陳忠,謝峰,鐘豐云,杜鴻,嚴(yán)永敏,錢暉
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胃癌組織中干細(xì)胞標(biāo)志物Sox2的表達(dá)及臨床意義
陳忠1,謝峰2,鐘豐云3,杜鴻4,嚴(yán)永敏5,錢暉5
目的探討胃癌組織中干細(xì)胞標(biāo)志物Sox2的表達(dá)及臨床意義。方法RT-PCR檢測(cè)60例胃癌患者癌組織(胃癌組)和癌旁組織(癌旁組)中Sox2 mRNA表達(dá),免疫組織化學(xué)法檢測(cè)2組Sox2蛋白表達(dá)水平。比較Sox2 mRNA陽(yáng)性表達(dá)情況在不同年齡、性別、腫瘤大小、組織類型、TNM分期、分化程度、浸潤(rùn)深度及淋巴結(jié)轉(zhuǎn)移中的差異。結(jié)果胃癌組Sox2 mRNA陽(yáng)性表達(dá)率53.3%(32/60)高于癌旁組的20.0%(12/60);Sox2 mRNA的相對(duì)表達(dá)強(qiáng)度高于癌旁組(0.724±0.209 vs. 0.256±0.065,P<0.01)。胃癌組Sox2蛋白的陽(yáng)性表達(dá)率50.0%(30/60)高于癌旁組的16.7%(10/60,P<0.01)。胃癌組Sox2 mRNA陽(yáng)性表達(dá)率臨床TNM分期(Ⅲ+Ⅳ)組高于(Ⅰ+Ⅱ)組,低、未分化組高于中、高分化組,浸潤(rùn)深度T3~T4組高于T1~T2組,淋巴結(jié)轉(zhuǎn)移組高于無(wú)轉(zhuǎn)移組(均P<0.05或P<0.01)。結(jié)論胃癌組織中Sox2高表達(dá)與其發(fā)生、侵襲、進(jìn)展及轉(zhuǎn)移有關(guān),可作為胃癌診療新的分子標(biāo)志物。
胃腫瘤;腫瘤干細(xì)胞;基因,腫瘤抑制;免疫組織化學(xué);逆轉(zhuǎn)錄聚合酶鏈反應(yīng);干細(xì)胞標(biāo)志物Sox2
干細(xì)胞標(biāo)志物Sox2(SRY-related HMG-box gene 2)是Sox基因超家族成員之一,屬于一類SRY (sex determination region of Y chromosome)相關(guān)基因家族,為干細(xì)胞轉(zhuǎn)錄因子[1]。Sox2通過(guò)自身高度保守的高遷移率族(HMG)區(qū)域與DNA產(chǎn)生交互作用,在調(diào)控胚胎和組織的分化發(fā)育、器官形成,維持胚胎干細(xì)胞(ESCs)的自我更新和多能性方面具有重要作用[2]。腫瘤干細(xì)胞(CSCs)被認(rèn)為是癌細(xì)胞的一個(gè)小的側(cè)群,具有類似ESCs能力,可介導(dǎo)腫瘤的發(fā)生、轉(zhuǎn)移及復(fù)發(fā)[3]。胃癌組織及細(xì)胞系中存在著胃癌干細(xì)胞樣細(xì)胞(GCSCs)[4]。Sox2為干細(xì)胞關(guān)鍵轉(zhuǎn)錄因子,越來(lái)越多的研究表明其與腫瘤發(fā)生密切相關(guān)[5]。其通過(guò)對(duì)靶基因的激活與抑制作用而參與腫瘤發(fā)生、增殖、侵襲及轉(zhuǎn)移,有關(guān)腫瘤中Sox2表達(dá)的研究結(jié)果并不一致。Sox2在胃癌、大腸癌及肺癌等中屬于癌基因還是抑癌基因尚存在爭(zhēng)議[6-10]。本研究旨在通過(guò)比較60例胃癌患者癌組織與癌旁組織中Sox2 mRNA及蛋白質(zhì)的表達(dá)水平差異,分析Sox2 mRNA表達(dá)與臨床病理參數(shù)的關(guān)系,探討其在胃癌發(fā)生、侵襲、進(jìn)展及轉(zhuǎn)移中的作用。
1.1一般資料選取2012年7月—2014年10月于蘇州永鼎醫(yī)院及蘇州大學(xué)附屬第二醫(yī)院普外科就診并經(jīng)病理組織學(xué)確診的60例胃癌患者,男38例,女22例,年齡31~81歲,中位年齡59歲。患者術(shù)前均未接受化療及放療。所有病理切片均經(jīng)2位病理科主治以上醫(yī)師進(jìn)行雙盲法核片,確定組織類型、臨床分期、分化程度及浸潤(rùn)深度。胃癌病理學(xué)分期依據(jù)2009年國(guó)際抗癌聯(lián)盟(UICC)第7版進(jìn)行TNM臨床分期。每例手術(shù)切除樣本均取約50 mg的癌組織(胃癌組)和癌旁組織(癌旁組,距癌變部位邊界>5 cm,且經(jīng)病理證實(shí)無(wú)癌變)標(biāo)本各3份;2份立即置于含0.1%DEPC(ddH2O配制)處理的1 mL PBS緩沖液中,-70℃保存,另1份用4%多聚甲醛固定,石蠟包埋。本研究經(jīng)2醫(yī)院醫(yī)學(xué)倫理委員會(huì)批準(zhǔn),患者均簽署知情同意書。
