李麗平，徐丹，葉俊，吳舜，馬松林( 華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬武漢中心醫(yī)院新洲院區(qū)，武漢430000； 華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬武漢中心醫(yī)院)

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miR-219-5p對(duì)胰腺癌細(xì)胞增殖、凋亡和侵襲的影響及機(jī)制

李麗平1，徐丹2，葉俊1，吳舜1，馬松林2
(1 華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬武漢中心醫(yī)院新洲院區(qū)，武漢430000；2 華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬武漢中心醫(yī)院)

目的 探討miR-219-5p對(duì)胰腺癌細(xì)胞增殖、凋亡和侵襲的影響及機(jī)制。方法 采用實(shí)時(shí)熒光定量PCR技術(shù)(qPCR)檢測(cè)miR-219-5p在胰腺癌細(xì)胞系Panc-1、AsPC-1及Bxpc-3和正常胰腺導(dǎo)管上皮細(xì)胞系HPDE6-C7中的表達(dá)。選取Panc-1細(xì)胞系，分成兩組，對(duì)照組(NC組)轉(zhuǎn)染陰性對(duì)照序列scramble，miR-219-5p組轉(zhuǎn)染miR-219-5p mimics,MTT實(shí)驗(yàn)和流式細(xì)胞儀分析兩組細(xì)胞增殖和凋亡情況，采用Transwell實(shí)驗(yàn)測(cè)定兩組細(xì)胞侵襲能力，Western blotting法檢測(cè)表皮生長(zhǎng)因子受體(EGFR)和裂解型Caspase 3(Cleaved Caspase-3)蛋白表達(dá)。結(jié)果 miR-219-5p在胰腺癌細(xì)胞系Panc-1、AsPC-1及Bxpc-3中的相對(duì)表達(dá)量低于正常胰腺導(dǎo)管上皮細(xì)胞系HPDE6-C7(P均<0.01)；miR-219-5p組在72、96及120 h 490 nm處的OD值低于NC組(P均<0.01)，細(xì)胞凋亡率高于NC組(P均<0.01)；miR-219-5p組、NC組細(xì)胞侵襲數(shù)分別為(75.3±6.9)、(172.5±10.5)個(gè)/視野，兩組細(xì)胞侵襲數(shù)比較，P<0.01；miR-219-5p組、NC組EGFR相對(duì)表達(dá)量分別為0.31±0.09、1.0，兩組比較，P<0.01；miR-219-5p組、NC組Cleaved Caspase 3蛋白相對(duì)表達(dá)量分別為2.7±0.15、1.0，兩組比較，P<0.01。結(jié)論 miR-219-5p在胰腺癌細(xì)胞系中呈低表達(dá)，miR-219-5p過(guò)表達(dá)可抑制胰腺癌細(xì)胞增殖和侵襲，并誘導(dǎo)細(xì)胞凋亡；其機(jī)制可能與miR-219-5p下調(diào)EGFR蛋白和上調(diào)Cleaved Caspase-3蛋白表達(dá)有關(guān)。

胰腺癌；細(xì)胞增殖；細(xì)胞侵襲；細(xì)胞凋亡；微小RNA-219-5p

MicroRNA(miRNA)是一類天然存在的長(zhǎng)19～25 nt的非編碼小分子RNA，通常與靶基因mRNA 3′UTR結(jié)合而阻止轉(zhuǎn)錄或降解mRNA[1]。miRNA在細(xì)胞分化、生長(zhǎng)、死亡等生長(zhǎng)發(fā)育過(guò)程中發(fā)揮重要作用[2]。目前已證實(shí)，miRNA的異常表達(dá)與多種疾病有關(guān)，特別是在腫瘤領(lǐng)域。多數(shù)miRNA在腫瘤組織中表達(dá)下降，可能作為癌基因或抑癌基因與腫瘤發(fā)生發(fā)展密切相關(guān)[3]。胰腺癌是消化系統(tǒng)常見(jiàn)的惡性腫瘤之一，具有預(yù)后差、易轉(zhuǎn)移、惡性程度高等特點(diǎn)，全球范圍內(nèi)胰腺癌在所有腫瘤中病死率占第八位，每年約有266 000例死于胰腺癌[4]。目前已發(fā)現(xiàn)有多種miRNA在胰腺癌中表達(dá)下調(diào)，如miRNA-96、miRNA-141等[5,6]。miRNA-96通過(guò)直接靶向KRAS降低胰腺癌細(xì)胞遷移和侵襲[5]。miR-141表達(dá)下調(diào)與胰腺癌腫瘤大小、TNM分期及轉(zhuǎn)移有關(guān)[6]。研究發(fā)現(xiàn)，miR-219-5p過(guò)表達(dá)抑制膠質(zhì)母細(xì)胞瘤等腫瘤細(xì)胞的增殖、侵襲、遷移過(guò)程[7]。miR-219-5p可能作為抑癌基因參與腫瘤的生長(zhǎng)過(guò)程，但對(duì)于miR-219-5p是否參與胰腺癌發(fā)展，目前尚不清楚。2016年1月～2017年1月，我們觀察了miR-219-5p在胰腺癌細(xì)胞系中表達(dá)及其對(duì)細(xì)胞增殖、侵襲、凋亡能力的影響，同時(shí)研究了對(duì)EGFR、Cleaved Caspase-3蛋白表達(dá)的調(diào)控作用?，F(xiàn)報(bào)告如下。

