張茂娜，張 弘,張 軍,張 雷

(1武漢大學(xué)人民醫(yī)院鄂州醫(yī)院病理科,鄂州 436000;2鄭州大學(xué)附屬河南省人民醫(yī)院病理科;*通訊作者,E-mail:zmn0306@126.com)

miR-451a過(guò)表達(dá)對(duì)宮頸癌細(xì)胞增殖和凋亡的影響及其分子機(jī)制

張茂娜1*，張 弘1,張 軍1,張 雷2

(1武漢大學(xué)人民醫(yī)院鄂州醫(yī)院病理科,鄂州 436000;2鄭州大學(xué)附屬河南省人民醫(yī)院病理科;*通訊作者,E-mail:zmn0306@126.com)

目的 觀察過(guò)表達(dá)miR-451a對(duì)宮頸癌細(xì)胞株SiHa和C33A增殖和凋亡的影響,探討其分子機(jī)制。 方法 以宮頸癌細(xì)胞株SiHa和C33A為研究對(duì)象,分為miR-NC組(脂質(zhì)體轉(zhuǎn)染miR-NC)和miR-451a組(脂質(zhì)體轉(zhuǎn)染miR-451a模擬物),qPCR檢測(cè)轉(zhuǎn)染48 h各組細(xì)胞miR-451a、巨噬細(xì)胞移動(dòng)抑制因子(MIF)和鈣結(jié)合蛋白39(CAB39)mRNA的表達(dá),Western blotting檢測(cè)各組細(xì)胞MIF、CAB39、p-PI3K和p-AKT蛋白的表達(dá),流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期和細(xì)胞凋亡,MTT法和平板克隆實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力。 結(jié)果 與miR-NC組相比,miR-451a組SiHa和C33A細(xì)胞中miR-451a表達(dá)均顯著增加(P<0.01),MIF和CAB39 mRNA及蛋白表達(dá)顯著減少,CAB39下游蛋白p-PI3K和p-AKT表達(dá)下調(diào)。miR-451a組細(xì)胞周期進(jìn)展被顯著抑制(P<0.05),細(xì)胞凋亡明顯增加(P<0.05),細(xì)胞的增殖能力顯著下降(P<0.05)。 結(jié)論 過(guò)表達(dá)miR-451a能夠抑制宮頸癌細(xì)胞株的增殖,促進(jìn)細(xì)胞凋亡,其機(jī)制可能與下調(diào)MIF、CAB39蛋白的表達(dá)有關(guān)。

微小RNA-451a; 宮頸癌; 細(xì)胞增殖; 細(xì)胞凋亡

宮頸癌是女性最常見的腫瘤之一,也是女性死亡率最高的腫瘤之一[1]。人乳頭瘤病毒(HPV)是宮頸癌主要的危險(xiǎn)因素,與90%以上宮頸癌的發(fā)生密切相關(guān),但不可否認(rèn)的是癌基因和抑癌基因的異常表達(dá)對(duì)宮頸癌的發(fā)生至關(guān)重要[2]。近年來(lái)宮頸癌的治療方面有了很大的進(jìn)步,仍有很多宮頸癌患者的預(yù)后并不理想。通過(guò)miRNA干擾基因表達(dá)模式的靶向治療日益成為宮頸癌研究的新方向[3]。miR-451a是一種新發(fā)現(xiàn)的在結(jié)腸癌、胃癌、食管癌等多種腫瘤中具有抑癌作用的miRNA[4-6]。然而,miR-451a在宮頸癌中的研究未見報(bào)道。本研究以宮頸癌細(xì)胞株SiHa和C33A為細(xì)胞模型,探討miR-451a對(duì)宮頸癌細(xì)胞增殖和凋亡的影響及可能的分子機(jī)制,為宮頸癌的防治提供新的治療策略。

1 材料與方法

1.1 細(xì)胞株及主要試劑

表1qPCR引物序列

Table1TheqPCRprimersequence

基因 引物序列(5′-3′) U6上游：TCCGATCGTGAAGCGTTC下游：GTGCAGGGTCCGAGGT miR?451a上游：TCCGATTGAGTCATTACCAT下游：GTGCAGGGTCCGAGGT GAPDH上游：CTGGGCTACACTGAGCACC下游：AAGTGGTCGTTGAGGGCAATG MIF上游：CCATGCCTATGTTCATCGTG下游：GAACAGCGGTGCAGGTAAGTG CAB39上游：TGAACCTGCTGCGAGACAAAA下游：TGCGTCTTGTTAGGATTGGCTA