1.2主要儀器及試劑Thermo Electron Hybaid PCR Express熱循環(huán)儀(英國(guó)HYBAID公司);Eppendorf Biophotometer核酸蛋白測(cè)定儀(德國(guó)Eppendorf公司);GelDoc凝膠成像系統(tǒng)、Trizol試劑(Life technologies公司);逆轉(zhuǎn)錄試劑盒(Vazyme公司);PCR試劑盒(TaKaRa公司);鼠抗人Sox2單克隆抗體(德國(guó)Milipore公司);免疫組織化學(xué)(SP)法試劑盒和DAB顯色劑(武漢博士德公司)。
1.3總RNA提取和逆轉(zhuǎn)錄用組織碾磨器把胃癌組織和癌旁組織分別研碎成細(xì)胞懸液,過(guò)濾后的單細(xì)胞懸液經(jīng)離心后沉淀,按Trizol試劑說(shuō)明書操作提取總RNA,核酸蛋白檢測(cè)儀檢測(cè)各樣本的總RNA濃度和純度,取吸光度(A260/280)在1.7~2.0之間的樣本用于后續(xù)試驗(yàn)。用瓊脂糖凝膠電泳檢測(cè)RNA完整性,紫外線燈下出現(xiàn)28S、18S和5S區(qū)帶,表明RNA的完整性好。按逆轉(zhuǎn)錄試劑盒說(shuō)明書操作進(jìn)行逆轉(zhuǎn)錄反應(yīng)合成cDNA,置-20℃保存。剩余總RNA置-70℃保存?zhèn)溆谩?/p>
1.4引物設(shè)計(jì)用Primer Premier 5.0引物設(shè)計(jì)軟件跨外顯子設(shè)計(jì)Sox2與β-actin基因(GenBank序列號(hào)分別為NM_ 003106.3、NM_001101.3)。Sox2序列:上游5′- ACACCAATCCCATCCACACT- 3′,下游5′- GCAAACTTCCTGCAAAGCTC-3′,產(chǎn)物長(zhǎng)度224 bp。β-actin引物序列:上游5′-CACGAAACTACCTTCAACTCC-3′,下游5′-CATACTCCTGCTTGCTGATC-3′,產(chǎn)物長(zhǎng)度265 bp。引物均由上海英濰捷基(上海)貿(mào)易有限公司合成。
1.5 RT-PCR檢測(cè)及半定量分析總反應(yīng)體系為20 μL,包括10×buffer(Mg2 +濃度為25 mmol/L)2.5 μL,10 mmol/L dNTP 0.5 μL,10 nmol/L上、下游引物各0.5 μL,5 U/μL Taq 酶0.2 μL,cDNA模板1 μL,ddH2O補(bǔ)足至20 μL。循環(huán)參數(shù):94℃5 min;94℃30 s,Sox2(60℃)、β-actin(56℃)30 s,72℃30 s,共35個(gè)循環(huán);72℃10 min。實(shí)驗(yàn)重復(fù)3次,取5 μL擴(kuò)增產(chǎn)物進(jìn)行10 g/L瓊脂糖凝膠電泳,溴化乙錠染色后用凝膠成像儀觀察拍照,并運(yùn)用Image tool分析軟件計(jì)算Sox2及β-actin條帶的A值,半定量分析胃癌組織及對(duì)應(yīng)癌旁組織Sox2 mRNA表達(dá)的相對(duì)強(qiáng)度(Sox2/β-actin),記錄3次結(jié)果進(jìn)行半定量分析。
1.6 SP法檢測(cè)Sox2蛋白表達(dá)Sox2抗體按1:100稀釋,PBS代替一抗作陰性對(duì)照,嚴(yán)格按SP試劑盒說(shuō)明書進(jìn)行操作。Sox2陽(yáng)性表達(dá)主要定位于細(xì)胞核,呈淡黃色、棕黃色及棕褐色顆粒,每張切片均由2名病理科中級(jí)以上醫(yī)師進(jìn)行雙盲法閱片,隨機(jī)讀取5個(gè)高倍鏡視野(×200)進(jìn)行觀察,采用染色強(qiáng)度結(jié)合陽(yáng)性細(xì)胞百分率雙評(píng)分進(jìn)行半定量計(jì)分,按陽(yáng)性細(xì)胞百分率評(píng)分:0分(無(wú)陽(yáng)性)、1分(<10%)、2分(10%~35%)、3分(>35%~75%)、4分(>75%);按染色強(qiáng)度評(píng)分:0分(不著色)、1分(淡黃色)、2分(棕黃色)、3分(棕褐色)。結(jié)果判定:兩種評(píng)分相乘所得的半定量計(jì)分0~1分為陰性, 2分為陽(yáng)性。
