王夢(mèng)潔，孟碧，李皓，高飛，葉婷，劉陽(yáng)晨

(1蚌埠醫(yī)學(xué)院研究生院，安徽蚌埠233030；2泰興市人民醫(yī)院)

轉(zhuǎn)染miR-124模擬物的宮頸癌細(xì)胞株Siha放射敏感性變化及其機(jī)制探討

王夢(mèng)潔1，2，孟碧1，2，李皓2，高飛2，葉婷2，劉陽(yáng)晨2

(1蚌埠醫(yī)學(xué)院研究生院，安徽蚌埠233030；2泰興市人民醫(yī)院)

目的觀察過(guò)表達(dá)微小RNA124(miR-124)對(duì)宮頸癌Siha細(xì)胞放射敏感性的影響，并探討其機(jī)制。方法將宮頸癌Siha細(xì)胞分為觀察組和對(duì)照組，觀察組轉(zhuǎn)染miR-124 模擬物，對(duì)照組不轉(zhuǎn)染。采用實(shí)時(shí)熒光定量PCR法檢測(cè)兩組細(xì)胞miR-124和信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子3(STAT3) mRNA表達(dá)量。采用克隆形成實(shí)驗(yàn)檢測(cè)兩組細(xì)胞放射敏感性。采用流式細(xì)胞術(shù)檢測(cè)兩組細(xì)胞周期分布和放射后細(xì)胞凋亡率。結(jié)果觀察組、對(duì)照組D0分別為1.062、1.542 Gy，Dq分別為1.605、1.970 Gy,SF2分別為0.527、0.685。觀察組相對(duì)于對(duì)照組的放射增敏比為1.451。轉(zhuǎn)染后24h觀察組、對(duì)照組miR-124相對(duì)表達(dá)量分別為89.3±13.6、1.0±0.1，STAT3 mRNA相對(duì)表達(dá)量分別為0.3±0.03、1.0±0.1，兩組相比，P均<0.05。觀察組G0/G1期細(xì)胞比例為82.49%±1.97%、S期細(xì)胞比例11.87%±1.38%、G2/M期細(xì)胞比例為5.64%±0.72%；對(duì)照組分別為74.58%±1.28%、19.88%±0.26%、5.54%±1.05%。觀察組G0/G1期比例高于對(duì)照組組，S期比例低于對(duì)照組(P均<0.05)，兩組G2/M期細(xì)胞比例相比P>0.05。觀察組、對(duì)照組細(xì)胞X線照射后細(xì)胞凋亡率分別為45.87%±3.16%、37.27%±0.87%，兩組相比，P<0.05。結(jié)論過(guò)表達(dá)miR-124可能提高宮頸癌Siha細(xì)胞的放射敏感性。其機(jī)制可能是過(guò)表達(dá)miR-124會(huì)抑制宮頸癌Siha細(xì)胞STAT3表達(dá)，進(jìn)而阻滯細(xì)胞周期于G0/G1期，促進(jìn)細(xì)胞凋亡，從而提高宮頸癌Siha細(xì)胞的放射敏感性。

宮頸癌細(xì)胞；微小RNA124；信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子3；放射敏感性；細(xì)胞凋亡；細(xì)胞周期

Abstract:ObjectiveTo observe the effect of microRNA124 (miR-124) overexpression on the radiosensitivity of cervical cancer Siha cells and its mechanism.MethodsThe cervical cancer Siha cells were divided into the observation group and control group. The miR-124 mimics were transfected into the observation group. The expression levels of miR-124 and signal transducer and activator of transcription 3 (STAT3) in the two groups were detected by real-time fluorescent quantitative PCR. Colony formation assay was used to detect the radiosensitivity of the two groups cells. Clone formation assay was used to detect the cell radiosensitivity. Flow cytometry was used to detect the cell cycle distritution and apoptosis rate after radiation.ResultsThe observation group and the control group had the D0 parameters of 1.062 Gy and 1.542 Gy, the Dq parameters of 1.605Gy and 1.970Gy, the SF2 parameters of 0.527 and 0.685, respectively. Compared with the control group, the radiation sensitization enhancement ratio (SER) was 1.451. At 24 h after transfection, the relative expression levels of miR-124 in the observation group and the control group were 89.3±13.6 and 1.0±0.1, respectively, and the relative expression levels of STAT3 mRNA were 0.3±0.03 and 1.0±0.1, respectively; significant difference was found between these two groups (allP<0.05). In the observation group, the percentage of cells in G0/G1phase was 82.49%±1.97%, was 11.87%±1.38% in S phase, and 5.64%±0.72% in G2/M phase；meanwhile, they were 74.58%±1.28%, 19.88%±0.26%, 5.54%±1.05%, respectively, in the control group. Compared with the control group, the percentage of cells in G0/G1phase was higher, while the percentage of cells in S phase was lower, and significant difference was found between these two groups (allP<0.05). Compared with the control group, the percentage of cells in G2/M phase was not significantly different (P>0.05). The apoptosis rates after X-irradiation were 45.87%±3.16% in observation group, versus 37.27%±0.87% in the control group, and significant difference was found between these two groups (P<0.05).ConclusionsOverexpression of miR-124 may enhance radiosensitivity of cervical cancer Siha cells. The mechanism may be that overexpression of miR-124 can inhibit the expression of STAT3 in cervical cancer Siha cells, and then induce the cell cycle arrest in G0/G1phase, promote the apoptosis, and thereby enhance the radiosensitivity of cervical cancer Siha cells.