1.2 miR-451a靶基因預(yù)測(cè)

本研究選擇microRNA.org、miRecords和PicTar 3種預(yù)測(cè)網(wǎng)站及參考文獻(xiàn)[7,8]預(yù)測(cè)miR-451a可能的靶基因。miR-451a與MIF mirSVR評(píng)分為-1.076 9,PhastCons評(píng)分為0.488 9;miR-451a與CAB39 mirSVR評(píng)分為-0.577 7,PhastCons評(píng)分為0.688 7;miR-451a可能的靶基因?yàn)镸IF和CAB39,具體的序列互補(bǔ)區(qū)域(見圖1)。

圖1 miR-451a與MIF、CAB39基因序列互補(bǔ)區(qū)域Figure 1 Complementary regions of miR-451a with gene sequence of MIF and CAB39

1.3 細(xì)胞培養(yǎng)與轉(zhuǎn)染

SiHa和C33A細(xì)胞株培養(yǎng)于含10%胎牛血清的RPMI-1640培養(yǎng)基中,在37 ℃、5% CO2飽和濕度培養(yǎng),取對(duì)數(shù)生長(zhǎng)期細(xì)胞,隨機(jī)將細(xì)胞分為miR-NC組(轉(zhuǎn)染miR-NC)和miR-451a組(轉(zhuǎn)染miR-451a)。轉(zhuǎn)染操作按照LipofectamineTM3000說(shuō)明書進(jìn)行,轉(zhuǎn)染6 h更換新鮮培養(yǎng)基,轉(zhuǎn)染48 h進(jìn)行后續(xù)實(shí)驗(yàn)。

1.4 qPCR檢測(cè)miR-451a、MIF和CAB39 mRNA相對(duì)表達(dá)量

提取各組細(xì)胞總RNA,逆轉(zhuǎn)錄后進(jìn)行qPCR檢測(cè)。miRNA檢測(cè)以U6為內(nèi)參,mRNA檢測(cè)以GAPDH為內(nèi)參。反應(yīng)體系和條件參照qPCR試劑盒進(jìn)行操作,Ct值均以2-ΔΔCt分析比較各組miRNA或mRNA表達(dá)差異。

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1.5 Western blotting檢測(cè)目的蛋白的表達(dá)

細(xì)胞轉(zhuǎn)染48 h后,提取細(xì)胞總蛋白,測(cè)定蛋白濃度,分別取適量蛋白樣品進(jìn)行聚丙烯酰氨凝膠電泳(SDS-PAGE),電轉(zhuǎn)至硝酸纖維膜上,5%脫脂牛奶室溫封閉,一抗4 ℃孵育過(guò)夜,二抗室溫孵育2 h,化學(xué)發(fā)光法顯影。

1.6 流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡

胰酶溶液消化收集細(xì)胞，70%乙醇固定4 ℃過(guò)夜，棄上清，50 μg/ml RNAse A溶液重懸，室溫下放置1 h，溴化丙啶染液4 ℃下避光孵育30 min，流式細(xì)胞儀檢測(cè)細(xì)胞周期。細(xì)胞凋亡分析根據(jù)Annexin V-FITC/PI細(xì)胞凋亡檢測(cè)試劑盒說(shuō)明書進(jìn)行操作。

1.7 MTT法檢測(cè)細(xì)胞增殖

在96孔板上以3×103個(gè)/孔接種轉(zhuǎn)染后的細(xì)胞,分別于第1,2,3,4,5天檢測(cè)細(xì)胞增殖情況。具體操作為：在每個(gè)時(shí)間點(diǎn),每孔加入20 μl MTT,繼續(xù)培養(yǎng)4 h,棄上清,加入150 μl二甲基亞砜振蕩15 min。酶標(biāo)儀檢測(cè)波長(zhǎng)在490 nm處的吸光值。

1.8 平板克隆實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力

在6孔板上以1×103個(gè)/孔接種轉(zhuǎn)染后的細(xì)胞,于培養(yǎng)箱內(nèi)培養(yǎng)10 d。取出后采用甲醇固定10 min,結(jié)晶紫染液染色30 min,洗去染色,倒置顯微鏡下隨機(jī)選取6個(gè)視野計(jì)數(shù),取平均值進(jìn)行統(tǒng)計(jì)學(xué)分析。