1.7統(tǒng)計(jì)學(xué)方法采用SPSS 13.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)處理。符合正態(tài)分布的計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示,2組間比較進(jìn)行配對(duì)t檢驗(yàn)。計(jì)數(shù)資料以例(%)表示,組間比較行χ2檢驗(yàn),以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2.1 2組Sox2 mRNA陽(yáng)性表達(dá)率比較胃癌組Sox2 mRNA的陽(yáng)性表達(dá)率(53.3%,32/60)高于癌旁組(20.0%,12/60,χ2=14.35,P<0.01),見(jiàn)圖1;胃癌組Sox2 mRNA相對(duì)表達(dá)強(qiáng)度顯著高于癌旁組(0.724± 0.209 vs. 0.256±0.065,t=16.56,P<0.01)。
2.2 2組間Sox2蛋白陽(yáng)性表達(dá)率比較胃癌組Sox2蛋白的陽(yáng)性表達(dá)率(50.0%,30/60)顯著高于癌旁組(16.7%,10/60,χ2=15.00,P<0.01),見(jiàn)圖2。
2.3胃癌組Sox2 mRNA陽(yáng)性表達(dá)與臨床病理參數(shù)的關(guān)系胃癌組Sox2 mRNA陽(yáng)性表達(dá)率臨床TNM分期(Ⅲ+Ⅳ)組高于(Ⅰ+Ⅱ)組,低、未分化組高于中、高分化組,浸潤(rùn)深度T3~T4組高于T1~T2組,淋巴結(jié)轉(zhuǎn)移組高于無(wú)轉(zhuǎn)移組(均P<0.05或P<0.01),見(jiàn)表1。
1~7:胃癌組(T)與對(duì)應(yīng)癌旁組(N);B:空白對(duì)照;M:DNA Marker DL 2 000Fig.1 Expression of Sox2 mRNA in gastric cancer and adjacent noncancerous tissues by agarose gel electrophoresis圖1 胃癌及癌旁組Sox2 mRNA表達(dá)的瓊脂糖凝膠電泳
Fig. 2 The positive expression of Sox2 protein in adjacent noncancerous and gastric cancer tissues(SP,×200)圖2 癌旁組和胃癌組Sox2蛋白陽(yáng)性表達(dá)(SP,×200)
Sox2基因定位于人類3號(hào)染色體(3q26.3-q27),屬于Sox超家族的SoxB1組,其編碼是由317個(gè)氨基酸組成。Sox2由氨基末端域、HMG區(qū)域及轉(zhuǎn)錄激活域3個(gè)主要區(qū)域組成。Sox2作為干細(xì)胞核心轉(zhuǎn)錄因子,協(xié)同Nanog、Oct3/4對(duì)維持胚胎干細(xì)胞多能性以及特定組織成體干細(xì)胞(adult stem cells,ASCs)自我更新的能力具有關(guān)鍵性的作用[6,11]。Sox2 在CSCs中表達(dá)水平相比較ASCs增高,在維持CSCs的永生及侵襲性過(guò)程中起重要作用[5]。近年來(lái)越來(lái)越多的研究表明,Sox2在多種實(shí)體瘤中異常表達(dá),如在乳腺癌[12]、食管癌[13]、膀胱癌[14]、前列腺癌[15]、肝癌[16]、卵巢上皮性癌[17]等中表達(dá)升高,可能具有癌基因的致瘤作用。本研究結(jié)果顯示,胃癌組中Sox2 mRNA及蛋白表達(dá)水平顯著高于癌旁組,另外,胃癌組Sox2 mRNA陽(yáng)性表達(dá)率臨床TNM分期(Ⅲ+Ⅳ)組高于(Ⅰ+Ⅱ)組,低、未分化組高于中、高分化組,浸潤(rùn)深度T3~T4組高于T1~T2組,淋巴結(jié)轉(zhuǎn)移組高于無(wú)轉(zhuǎn)移組,證實(shí)Sox2參與了胃癌的發(fā)生、發(fā)展、侵襲及轉(zhuǎn)移過(guò)程。有研究認(rèn)為,Sox2參與胰腺癌的侵襲與轉(zhuǎn)移,敲除胰腺癌細(xì)胞中Sox2后可經(jīng)由p21Cip1和p27Kip1轉(zhuǎn)錄誘導(dǎo),進(jìn)而通過(guò)細(xì)胞周期停滯(非凋亡)而導(dǎo)致細(xì)胞生長(zhǎng)受抑制[5]。
Tab. 1 Relationship between Sox2 expression andclinicopathological parameters in 60 gastric cancer patients表1 胃癌組Sox2表達(dá)與臨床病理參數(shù)的關(guān)系
