抑制miR-421表達(dá)增強(qiáng)宮頸癌細(xì)胞放療敏感性*

潘雨露， 吳淑霞， 石翠格， 任興業(yè)△

(1濟(jì)南市第五人民醫(yī)院婦產(chǎn)科， 山東 濟(jì)南 250022； 2中國(guó)計(jì)劃生育研究所細(xì)胞生物學(xué)實(shí)驗(yàn)室， 北京 100081)

抑制miR-421表達(dá)增強(qiáng)宮頸癌細(xì)胞放療敏感性*

潘雨露1， 吳淑霞1， 石翠格2， 任興業(yè)1△

(1濟(jì)南市第五人民醫(yī)院婦產(chǎn)科， 山東 濟(jì)南 250022；2中國(guó)計(jì)劃生育研究所細(xì)胞生物學(xué)實(shí)驗(yàn)室， 北京 100081)

目的： 研究抑制miR-421表達(dá)增強(qiáng)宮頸癌細(xì)胞放療敏感性的分子機(jī)制。方法: 運(yùn)用脂質(zhì)體2000將miR-421 inhibitor轉(zhuǎn)染4株宮頸癌細(xì)胞系HeLa、SiHa、C33A和CaSki，以轉(zhuǎn)染陰性核苷酸為對(duì)照，real-time PCR檢測(cè)子宮內(nèi)膜上皮細(xì)胞ESC細(xì)胞和4株宮頸癌細(xì)胞中miR-421的表達(dá)水平；轉(zhuǎn)染的細(xì)胞經(jīng)不同劑量電離輻射(0、2、4、6和8 Gy)處理48 h后運(yùn)用MTT法、ELISA和Annexin V-FITC/PI染色法聯(lián)合流式細(xì)胞術(shù)分別測(cè)定細(xì)胞活力、乳酸脫氫酶(LDH)漏出率和細(xì)胞凋亡率；運(yùn)用Western blot檢測(cè)細(xì)胞中cleaved caspase-9、caspase-9、cleaved PARP、PARP、Bcl-2和Bax蛋白的水平。結(jié)果: miR421在ESC細(xì)胞中低表達(dá)，而在4株宮頸癌細(xì)胞中高表達(dá)，轉(zhuǎn)染miR-421 inhibitor后miR-421顯著低于陰性對(duì)照組(P<0.05)。相同條件下，輻照后低表達(dá)miR-421的宮頸癌細(xì)胞活力和LDH漏出率顯著低于陰性對(duì)照組，72 h的細(xì)胞凋亡率明顯增加(P<0.05)。Western blot結(jié)果顯示，電離輻射后與陰性對(duì)照組比較，低表達(dá)miR-421的宮頸癌細(xì)胞中cleaved caspase-9、cleaved PARP和Bcl-2的蛋白水平明顯增高，而B(niǎo)ax蛋白水平顯著降低(P<0.05)。結(jié)論: miR-421在正常子宮內(nèi)膜上皮ESC細(xì)胞中低表達(dá)，而在宮頸癌細(xì)胞系中高表達(dá)；下調(diào)miR-421表達(dá)能抑制宮頸癌細(xì)胞的活力，顯著增強(qiáng)宮頸癌細(xì)胞的輻射敏感性，其機(jī)制至少部分通過(guò)激活caspase-9細(xì)胞凋亡通路進(jìn)而促進(jìn)抗凋亡蛋白Bcl-2表達(dá)和抑制促凋亡蛋白Bax表達(dá)而實(shí)現(xiàn)。

宮頸癌； miR-421； 電離輻射； 放療敏感性； 細(xì)胞凋亡

宮頸癌是一種嚴(yán)重威脅婦女健康與生命的惡性腫瘤，其發(fā)病率和死亡率位居所有腫瘤的第2位。資料顯示全球每年約有50萬(wàn)新增病例，其中我國(guó)每年有約13.1萬(wàn)宮頸癌患者[1-2]，發(fā)病率和死亡率呈逐年增加及年輕化趨勢(shì)[3]。目前手術(shù)和放療是宮頸癌的主要治療方案，但轉(zhuǎn)移、放療耐受的宮頸癌卻很難徹底根治。因此,尋找和研發(fā)增強(qiáng)宮頸癌放療敏感性的靶向藥物迫在眉睫。

