趙紅丹,郭康,宋士軍,高明
[摘要]目的? ?探究miR-145-5p靶向調(diào)控[STBX]SMAD4基因?qū)κ彻荀[狀細(xì)胞癌細(xì)胞增殖和凋亡的作用及其機(jī)制。方法? ?收集68例食管鱗狀細(xì)胞癌病人癌組織及其癌旁正常組織,采用免疫組織化學(xué)方法檢測(cè)SMAD4蛋白陽(yáng)性表達(dá)率,應(yīng)用實(shí)時(shí)定量聚合酶鏈反應(yīng)(qRT-PCR)技術(shù)檢測(cè)食管鱗狀細(xì)胞癌及其癌旁正常組織中miR-145-5p和[STBX]SMAD4表達(dá)水平。篩選合適食管鱗狀細(xì)胞癌細(xì)胞株分為Blank組、NC組、miR-145-5p mimic組、miR-145-5p inhibitor組、siRNA-SMAD4組和miR-145-5p inhibitor+siRNA-SMAD4組。生物學(xué)網(wǎng)站和雙熒光素酶實(shí)驗(yàn)驗(yàn)證miR-145-5p和[STBX]SMAD4的靶向關(guān)系。qRT-PCR檢測(cè)miR-145-5p、[STBX]SMAD4、TGF-β、Bcl-2和Bax的mRNA表達(dá)水平,Western Blot分別檢測(cè)SMAD4、TGF-β、Bcl-2和Bax的蛋白的表達(dá)水平。CCK8方法檢測(cè)細(xì)胞增殖活力;流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率。 結(jié)果? ?免疫組織化學(xué)檢測(cè)顯示,SMAD4陽(yáng)性顆粒主要表達(dá)于細(xì)胞漿或細(xì)胞核,且與癌旁正常組織相比,食管鱗狀細(xì)胞癌組織中SMAD4陽(yáng)性率明顯升高(χ2=14.251,P<0.05);而qRT-PCR顯示miR-145-5p表達(dá)水平顯著降低(t=109.800,P<0.05)。相比其他細(xì)胞株,ECA-109細(xì)胞中的miR-145-5p表達(dá)量最低(F=48.000,P<0.05),選用于后續(xù)實(shí)驗(yàn)。雙熒光素酶報(bào)告基因?qū)嶒?yàn)顯示,miR-145-5p可靶向調(diào)控[STBX]SMAD4(t=21.820,P<0.05)。與Blank組相比,NC組的各項(xiàng)指標(biāo)無(wú)顯著差異(P均>0.05);與Blank組和NC組相比,miR-145-5p mimic組miR-145-5p和Bax表達(dá)增加,SMAD4、TGF-β和Bcl-2表達(dá)降低,細(xì)胞增殖能力明顯下降,細(xì)胞凋亡率顯著上升(P均<0.05);siRNA-SMAD4組miR-145-5p表達(dá)無(wú)明顯變化,Bax表達(dá)增加,SMAD4、TGF-β和Bcl-2表達(dá)降低,細(xì)胞增殖能力明顯下降,細(xì)胞凋亡率顯著上升(P均<0.05);miR-145-5p inhibitor組miR-145-5p和Bax表達(dá)降低,SMAD4、TGF-β和Bcl-2表達(dá)上升,細(xì)胞增殖能力明顯上升,細(xì)胞凋亡率顯著下降(P均<0.05);而miR-145-5p inhibitor+siRNA-SMAD4組與Blank組和NC組相比組間差異無(wú)顯著性(P均>0.05)。結(jié)論? ?miR-145-5p高表達(dá)可通過(guò)靶向抑制[STBX]SMAD4基因表達(dá),抑制TGF-β/Smad4信號(hào)通路的激活,抑制食管鱗狀細(xì)胞癌細(xì)胞增殖并促進(jìn)細(xì)胞凋亡。
[關(guān)鍵詞]食管鱗癌;微RNAs;Smad4蛋白質(zhì);TGF-β/Smad4信號(hào)通路;細(xì)胞增殖;細(xì)胞凋亡
[中圖分類號(hào)]R735.1;R342.2
[文獻(xiàn)標(biāo)志碼]A
[文章編號(hào)]2096-5532(2022)04-0577-07
doi:10.11712/jms.2096-5532.2022.58.146[開(kāi)放科學(xué)(資源服務(wù))標(biāo)識(shí)碼(OSID)]
[網(wǎng)絡(luò)出版]https://kns.cnki.net/kcms/detail/37.1517.R.20220822.1544.004.html;[JY]2022-08-2309:48:05
EFFECT OF MIR-145-5P ON THE PROLIFERATION AND APOPTOSIS OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA CELLS BY TARGETING THE [STHX]SMAD4 GENE
ZHAO Hongdan, GUO Kang, SONG Shijun, GAO Ming
(Department of Oncology, the Third Affiliated Hospital of Xinxiang Medical University, Xinxiang 453000, China)
[ABSTRACT] Objective[WTBZ] To investigate the effect of miR-145-5p on the proliferation and apoptosis of esophageal squamous cell carcinoma cells through targeted regulation of the [STBX]SMAD4 gene and its mechanism.