Keywords: cervical carcinoma cell; microRNA-124; signal transducer and activator of transcription 3; radiosensitivity; apoptosis; cell cycle

放療抵抗是導(dǎo)致腫瘤放療失敗的主要原因[1,2]。放療主要是通過(guò)損傷癌細(xì)胞的DNA，進(jìn)而導(dǎo)致癌細(xì)胞發(fā)生凋亡和不可逆性細(xì)胞周期阻滯[3,4]。miRNA在這一過(guò)程中起重要作用[5,6]。miR-124是一種高度保守的miRNA，在多種實(shí)體腫瘤中低表達(dá)，如頭頸部鱗狀細(xì)胞癌、膠質(zhì)細(xì)胞瘤、非小細(xì)胞肺癌、乳腺癌、肝癌等[7～11]，miR-124在腫瘤的發(fā)生發(fā)展過(guò)程中起著類(lèi)似于抑癌基因的作用。過(guò)表達(dá)miR-124可以提高上述腫瘤組織對(duì)放療的敏感性。但是宮頸癌細(xì)胞中miR-124表達(dá)情況及過(guò)表達(dá)miR-124對(duì)宮頸癌Siha細(xì)胞放射敏感性的影響尚未見(jiàn)文獻(xiàn)報(bào)道。本課題組前期研究中發(fā)現(xiàn)宮頸癌Siha細(xì)胞中miR-124表達(dá)下降。本研究觀察了過(guò)表達(dá)miR-124對(duì)宮頸癌SiHa細(xì)胞放射敏感性的影響，并探討其機(jī)制。現(xiàn)報(bào)告如下。

1 材料與方法

1.1 細(xì)胞株、材料及試劑 宮頸癌SiHa 細(xì)胞購(gòu)自上海中科院細(xì)胞庫(kù)，用RPMI-1640培養(yǎng)基(含10%胎牛血清)，置于37°C、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)，待細(xì)胞融合度達(dá)85%左右后經(jīng)胰酶消化傳代。RPMI-1640培養(yǎng)液、胎牛血清購(gòu)自美國(guó)Gibco公司；PBS、0.1%結(jié)晶紫染色液購(gòu)自美國(guó)Solarbio公司；細(xì)胞培養(yǎng)瓶、培養(yǎng)板購(gòu)自美國(guó)Corning公司；miR-124模擬物(序列：5′-UAAGGCACGCGGUGAAUGCCCAUUCACCGCGUGCCUUAUU-3′)購(gòu)自蘇州吉瑪基因股份有限公司；Lipofectamine2000、Trizol購(gòu)自美國(guó)Invitrogen公司；RT-PCR 試劑盒購(gòu)自日本TaKaRa公司；胰酶細(xì)胞消化液、青霉素-鏈霉素溶液、細(xì)胞周期與細(xì)胞凋亡檢測(cè)試劑盒、Annexin V-FITC凋亡檢測(cè)試劑盒購(gòu)自上海碧云天生物技術(shù)有限公司。

1.2 Siha細(xì)胞分組、miR-124模擬物轉(zhuǎn)染 對(duì)數(shù)期生長(zhǎng)期的Siha細(xì)胞，消化重懸后以4×105/孔鋪于6孔板。設(shè)觀察組和對(duì)照組。觀察組細(xì)胞貼壁后按照Lipofectamine2000轉(zhuǎn)染試劑盒說(shuō)明書(shū)操作，用30 nmol/L的miR-124 模擬物轉(zhuǎn)染Siha細(xì)胞6 h，然后替換為完全培養(yǎng)基培養(yǎng)繼續(xù)培養(yǎng)48 h；對(duì)照組不轉(zhuǎn)染，一直用完全培養(yǎng)基培養(yǎng)。