微小RNAs(microRNAs, miRNAs，miR)是一類(lèi)長(zhǎng)度約21～25 nt的非編碼小RNA分子，在細(xì)胞增殖、分化、凋亡和腫瘤發(fā)生等生理或病理學(xué)進(jìn)程中扮演著重要的角色[4]。大量的研究表明miRNAs的異常表達(dá)與多種腫瘤的發(fā)生發(fā)展密切相關(guān)[5-7]。已有研究證實(shí)，miR-210[8]、miR-193b[9]和miR-221/miR-222[10]異常表達(dá)與多種腫瘤細(xì)胞的放療耐受有關(guān)。研究證實(shí)miR-421在神經(jīng)母細(xì)胞瘤、胰腺癌、前列腺癌等惡性腫瘤中高表達(dá)[11-12]，表明miR-421是一種腫瘤促進(jìn)因子。Zhou等[13]發(fā)現(xiàn)下調(diào)miR-421表達(dá)顯著抑制胃癌細(xì)胞異種移植腫瘤的生長(zhǎng)。最新研究發(fā)現(xiàn)miR-421在MCF-7/ADR耐藥細(xì)胞中的表達(dá)明顯降低[14]。目前，miR-421在宮頸癌發(fā)生發(fā)展中的生物學(xué)功能以及下調(diào)miR-421的表達(dá)能否增強(qiáng)宮頸癌細(xì)胞的放療敏感性尚無(wú)報(bào)道。本研究旨在觀察下調(diào)miR-421表達(dá)對(duì)宮頸癌細(xì)胞放療敏感性的影響，并探討其分子機(jī)制，為以miR-421為靶點(diǎn)聯(lián)合放療的宮頸癌治療方案提供科學(xué)依據(jù)。

材 料 和 方 法

1 細(xì)胞系與試劑

子宮內(nèi)膜上皮細(xì)胞系ESC購(gòu)自ATCC；宮頸癌細(xì)胞系HeLa、SiHa、C33A和CaSki購(gòu)自中國(guó)科學(xué)院上海細(xì)胞生物研究所；DMEM培養(yǎng)基(Gibco)、胎牛血清(fetal bovine serum，F(xiàn)BS)和胰酶購(gòu)自北京同立海源生物科技有限公司；雙抗、MTT和DMSO購(gòu)自Sigma；乳酸脫氫酶(lactate dehydrogenase，LDH) ELISA試劑盒和Annexin V-FITC/PI細(xì)胞凋亡檢測(cè)試劑盒購(gòu)自南京博泰生物技術(shù)有限公司；miR-421 inhibitor和陰性對(duì)照核苷酸片段購(gòu)自Sigma；microRNA提取試劑盒和TaqMan? miRNA試劑盒購(gòu)自Applied Biosystems；脂質(zhì)體2000購(gòu)自Invitrogen；抗cleaved caspase-9、caspase-9、cleaved PARP和PARP抗體購(gòu)自Cell Signaling Technology；抗Bcl-2和Bax抗體購(gòu)自Abcam；抗β-actin抗體購(gòu)自Santa Curz；BCA試劑盒購(gòu)自Thermo。

2 方法

2.1 細(xì)胞培養(yǎng) ESC、HeLa、SiHa、C33A和CaSki細(xì)胞培養(yǎng)于含10% FBS、1%青霉素和1%鏈霉素的DMEM培養(yǎng)基中，置于37 ℃、5% CO2及95%濕度的培養(yǎng)箱，細(xì)胞融合度達(dá)到80%～90%時(shí)進(jìn)行細(xì)胞傳代。

2.2 細(xì)胞轉(zhuǎn)染 將生長(zhǎng)良好的HeLa、C33A、SiHa和CaSki細(xì)胞消化計(jì)數(shù)后，以細(xì)胞密度為每孔2.5×104個(gè)接種到6孔板中，置于CO2培養(yǎng)箱中待細(xì)胞融合度達(dá)到60%時(shí)按照脂質(zhì)體2000說(shuō)明書(shū)進(jìn)行miR-421 inhibitor細(xì)胞轉(zhuǎn)染。轉(zhuǎn)染的細(xì)胞培養(yǎng)4 h后換成完全培養(yǎng)基；以轉(zhuǎn)染陰性對(duì)照核苷酸片段為陰性對(duì)照，細(xì)胞置于培養(yǎng)箱中培養(yǎng)用于后續(xù)實(shí)驗(yàn)。

