劉宇亭　沈祥國　蘇長青　孫斌　李兆申　徐燦

200433　上海，第二軍醫(yī)大學(xué)長海醫(yī)院消化內(nèi)科(劉宇亭、沈祥國、李兆申、徐燦)；第二軍醫(yī)大學(xué)東方肝膽外科醫(yī)院分子腫瘤研究室(蘇長青、孫斌)；東部戰(zhàn)區(qū)空軍機(jī)關(guān)醫(yī)院(劉宇亭)

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·論著·

聯(lián)合使用miR-34a及miR-let7對胰腺癌細(xì)胞生物學(xué)特性的影響

劉宇亭沈祥國蘇長青孫斌李兆申徐燦

200433上海，第二軍醫(yī)大學(xué)長海醫(yī)院消化內(nèi)科(劉宇亭、沈祥國、李兆申、徐燦)；第二軍醫(yī)大學(xué)東方肝膽外科醫(yī)院分子腫瘤研究室(蘇長青、孫斌)；東部戰(zhàn)區(qū)空軍機(jī)關(guān)醫(yī)院(劉宇亭)

【摘要】目的探討聯(lián)合使用兩種具有抑癌作用的microRNA(miRNA)同時轉(zhuǎn)染人胰腺癌細(xì)胞對其生物學(xué)特性的影響。方法采用脂質(zhì)體法將miR-34a及miR-let7單獨(dú)或同時轉(zhuǎn)染胰腺癌PANC1、SW1990細(xì)胞及正常胰腺腺泡AR42J細(xì)胞，以轉(zhuǎn)染陰性對照miRNA(miR-NC)組及未轉(zhuǎn)染組作為對照。應(yīng)用qRT-PCR法檢測各組細(xì)胞miR-34a及miR-let7的表達(dá)，MTT法檢測細(xì)胞增殖，Transwell小室檢測細(xì)胞遷移和侵襲能力，流式細(xì)胞儀檢測細(xì)胞凋亡。結(jié)果MiR-34a轉(zhuǎn)染組、miR-let7轉(zhuǎn)染組、雙轉(zhuǎn)染組細(xì)胞的miR-34a、miR-let7表達(dá)水平均較miR-NC轉(zhuǎn)染組及未轉(zhuǎn)染組顯著上調(diào)，差異有統(tǒng)計學(xué)意義(P值均<0.05)，表明miRNA成功轉(zhuǎn)染了細(xì)胞。雙轉(zhuǎn)染組的PANC1、SW1990細(xì)胞增殖活性分別為(0.665±0.010)%、(0.638±0.030)%，較miR-NC轉(zhuǎn)染組的(0.974±0.030)%、(0.971±0.050)%，miR-let7轉(zhuǎn)染組的(0.888±0.050)%、(0.863±0.060)%顯著被抑制，差異有統(tǒng)計學(xué)意義(P值均<0.05)，較miR-34a轉(zhuǎn)染組的(0.795±0.060)%、(0.793±0.060)%下降，但差異無統(tǒng)計學(xué)意義。AR42J細(xì)胞的增殖活性無顯著變化，差異無統(tǒng)計學(xué)意義。PANC1細(xì)胞miR-34a轉(zhuǎn)染組、miR-let7轉(zhuǎn)染組的穿膜細(xì)胞數(shù)分別為(103.70±3.28)、(100.70±1.76)個/200倍視野，較miR-NC轉(zhuǎn)染組的(231.30±2.60)個/200倍視野及未轉(zhuǎn)染組的(153.70±2.60)個/200倍視野顯著減少，雙轉(zhuǎn)染組穿膜細(xì)胞數(shù)為(61.67±3.18)個/200倍視野，又較兩個單轉(zhuǎn)染組顯著減少，差異均有統(tǒng)計學(xué)意義(P值均<0.01)。細(xì)胞遷移實(shí)驗(yàn)與侵襲實(shí)驗(yàn)的結(jié)果一致。SW1990細(xì)胞侵襲及遷移能力的變化與PANC1一致。PANC1細(xì)胞的miR-34a轉(zhuǎn)染組、miR-let7轉(zhuǎn)染組、雙轉(zhuǎn)染組、miR-NC轉(zhuǎn)染組、未轉(zhuǎn)染組的細(xì)胞凋亡率分別為(16.66±1.27)%、(15.46±0.33)%、(23.35±1.80)%、(9.33±0.31)%、(8.83±0.36)%。兩單轉(zhuǎn)染組的細(xì)胞凋亡率較miR-NC轉(zhuǎn)染組、未轉(zhuǎn)染組顯著增加；雙轉(zhuǎn)染組又較miR-let7組顯著增加，差異有統(tǒng)計學(xué)意義(P值均<0.05)，而雙轉(zhuǎn)染組與miR-34a轉(zhuǎn)染組的差異無統(tǒng)計學(xué)意義。結(jié)論雙轉(zhuǎn)染的胰腺癌細(xì)胞增殖活性、細(xì)胞遷移和侵襲力均較單轉(zhuǎn)染細(xì)胞顯著下降，而細(xì)胞凋亡率較單轉(zhuǎn)染細(xì)胞顯著增加，雙轉(zhuǎn)染能夠發(fā)揮更加顯著的協(xié)同抗腫瘤作用。