但目前國(guó)內(nèi)外學(xué)者對(duì)胃癌等腫瘤中Sox2 mRNA及蛋白表達(dá)的具體作用尚存在爭(zhēng)議。有研究認(rèn)為Sox2具有癌基因作用,能促進(jìn)腫瘤的發(fā)生和發(fā)展[18];但另有研究表明其具有抑癌基因的功能,能抑制腫瘤的發(fā)生和發(fā)展,并認(rèn)為這可能與腫瘤組織異質(zhì)性及抗體特異性等因素的差異有關(guān)[19]。Matsuoka等[18]研究顯示,胃癌組中干細(xì)胞標(biāo)志物Sox2的陽(yáng)性表達(dá)與胃癌發(fā)生相關(guān),并與其浸潤(rùn)深度、TNM分期及淋巴結(jié)轉(zhuǎn)移有關(guān),與本研究結(jié)果基本一致。此外,本研究結(jié)果顯示,Sox2 mRNA表達(dá)還與其分化程度相關(guān),低、未分化組高于中、高分化組,提示Sox2表達(dá)升高的腫瘤細(xì)胞分化程度較低[6];Sox2的陽(yáng)性表達(dá)與其去分化狀態(tài)有關(guān)[20]。另有研究認(rèn)為,Sox2是多效性的原癌基因,其能誘導(dǎo)鱗癌標(biāo)記腫瘤相關(guān)因子p63和角蛋白6的表達(dá),調(diào)控鱗癌發(fā)生,并與CSCs的分化、侵襲與遷移相關(guān)[9]。Sox2可通過(guò)調(diào)控WNT/β-catenin信號(hào)通路使上皮細(xì)胞發(fā)生間質(zhì)轉(zhuǎn)化(epithelial-mensenchymal transition,EMT)[10]。Sox2是多種類型腫瘤中的譜系生存性癌基因,可促進(jìn)腫瘤發(fā)生;Sox2可通過(guò)上調(diào)Snail、Slug和Twist這3種主要蛋白來(lái)促進(jìn)EMT及胃癌擴(kuò)散與轉(zhuǎn)移,Sox2過(guò)表達(dá)能通過(guò)CyclinD3(CCND3)轉(zhuǎn)錄誘導(dǎo)而促進(jìn)細(xì)胞過(guò)度增殖[21]。Hütz等[7]研究證實(shí),胃癌組中Sox2具有癌基因作用。Tian等[22]研究顯示,從胃癌組中成功檢測(cè)出高表達(dá)的Sox2,癌組織中Sox2的陽(yáng)性表達(dá)率顯著高于癌旁組,其陽(yáng)性表達(dá)率與胃癌分化程度及有無(wú)淋巴結(jié)轉(zhuǎn)移相關(guān),與本研究結(jié)果相符。
綜上所述,本研究從轉(zhuǎn)錄及翻譯水平上檢測(cè)了CSCs標(biāo)志物Sox2在胃癌發(fā)生進(jìn)展中的癌基因作用,Sox2可作為胃癌基因診斷及靶向治療潛在的新的分子標(biāo)志物?;赟ox2在GCSCs形成腫瘤過(guò)程中的重要作用,進(jìn)一步研究Sox2在胃癌侵襲轉(zhuǎn)移、增殖及致瘤過(guò)程中的作用機(jī)制具有重要的臨床意義。
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(2015-11-05收稿2016-01-04修回)(本文編輯陸榮展)
Expression and clinical significance of stem cell marker Sox2 in human gastric cancer
CHEN Zhong1,XIE Feng2,ZHONG Fengyun3,DU Hong4,YAN Yongmin5,QIAN Hui5
1 Clinical Laboratory,2 General Surgery,Suzhou Yongding Hospital,Suzhou 215200,China;3 General Surgery,4 Clinical Laboratory,the 2nd Affiliated Hospital of Soochow University;5 School of Medicine,Jiangsu Key Laboratory of Medical Science and Laboratory Medicine,Jiangsu University
Objective To detect the expression of stem cell marker Sox2 in gastric cancer(GC). Methods The mRNA and protein expressions of Sox2 in paired primary tumor tissues and their matching,adjacent non-cancerous tissues in a series of 60 cases of human GC were examined by reverse transcription-PCR(RT-PCR)and immunohistochemistry (IHC).