Methods Cancerous tissue samples and adjacent tissue samples were collected from 68 patients with esophageal squamous cell carcinoma. Immunohistochemistry was used to measure the positive expression rate of SMAD4 protein, and quantitative real-time PCR was used to measure the expression level of miR-145-5p and SMAD4 in esophageal squamous cell carcinoma tissue and adjacent tissue. Suitable esophageal squamous cell carcinoma cell lines were screened out and divided into Blank group, NC group, miR-145-5p mimic group, miR-145-5p inhibitor group, siRNA-SMAD4 group, and miR-145-5p inhibitor+siRNA-SMAD4 group. Biological websites and dual-luciferase reporter assay were used to verify the targeting relationship between miR-145-5p and SMAD4. Quantitative real-time PCR was used to measure the mRNA expression levels of miR-145-5p, [STBX]SMAD4, TGF-β, Bcl-2, and Bax, and Western Blot was used to measure the protein expression levels of SMAD4, TGF-β, Bcl-2, and Bax. CCK8 assay was used to measure cell proliferation activity, and flow cytometry was used to measure cell apoptosis rate.
Results Immunohistochemistry showed that SMAD4 was mainly expressed in cytoplasm or nucleus, and the positive rate of SMAD4 in esophageal squamous cell carcinoma tissue was [JP]significantly higher than that in normal adjacent tissue (χ2=14.251,P<0.05), while quantitative real-time PCR showed a significant reduction in the expression level of miR-145-5p (t=109.800,P<0.05). Compared with the other cell lines, ECA-109 cells had the lowest expression level of miR-145-5p (F=48.000,P<0.05) and were then selected for subsequent experiments. Dual-luciferase reporter assay showed that miR-145-5p had a targeted regulatory effect on SMAD4 (t=21.820,P<0.05). There were no significant differences in related indices between the Blank group and the NC group (all P>0.05); compared with the Blank group and the NC group, the miR-145-5p mimic group had significant increases in the expression of miR-145-5p and Bax, significant reductions in the expression of SMAD4, TGF-β, and Bcl-2, a significant reduction in cell proliferative capacity, and a significant increase in cell apoptosis rate (all P<0.05). Compared with the Blank group and the NC group, the siRNA-SMAD4 group showed no significant change in the expression of miR-145-5p and had a significant increase in the expression of Bax, significant reductions in the expression of SMAD4, TGF-β, and Bcl-2, a significant reduction in cell proliferative capacity, and a significant increase in cell apoptosis rate (all P<0.05). Compared with the Blank group and the NC group, the miR-145-5p inhibitor group had significant reductions in the expression of miR-145-5p and Bax, significant increases in the expression of SMAD4, TGF-β, and Bcl-2, a significant increase in cell proliferative capacity, and a significant reduction in cell apoptosis rate (all P<0.05). There were no significant differences in these indices between the miR-145-5p inhibitor+siRNA-SMAD4 group and the Blank/NC groups (all P>0.05).
Conclusion The high expression of miR-145-5p can inhibit activation of the TGF-β/Smad4 signaling pathway through targeted inhibition of [STBX]SMAD4 gene expression, and it can also inhibit the proliferation of esophageal squamous cell carcinoma cells and promote cell apoptosis.