1.3 兩組細(xì)胞放射敏感性觀察 兩組細(xì)胞轉(zhuǎn)染后用胰酶消化、重懸，在6孔板上分別以200、500、1 000、3 000、5 000/孔鋪板，然后用0、2、4、6、8 Gy的X線照射，每個(gè)照射劑量和細(xì)胞數(shù)目的組合設(shè)3孔。照射方法：用6MV X射線直線加速器，大機(jī)頭旋轉(zhuǎn)180°從下往上垂直照射細(xì)胞，并在培養(yǎng)板下方加墊1 cm厚的凡士林膠，源皮距為100 cm。繼續(xù)培養(yǎng)一至兩周，待6孔板涌現(xiàn)肉眼可見(jiàn)的細(xì)胞集落時(shí)，PBS洗滌2次，乙醇固定15 min，再用PBS洗滌2次，0.1%的結(jié)晶紫染色20 min，在顯微鏡下計(jì)數(shù)細(xì)胞個(gè)數(shù)≥50的單克隆集落，根據(jù)公式：克隆形成率(PE)=克隆數(shù)/接種細(xì)胞數(shù)×100%；存活分?jǐn)?shù)(SF)=克隆數(shù)/(接種細(xì)胞數(shù)×未照射的PE)×100%，計(jì)算各組的細(xì)胞存活分?jǐn)?shù)SF，用GraphPad Prime 5.0軟件構(gòu)建Siha細(xì)胞劑量存活曲線，根據(jù)公式：y=1-(1-exp{-k×x} )^N擬合成多靶單擊模型，得到K和N這兩個(gè)參數(shù)，用公式[K=1/D0；lnN=Dq/D0；SER= D0(對(duì)照組)/D0(觀察組)]計(jì)算平均致死量(D0)、準(zhǔn)閾劑量(Dq)、照射劑量2 Gy下細(xì)胞存活分?jǐn)?shù)(SF2)和放射增敏比(SER)、。

1.4 兩組細(xì)胞miR-124、STAT3 mRNA檢測(cè) 用RT-PCR法檢測(cè)兩組細(xì)胞miR-124、STAT3 mRNA，操作按試劑盒說(shuō)明書(shū)進(jìn)行。每組測(cè)3孔。以U6為miR-124的內(nèi)參基因，GAPDH為STAT3的內(nèi)參基因。miR-124正向引物5′-GCTAAGGCACGCGGTG-3′，反向引物5′-GTGCAGGGTCCGAGGT-3′；U6正向引物5′-CTCGCTTCGGCAGCACATATACT-3′，反向引物 5′-ACGCTTCACGAATTTGCGTGTC-3′；STAT3正向引物5′-GAAGGACATCAGCGGTAAGA-3′，反向引物5′-AGATAGACCAGTGGAGACAC-3′；GAPDH正向引物5′-GGAGTCCACTGGCGTCTT-3′，反向引物 5′-GAGTCCTTCCACGATACCAA-3′。用2-ΔΔCt表示miR-124、STAT3 mRNA相對(duì)表達(dá)量。

1.5 兩組細(xì)胞周期分布觀察 采用流式細(xì)胞術(shù)。取兩組轉(zhuǎn)染后細(xì)胞，胰酶消化、重懸、離心后用預(yù)冷的PBS洗滌，再置于預(yù)冷的75%乙醇中固定，4 ℃過(guò)夜。再用預(yù)冷的PBS洗滌，碘化丙啶染色液染色，后37 ℃下避光孵育30 min，用流式細(xì)胞儀檢測(cè)細(xì)胞周期分布。

1.6 兩組細(xì)胞細(xì)胞凋亡率測(cè)算 采用流式細(xì)胞術(shù)。取兩組轉(zhuǎn)染后細(xì)胞，用8 Gy 劑量X線照射，繼續(xù)培養(yǎng)48 h，胰酶消化重懸后計(jì)數(shù)8萬(wàn)個(gè)左右細(xì)胞，離心后加入Annexin V-FITC結(jié)合液，充分重懸后再加Annexin V-FITC混勻，最后加碘化丙啶染色，混勻后室溫下避光孵育20 min，用流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率。

2 結(jié)果

2.1 兩組細(xì)胞放射敏感性比較 觀察組、對(duì)照組D0分別為1.062、1.542 Gy，Dq分別為1.605、1.970 Gy,SF2分別為0.527、0.685。觀察組相對(duì)于對(duì)照組的SER為1.451。