χ2test was used to analyze the correlation of Sox2 expression with clinicopathological parameters of GC tissues including age,gender,tumor size,histological type,TNM stage,differentiation degree,depth of invasion and lymph node metastasis. Results RT-PCR results showed that the positive rate of Sox2 expression was significantly increased in gastric tumor tissues(53.3%,32/60)compared with that of matching,adjacent non-cancerous tissues(20.0%,12/60,P<0.01). Semi-quantitative analysis showed that the relative intensity of Sox2 mRNA expression was significantly higher in gastric cancer tissues(0.724±0.209)than that in tissues adjacent to carcinoma(0.256±0.065,P<0.01). The positive expression of Sox2 was significantly higher in gastric tumor tissues(50.0%,30/60)than that of matching,adjacent non-cancerous tissues (16.7%,10/60,P<0.01). The positive expression of Sox2 was significantly higher in gastric tumor patients with TNM stage(Ⅲ+Ⅳ)than that of TNM stage(Ⅰ+Ⅱ). The positive expression of Sox2 was significantly higher in gastric tumor patients with low differentiation and undifferentiated tumor cells than that of patients with middle and high differented cells. The positive expression of Sox2 was also significantly higher in gastric tumor patients with the depth of invasion T3-T4than that of patients with T1-T2. The positive expression of Sox2 was significantly higher in gastric tumor patients with lymph node metastasis than that of patients without lymph node metastasis(P<0.05 or P<0.01). Conclusion The elevated expression of Sox2 is associated with the initiation,invasion,progression,and metastasis of GC. Sox2 may serve as a novel diagnostic and therapeutic marker for human GC.
stomach neoplasms;neoplastic stem cells;genes,tumor suppressor;immunohistochemistry;reverse transcriptase polymerase chain reaction;SRY-related HMG-box gene 2
R735.2
A
10.11958/20150286
蘇州市科技發(fā)展計(jì)劃項(xiàng)目(SYSD2012048)
1蘇州永鼎醫(yī)院檢驗(yàn)科(郵編215200),2普通外科;3蘇州大學(xué)附屬第二醫(yī)院普通外科,4檢驗(yàn)科;5江蘇大學(xué)醫(yī)學(xué)院,江蘇省檢驗(yàn)醫(yī)學(xué)重點(diǎn)實(shí)驗(yàn)室
陳忠(1968),男,副主任技師,碩士,主要從事血液及腫瘤分子生物學(xué)診斷研究