[KEY WORDS]esophageal squamous cell carcinoma; microRNAs; Smad4 protein; TGF-β/Smad4 signaling pathway; cell proliferation; apoptosis
食管癌作為世界常見(jiàn)六大惡性腫瘤之一,具有顯著的地域分布差異[1-2]。我國(guó)是食管癌高病死率國(guó)家,其中90%以上為鱗狀細(xì)胞癌[3-4]。食管癌的發(fā)病與飲食習(xí)慣、生活環(huán)境、遺傳因素、種族差異、地理分布等均有密切關(guān)系[5-7]。食管癌的發(fā)病過(guò)程涉及基因的遺傳學(xué)改變,包括原癌基因的激活和抑癌基因的失活[8-10]。[STBX]SMAD4最早以胰腺癌的抑癌基因被發(fā)現(xiàn),后續(xù)研究證實(shí)其表達(dá)缺失與人類多種惡性腫瘤的進(jìn)程相關(guān)[11]。同時(shí),微小RNAs(miRNA)作為一種非編碼的內(nèi)源性單鏈小分子RNA,已經(jīng)被證實(shí)可以通過(guò)靶向調(diào)節(jié)mRNA的降解或抑制該類靶基因的翻譯,進(jìn)而激活或抑制下游信號(hào)通路,參與并介導(dǎo)腫瘤細(xì)胞的生長(zhǎng)、增殖和凋亡等行為[12-13]。鑒于此,本研究通過(guò)探究miR-145-5p是否可以通過(guò)靶向調(diào)控[STBX]SMAD4基因介導(dǎo)TGF-β/Smad4信號(hào)通路,進(jìn)而參與食管鱗狀細(xì)胞癌細(xì)胞增殖和凋亡,并闡述其關(guān)聯(lián)作用機(jī)制,以期為食管鱗狀細(xì)胞癌的早期診療和預(yù)后評(píng)估提供新的分子標(biāo)志物。
1資料和方法
1.1實(shí)驗(yàn)材料
1.1.1組織標(biāo)本收集新鄉(xiāng)醫(yī)學(xué)院第三附屬醫(yī)院2016年3月—2019年3月經(jīng)病理確診的食管鱗狀細(xì)胞癌病人68例,男40例,女28例,年齡34~78歲,平均(57.48±6.88)歲。所有病例均未接受放化療;既往無(wú)除食管鱗狀細(xì)胞癌之外的惡性腫瘤病史;既往無(wú)糖尿病、高血壓及嚴(yán)重心臟病等病史;無(wú)家族遺傳性病史。取食管鱗狀細(xì)胞癌組織為實(shí)驗(yàn)組,同時(shí)取相應(yīng)癌旁正常組織作為對(duì)照組。采集標(biāo)本分為兩份,一份液氮保存,一份甲醛固定備用。本研究通過(guò)醫(yī)院倫理委員會(huì)評(píng)審批準(zhǔn),所有受試者均簽署知情同意書。
1.1.2主要試劑與儀器食管鱗狀細(xì)胞癌細(xì)胞株TE-1、TE-13、ECA-109和TTN(購(gòu)買于中國(guó)科學(xué)院上海細(xì)胞庫(kù)),目的質(zhì)粒均購(gòu)自上海生工生物工程技術(shù)服務(wù)有限公司(上海,中國(guó)),Lipofectamin 2000試劑盒、RNA提取試劑盒(Invitrogen公司,USA),Prime Script RT試劑盒(寶日醫(yī)生物科技公司,北京),PCR試劑盒(北京天根生化科技有限公司),GAPDH(abcam公司,英國(guó)),ECL試劑盒(Bio-Rad,美國(guó)),CCK8試劑、Annexin-V-FITC細(xì)胞凋亡檢測(cè)試劑盒(碧云天生物技術(shù)公司,上海);成像分析儀(Image Reader,Bio-Rad,美國(guó)),酶標(biāo)儀(賽默飛世爾科技有限公司,美國(guó))。
1.2實(shí)驗(yàn)方法