1 材料與方法

1.1 材料 胰腺癌細(xì)胞系Panc-1、AsPC-1、Bxpc-3及正常胰腺導(dǎo)管上皮細(xì)胞系HPDE6-C7均購(gòu)自武漢大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)醫(yī)學(xué)中心，RPMI1640培養(yǎng)基、胎牛血清、胰蛋白酶及TRIzol均購(gòu)自美國(guó)BD公司，EGFR、Caspase3及GAPDH均購(gòu)自Cell signaling公司，羊抗兔二抗購(gòu)自武漢博士德生物科技有限公司，miRNA-219-5p mimics及scramble均由上海吉瑪生物科技有限公司合成。

1.2 細(xì)胞培養(yǎng)、轉(zhuǎn)染及分組 將胰腺癌細(xì)胞系Panc-1、AsPC-1、Bxpc-3及正常胰腺導(dǎo)管上皮細(xì)胞系HPDE6-C7加入到RPMI1640培養(yǎng)基，于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)，經(jīng)48 h后消化傳代。將Panc-1細(xì)胞系分成兩組，NC組和miR-219-5p組，采用Lipofectamine 2000 reagent (Invitrogen, USA)分別轉(zhuǎn)染miRNA-219-5p scramble及mimics，miR-219 mimics 轉(zhuǎn)染序列：miR-219-5p mimics sense 5′-AAAAGAATTCCCACTTCCCACTCCAGACATT-3′，antisense 5′-AAAGCGGCCGCCCCTCACTTCTCCGTAACCC-3′。

1.3 miR-219-5p表達(dá)檢測(cè) 采用All-in-One microRNA抽提試劑盒提取Panc-1、AsPC-1、Bxpc-3及HPDE6-C7細(xì)胞系的miRNAs，ABI Prism 7700 system 的SYBR Green Reagents (TaKaRa, Tokyo, Japan)定量real-time PCR(qRT-PCR)，在ABI 7500實(shí)時(shí)定量PCR儀中，以U6小核RNA作為內(nèi)參，使用2-ΔΔCt方法定量，量化miR-219-5p相對(duì)表達(dá)水平。

1.4 細(xì)胞增殖情況檢測(cè) 采用MTT法。將NC組和miR-219-5p組細(xì)胞經(jīng)培養(yǎng)后消化成單細(xì)胞懸液，按2×103/孔接種于96孔板上，按200 μL每孔標(biāo)準(zhǔn)培養(yǎng)，在培養(yǎng)后0、24、48、72、96及120 h后，按每孔20 μL的標(biāo)準(zhǔn)加入MTT溶液，用酶標(biāo)儀在490 nm波長(zhǎng)處測(cè)定各孔的吸光度，繪制MTT增殖曲線。

1.5 細(xì)胞凋亡率測(cè)算 采用流式細(xì)胞術(shù)。用Annexin V/PI 染色檢測(cè)，將NC組和miR-219-5p組細(xì)胞消化成單細(xì)胞懸液后，PBS清洗2次，并使用Binding Buffer重懸，加入相應(yīng)比例的Annexin V抗體，避光染色10 min后加入適量PBS溶液及PI染料，流式細(xì)胞儀檢測(cè)Annexin V陽(yáng)性細(xì)胞比例確定細(xì)胞凋亡情況。

