢火蟲熒光素酶活性。然后加入100 μL Stop & GloTM試劑，立即檢測海腎熒光素酶活性。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熒光素酶活性。結果顯示，陰性對照組相對熒光素酶活性為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組相對熒光素酶活性明顯低于其他兩組(P均<0.01)。

2.6 細胞凋亡檢測 取SGC7901細胞隨機分為陰性對照組、miR- 34a- 5p mimic組、pcDNA3.1- Bcl- 2組、miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組，陰性對照組轉染scramble+pcDNA3.1- 空載體，miR- 34a- 5p mimic組轉染miR- 34a- 5p mimic，pcDNA3.1- Bcl- 2組轉染pcDNA3.1- Bcl- 2，miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組轉染miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2。各組轉染48 h，用不含EDTA的胰酶消化后收集細胞，預冷1×PBS洗滌，1×Binding Buffer懸浮，加入Annexin V- FITC，室溫避光孵育15 min，PI標記后流式細胞儀檢測各組細胞凋亡率。結果顯示，陰性對照組細胞凋亡率為(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組細胞凋亡率明顯高于陰性對照組，pcDNA3.1- Bcl- 2組、miR- 34a- 5p mimic+pcDNA3.1- Bcl- 2組細胞凋亡率明顯低于陰性對照組及miR- 34a- 5p mimic組，組間比較P均<0.01。

3 討論

miRNA是高度保守的內源性非編碼小分子RNA，由20～23個核苷酸組成，在轉錄后水平調控基因表達[12]。miRNA可加速降解和(或)阻斷其靶基因的翻譯及誘導轉錄后基因表達，從而參與調節(jié)細胞增殖、分化、代謝和凋亡等各種生物學過程[13]，且與多種腫瘤的發(fā)生密切相關[14～17]。miR- 34a- 5p是miRNA中比較重要的一種。Sun等[18]研究發(fā)現，與癌旁正常肝組織相比，miR- 34a- 5p在肝癌組織中低表達；上調肝癌細胞中miR- 34a- 5p表達可抑制肝癌細胞增殖，促進肝癌細胞凋亡；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，在多發(fā)性骨髓瘤干細胞中過表達miR- 34a- 5p可抑制骨髓瘤細胞增殖和克隆形成，并促進細胞凋亡，在多發(fā)性骨髓瘤的發(fā)生、發(fā)展過程中發(fā)揮負性調控作用。但是，關于miR- 34a- 5p與胃腺癌的關系鮮見報道。本研究結果顯示，與人正常胃黏膜上皮細胞RGM- 1相比，人胃腺癌細胞SGC7901 miR- 34a- 5p低表達，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。進一步對miR- 34a- 5p在胃腺癌細胞凋亡中的作用進行研究，發(fā)現轉染miR- 34a- 5p mimic上調miR- 34a- 5p表達可促進胃腺癌細胞凋亡，提示miR- 34a- 5p可能通過促進胃腺癌細胞凋亡，在胃腺癌的發(fā)生、發(fā)展中發(fā)揮抑癌基因作用。

Bcl- 2基因家族與細胞凋亡密切相關，根據其功能和結構分為兩類：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]。越來越多的研究表明，Bcl- 2信號通路在細胞增殖與凋亡過程中發(fā)揮重要作用[21]。Chen等[22]報道，miR- 744可直接靶向調控Bcl- 2，在宮頸癌細胞中過表達miR- 744可下調Bcl- 2表達，并激活Caspase- 3表達，從而抑制宮頸癌細胞增殖，促進宮頸癌細胞凋亡，對宮頸癌的發(fā)生、發(fā)展起負性調控作用。季濤等[23]研究發(fā)現，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陽性表達率呈逐漸升高趨勢，同時細胞凋亡率呈逐漸降低趨勢，提示Bcl- 2可能作為癌基因，通過使細胞凋亡受抑，參與胃癌的發(fā)生、發(fā)展。本研究結果發(fā)現，與人正常胃黏膜上皮細胞RGM- 1相比，Bcl- 2在人胃腺癌細胞SGC7901中高表達，提示Bcl- 2可能作為癌基因參與胃腺癌的發(fā)生、發(fā)展。為進一步驗證Bcl- 2在胃腺癌中的表達調控機制，我們采用miRNA數據庫對miR- 34a- 5p的下游靶基因進行預測，結果發(fā)現miR- 34a- 5p可與Bcl- 2的3′- UTR結合，提示miR- 34a- 5p可能靶向調控Bcl- 2表達。我們進一步在人胃腺癌細胞SGC7901中轉染miR- 34a- 5p mimic以上調miR- 34a- 5p表達，結果發(fā)現，過表達miR- 34a- 5p可抑制Bcl- 2在轉錄和蛋白水平上的表達，進一步驗證了miR- 34a- 5p可對Bcl- 2表達具有調控作用。細胞凋亡檢測結果顯示，轉染pcDNA3.1- Bcl- 2以進一步上調Bcl- 2表達，可顯著抑制人胃腺癌細胞SGC7901凋亡，提示Bcl- 2可能通過抑制胃腺癌細胞凋亡，在胃腺癌的發(fā)生、發(fā)展中發(fā)揮癌基因作用。為驗證miR- 34a- 5p是否通過調控Bcl- 2的表達參與胃腺癌的凋亡過程，我們在人胃腺癌細胞SGC7901中同時轉染miR- 34a- 5p mimic和pcDNA3.1- Bcl- 2，結果顯示，共同過表達miR- 34a- 5p和Bcl- 2可逆轉miR- 34a- 5p促進胃腺癌細胞凋亡的作用，進而使胃腺癌細胞凋亡受抑。提示miR- 34a- 5p靶向作用于Bcl- 2，通過下調Bcl- 2表達促進胃腺癌細胞凋亡。但是，當胃腺癌細胞共同轉染miR- 34a- 5p mimic和pcDNA3.1- Bcl- 2時，解除了miR- 34a- 5p對Bcl- 2表達的抑制作用，從而逆轉了miR- 34a- 5p促進胃腺癌細胞凋亡的作用。

綜上所述，miR- 34a- 5p通過靶向調控Bcl- 2參與胃腺癌細胞凋亡，進而參與胃腺癌的發(fā)生、發(fā)展。

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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)自然科學基金面上項目(2016D01C347)。

朱金峰(1980- )，男，主治醫(yī)師，主要研究方向為胃腸腫瘤的診治。E- mail: zjf122@hotmail.com

王海江(1963- )，男，主任醫(yī)師，主要研究方向為胃腸腫瘤的診治。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)