【關(guān)鍵詞】胰腺腫瘤；細(xì)胞系，腫瘤；miR-34a；miR-let7；轉(zhuǎn)染；分子生物學(xué)

胰腺癌惡性程度高,手術(shù)切除率低，預(yù)后差,總體5年生存率不到5%[1]。腫瘤基因治療及免疫治療的出現(xiàn)為人們攻克胰腺癌提供了新的思路和視野。microRNA(miRNA)是一種高度保守的短鏈非編碼RNA，能夠在轉(zhuǎn)錄后水平調(diào)節(jié)基因的表達(dá)。到目前為止，已有超過1800余種miRNA被陸續(xù)報道[2-4]，且大量的研究已經(jīng)證實(shí)miRNA的異常表達(dá)與腫瘤的發(fā)生相關(guān)[5]，從而為腫瘤的治療提供了新的方向。有研究報道，miR-34a的靶基因在調(diào)節(jié)細(xì)胞凋亡、細(xì)胞周期阻滯、DNA修復(fù)、血管生成等方面有非常重要的作用，在胰腺癌中表達(dá)缺失或者明顯下調(diào)，發(fā)揮抑癌基因的作用[6-7]。miR-let7存在于正常胰腺腺泡細(xì)胞，在低分化胰腺癌組織中表達(dá)缺失，提高胰腺癌細(xì)胞株miR-let7的表達(dá)水平可顯著抑制癌細(xì)胞增殖[8]。因此本研究聯(lián)合使用兩種miRNA轉(zhuǎn)染胰腺癌細(xì)胞，觀察其對胰腺癌細(xì)胞增殖的影響，為胰腺癌的治療提供新的思路。

材料與方法

一、材料

人胰腺癌細(xì)胞系PANC1、SW1990細(xì)胞及正常胰腺腺泡細(xì)胞AR42J由第二軍醫(yī)大學(xué)附屬長海醫(yī)院消化內(nèi)科實(shí)驗(yàn)室提供。高糖DMEM培養(yǎng)基和胎牛血清購自美國Gibico公司，miR-34a、miR-let7 mimic及陰性對照miRNA (miR-NC)由廣東銳博技術(shù)有限公司合成，LipofectamineTM2000試劑和RNA抽提試劑Trizol購自美國Invitrogen公司，實(shí)時熒光定量PCR試劑盒購自Applied Biosystems公司，MTT試劑盒購自美國Sigama公司，Transwell小室購自美國Millipore公司，聚碳酸酯膜基質(zhì)膠和細(xì)胞凋亡檢測試劑盒購自美國BD公司。