1.9 統(tǒng)計(jì)學(xué)分析

實(shí)驗(yàn)數(shù)據(jù)均以均數(shù)±標(biāo)準(zhǔn)差表示,組間比較采用t檢驗(yàn),所有數(shù)據(jù)均在SPSS18.0統(tǒng)計(jì)軟件中進(jìn)行統(tǒng)計(jì)。P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 miR-451a轉(zhuǎn)染效率測(cè)定及各組細(xì)胞中MIF和CAB39 mRNA的表達(dá)

miR-NC組和miR-451a組SiHa細(xì)胞中miR-451a相對(duì)表達(dá)量分別為1.09±0.52和8 223.46±1 324.91(t=12.410,P<0.001),C33A細(xì)胞miR-451a相對(duì)表達(dá)量分別為1.04±0.33和10 930.67±3 302.61(t=6.619,P=0.001)。與miR-NC組相比,miR-451a組細(xì)胞中MIF和CAB39 mRNA的表達(dá)顯著下降(見表2)。

細(xì)胞分組MIFCAB39SiHa對(duì)照組 103±027 101±016實(shí)驗(yàn)組 057±012? 053±024?C33A對(duì)照組 109±055 104±035實(shí)驗(yàn)組 032±020? 037±008??

與對(duì)照組比較,*P<0.05,**P<0.01

2.2 過(guò)表達(dá)miR-451a對(duì)靶蛋白表達(dá)的影響

Western blotting結(jié)果顯示,與miR-NC組相比,轉(zhuǎn)染miR-451a后,MIF和CAB39蛋白的表達(dá)水平顯著降低,CAB39下游蛋白p-PI3K和p-AKT表達(dá)明顯下調(diào)(見圖2)。

2.3 過(guò)表達(dá)miR-451a對(duì)細(xì)胞周期和細(xì)胞凋亡的影響

與miR-NC組相比,miR-451a組細(xì)胞在S期和G2/M期的比例明顯下降,G0/G1期細(xì)胞的比例顯著增加(P<0.05,見表3),提示細(xì)胞被阻滯在G0/G1期。miR-NC組和miR-451a組SiHa細(xì)胞凋亡率分別為6.72%±0.87%和26.07%±7.22%(t=5.327,P=0.002),C33A細(xì)胞凋亡率分別為8.13%±1.67%和17.77%±2.42%(t=6.560,P=0.001),過(guò)表達(dá)miR-451a顯著促進(jìn)宮頸癌細(xì)胞的凋亡。

1.miR-NC組；2.miR-451a組圖2 過(guò)表達(dá)miR-451a對(duì)靶蛋白及下游蛋白的影響Figure 2 Effect of miR-451a overexpression on target protein and downstream protein

細(xì)胞株組別 G0/G1 SG2/MSiHa對(duì)照組4481±3103216±2202304±185實(shí)驗(yàn)組6693±486??2144±156??1163±432??C33A對(duì)照組4153±3672814±3423033±228實(shí)驗(yàn)組5882±351??2215±285?1903±241??

與對(duì)照組比較，*P<0.05,**P<0.01

2.4 過(guò)表達(dá)miR-451a對(duì)細(xì)胞增殖的影響

MTT檢測(cè)結(jié)果顯示,與對(duì)照組相比,miR-451a組的細(xì)胞增殖能力明顯降低(P<0.05,見圖3)。

與miR-NC組相比,*P<0.05圖3 過(guò)表達(dá)miR-451a對(duì)宮頸癌細(xì)胞增殖的影響Figure 3 Effect of miR-451a over expression on the proliferation of cervical cancer cells

2.5 過(guò)表達(dá)miR-451a對(duì)細(xì)胞克隆形成的影響

miR-NC組和miR-451a組SiHa細(xì)胞形成的克隆數(shù)分別為206.08±31.25和140.17±30.4(t=3.024,P=0.023),C33A細(xì)胞形成的克隆數(shù)分別為138.77±17.24和82.75±17.86(t=4.513,P=0.004)。與對(duì)照組相比,轉(zhuǎn)染miR-451a后的宮頸癌細(xì)胞形成的克隆數(shù)明顯減少(P<0.05)。