1.6 細(xì)胞侵襲情況檢測(cè) 采用Transwell法。NC組和miR-219-5p組各取2×104個(gè)細(xì)胞，接種于碳酸磷脂表面，于37 ℃下培養(yǎng)24 h，用1%多聚甲醛與膜下面的細(xì)胞結(jié)合并用0.2%結(jié)晶紫溶液染色，隨機(jī)取10個(gè)視野(200×),計(jì)算穿過(guò)膜的細(xì)胞數(shù)量，實(shí)驗(yàn)重復(fù)3次，取平均值。

1.7 EGFR和Cleaved Caspase-3蛋白表達(dá)檢測(cè) 采用Western blotting法。將NC組和miR-219-5p組細(xì)胞經(jīng)RIPA細(xì)胞裂解液冰上裂解30 min后，變性、上樣，以每孔30 μg總蛋白上樣，濃縮膠80 V電泳40 min，分離膠100 V電泳2 h。常規(guī)濕法轉(zhuǎn)膜，加入EGFR、Cleaved Caspase-3及GAPDH一抗，濃度為1∶300,一抗孵育過(guò)夜，二抗(1∶500)于37 ℃孵育4 h，PBST漂洗3次，ECL液顯影，Quantity One 1-D分析軟件對(duì)蛋白質(zhì)印跡條帶進(jìn)行定量。目的蛋白相對(duì)表達(dá)量=目的蛋白測(cè)定值/ GAPDH，實(shí)驗(yàn)重復(fù)3次，取平均值。

2 結(jié)果

2.1 不同胰腺癌細(xì)胞系中miR-219-5p表達(dá)比較 qPCR結(jié)果顯示，胰腺癌細(xì)胞系Panc-1、AsPC-1及Bxpc-3 miR-219-5p的相對(duì)表達(dá)量分別為0.27±0.023、0.33±0.029、0.43±0.033，正常胰腺導(dǎo)管上皮細(xì)胞系HPDE6-C7中miR-219-5p相對(duì)表達(dá)量為1.0，胰腺癌細(xì)胞系Panc-1、AsPC-1及Bxpc-3中miR-219-5p的相對(duì)表達(dá)量均低于正常胰腺導(dǎo)管上皮細(xì)胞系HPDE6-C7(P均<0.01)。

2.2 各組細(xì)胞增殖情況及細(xì)胞凋亡率比較 qPCR測(cè)定miR-219-5p組miR-219-5p相對(duì)表達(dá)量為10.25±0.45，NC組為1.0，兩組比較，P<0.01。MTT實(shí)驗(yàn)檢測(cè)結(jié)果顯示，miR-219-5p組與NC組0 h時(shí)490 nm處OD值分別為0.17±0.02、0.16±0.04，兩組比較，P>0.05；24 h時(shí)OD值分別為0.28±0.05、0.27±0.04，兩組比較，P>0.05；48 h時(shí)OD值分別為0.43±0.07、0.66±0.09，兩組比較，P>0.05；72 h時(shí)OD值分別為0.73±0.09、1.49±0.13，兩組比較，P<0.05；96 h時(shí)OD值分別為1.29±0.17、2.45±0.26，兩組比較，P<0.01；120 h時(shí)OD值分別為2.22±0.21、3.83±0.29，兩組比較，P<0.001。流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示，miR-219-5p組、NC組細(xì)胞凋亡率分別為(15.75±2.42)%、(5.5±0.70)%，兩組細(xì)胞凋亡率比較，P<0.01。

2.3 miR-219-5p組、NC組細(xì)胞侵襲情況比較 Transwell實(shí)驗(yàn)顯示，200倍視野下，miR-219-5p組、NC組穿膜細(xì)胞數(shù)分別為(75.3 ± 6.9)、(172.5±10.5)個(gè)/視野，兩組比較，P<0.01。

2.4 miR-219-5p組、NC組EGFR和Cleaved Caspase-3蛋白表達(dá)比較 miR-219-5p組、NC組EGFR相對(duì)表達(dá)量分別為0.31±0.09、1.0，兩組比較，P<0.01；miR-219-5p組、NC組Cleaved Caspase-3蛋白相對(duì)表達(dá)量分別為2.7±0.15、1.0，兩組比較，P<0.01。