2.3 Real-time PCR 轉(zhuǎn)染的細(xì)胞培養(yǎng)48 h時(shí)刮下細(xì)胞、離心收集，按照mirVanaTMmiRNA分離試劑盒提取細(xì)胞miRNA；運(yùn)用TaqMan? miRNA檢測(cè)試劑盒檢測(cè)miR-421的表達(dá)水平，采用SYBR GreenⅡ熒光染料法和IQ5TMReal-Time PCR Detection System(Bio-Rad)進(jìn)行real-time PCR數(shù)據(jù)分析，內(nèi)參照采用U6，miR-421的相對(duì)表達(dá)量用2-ΔΔCt表示。進(jìn)行3次獨(dú)立重復(fù)實(shí)驗(yàn)。

2.4 電離輻射 采用X射線照射，放射劑量率為2.0 Gy/min。運(yùn)用0.5 mm鋁過(guò)濾器對(duì)X射線進(jìn)行過(guò)濾。實(shí)驗(yàn)分為3種細(xì)胞：未轉(zhuǎn)染對(duì)照細(xì)胞、轉(zhuǎn)染miR-421 inhibitor的細(xì)胞和陰性對(duì)照細(xì)胞。細(xì)胞經(jīng)不同劑量(0、2、4、6和8 Gy)輻射處理，用于后續(xù)研究。

2.5 MTT實(shí)驗(yàn)檢測(cè)細(xì)胞活性 放射處理的細(xì)胞經(jīng)消化計(jì)數(shù)后，以細(xì)胞密度為每孔2.5×104個(gè)接種到96孔板中，置于 CO2培養(yǎng)箱中培養(yǎng)48 h后進(jìn)行MTT實(shí)驗(yàn)，同時(shí)設(shè)空白對(duì)照組。每孔加入20 μL 5 g/L MTT，37 ℃繼續(xù)培養(yǎng)4 h，棄掉培養(yǎng)液，每孔加入150 μL DMSO，置于搖床上室溫振蕩5 min，用酶標(biāo)儀測(cè)定490 nm處的吸光度(A)值，按下式計(jì)算細(xì)胞活力抑制率：細(xì)胞增殖抑制率(%)=(對(duì)照組A值-實(shí)驗(yàn)組A值)/實(shí)驗(yàn)組A值×100%。

2.6 測(cè)定LDH漏出率 放射處理的細(xì)胞以每孔2.5×104個(gè)接種于96孔板中，同時(shí)設(shè)細(xì)胞自然釋放對(duì)照組(DMEM培養(yǎng)基)和最大釋放對(duì)照組(0.8% Triton X-100)，每組各設(shè)5個(gè)復(fù)孔。37 ℃、5% CO2溫箱中培養(yǎng)48 h，最大釋放孔在結(jié)束培養(yǎng)前加入終濃度為0.8%的Triton X-100，作用45 min。分別從對(duì)應(yīng)的培養(yǎng)板中取100 μL上清加入ELISA板中，37 ℃孵育10 min后, 加入新鮮配制的100 μL LDH底物溶液，室溫避光反應(yīng)15 min，每孔加入1 mol/L 檸檬酸終止液30 μL，用酶標(biāo)儀在490 nm 處讀取各孔A值, 并計(jì)算LDH漏出率：LDH漏出率(%)=(實(shí)驗(yàn)組A值-自然釋放對(duì)照組A值)/(最大釋放對(duì)照組A值-自然釋放對(duì)照組A值)×100%。