1.2.1免疫組化檢測(cè)SMAD4陽(yáng)性率組織標(biāo)本常規(guī)脫水、石蠟包埋,制作4 μm連續(xù)切片。參照試劑盒說(shuō)明進(jìn)行檢測(cè),其中,采用體積分?jǐn)?shù)0.03的H2O2阻斷內(nèi)源性過(guò)氧化物酶活性,滴加非免疫山羊血清封閉;滴加鼠抗人SMAD4(1∶100)4 ℃冰箱內(nèi)孵育過(guò)夜。次日復(fù)溫后,加入生物素標(biāo)記的山羊抗兔二抗IgG,37 ℃孵育30 min。應(yīng)用含體積分?jǐn)?shù)0.01 Tween20的PBS洗滌后,滴加辣根過(guò)氧化物酶-鏈霉卵白素復(fù)合物進(jìn)行反應(yīng)。每張切片隨機(jī)選取5個(gè)視野(200倍)分別計(jì)數(shù)其陽(yáng)性細(xì)胞,并取平均值。判定標(biāo)準(zhǔn):陽(yáng)性細(xì)胞≤10%為1分,11%~50%為2分,51%~80%為3分,≥81%為4分。
1.2.2食管鱗狀細(xì)胞癌細(xì)胞株篩選選擇食管鱗狀細(xì)胞癌細(xì)胞株TE-1、TE-13、ECA-109和TTN,將以上4種細(xì)胞株置培養(yǎng)液中培養(yǎng),每24 h更換培養(yǎng)液,進(jìn)行傳代培養(yǎng),并采用實(shí)時(shí)定量聚合酶鏈反應(yīng)(qRT-PCR)方法篩選出miR-145-5p表達(dá)水平最低的細(xì)胞系用于后續(xù)實(shí)驗(yàn)。
1.2.3細(xì)胞分組與轉(zhuǎn)染選擇培養(yǎng)的人食管鱗狀細(xì)胞癌ECA-109細(xì)胞,將其分為6組:Blank組(不進(jìn)行任何處理,a組)、NC組(轉(zhuǎn)染陰性質(zhì)粒,b組)、miR-145-5p mimic組(轉(zhuǎn)染miR-145-5p mimic質(zhì)粒,c組)、miR-145-5p inhibitor組(轉(zhuǎn)染miR-145-5p inhibitor質(zhì)粒,d組)、siRNA-SMAD4組(轉(zhuǎn)染[STBX]SMAD4 siRNA質(zhì)粒,e組)和miR-145-5p inhibi-tor+siRNA-SMAD4組(共轉(zhuǎn)染siRNA-[STBX]SMAD4質(zhì)粒和miR-145-5p mimic質(zhì)粒,f組)。將篩選細(xì)胞以每孔3×105個(gè)的密度接種于6孔板中,待細(xì)胞融合度達(dá)80%時(shí),使用Lipofectamin 2000試劑盒進(jìn)行轉(zhuǎn)染。用250 μL無(wú)血清Opti-MEM培養(yǎng)液分別稀釋目的質(zhì)粒和Lipofectamine 2000。室溫下靜置5 min,兩者混合均勻,放置20 min后將混合液加到培養(yǎng)孔中,置于37 ℃、含體積分?jǐn)?shù)0.05 CO2培養(yǎng)箱中培養(yǎng),6 h后將培養(yǎng)液更換成完全培養(yǎng)液,繼續(xù)培養(yǎng)48 h后,收集細(xì)胞。
1.2.4雙熒光素酶報(bào)告實(shí)驗(yàn)登錄生物學(xué)預(yù)測(cè)網(wǎng)站(http://www.microrna.org和http://www.targetscan.org/vert_72/)進(jìn)行miR-145-5p的靶基因預(yù)測(cè),驗(yàn)證[STBX]SMAD4是miR-145-5p的直接靶點(diǎn)。人工合成[STBX]SMAD4基因3′UTR野生型(WT)和突變型(MUT)序列。構(gòu)建熒光素酶報(bào)告載體,將測(cè)序正確的熒光素酶報(bào)告質(zhì)粒WT、MUT分別與miR-145-5p mimic和miR-145-5p NC共轉(zhuǎn)染至食管鱗狀細(xì)胞癌細(xì)胞中。轉(zhuǎn)染48 h后收集并裂解細(xì)胞,分別使用熒光素酶檢測(cè)試劑盒檢測(cè)熒光素酶活性。