二、方法

1．細(xì)胞培養(yǎng)及miRNA轉(zhuǎn)染：胰腺癌PANC1細(xì)胞及SW1990細(xì)胞由高糖DMEM培養(yǎng)基(含10%FBS)培養(yǎng)，正常胰腺腺泡細(xì)胞AR42J由1640(含10% FBS)培養(yǎng)基培養(yǎng)，取對數(shù)生長期細(xì)胞接種于培養(yǎng)板內(nèi)。實(shí)驗(yàn)分5組：miR-34a轉(zhuǎn)染組、miR-let7轉(zhuǎn)染組、miR-34a和miR-let7雙轉(zhuǎn)染組、miR-NC轉(zhuǎn)染組及未轉(zhuǎn)染對照組，每組設(shè)6個復(fù)孔，按miRNA mimics及LipofectamineTM2000試劑說明書操作。轉(zhuǎn)染后用無血清培養(yǎng)基饑餓處理細(xì)胞4 h，更換含10%FBS的DMEM培養(yǎng)液繼續(xù)培養(yǎng)24 h進(jìn)行下一步實(shí)驗(yàn)。

2．qRT-PCR法檢測細(xì)胞miRNA表達(dá)：取上述各組轉(zhuǎn)染24 h細(xì)胞，應(yīng)用Trizol收集細(xì)胞總RNA，在紫外分光光度儀上檢測RNA濃度及純度后采用逆轉(zhuǎn)錄試劑盒反轉(zhuǎn)錄獲得cDNA，采用實(shí)時熒光定量PCR試劑盒進(jìn)行擴(kuò)增，以U6為內(nèi)參。PCR反應(yīng)條件：95℃ 10 min，95℃ 15 s、60℃ 30 s、72℃ 30 s，40個循環(huán)。所有反應(yīng)設(shè)3個復(fù)孔。由儀器自帶軟件獲取Ct值，以公式2-ΔΔCt計算mRNA相對表達(dá)量。

3．MTT法檢測細(xì)胞增殖活性：取上述各組轉(zhuǎn)染24 h細(xì)胞，加MTT 20 μl繼續(xù)培養(yǎng)4 h，加DMSO 150 ml終止反應(yīng)，上酶標(biāo)儀檢測490 nm處吸光度值(A490值)，以單加培養(yǎng)液的孔調(diào)零。細(xì)胞增殖率=實(shí)驗(yàn)組A490值/對照組A490值×100%。實(shí)驗(yàn)重復(fù)3次，取均值。

4．Transwell小室檢測細(xì)胞的遷移、侵襲力：預(yù)先將基質(zhì)膠與DMEM 培養(yǎng)液按1∶5混合稀釋后，以100 μl/室鋪入小室隔膜上，37℃過夜風(fēng)干、凝固。取上述各組轉(zhuǎn)染細(xì)胞，上室加4×104個細(xì)胞，容積200 μl。下室加500 μl含20% FBS的DMEM培養(yǎng)液，培養(yǎng)24 h后用結(jié)晶紫染色20～30 min，PBS洗滌后用棉簽輕輕擦拭小室內(nèi)未穿膜細(xì)胞，顯微鏡下取3個200倍視野，計數(shù)穿膜細(xì)胞數(shù)。實(shí)驗(yàn)重復(fù)3次，取均值。參考駱廣濤等[9]方法，檢測細(xì)胞遷移能力，Transwell小室隔膜不鋪基質(zhì)膠。

5．流式細(xì)胞儀檢測細(xì)胞凋亡：取對數(shù)生長期各組轉(zhuǎn)染細(xì)胞，用不含EDTA的胰酶消化，收集細(xì)胞后用預(yù)冷PBS洗兩遍，1 500 r/min離心10 min，用Binding Buffer重懸，置冰上加入Annexin V輕混勻后加入PI染色，上流式細(xì)胞儀測細(xì)胞凋亡。實(shí)驗(yàn)重復(fù)3次。

三、統(tǒng)計學(xué)處理

結(jié)果

一、MiRNA轉(zhuǎn)染細(xì)胞的鑒定

PANC1、SW1990、AR42J細(xì)胞轉(zhuǎn)染miRNA后，miR-34a轉(zhuǎn)染組、雙轉(zhuǎn)染組細(xì)胞的miR-34a表達(dá)水平均較miR-let7轉(zhuǎn)染組、miR-NC轉(zhuǎn)染組及未轉(zhuǎn)染組顯著上調(diào)；miR-let7轉(zhuǎn)染組、雙轉(zhuǎn)染組細(xì)胞的miR-let7表達(dá)水平均較miR-34a轉(zhuǎn)染組、miR-NC轉(zhuǎn)染組、未轉(zhuǎn)染組顯著上調(diào)，差異均有統(tǒng)計學(xué)意義(P值均<0.05)。見表1、2。表明miRNA成功轉(zhuǎn)染了細(xì)胞。