2.7 細(xì)胞凋亡的檢測(cè) 轉(zhuǎn)染的細(xì)胞消化計(jì)數(shù)后接種到6孔板中，培養(yǎng)24 h后經(jīng)5 Gy輻射處理；細(xì)胞培養(yǎng)72 h后棄掉培養(yǎng)基，PBS洗滌，細(xì)胞經(jīng)無(wú)EDTA的胰酶消化收集細(xì)胞，加入PBS制成細(xì)胞懸液。按照Annexin V試劑盒說(shuō)明書(shū)操作，先加入500 μL的binding buffer重懸細(xì)胞，再加入5 μL FITC標(biāo)記的Annexin V和5 μL PI混勻，室溫下避光孵育15 min，流式細(xì)胞儀檢測(cè)細(xì)胞凋亡。

2.8 Western blot實(shí)驗(yàn) 照射處理的細(xì)胞經(jīng)消化，離心收集細(xì)胞，加入RIPA(50 mmol/L Tris-HCl，pH 7.5，150 mmol/L NaCl，1% NP-40，0.5%脫氧膽酸鈉，0.1% SDS)重懸細(xì)胞，超聲破碎、12 000 r/min，4 ℃離心10 min，按照BCA試劑盒說(shuō)明書(shū)測(cè)定蛋白濃度。取30 μg進(jìn)行SDS-PAGE，將蛋白轉(zhuǎn)移到PVDF膜上，5%脫脂奶粉室溫封閉，孵育 I 抗(cleaved caspase-9、caspase-9、cleaved PARP、PARP、Bcl-2和Bax抗體)，以β-actin為內(nèi)參照，4 ℃過(guò)夜。洗膜、孵育 II 抗，室溫1 h。ECL顯影后掃描，蛋白相對(duì)表達(dá)量經(jīng)內(nèi)參照校正后經(jīng)ImageJ軟件分析。

3 統(tǒng)計(jì)學(xué)處理

每個(gè)實(shí)驗(yàn)進(jìn)行3次獨(dú)立重復(fù)試驗(yàn)。應(yīng)用SPSS 17.0統(tǒng)計(jì)軟件進(jìn)行相關(guān)數(shù)據(jù)分析，結(jié)果用平均值±標(biāo)準(zhǔn)差(mean±SD)表示，兩組間的比較采用t檢驗(yàn)，多組間的比較采用單因素方差分析(one-way ANOVA)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 miR-421在宮頸癌細(xì)胞中的表達(dá)

如圖1所示，miR-421在HeLa、SiHa、C33A和CaSki細(xì)胞中高表達(dá)，而在ESC細(xì)胞中低表達(dá)，分別與4株宮頸癌細(xì)胞比較差異具有統(tǒng)計(jì)學(xué)意義(P<0.01)；當(dāng)宮頸癌細(xì)胞轉(zhuǎn)染miR421 inhibitor后miR-421顯著降低，與陰性對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P<0.01)。

Figure 1.The level of miR-421 expression was detected by real-time PCR in the cervical cancer cells. Mean±SD.n=3.**P<0.01vsESC;##P<0.01vsnegative control.

圖1 Real-time PCR檢測(cè)宮頸癌細(xì)胞中miR-421的表達(dá)水平

2 電離輻射對(duì)低表達(dá)miR-421宮頸癌細(xì)胞活性的影響

如圖2所示，隨著輻射劑量的增加4株宮頸癌細(xì)胞活性逐漸降低；相同條件下，轉(zhuǎn)染miR-421 inhibitor的宮頸癌細(xì)胞經(jīng)不同劑量電離輻射后，細(xì)胞活性顯著低于陰性對(duì)照組，其差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。這提示抑制miR-421的表達(dá)可提高宮頸癌細(xì)胞對(duì)電離輻射的敏感性。

3 電離輻射對(duì)低表達(dá)miR-421宮頸癌細(xì)胞LDH漏出率的影響

如圖3所示，隨著輻射劑量的增加，4株宮頸癌細(xì)胞中LDH漏出率逐漸降低；相同條件下，轉(zhuǎn)染miR-421 inhibitor的宮頸癌細(xì)胞經(jīng)不同劑量電離輻射后，LDH漏出率顯著低于陰性對(duì)照組，其差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)果提示抑制miR-421表達(dá)能抑制宮頸癌細(xì)胞增殖以及明顯提高宮頸癌細(xì)胞對(duì)電離輻射敏感性。