1.2.5qRT-PCR檢測(cè)各組相關(guān)基因的mRNA表達(dá)取新鮮組織樣本采用RNA提取試劑盒提取總RNA,使用Prime Script RT試劑盒將RNA逆轉(zhuǎn)錄成cDNA,逆轉(zhuǎn)錄體系11.5 μL。設(shè)計(jì)miR-145-5p、[STBX]SMAD4、TGF-β、Bcl-2、Bax、U6和GAPDH引物,交由TAKARA公司合成。按照PCR試劑盒操作步驟進(jìn)行qRT-PCR反應(yīng),反應(yīng)條件為:95 ℃預(yù)變性5 min后,95 ℃、 40 s,57 ℃、40 s,72 ℃、40 s,循環(huán)40次,72 ℃延伸10 min,4 ℃、5 min。miR-145-5p的相對(duì)水平以[STBX]U6為內(nèi)參照,[STBX]SMAD4、TGF-β、Bcl-2和Bax相對(duì)表達(dá)水平以GAPDH基因作為內(nèi)參照。采用2-△△Ct法計(jì)算目的基因 mRNA的相對(duì)轉(zhuǎn)錄水平。該實(shí)驗(yàn)步驟同樣適用于細(xì)胞實(shí)驗(yàn)。
1.2.6Western Blot檢測(cè)各組相關(guān)基因的蛋白表達(dá)
收集轉(zhuǎn)染48 h后各組細(xì)胞,以預(yù)冷PBS洗滌細(xì)胞3次,加入全蛋白裂解液后置冰上裂解30 min,收獲蛋白。BCA法定量抽提蛋白后,于80 V泳道進(jìn)行SDS-PAGE,待樣品跑至分離膠時(shí)改用120 V電壓繼續(xù)電泳。電泳結(jié)束后,將蛋白轉(zhuǎn)移至NC膜;置入50 g/L 脫脂奶粉溶液中室溫孵育60 min。TBST洗膜3次后,加入一抗多克隆抗體SMAD4、TGF-β、Bcl-2、Bax和GAPDH于4 ℃過(guò)夜;TBST洗膜3次后,加入二抗(辣根過(guò)氧化酶標(biāo)記的羊抗兔IgG)室溫下孵育60 min。TBST洗膜3次后用ECL試劑盒進(jìn)行發(fā)光反應(yīng),濾紙吸去膜表面多余底物溶液,置于成像分析儀中顯影成像。用Quantity One軟件分析條帶灰度值,計(jì)算目的基因蛋白的相對(duì)表達(dá)量。每組3個(gè)樣本,設(shè)3個(gè)平行孔,實(shí)驗(yàn)重復(fù)3次。
1.2.7CCK8法檢測(cè)各組細(xì)胞增殖能力的變化情況
將轉(zhuǎn)染12 h后的各組細(xì)胞鋪入96孔培養(yǎng)板,調(diào)整細(xì)胞密度為2×106/L,每孔加入100 μL細(xì)胞培養(yǎng)液。將培養(yǎng)板置于室溫下培養(yǎng),分別檢測(cè)24、48、72、96 h的細(xì)胞活力。每孔加10 μL的CCK8試劑,37 ℃孵育2 h,采用酶標(biāo)儀檢測(cè)450 nm波長(zhǎng)處的光密度值(OD)。以時(shí)間點(diǎn)為橫坐標(biāo),OD值為縱坐標(biāo)繪制細(xì)胞活力曲線圖。每組3個(gè)樣本,設(shè)3個(gè)平行孔,實(shí)驗(yàn)重復(fù)3次。
1.2.8流式細(xì)胞儀檢測(cè)各組細(xì)胞的周期和凋亡率
按照Annexin-V-FITC細(xì)胞凋亡檢測(cè)試劑盒說(shuō)明檢測(cè)細(xì)胞凋亡。取各組轉(zhuǎn)染48 h后的細(xì)胞,每100 μL染液重懸1×106個(gè)細(xì)胞,振蕩混勻,加入5 μL的 Annexin-V-FITC溶液,振蕩混勻。在室溫下孵育15 min后,加入5 μL的 PI后輕輕混勻,于4 ℃下避光孵育5 min。在1 h內(nèi)上流式細(xì)胞儀檢測(cè),在激發(fā)波長(zhǎng)488 nm下檢測(cè)細(xì)胞凋亡率。每組3個(gè)樣本,設(shè)3個(gè)平行孔,實(shí)驗(yàn)重復(fù)3次。
1.3統(tǒng)計(jì)學(xué)方法
采用SPSS 21.0軟件包進(jìn)行統(tǒng)計(jì)學(xué)處理。計(jì)量資料采用[AKx-D]±s形式表示,兩組均數(shù)的比較采用Students t檢驗(yàn),率的比較采用卡方檢驗(yàn),多組間均數(shù)的比較采用單因素方差分析。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2結(jié)果
2.1不同組織SMAD4蛋白及miR-145-5p mRNA表達(dá)比較