二、各組轉(zhuǎn)染細(xì)胞增殖的變化

雙轉(zhuǎn)染組的PANC1、SW1990細(xì)胞增殖活性較miR-NC轉(zhuǎn)染組、miR-let7轉(zhuǎn)染組顯著被抑制，差異均有統(tǒng)計學(xué)意義(P值均<0.05)，而與miR-34a轉(zhuǎn)染組的差異無統(tǒng)計學(xué)意義。AR42J細(xì)胞各轉(zhuǎn)染組間的增殖活性無顯著變化，差異無統(tǒng)計學(xué)意義(表3)。

表1　3種細(xì)胞系中各轉(zhuǎn)染組miR-34a的相對表達(dá)量

表2　3種細(xì)胞系中各轉(zhuǎn)染組miR-let7的相對表達(dá)量

三、各組轉(zhuǎn)染細(xì)胞遷移和侵襲力的變化

胰腺癌PANC1細(xì)胞及SW1990細(xì)胞的miR-34a轉(zhuǎn)染組及miR-let7轉(zhuǎn)染組的穿膜細(xì)胞數(shù)較miR-NC轉(zhuǎn)染組及未轉(zhuǎn)染組顯著減少，雙轉(zhuǎn)染組的穿膜細(xì)胞數(shù)又較兩個單轉(zhuǎn)染組顯著減少，差異均有統(tǒng)計學(xué)意義(P值均<0.01)。見圖1、2，表4、5。提示同時轉(zhuǎn)染miR-34a及miR-let7能夠更顯著地抑制胰腺癌細(xì)胞的侵襲及遷移能力。

四、各組轉(zhuǎn)染細(xì)胞的凋亡變化

胰腺癌PANC1細(xì)胞的miR-34a轉(zhuǎn)染組、miR-let7轉(zhuǎn)染組、雙轉(zhuǎn)染組、miR-NC轉(zhuǎn)染組、未轉(zhuǎn)染組的細(xì)胞凋亡率分別為(16.66±1.27)%、(15.46±0.33)%、(23.35±1.80)%、(9.33±0.31)%、(8.83±0.36)%(圖3)。miR-34a轉(zhuǎn)染組、miR-let7轉(zhuǎn)染組的細(xì)胞凋亡率較miR-NC轉(zhuǎn)染組、未轉(zhuǎn)染組顯著增加；雙轉(zhuǎn)染組又較miR-let7轉(zhuǎn)染組顯著增加，差異有統(tǒng)計學(xué)意義(P值均<0.05)，而與miR-34a轉(zhuǎn)染組的差異無統(tǒng)計學(xué)意義，提示雙轉(zhuǎn)染兩種miRNA能進(jìn)一步提高胰腺癌細(xì)胞的凋亡率。

表3　3種細(xì)胞系中各轉(zhuǎn)染組細(xì)胞增殖活性±s)

圖1　miR-34a轉(zhuǎn)染組(1A)、miR-let7轉(zhuǎn)染組(1B)、雙轉(zhuǎn)染組(1C)、miR-NC轉(zhuǎn)染組(1D)、未轉(zhuǎn)染組(1E)PANC1細(xì)胞的遷移(上)和侵襲能力(下)變化(×200)

圖2　miR-34a轉(zhuǎn)染組(2A)、miR-let7轉(zhuǎn)染組(2B)、雙轉(zhuǎn)染組(2C)、miR-NC轉(zhuǎn)染組(2D)、未轉(zhuǎn)染組(2E)SW1990細(xì)胞的遷移(上)和侵襲能力(下)變化(×200)