4 電離輻射對(duì)低表達(dá)miR-421宮頸癌細(xì)胞凋亡的影響

如圖4所示，5 Gy電離輻射能誘導(dǎo)4株宮頸癌細(xì)胞凋亡，HeLa、SiHa、C33A和CaSki細(xì)胞凋亡率分別為(24.7±3.3)%、(20.8±2.8)%、(19.9±3.0)%和(18.9±2.2)%；相同條件下，下調(diào)miR-421表達(dá)后HeLa、SiHa、C33A和CaSki細(xì)胞凋亡率分別為(46.1±5.5)%、(68.7±7.8)%、(56.1±6.5)%和(45.7±4.8)%，均顯著低于陰性對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。以上結(jié)果表明，抑制miR-421表達(dá)可促進(jìn)電離輻射誘導(dǎo)的宮頸癌細(xì)胞凋亡。

Figure 2.The effect of various doses of ionizing radiation on the viability of cervical cancer cells with miR-421 low expression. Mean±SD.n=3.*P<0.05,**P<0.01vsnegative control.

圖2 不同劑量電離輻射對(duì)低表達(dá)miR-421宮頸癌細(xì)胞活力的影響

Figure 3.The effect of miR-421 down-regulation on the LDH leakage rate of cervical cancer cells. Mean±SD.n=3.*P<0.05,**P<0.01vsnegative control.

圖3 下調(diào)miR-421表達(dá)對(duì)宮頸癌細(xì)胞LDH漏出率的影響

Figure 4.The effect of ionizing radiation on apoptosis of the cervical cancer cells with miR-421 low expression. Mean±SD.n=3.**P<0.01vsnegative control.

圖4 電離輻射對(duì)低表達(dá)miR-421宮頸癌細(xì)胞凋亡的影響

5 電離輻射對(duì)miR-421低表達(dá)宮頸癌細(xì)胞凋亡通路的影響

Western blot結(jié)果顯示，抑制miR-421表達(dá)促進(jìn)caspase-9和PARP蛋白發(fā)生裂解，提示caspase-9通路被激活；促凋亡蛋白Bcl-2的表達(dá)水平顯著增加，而抗凋亡蛋白Bax的表達(dá)水平顯著降低，提示抑制miR-421表達(dá)激活細(xì)胞凋亡通路，電離輻射能增強(qiáng)miR-421下調(diào)介導(dǎo)的細(xì)胞凋亡通路活性，調(diào)控Bcl-2/Bax表達(dá)，從而促進(jìn)細(xì)胞凋亡，見(jiàn)圖5、表1。本研究結(jié)果提示，抑制miR-421表達(dá)可能通過(guò)激活細(xì)胞凋亡通路誘導(dǎo)細(xì)胞凋亡從而增強(qiáng)宮頸癌細(xì)胞對(duì)放療的敏感性。

討 論

宮頸癌細(xì)胞對(duì)放療產(chǎn)生耐受?chē)?yán)重影響患者預(yù)后和生存率，鱗狀宮頸癌是宮頸癌的主要類(lèi)型，80%的宮頸癌患者屬于鱗狀宮頸癌，這類(lèi)腫瘤對(duì)放療敏感，但是宮頸癌細(xì)胞群中的小部分惡性腺瘤或腫瘤干細(xì)胞對(duì)放療耐受是導(dǎo)致腫瘤復(fù)發(fā)和預(yù)后不良的主要原因[15-16]。研究發(fā)現(xiàn)局部或遠(yuǎn)距離轉(zhuǎn)移性宮頸癌患者對(duì)放療產(chǎn)生抗性，導(dǎo)致預(yù)后不良[15]。因此，增強(qiáng)宮頸癌細(xì)胞的放療敏感性是改善患者預(yù)后的重要手段，尋找和研發(fā)增強(qiáng)腫瘤細(xì)胞對(duì)放療敏感性藥物迫在眉睫。