2.2 兩組細(xì)胞miR-124、STAT3 mRNA相對(duì)表達(dá)量比較 轉(zhuǎn)染后24 h在熒光倒置顯微鏡下觀察，轉(zhuǎn)染效率接近100%。觀察組、對(duì)照組miR-124相對(duì)表達(dá)量分別為89.3±13.6、1.0±0.1，STAT3 mRNA相對(duì)表達(dá)量分別為0.3±0.03、1.0±0.1，兩組相比，P均<0.05。

2.3 兩組細(xì)胞周期分布情況比較 觀察組G0/G1期細(xì)胞比例為82.49%±1.97%、S期細(xì)胞比例為11.87%±1.38%、G2/M期細(xì)胞比例為5.64%±0.72%；對(duì)照組分別為74.58%±1.28%、19.88%±0.26%、5.54%±1.05%。觀察組G0/G1期比例高于對(duì)照組組，S期比例低于對(duì)照組(P均<0.05)，兩組G2/M期細(xì)胞比例相比P>0.05。

2.4 兩組細(xì)胞X線照射后細(xì)胞凋亡率比較 觀察組、對(duì)照組細(xì)胞X線照射后細(xì)胞凋亡率分別為45.87%±3.16%、37.27%±0.87%，兩組相比，P<0.05。

3 討論

宮頸癌是我國(guó)女性生殖系統(tǒng)中最常見(jiàn)的惡性腫瘤，放療是治療宮頸癌的主要手段，放療抵抗已成為影響宮頸癌患者治療的關(guān)鍵問(wèn)題。部分患者出現(xiàn)放療抵抗，導(dǎo)致癌癥復(fù)發(fā)、轉(zhuǎn)移，甚至死亡。癌細(xì)胞對(duì)放射的敏感性是影響放療療效的關(guān)鍵因素，若癌細(xì)胞對(duì)放射的敏感性高，則較小的劑量就能達(dá)到較好的療效。反之，若癌細(xì)胞對(duì)放射抵抗，則療效將遠(yuǎn)低于預(yù)期目標(biāo)。因此需要一種安全有效的放療增敏劑，提高癌細(xì)胞對(duì)放射的敏感性，力求在減少放射相關(guān)不良反應(yīng)的同時(shí)達(dá)到最好的療效。倪猛等[12]發(fā)現(xiàn)miR-96可通過(guò)負(fù)調(diào)控HERG1表達(dá)增強(qiáng)胰腺癌PANC-1細(xì)胞的放射敏感性，這為提高胰腺癌臨床放療療效提供了新思路。越來(lái)越多的研究顯示，某些miRNA與腫瘤細(xì)胞的放射抵抗相關(guān)。本研究中我們通過(guò)克隆形成實(shí)驗(yàn)繪制Siha細(xì)胞的存活曲線，比較觀察組和對(duì)照組細(xì)胞的放射敏感性，發(fā)現(xiàn)過(guò)表達(dá)miR-124可降低Siha細(xì)胞克隆形成能力。觀察組和對(duì)照組D0分別為1.062、1.542 Gy，表明過(guò)表達(dá)miR-124可提高了Siha細(xì)胞對(duì)放射損傷敏感度；觀察組和對(duì)照組Dq分別為1.605、1.970 Gy，表明過(guò)表達(dá)miR-124降低了Siha細(xì)胞對(duì)放射后的亞損傷修復(fù)能力。我們的結(jié)果表明miR-124可增強(qiáng)Siha細(xì)胞的放射敏感性，SER可達(dá)到1.451，這與Hao等[13]發(fā)現(xiàn)miR-124可增加非小細(xì)胞肺癌細(xì)胞放射敏感性的研究結(jié)果一致。

本研究結(jié)果顯示，過(guò)表達(dá)miR-124的制Siha細(xì)胞G0/G1期細(xì)胞比例增高，而S期細(xì)胞比例降低，細(xì)胞發(fā)生G1期阻滯，這會(huì)導(dǎo)致DNA復(fù)制減少，細(xì)胞分裂速度減慢，進(jìn)而抑制Siha細(xì)胞增殖能力。而且就放射敏感性而言，處于S期的細(xì)胞放射敏感性最差，這可能是觀察組細(xì)胞放射敏感性較高的原因。目前為止絕大多數(shù)研究結(jié)果表明G1期阻滯的程度與細(xì)胞放射敏感性呈正相關(guān)，我們的實(shí)驗(yàn)結(jié)果也提示G1期阻滯能增加宮頸癌Siha細(xì)胞的放射敏感性[14]。除細(xì)胞周期阻滯外，凋亡也與宮頸癌放療敏感性密切相關(guān)，細(xì)胞凋亡是放射誘導(dǎo)的宮頸癌細(xì)胞死亡的主要方式，也是評(píng)價(jià)宮頸癌放射敏感性的重要指標(biāo)之一[15]。細(xì)胞對(duì)放射的敏感性與其凋亡的反應(yīng)程度一致，放射誘導(dǎo)的凋亡越高, 則表示放射敏感性越高，反之亦然。在本研究中，兩組細(xì)胞經(jīng)8Gy X線照射后48 h，觀察組細(xì)胞凋亡率明顯高于對(duì)照組，因此我們推論miR-124可能通過(guò)阻滯細(xì)胞周期于G0/G1期和促進(jìn)放射誘導(dǎo)的凋亡來(lái)提高Siha細(xì)胞的放射敏感性。