免疫組化檢測(cè)顯示,在食管癌組織中SMAD4蛋白陽(yáng)性表達(dá)呈黃色或棕黃色顆粒,主要分布于細(xì)胞漿或細(xì)胞核。SMAD4蛋白在食管癌組織(44/68)中表達(dá)顯著高于在正常癌旁組織(22/68)中表達(dá),差異有顯著性(χ2=14.251,P<0.05)。見(jiàn)圖1A、B。qRT-PCR檢測(cè)顯示,與正常癌旁組織相比,食管癌組織中miR-145-5p的表達(dá)水平明顯下降,差異有顯著性(t=109.800,P<0.05)。見(jiàn)圖1C。
2.2雙熒光素酶報(bào)告基因?qū)嶒?yàn)
生物學(xué)的預(yù)測(cè)網(wǎng)站microRNA.org顯示,miR-145-5p和[STBX]SMAD4基因間存在結(jié)合位點(diǎn)(圖2A)。雙熒光素酶報(bào)告基因系統(tǒng)結(jié)果顯示,與對(duì)照組相比,miR-145-5p mimic和SMAD4-wild共轉(zhuǎn)染組熒光素酶熒光信號(hào)明顯下降(t=21.820,P<0.05),而SMAD4-mutant組熒光素酶信號(hào)無(wú)明顯變化(t=0.775,P>0.05)。提示miR-145-5p能夠特異性靶向結(jié)合[STBX]SMAD4基因(圖2B)。
2.3食管鱗狀細(xì)胞癌細(xì)胞株篩選
qRT-PCR檢測(cè)食管鱗狀細(xì)胞癌細(xì)胞株TE-1、TE-13、ECA-109和TTN中miR-145-5p表達(dá)量顯示,與其他3種細(xì)胞株相比較,ECA-109細(xì)胞中的miR-145-5p表達(dá)量最低(F=48.000,P<0.05)。篩選出ECA-109細(xì)胞株用于后續(xù)實(shí)驗(yàn)。見(jiàn)圖3。
2.4各組細(xì)胞中相關(guān)基因mRNA和蛋白表達(dá)比較
qRT-PCR和Western Blot的檢測(cè)結(jié)果顯示,Blank組和NC組各基因mRNA和蛋白表達(dá)比較均無(wú)明顯差異(F=2.023、0.557, P 均>0.05)。與Blank組和NC組細(xì)胞比較,miR-145-5p mimic組miR-145-5p和Bax的mRNA和蛋白相對(duì)表達(dá)量增加(F=38.240~118.600, P均<0.05),[STBX]SMAD4、TGF-β和[STBX]Bcl-2的mRNA和蛋白相對(duì)表達(dá)量顯著降低(F=24.820~156.200, P均<0.05);siRNA-SMAD4組miR-145-5p的表達(dá)無(wú)明顯變化(F=0.937,P>0.05),Bax的mRNA和蛋白相對(duì)表達(dá)水平顯著增加(F=38.27、148.200, P均<0.05),[STBX]SMAD4、TGF-β和Bcl-2的mRNA和蛋白相對(duì)表達(dá)量降低(F=27.470~134.900, P均<0.05);miR-145-5p inhibitor組miR-145-5p以及Bax的mRNA和蛋白相對(duì)表達(dá)量明顯降低(F=65.550~82.320,P均<0.05),[STBX]SMAD4、TGF-β和Bcl-2的mRNA和蛋白相對(duì)表達(dá)水平上升(F=35.350~101.400, P均<0.05)。而miR-145-5p inhibitor+siRNA-SMAD4組與Blank組和NC組相比較差異均無(wú)顯著性(P均>0.05);且與siRNA-SMAD4組相比,miR-145-5p inhibitor+siRNA-SMAD4組[STBX]SMAD4、TGF-β和Bcl-2的mRNA和蛋白相對(duì)表達(dá)量上升(t=15.920~24.020, P均<0.05),miR-145-5p和Bax的mRNA和蛋白相對(duì)表達(dá)量降低(t=6.325~14.720,P均<0.05)。見(jiàn)圖4A、B。
2.5CCK8法檢測(cè)各組細(xì)胞增殖活力