大量研究表明microRNA異常表達(dá)與腫瘤細(xì)胞放療耐受密切相關(guān)。研究發(fā)現(xiàn)let-7 miRNA過(guò)表達(dá)通過(guò)調(diào)控RAS和DNA損傷修復(fù)基因的表達(dá)增強(qiáng)肺癌細(xì)胞的放療敏感性[17]；miR-218低表達(dá)與宮頸癌惡性程度和預(yù)后不良相關(guān)[18]。因此，以micoRNA為靶點(diǎn)研發(fā)特異性增強(qiáng)宮頸癌細(xì)胞的放療敏感性是宮頸癌治療的熱點(diǎn)研究領(lǐng)域。目前以miR-34為靶點(diǎn)的原發(fā)性肝癌靶向藥物(NCT01829971)和以miR-122為靶點(diǎn)的慢性丙型肝炎靶向藥物(NCT01872936)正在進(jìn)行臨床I期和II期試驗(yàn)[19-20]。研究發(fā)現(xiàn)miR-421在胃癌組織中的表達(dá)水平顯著高于正常非癌旁組織，其在早期胃癌發(fā)生過(guò)程中扮演著重要作用[21]。本研究結(jié)果顯示，miR-421在正常子宮內(nèi)膜上皮細(xì)胞ESC中低表達(dá)，而在4株宮頸癌細(xì)胞系中高表達(dá)，提示miR-421在宮頸癌發(fā)生發(fā)展中具有重要作用。最新研究發(fā)現(xiàn)miR-421低表達(dá)與MCF-7/ADR細(xì)胞耐藥密切相關(guān)[14]。本研究發(fā)現(xiàn)，低表達(dá)miR-421的宮頸癌細(xì)胞經(jīng)電離輻射后細(xì)胞活性和LDH漏出率顯著低于陰性對(duì)照組，提示抑制miR-421表達(dá)能增強(qiáng)宮頸癌細(xì)胞的放療敏感性。

Figure 5.The effect of ionizing radiation on apoptosis pathway in the cervical cancer cells with miR-421 low expression.

圖5 電離輻射對(duì)miR-421低表達(dá)宮頸癌細(xì)胞凋亡通路的影響

表1 電離輻射對(duì)miR-421低表達(dá)宮頸癌細(xì)胞中細(xì)胞凋亡相關(guān)蛋白表達(dá)的影響

Table 1.The effect of ionizing radiation on expression of apoptosis-associated proteins in the cervical cancer cells with miR-421 low expression (Mean±SD.n=3)

Cellline GroupCleavedcaspase-9Caspase-9CleavedPARPPARPBcl-2BaxHeLaNegativecontrol0.17±0.021.02±0.120.34±0.051.10±0.060.31±0.021.05±0.07miR-421inhibition0.86±0.11**1.13±0.091.16±0.07**1.14±0.031.20±0.08**0.18±0.03**SiHaNegativecontrol0.11±0.010.90±0.070.25±0.070.89±0.110.58±0.081.82±0.11miR-421inhibition0.76±0.04**0.93±0.081.07±0.04**1.02±0.091.04±0.10*0.20±0.05**C33ANegativecontrol0.15±0.080.82±0.020.37±0.081.12±0.050.40±0.030.73±0.02miR-421inhibition0.88±0.05**0.89±0.072.04±0.22**1.08±0.081.42±0.10**0.12±0.08**CaSkiNegativecontrol0.09±0.010.60±0.050.18±0.060.70±0.070.08±0.000.66±0.02miR-421inhibition0.67±0.03**0.58±0.092.15±0.12**0.75±0.012.18±0.14**0.11±0.01**

*P<0.05，**P<0.01vsnegative control.