本研究發(fā)現(xiàn)轉(zhuǎn)染miR-124模擬物可提高Siha細(xì)胞中miR-124的表達(dá)，同時(shí)STAT3的表達(dá)明顯降低。在之前的研究中我們通過(guò)熒光素酶報(bào)告基因?qū)嶒?yàn)已證明STAT3為miR-124的直接靶基因。STAT3在多種癌組織中高表達(dá)，參與細(xì)胞生長(zhǎng)、分化、凋亡等多種生理功能的調(diào)控 ,與腫瘤的增殖、分化、細(xì)胞凋亡及預(yù)后密切相關(guān)[16～18]。關(guān)于STAT3對(duì)放射抵抗的作用機(jī)制可能是激活STAT3可靶向調(diào)節(jié)與細(xì)胞惡性轉(zhuǎn)化相關(guān)的基因表達(dá)[19]。一方面STAT3可通過(guò)激活下游抗凋亡的基因、調(diào)控細(xì)胞周期的基因或血管生成基因表達(dá)，影響細(xì)胞生長(zhǎng)和凋亡，導(dǎo)致腫瘤產(chǎn)生放射抵抗[20,21]。另一方面STAT3也可激活乏氧誘導(dǎo)因子 1 (HIF-1)基因，誘發(fā)腫瘤細(xì)胞自噬，而達(dá)到提高放射敏感性的目的[22]。如STAT3可直接調(diào)節(jié)細(xì)胞周期蛋白D1(Cyclin D1)基因表達(dá)，使細(xì)胞周期發(fā)生S期阻滯，DNA復(fù)制速度加快，促使細(xì)胞增殖、癌變等。CyclinD1表達(dá)水平已被證明與癌細(xì)胞放療敏感性有關(guān)[23,24]。抑制STAT3基因也可調(diào)節(jié)下游B淋巴細(xì)胞瘤-2(Bcl-2)抗凋亡的表達(dá)，促進(jìn)放射處理后細(xì)胞的凋亡，逆轉(zhuǎn)癌細(xì)胞的放射抵抗性[25]。這與我們的研究結(jié)果一致。我們推測(cè)miR-124可能是通過(guò)靶向調(diào)節(jié)STAT3表達(dá)，阻滯細(xì)胞周期于G0/G1期，并促進(jìn)放射誘導(dǎo)的調(diào)亡，進(jìn)而增加Siha細(xì)胞的放射敏感性。但這有待于我們進(jìn)一步深入研究證實(shí)。

綜上所述，過(guò)表達(dá)miR-124可增加宮頸癌Siha細(xì)胞的放射敏感性，抑制宮頸癌Siha細(xì)胞STAT3表達(dá)可能是其作用機(jī)制。miR-124、STAT3可能是宮頸癌放療中潛在的放射增敏劑，這需要通過(guò)大量臨床研究進(jìn)一步確定miR-124和STAT3的臨床意義。本研究為研究宮頸癌細(xì)胞輻射抵抗機(jī)制提供新的證據(jù)，可能發(fā)現(xiàn)了一個(gè)增加宮頸癌放射敏感性的潛在靶點(diǎn)。

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Changes of radiosensitivity of cervical cancer cell line Siha transfected with miR-124 mimics

WANGMengjie1,MENGBi,LIHao,GAOFei,YETing,LIUYangchen

(1GraduateSchoolofBengbuMedicalCollege,Bengbu233003,China)

10.3969/j.issn.1002-266X.2017.35.003

R737.3

A

1002-266X(2017)35-0008-04

2017-03-22)

蚌埠醫(yī)學(xué)院研究生科研創(chuàng)新計(jì)劃(Byycx1624)。

王夢(mèng)潔(1991-)，女，在讀研究生，主要研究方向?yàn)槟[瘤放射治療。E-mail: wangmengjiemj@163.com

劉陽(yáng)晨(1969-)，男，主任醫(yī)師，碩士生導(dǎo)師，主要研究方向?yàn)槟[瘤綜合治療。E-mail: liuyctx@163.com