CCK8檢測(cè)結(jié)果顯示,在轉(zhuǎn)染24 h各組細(xì)胞增殖能力均無(wú)明顯差異,且NC組和Blank組相比各時(shí)間點(diǎn)細(xì)胞活力均無(wú)明顯差異(P 均>0.05);在48、72和96 h時(shí),與Blank組和NC組相比,miR-145-5p mimic組與siRNA-SMAD4組細(xì)胞活力均顯著下降(F=10.770~22.340,P均<0.05);miR-145-5p inhibitor組細(xì)胞活力顯著升高(F=5.814~15.810,P<0.05);miR-145-5p inhibitor+siRNA-SMAD4組與siRNA-SMAD4組比較細(xì)胞活力顯著增加(P均<0.05),而與NC組和Blank組相比無(wú)明顯差異(P>0.05)。見(jiàn)圖5。
2.6流式細(xì)胞術(shù)檢測(cè)各組細(xì)胞凋亡率
細(xì)胞凋亡實(shí)驗(yàn)的結(jié)果表明,轉(zhuǎn)染后NC組與Blank組相比凋亡率無(wú)明顯差異(t=0.537,P>0.05);與Blank組和NC組相比,miR-145-5p mimic組與siRNA-SMAD4組細(xì)胞凋亡率顯著升高(F=136.900、151.000, P均<0.05);miR-145-5p inhibitor組凋亡率顯著降低(F=18.000,P<0.05);而miR-145-5p inhibitor+siRNA-SMAD4組的細(xì)胞凋亡率與siRNA-SMAD4組相比較明顯降低(P<0.05),與NC組和Blank組相比無(wú)明顯差異(F=1.355,P>0.05)。見(jiàn)圖6。
3討論
食管癌的病因及發(fā)病機(jī)制目前尚不明確。我國(guó)食管癌發(fā)病率高,其5年生存率低、預(yù)后不良[14]。作為食管癌的主要病理類型,食管鱗狀細(xì)胞癌的診治方案研發(fā)日益受到重視[15-16]。本研究通過(guò)體內(nèi)外實(shí)驗(yàn)研究miR-145-5p靶向調(diào)控[STBX]SMAD4基因介導(dǎo)TGF-β/Smad4信號(hào)通路對(duì)食管鱗狀細(xì)胞癌細(xì)胞增殖、凋亡的作用機(jī)制。
食管鱗狀細(xì)胞癌中促癌作用的基因激活與抑癌作用的基因失活,及其相互作用的失衡是腫瘤發(fā)展的分子基礎(chǔ)[17]。近年來(lái),miRNA在腫瘤進(jìn)程中的作用日益突出[18-19]。既往研究顯示,在食管鱗狀細(xì)胞癌中異常表達(dá)的miRNA通過(guò)調(diào)控某些關(guān)鍵原癌基因或抑癌基因介導(dǎo)腫瘤發(fā)生[20-21]。尋找食管鱗狀細(xì)胞癌中異常表達(dá)的miRNA并預(yù)測(cè)及驗(yàn)證下游靶基因,對(duì)于食管鱗狀細(xì)胞癌基礎(chǔ)研究和臨床應(yīng)用具有重要價(jià)值。例如孟利峰等[22]研究表明,miR-149-5p可能通過(guò)調(diào)控Aurora-B表達(dá)參與食管鱗狀細(xì)胞癌發(fā)展進(jìn)程。CUI等[23]發(fā)現(xiàn),miR-34a可通過(guò)抑制PLCE1發(fā)揮其抗癌作用,miR-34a/PLCE1軸可參與食管鱗狀細(xì)胞癌轉(zhuǎn)移,為食管鱗狀細(xì)胞癌治療提供了新的候選靶點(diǎn)?;诩韧愃蒲芯浚芯恐跷覀兗僭O(shè)miR-145-5p可能通過(guò)靶向調(diào)控[STBX]SMAD4基因表達(dá)進(jìn)而參與食管鱗狀細(xì)胞癌生物學(xué)行為。