3 討論

miRNA是一類長(zhǎng)度約為22個(gè)核苷酸的小分子非編碼RNA,主要通過(guò)與靶基因mRNA的3′UTR區(qū)域特異性結(jié)合,引起mRNA翻譯抑制或直接導(dǎo)致其降解發(fā)揮干擾作用[9]。miRNA的低表達(dá)與多種腫瘤的發(fā)生發(fā)展密切相關(guān),近年來(lái),通過(guò)過(guò)表達(dá)miRNA治療腫瘤的研究逐漸成為熱點(diǎn)[10]。miR-451a是近年來(lái)研究較多的miRNA之一,已被證實(shí)與多種腫瘤的生物學(xué)行為密切相關(guān),miR-451a可通過(guò)干擾Ywhaz基因的表達(dá),調(diào)節(jié)FoxO3蛋白在胞核內(nèi)的聚集,抑制結(jié)腸癌細(xì)胞的生長(zhǎng)[4];miR-451a可作為胃癌潛在的預(yù)后標(biāo)志物和腫瘤抑制因子[5];miR-451a可通過(guò)靶向作用于CDKN2D和MAP3K1,抑制食管癌細(xì)胞的增殖[6];miR-451a也可抑制肺癌細(xì)胞的增殖、遷移和侵襲[11];miR-451a亦可通過(guò)靶向干擾Bcl-2基因的表達(dá),影響caspase3蛋白的表達(dá),加速乳腺癌細(xì)胞的凋亡,改善乳腺癌細(xì)胞對(duì)紫杉醇的耐藥性[12];miR-451a的表達(dá)水平與骨肉瘤的預(yù)后密切相關(guān),通過(guò)靶向干擾CXCL16基因的表達(dá),抑制骨肉瘤細(xì)胞的生長(zhǎng)和侵襲[13]。miR-451a對(duì)宮頸癌生長(zhǎng)的作用至今仍未明確。

巨噬細(xì)胞移動(dòng)抑制因子(MIF)最早發(fā)現(xiàn)于活化的T淋巴細(xì)胞,MIF通過(guò)促進(jìn)腫瘤細(xì)胞的增殖、分化、抑制p53依賴性的細(xì)胞凋亡,參與腫瘤的發(fā)生發(fā)展[14]。鈣結(jié)合蛋白39(CAB39)參與PI3K/AKT信號(hào)通路的活化,最終導(dǎo)致PI3K和AKT的磷酸化,促進(jìn)細(xì)胞周期的進(jìn)展,抑制細(xì)胞凋亡[15]。CAB39在宮頸癌細(xì)胞中的研究尚未見文獻(xiàn)報(bào)道。本研究顯示,過(guò)表達(dá)miR-451a后,宮頸癌細(xì)胞中MIF和CAB39蛋白的表達(dá)水平顯著下降,CAB39下游蛋白p-PI3K和p-AKT表達(dá)下調(diào),引起宮頸癌細(xì)胞周期G1的阻滯和細(xì)胞凋亡的顯著增加,導(dǎo)致宮頸癌細(xì)胞的增殖能力顯著下降。以上現(xiàn)象提示miR-451a可能通過(guò)靶向干擾MIF和CAB39基因的表達(dá),抑制宮頸癌細(xì)胞的增殖,促進(jìn)細(xì)胞凋亡。MIF和CAB39基因與腫瘤細(xì)胞遷移和侵襲能力的獲得密切相關(guān)[7,16],本研究下一步將重點(diǎn)關(guān)注miR-451a干擾MIF和CAB39基因表達(dá)后對(duì)宮頸癌細(xì)胞上皮間充質(zhì)轉(zhuǎn)化(EMT)過(guò)程的影響,探討其可能的分子機(jī)制。同時(shí),本研究也將通過(guò)收集臨床宮頸癌組織表達(dá),檢測(cè)miR-451a的表達(dá),研究其在診療及預(yù)后的生物標(biāo)志物價(jià)值。

綜上所述,過(guò)表達(dá)miR-451a后,宮頸癌細(xì)胞增殖能力顯著下降,細(xì)胞凋亡明顯增加,其可能的分子機(jī)制為miR-451a干擾MIF和CAB39蛋白的表達(dá),這為臨床治療宮頸癌提供了新靶點(diǎn)。

[1] Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017[J]. CA Cancer J Clin,2017,67(1):7-30.

[2] Patadji S, Li Z, Pradhan D,etal. Significance of high-risk HPV detection in women with atypical glandular cells on Pap testing: Analysis of 1857 cases from an academic institution[J]. Cancer,2016, 125(3):205-211.

[3] Gonzalez-Quintana V, Palma-Berre L, Campos-Parra AD,etal. MicroRNAs are involved in cervical cancer development, progression, clinical outcome and improvement treatment response (Review)[J]. Oncol Rep,2016,35(1):3-12.

[4] Li Y, Wang J, Dai X,etal. miR-451 regulates FoxO3 nuclear accumulation through Ywhaz in human colorectal cancer[J]. Am J Transl Res,2015,7(12):2775-2785.

[5] Su Z, Zhao J, Rong Z,etal. MiR-451, a potential prognostic biomarker and tumor suppressor for gastric cancer[J]. Int J Clin Exp Pathol,2015,8(8):9154-9160.