3 討論

胰腺癌是常見(jiàn)的惡性腫瘤之一，在癌癥病死率中位列第四[8]。目前針對(duì)胰腺癌的治療以傳統(tǒng)手術(shù)、放療、化療為主，但缺乏更有效的治療方式[9]。miRNA是一類與腫瘤密切相關(guān)的小分子RNA，目前已有多個(gè)miRNA被證實(shí)參與胰腺癌細(xì)胞轉(zhuǎn)移、增殖、凋亡等過(guò)程，如miRNA-96、miRNA-141等[5,6]。miRNA-219-5p已被證實(shí)與多種腫瘤發(fā)生發(fā)展有關(guān)，通過(guò)靶向雌激素受體抑制甲狀腺乳頭狀癌細(xì)胞增殖、遷移，促進(jìn)凋亡[10]；通過(guò)靶向PRKCI抑制舌鱗狀細(xì)胞癌生長(zhǎng)和轉(zhuǎn)移[11]等。胰腺癌特異型性miRNAs及其靶點(diǎn)的識(shí)別有助于了解其在腫瘤發(fā)生中的作用。本研究發(fā)現(xiàn)miR-219-5p在胰腺癌細(xì)胞系中表達(dá)下降，過(guò)表達(dá)后能明顯抑制胰腺癌細(xì)胞增殖和侵襲能力，并誘導(dǎo)細(xì)胞凋亡，這一機(jī)制是通過(guò)降低EGFR、提高Cleaved Caspase-3蛋白表達(dá)實(shí)現(xiàn)的。

信號(hào)轉(zhuǎn)導(dǎo)通路紊亂可致細(xì)胞增殖失控，是腫瘤發(fā)生發(fā)展的重要原因[12]。PI3K/Akt、Ras/Raf/MEK信號(hào)轉(zhuǎn)導(dǎo)通路通常在細(xì)胞內(nèi)發(fā)揮抑制凋亡、促進(jìn)增殖的作用，其過(guò)度激活極易導(dǎo)致腫瘤發(fā)生，他們往往是由發(fā)生基因突變的信號(hào)通路組成因子或上游的受體酪氨酸激酶所介導(dǎo)[13]。受體酪氨酸激酶包括EGFR、血小板衍化生長(zhǎng)因子、纖維細(xì)胞生長(zhǎng)因子、胰島素受體等[14]。研究顯示，45%的膠質(zhì)母細(xì)胞瘤患者受體酪氨酸激酶介導(dǎo)的信號(hào)通路被激活是由于EGFR表達(dá)失調(diào)所致[7]。EGFR在胰腺癌等多種實(shí)體瘤中表達(dá)過(guò)度的現(xiàn)象廣泛存在，表明其表達(dá)異常與腫瘤細(xì)胞增殖、轉(zhuǎn)移、侵襲有關(guān)[15]。EGFR異常表達(dá)可能由miRNA失調(diào)引起，且已被驗(yàn)證為多個(gè)miRNA的靶基因。如在膠質(zhì)母細(xì)胞瘤中，miRNA-7通過(guò)抑制EGFR表達(dá)和阻礙PI3K/Akt信號(hào)通路激活，從而降低細(xì)胞存活率和侵襲能力[16]。更重要的是，在膠質(zhì)母細(xì)胞瘤中miR-219-5p通過(guò)直接綁定結(jié)合EGFR 3′UTR位點(diǎn)而降低EGFR表達(dá)，使EGFR介導(dǎo)的PI3K/Akt、Ras/Raf/MEK信號(hào)途徑受抑制，由此降低細(xì)胞增殖、生長(zhǎng)和轉(zhuǎn)移能力[7]。本研究中，miR-219-5p過(guò)表達(dá)可引起EGFR蛋白水平降低，我們猜測(cè)在胰腺癌中miR-219-5p是直接與EGFR基因3′UTR位點(diǎn)特異性結(jié)合，負(fù)調(diào)控EGFR轉(zhuǎn)錄后水平，抑制了PI3K/Akt、Ras/Raf/MEK信號(hào)通路，因而減弱細(xì)胞增殖和侵襲能力。