細(xì)胞系miR-34a轉(zhuǎn)染組miR-let7轉(zhuǎn)染組雙轉(zhuǎn)染組miR-NC轉(zhuǎn)染組未轉(zhuǎn)染組PANC1103.70±3.28100.70±1.7661.67±3.18231.30±2.60153.70±2.60SW199094.67±3.18117.00±4.5051.00±2.08131.30±4.37160.70±4.63

表5　細(xì)胞遷移實(shí)驗(yàn)中各轉(zhuǎn)染組穿過小室膜的細(xì)胞數(shù)(個/200倍視野，±s)

圖3　PANC1細(xì)胞的miR-34a轉(zhuǎn)染組(3A)、miR-let7轉(zhuǎn)染組(3B)、雙轉(zhuǎn)染組(3C)、miR-NC轉(zhuǎn)染組(3D)、未轉(zhuǎn)染組(3E)的細(xì)胞凋亡

討論

近年來隨著分子生物學(xué)領(lǐng)域的快速發(fā)展，胰腺癌的早診、早治已受到廣泛重視，針對胰腺癌分子水平的生物治療技術(shù)也取得了一定進(jìn)展，為胰腺癌的治療提供了更新的理論基礎(chǔ)和實(shí)踐工具。盡管如此，多數(shù)新療法依然未得到廣泛認(rèn)可，致使胰腺癌的早診、早治依然停留在臨床前研究階段，胰腺癌患者的生存率并無顯著改善[10]。究其原因，一方面在于腫瘤的發(fā)生、發(fā)展機(jī)制并不十分明確，另一方面在于多數(shù)分子靶向治療往往針對單一靶點(diǎn)，而未形成協(xié)同的抗腫瘤治療網(wǎng)絡(luò)，以致抗腫瘤效果不理想。

MicroRNA是一種內(nèi)源性的小分子非編碼RNA，在轉(zhuǎn)錄后水平對細(xì)胞新陳代謝的多個環(huán)節(jié)進(jìn)行調(diào)控。其表達(dá)譜的改變在胰腺癌細(xì)胞的增殖、凋亡、侵襲、分化等過程中發(fā)揮重要作用，與胰腺癌的發(fā)生、發(fā)展、預(yù)后等有著密切的聯(lián)系。有些miRNA，例如miR-34a、miR-203、miR-124、miR-let7在胰腺癌中表達(dá)降低，發(fā)揮抑癌基因作用[11-12]，而另外一些miRNA，如miR-10a、miR-10b、miR-198、miR-21在胰腺癌組織中高表達(dá)，發(fā)揮癌基因的作用[13-14]。文獻(xiàn)報道，miR-34a在胰腺癌中表達(dá)缺失或明顯下調(diào)發(fā)揮著抑癌基因的作用，其靶基因CDK4、cyclins及E2F在調(diào)節(jié)細(xì)胞周期、細(xì)胞凋亡、DNA修復(fù)中有非常重要的作用[6,15-16]。Lize等[16]報道，miR-let7c及miR-let7f表達(dá)在人胰腺癌的穿刺標(biāo)本中明顯降低，提高胰腺癌細(xì)胞株miR-let7的表達(dá)可顯著抑制癌細(xì)胞增殖[8]。miR-let7還與結(jié)腸癌、肝癌等有關(guān)[17]，能夠抑制腫瘤細(xì)胞增殖，其作用可能與Ras、HMGA2和CRD-BP/IMP1的表達(dá)相關(guān)[18-20]。將miR-34a作用于胰腺癌細(xì)胞可抑制其皮下移植瘤的生長[21]，利用質(zhì)?；蚵《緮y帶miRNA感染胰腺癌細(xì)胞增強(qiáng)其miR-let7的表達(dá)，能顯著抑制癌細(xì)胞的增殖[8]。Lou等[22]報道，通過溶瘤腺病毒共表達(dá)miR-34a及IL-24能夠發(fā)揮協(xié)同抗腫瘤活性，顯著提高二者抗腫瘤作用。因此，本研究同時使用兩種具有抑癌作用的miR-34a及miR-let7 轉(zhuǎn)染胰腺癌細(xì)胞，結(jié)果顯示雙轉(zhuǎn)染細(xì)胞增殖活性、細(xì)胞遷移和侵襲力均較單轉(zhuǎn)染細(xì)胞顯著下降，而細(xì)胞凋亡率較單轉(zhuǎn)染細(xì)胞顯著增加，能夠發(fā)揮更加顯著的協(xié)同抗腫瘤作用，為胰腺癌的治療提供新的思路。