[6] Zang WQ, Yang X, Wang T,etal. MiR-451 inhibits proliferation of esophageal carcinoma cell line EC9706 by targeting CDKN2D and MAP3K1[J]. World J Gastroenterol,2015,21(19):5867-5876.

[7] Guo R, Gu J, Zhang Z,etal. MiR-451 promotes cell proliferation and metastasis in pancreatic cancer through targeting CAB39[J]. Biomed Res Int,2017,2017:2381482.

[8] Liu W, Liu SY, He YB,etal. MiR-451 suppresses proliferation, migration and promotes apoptosis of the human osteosarcoma by targeting macrophage migration inhibitory factor[J]. Biomed Pharmacother,2017,87:621-627.

[9] Chitkara D, Mittal A, Mahato RI. miRNAs in pancreatic cancer: therapeutic potential, delivery challenges and strategies[J]. Adv Drug Deliv Rev,2015,81:34-52.

[10] Zhang LF, Jiang S, Liu MF. MicroRNA regulation and analytical methods in cancer cell metabolism[J]. Cell Mol Life Sci,2017, 74(16):2929-2941.

[11] Yin P, Peng R, Peng H,etal. MiR-451 suppresses cell proliferation and metastasis in A549 lung cancer cells[J]. Mol Biotechnol,2015,57(1):1-11.

[12] Gu X, Li JY, Guo J,etal. Influence of mir-451 on drug resistances of paclitaxel-resistant breast cancer cell line[J]. Med Sci Monit,2015,21:3291-3297.

[13] Zhang F, Huang W, Sheng M,etal. MiR-451 inhibits cell growth and invasion by targeting CXCL16 and is associated with prognosis of osteosarcoma patients[J]. Tumour Biol,2015,36(3):2041-2048.

[14] O’Reilly C, Doroudian M, Mawhinney L,etal. Targeting MIF in cancer: therapeutic strategies, current developments, and future opportunities[J]. Med Res Rev,2016,36(3):440-460.

[15] Tian Y, Nan Y, Han L,etal. MicroRNA miR-451 downregulates the PI3K/AKT pathway through CAB39 in human glioma[J]. Int J Oncol,2012,40(4):1105-1112.

[16] Richard V, Kindt N, Saussez S. Macrophage migration inhibitory factor involvement in breast cancer [J]. Int J Oncol,2015,47(5):1627-1633.

EffectofmiR-451aoverexpressiononproliferationandapoptosisofcervicalcancercellsanditsmolecularmechanism

ZHANG Maona1*,ZHANG Hong1,ZHANG Jun1,ZHANG Lei2

(1DepartmentofPathology,EzhouHospital,People’sHospital,WuhanUniversity,Ezhou436000,China;2DepartmentofPathology,People’sHospitalofHenanProvince,ZhengzhouUniversity;*Correspondingauthor,E-mail:zmn0306@126.com)

ObjectiveTo explore the effect of miR-451a overexpression on the proliferation and apoptosis of cervical cancer cell lines SiHa and C33A and its possible molecular mechanisms.MethodsThe cervical cancer cell lines were divided into miR-NC group(transfected with miR-NC) and miR-451a group(transfected with miR-451a mimics). The expression of miR-451a, macrophage migration inhibitory factor(MIF) and calcium binding protein 39(CAB39) mRNA was detected by qPCR. The expression of MIF, CAB39, p-PI3K and p-AKT protein was analyzed by Western blotting. Cell cycle and apoptosis were detected by flow cytometry. MTT assay and plate cloning experiment were used to detect the cell proliferation ability.ResultsCompared with miR-NC group, the expression of miR-451a was significantly increased in miR-451a group(P<0.01), while the expression of MIF and CAB39 mRNA was significantly decreased(P<0.05), and the expression of MIF, CAB39, p-PI3K and p-AKT protein were significantly decreased. The cell cycle progression was significantly inhibited in miR-451a group(P<0.05), the apoptosis rate was significantly increased(P<0.05), and the cell proliferation ability was significantly decreased(P<0.05).ConclusionOverexpression of miR-451a can inhibit the proliferation of cervical cancer cells and promote the apoptosis, which may be related to down-regulation of MIF and CAB39 protein.

microRNA-451a; cervical cancer; cell proliferation; cell apoptosis

張茂娜,女,1987-03生,碩士研究生,E-mail:zmn0306@126.com

2017-07-29

R737.33

A

1007-6611(2017)11-1145-04

10.13753/j.issn.1007-6611.2017.11.012