電離輻射殺死腫瘤細(xì)胞可能的分子機(jī)制在于激活細(xì)胞凋亡通路誘導(dǎo)細(xì)胞凋亡[22]。凋亡相關(guān)基因表達(dá)異?；蚬δ苋笔?dǎo)致細(xì)胞對(duì)外界刺激失敏，從而產(chǎn)生抗性。本研究發(fā)現(xiàn)miR-421低表達(dá)的宮頸癌細(xì)胞發(fā)生了細(xì)胞凋亡，經(jīng)電離輻射后細(xì)胞凋亡率顯著高于陰性對(duì)照組細(xì)胞，提示抑制miR-421表達(dá)能增強(qiáng)電離輻射介導(dǎo)的細(xì)胞凋亡，宮頸癌細(xì)胞對(duì)電離輻射的敏感性增加。死亡受體和線粒體介導(dǎo)的細(xì)胞凋亡是細(xì)胞凋亡主要的信號(hào)通路，caspase家族在細(xì)胞凋亡中具有重要作用，胞外死亡受體與相應(yīng)配體結(jié)合、DNA損傷、缺氧及生長(zhǎng)因子等激活caspase通路，導(dǎo)致細(xì)胞內(nèi)重要蛋白質(zhì)降解從而誘導(dǎo)細(xì)胞凋亡[23]。本研究發(fā)現(xiàn)miR-421低表達(dá)促進(jìn)caspase-9和PARP蛋白降解，提示caspase通路被激活；經(jīng)電離輻射后cleaved caspase-9和cleaved PARP蛋白表達(dá)水平顯著升高，抗凋亡蛋白Blc-2表達(dá)升高，而促凋亡蛋白Bax表達(dá)降低，細(xì)胞凋亡率明顯增高，提示抑制miR-421表達(dá)通過(guò)激活細(xì)胞凋亡通路誘導(dǎo)細(xì)胞凋亡，最終增強(qiáng)宮頸癌細(xì)胞對(duì)放療敏感性?；谝陨涎芯浚詍iR-421為靶向藥物聯(lián)合放療有望成為miR-421高表達(dá)宮頸癌患者的新型治療方案。

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(責(zé)任編輯： 盧 萍， 羅 森)

Inhibition of miR-421 expression enhances radiosensitivity of cervical cancer cells

PAN Yu-lu1, WU Shu-xia1, SHI Cui-ge2, REN Xing-ye1

(1DepartmentofObstetrics&Gynecology,TheFifthPeople’sHospitalofJinan,Jinan250022,China;2LaboratoryofCellBiology,InstituteofFamilyPlanninginChina,Beijing100081,China.E-mail:wuyuanrenxingye@163.com)

AIM: To investigate the molecular mechanism of inhibition of miR-421 expression promoting radiosensitivity in the cervical cancer cells. METHODS: Cervical cancer lines HeLa, SiHa, C33A and CaSki were transfected with miR-421 inhibitor or negative control nucleotide using Lipofectamine 2000 kit, and the levels of miR-421 expression in the cervical cancer lines and endometrial epithelium cell line ESC were detected by real-time PCR. These cells with transfection were exposed to various doses of X-ray (0, 2, 4, 6 and 8 Gy). After 48 h, the cell viability, LDH leakage rate and apoptotic rate were measured respectively by MTT assay, ELISA and flow cytometry with Annexin V-FITC/PI staining. The protein levels of cleaved caspase-9, caspase-9, cleaved PARP, PARP, Bcl-2 and Bax were monitored by Western blot. RESULTS: Low miR-421 levels was found in the ESC cells, while high miR-421 levels were observed in the HeLa, SiHa, C33A and CaSki cells. The level of miR-421 in the cells transfected with miR-421 inhibitor was significantly lower than that in negative control group (P<0.05). The viability and LDH leakage rate of the cervical cancer cells with low miR-421 expression were notablely lower than those in negative control group, and the apoptotic rate at 72 h was remarkablely increased (P<0.05) under the same conditions. The results of Western blot indicated that, after exposure to ionizing radiation, the protein levels of cleaved caspase-9, cleaved PARP and Bcl-2 were significantly increased, while the protein level of Bax was significantly decreased in the cervical cancer cells with low miR-421 expression compared with negative control group (P<0.05). CONCLUSION: miR-421 is lowly expressed in the normal endometrial epithelial cells, but highly expressed in the cervical cancer cells. Down-regulation of miR-421 expression significantly inhibits the growth and enhances the radiosensitivity of cervical cancer cells at least partly via activating caspase-9 apoptosis pathway, thus promoting Bcl-2 and inhibiting Bax expression.

Cervical cancer; miR-421; Ionizing radiation; Radiosensitivity; Apoptosis

1000- 4718(2017)05- 0798- 07

2016- 11- 02

2017- 01- 16

山東省醫(yī)藥衛(wèi)生資助項(xiàng)目(No. 2013WS0010)

R730.23

A

10.3969/j.issn.1000- 4718.2017.05.006

雜志網(wǎng)址： http://www.cjpp.net

△通訊作者 Tel: 0531-87197046; E-mail: wuyuanrenxingye@163.com