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(本文編輯：屠振興)

Effect of the combination of miR-34a and miR-let7 on the biological properties of pancreatic cancer cells

LiuYuting,ShenXiangguo,SuChangqing,SunBin,LiZhaoshen,XuCan.DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433China

【Abstract】ObjectiveTo investigate the influence on biological characteristics in human pancreatic cancer cells after beding transfected by two anti-carcinoma miRNAs at the same time. MethodsPancreatic cancer cells PANC1, SW1990 and normal pancreatic cells AR42J were transfected by miR-34a and(or) miR-let7 by liposome. Cells transfected with negative control miRNA (miR-NC) and untransfected were as controls. The expression of miR-34a and miR-let7 were detected by real-time fluorescent quantitative RT-PCR. The cell proliferation was detected by MTT test and the migration and invasion were evaluated by transwell assay. The apoptosis rate was measured by flow cytometric analysis. ResultsAfter being transfected with miRNAs, the expression of miR-34a and miR-let7 in double transfection group (miR-34a and miR-let7 were transfected at the same time). miR-34a transfection group, miR-let7 transfection group was significantly up-regulated than those in miRNA-NC transfection group and untransfected group in PANC1 cells, SW1990 cells and AR42J cells, repectively. The difference which was statistically significant(P<0.05)indicating that cells were successfully transfected. The cell proliferation in double transfection group of PANC1 cells and SW1990 cells were (0.665±0.01, 0.6375±0.03), which were significantly inhibited compared with (0.974±0.03, 0.971±0.05) in miR-NC group and (0.8875±0.05, 0.8625±0.06) in miR-let7 group. The difference was statistically significant(P<0.05). The cell proliferation activity in double transfection group was lower than those in miR-34a group (0.795±0.06, 0.7925±0.06), but did not have statistically significant difference. There was no significant change in AR42J cells. Cell invasion assay showed that the number of PANC1 cells permeating substrate membrane in miR34a group (103.7±3.28) and miR-let7 group (100.7±1.76) were significantly fewer than miR-NC group (231.3±2.6) and untransfected group (153.7±2.6). The number of cells permeating substrate membrane in double transfection group(61.67±3.18)was fewer than miR-34a group and miR-let7 group, respectively. The difference was statistically significant(P<0.01).The migration test had consistent results with invasion test. The changes of invasion and migration in SW1990 cells were similar to those in PANC1 cells. The apoptosis rate of PANC1 cells in miR-34a group, miR-let7 group, double transfection group, miR-NC group and untransfected group was (16.66±1.27)%,(15.46±0.33)%,(23.35±1.80)%,(9.33±0.31)% and (8.83±0.36)% respectively. Single transfection group had higher apoptosis rate than miR-NC group and untransfected group (P<0.05). Double transfection group had a significantly higher apoptosis rate than miR-let7 group (P<0.05), while there was no significant difference between double transfection group and miR-34a group. ConclusionsThe cell proliferation, invasion and migration in double miRNAs transfected pancreatic cancer cells were significantly down-regulated compared with those in single miRNA transfected cells, while apoptosis rate in double miRNAs transfection group was higher than single miRNA transfection group. Thus, the combination of two anti-cancer miRNAs may exert a more significant synergistic antitumor effect.

【Key words】Pancreatic neoplasms;Cell line, tumor;miR-34a;miR-let7;Transfection;Molecular biology

(收稿日期：2015-12-15)

Corresponding author:Xu Can, Email: xxcc211@126.com

基金項目：國家自然科學(xué)基金面上項目(81372673)；上海市浦江人才計劃項目(14PJD004)

通信作者：徐燦，Email: xxcc211@126.com

DOI:10.3760/cma.j.issn.1674-1935.2016.02.004

Fund program：National Natural Sceince Foundation of China(81372673)；Shanghai Pujiang Talent Program Item(14PJD004)