細(xì)胞凋亡是細(xì)胞程序性死亡，是調(diào)控機(jī)體發(fā)育、清除受損細(xì)胞、維持內(nèi)環(huán)境的重要機(jī)制，其失衡是腫瘤發(fā)生發(fā)展的一大重要因素。細(xì)胞凋亡主要通過(guò)死亡受體介導(dǎo)的細(xì)胞凋亡途徑、線粒體途徑、內(nèi)質(zhì)網(wǎng)途徑三種方式[17]。Caspase-3是這三種通路的關(guān)鍵執(zhí)行酶，細(xì)胞在受到刺激信號(hào)后經(jīng)過(guò)一系列級(jí)聯(lián)反應(yīng)使無(wú)活性的Procaspase-3被激活，裂解為有活性的Caspase-3，最終誘導(dǎo)細(xì)胞凋亡[18]。本研究中，miR-219-5p過(guò)表達(dá)誘導(dǎo)胰腺癌細(xì)胞凋亡，同時(shí)Cleaved Caspase-3表達(dá)量上升，說(shuō)明Caspase-3被激活，細(xì)胞凋亡被誘導(dǎo)。我們猜測(cè)，miR-219-5p誘導(dǎo)細(xì)胞凋亡可能是通過(guò)死亡受體途徑、線粒體途徑或內(nèi)質(zhì)網(wǎng)途徑，亦可能同時(shí)執(zhí)行兩種或三種途徑，但具體的作用途徑還需進(jìn)一步研究。

綜上所述，miR-219-5p在胰腺癌中作為抑癌基因是通過(guò)上調(diào)EGFR蛋白、下調(diào)Caspase-3蛋白參與抑制細(xì)胞增殖和侵襲、促進(jìn)細(xì)胞凋亡。該基因功能的鑒定有望為胰腺癌靶向治療提供新的靶點(diǎn)，為腫瘤治療提供新的途徑。

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Effects of miR-219-5p on proliferation, apoptosis, and invasion of pancreatic carcinoma cells

LILiping1,XUDan,YEJun,WUShun,MASonglin

(1TheCentralHospitalofWuhanAffiliatedtoTongjiMedicalCollegeofHuazhongUniversityofScienceandTechnology,Wuhan430000,China)

Objective To investigate the effects of miRNA-219-5p on the proliferation, apoptosis, and invasion of pancreatic carcinoma cells and its mechanism. Methods Real-time fluorescence quantitative PCR (qPCR) was carried out to determine miR-219-5p expression in the pancreatic carcinoma cell lines Panc-1, AsPC-1, and Bxpc-3 and normal pancreatic ductal epithelial cell line HPDE6-C7. Further, Panc-1 cells were chosen and were divided into two groups: the normal control group (NC group) (transfection with miR-219-5p scramble) and miR-219-5p group (transfection with miR-219-5p mimics). The cell proliferation and apoptosis were detected by MTT assay and flow cytometry. The invasion ability was checked by transwell assay. The expression of epidermal growth factor receptor (EGFR) and Cleaved Caspase-3 proteins was examined by Western blotting. Results MiR-219-5p expression was lower in pancreatic carcinoma cell lines Panc-1, AsPC-1, and Bxpc-3, as compared with that in the pancreatic ductal epithelial cell line HPDE6-C7 (allP<0.01). The OD490nm of the miR-219-5p group was significantly lower than that of the NC group at 72, 96, and 120 h (allP<0.01); the apoptosis rate was higher (allP<0.01). The invasive cells were (75.3±6.9)/Hp in the miR-219-5p group, which was significantly lower than that [(172.5±10.5)/Hp] in the NC group (P<0.01). The expressive level of EGFR in the miR-219-5p group was significantly lower than NC group (0.31±0.09 vs. 1.0,P<0.01). The expression of Cleaved Caspase-3 was up-regulated in the miR-219-5p group as compared with that of NC group (2.7±0.15 vs. 1.0,P<0.01).Conclusions The miR-219-5p is lowly expressed in pancreatic carcinoma cell lines. The over-expression of miR-219-5p can inhibit the proliferation and invasion of pancreatic carcinoma cells, and induce the apoptosis, possibly though the down-regulated protein expression of EGFR and up-regulated protein expression of Cleaved Caspase-3.

pancreatic carcinoma; cell proliferation; cell invasion; apoptosis; miRNA-219-5p

湖北省自然科學(xué)基金面上項(xiàng)目(CFB578)；武漢市衛(wèi)計(jì)委課題(WX14A04)。

李麗平(1977-)，在讀碩士，住院醫(yī)師，主要研究方向?yàn)橐认倌[瘤的基礎(chǔ)與臨床。E-mail:lipingli2017@126.com

馬松林(1979-)，碩士，主治醫(yī)師，主要研究方向?yàn)橐认倌[瘤的基礎(chǔ)與臨床。E-mail:songlinma8887@sina.com

10.3969/j.issn.1002-266X.2017.25.004

R735.9

A

1002-266X(2017)25-0012-04

2017-04-01)