本研究對(duì)食管鱗狀細(xì)胞癌組織及其癌旁正常組織免疫組織化學(xué)檢測(cè)結(jié)果顯示,SMAD4陽(yáng)性顆粒主要表達(dá)于細(xì)胞漿或細(xì)胞核,且與癌旁正常組織相比,食管鱗狀細(xì)胞癌組織中SMAD4陽(yáng)性率明顯升高;同時(shí),qRT-PCR顯示食管鱗狀細(xì)胞癌組織miR-145-5p表達(dá)水平顯著降低,[STBX]SMAD4表達(dá)水平則顯著增加。本文組織實(shí)驗(yàn)結(jié)果初步提示,miR-145-5p在食管鱗狀細(xì)胞癌中低表達(dá),SMAD4高表達(dá),推測(cè)miR-145-5p高表達(dá)可通過(guò)靶向抑制[STBX]SMAD4基因表達(dá)進(jìn)而發(fā)揮抑癌作用。在細(xì)胞實(shí)驗(yàn)前的細(xì)胞株篩選結(jié)果顯示,ECA-109細(xì)胞中的miR-145-5p表達(dá)量最低,可用于后續(xù)實(shí)驗(yàn)。同時(shí),要了解miRNA的作用機(jī)制,關(guān)鍵在于鑒定靶基因[24-26]。本研究熒光素酶報(bào)告基因?qū)嶒?yàn)結(jié)果顯示,miR-145-5p可靶向調(diào)控[STBX]SMAD4。SMAD4已被報(bào)道在諸如胰腺腫瘤、宮頸癌、乳癌中異常表達(dá)[27-31]。為了解miR-145-5p及[STBX]SMAD4對(duì)食管鱗狀細(xì)胞癌生物學(xué)功能的影響,本實(shí)驗(yàn)基于不同轉(zhuǎn)染組別,應(yīng)用qRT-PCR、Western Blot、CCK8實(shí)驗(yàn)和流式細(xì)胞術(shù)檢測(cè)分別檢測(cè)關(guān)聯(lián)基因mRNA及蛋白表達(dá)、細(xì)胞增殖活力和凋亡率等指標(biāo)。結(jié)果顯示,與空白對(duì)照和陰性對(duì)照細(xì)胞相比,上調(diào)miR-145-5p表達(dá)可促進(jìn)促凋亡因子Bax表達(dá)增加,抑制SMAD4、TGF-β和抑制凋亡因子Bcl-2表達(dá),并下調(diào)細(xì)胞增殖活力且提升細(xì)胞凋亡率;且抑制[STBX]SMAD4基因表達(dá)可發(fā)揮同樣作用;而下調(diào)miR-145-5p表達(dá)則導(dǎo)致Bax表達(dá)降低,SMAD4、TGF-β和Bcl-2的表達(dá)上升,細(xì)胞增殖能力上升,細(xì)胞凋亡率下降。同時(shí),值得注意的是,在下調(diào)miR-145-5p表達(dá)水平同時(shí)抑制[STBX]SMAD4基因表達(dá)則逆轉(zhuǎn)了抑制siRNA-[STBX]SMAD4表達(dá)處理細(xì)胞的積極作用,導(dǎo)致細(xì)胞增殖能力的提升和凋亡率的下降。上述結(jié)果提示,上調(diào)miR-145-5p或過(guò)表達(dá)[STBX]SMAD4基因,可抑制TGF-β/Smad4信號(hào)通路的激活,且過(guò)表達(dá)miR-145-5p能夠靶向抑制[STBX]SMAD4基因而抑制食管癌細(xì)胞增殖能力、促進(jìn)細(xì)胞凋亡。
綜上所述,本研究對(duì)miR-145-5p以及[STBX]SMAD4基因在食管鱗狀細(xì)胞癌中作用研究的結(jié)果顯示,[STBX]SMAD4為miR-145-5p的下游靶基因,上調(diào)miR-145-5p表達(dá)可以通過(guò)下調(diào)[STBX]SMAD4基因表達(dá),抑制TGF-β/Smad4信號(hào)通路的激活,進(jìn)而在調(diào)控腫瘤生物學(xué)行為中發(fā)揮重要作用。本研究為今后食管鱗狀細(xì)胞癌的早期診治和預(yù)后評(píng)估提供了新型分子標(biāo)志物和機(jī)制通路解釋。然而,本研究機(jī)制探究單純基于細(xì)胞實(shí)驗(yàn),缺乏進(jìn)一步驗(yàn)證試驗(yàn),未來(lái)可通過(guò)動(dòng)物實(shí)驗(yàn)進(jìn)一步驗(yàn)證細(xì)胞實(shí)驗(yàn)的結(jié)果,并確認(rèn)下游潛在信號(hào)通路,從而為后續(xù)機(jī)制及臨床轉(zhuǎn)化研究提供理論指導(dǎo)。
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青島大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)2022年4期