馬松林，張姮，廖宇圣，徐丹，吳杰

(華中科技大學(xué)同濟醫(yī)學(xué)院附屬武漢市中心醫(yī)院，武漢430014)

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Wnt3a對結(jié)腸癌SW480細(xì)胞增殖的影響及作用機制

馬松林，張姮，廖宇圣，徐丹，吳杰

(華中科技大學(xué)同濟醫(yī)學(xué)院附屬武漢市中心醫(yī)院，武漢430014)

目的 探討Wnt3a對結(jié)腸癌SW480細(xì)胞增殖的調(diào)控效應(yīng)及其作用機制。方法 將培養(yǎng)好的結(jié)腸癌細(xì)胞SW480隨機分為對照組和Wnt3a組，對照組僅給予DMEM培養(yǎng)基處理，Wnt3a組給予100 ng/mL Wnt3a培養(yǎng)。用MTT法檢測細(xì)胞增殖情況，計算相對活細(xì)胞百分比；用平板克隆形成實驗檢測細(xì)胞克隆形成率(PE)；用流式細(xì)胞術(shù)檢測細(xì)胞周期分布；用Western blotting法檢測細(xì)胞內(nèi)醛脫氫酶1B1(ALDH1B1)蛋白表達，RT-PCR技術(shù)檢測ALDH1B1 mRNA表達。結(jié)果 Wnt3a誘導(dǎo)24、48、72 h，Wnt3a組相對活細(xì)胞百分比高于對照組(P均<0.05)。Wnt3a誘導(dǎo)7 d，Wnt3a組PE高于對照組(P<0.05)。Wnt3a誘導(dǎo)24 h，Wnt3a組G1期細(xì)胞百分比低于對照組，G2期細(xì)胞百分比高于對照組(P均<0.05)。Wnt3a誘導(dǎo)48 h，Wnt3a組ALDH1B1蛋白、mRNA相對表達量高于對照組(P均<0.05)。結(jié)論 Wnt3a可能通過誘導(dǎo)ALDH1B1高表達促進結(jié)腸癌SW480細(xì)胞增殖。

結(jié)腸腫瘤；SW480細(xì)胞；Wnt3a；醛脫氫酶1B1；細(xì)胞增殖

結(jié)腸癌是最常見的惡性腫瘤之一。據(jù)調(diào)查，2014年美國有結(jié)腸癌新增病例136 830例，因結(jié)腸癌死亡50 310例[1]。結(jié)腸癌的發(fā)病與幾種致瘤信號通路的異常激活密切相關(guān)。其中，Wnt/β-catenin信號通路被認(rèn)為是結(jié)腸癌發(fā)病的重要原因之一[2]。Wnt/β-catenin信號的異常激活引導(dǎo)結(jié)腸正常細(xì)胞向癌細(xì)胞轉(zhuǎn)變、促進結(jié)腸癌細(xì)胞向結(jié)腸基底細(xì)胞侵襲[2～4]。Wnt3a是Wnt信號蛋白的重要成員。研究表明，Wnt3a能促進結(jié)腸癌腫瘤組織血管形成[5,6]，然而其對結(jié)腸癌細(xì)胞的增殖調(diào)控效應(yīng)及其作用機制目前依然未知。2015年1月～2016年1月，本研究就Wnt3a對結(jié)腸癌細(xì)胞SW480增殖的調(diào)控作用及其分子機制進行了探討。

1 材料與方法

1.1 材料 DMEM培養(yǎng)基、胎牛血清(FBS)、胰蛋白酶(購自美國Hyclone公司)，TRIzol(美國Invitrogen公司)，醛脫氫酶1B1抗體(anti-ALDH1B1)和anti-GAPDH(美國Santa Cruz公司)，HRP標(biāo)記的二抗(英國Abcam公司)，人類結(jié)腸癌細(xì)胞SW480(美國ATCC細(xì)胞庫)。

1.2 細(xì)胞培養(yǎng)及分組 結(jié)腸癌SW480細(xì)胞均采用DMEM培養(yǎng)基(添加10% FBS以及抗生素)于37 ℃、5%二氧化碳、飽和濕度的恒溫細(xì)胞培養(yǎng)箱中培養(yǎng)。2～3 d傳代1次，將處于對數(shù)生長期的細(xì)胞經(jīng)消化分散后計數(shù)，制成細(xì)胞懸液。將SW480細(xì)胞隨機分為對照組和Wnt3a組。對照組僅給予DMEM培養(yǎng)基處理，Wnt3a組給予100 ng/mL Wnt3a培養(yǎng)24 h。

1.3 細(xì)胞增殖情況檢測 采用MTT法。Wnt3a誘導(dǎo)24、48、72 h，分別取SW480細(xì)胞按每孔10 000個接種于12孔板中，培養(yǎng)24 h 后添加5 mg/mL的MTT溶液40 μL，孵育4 h 后每孔加DMSO 200 μL。用酶標(biāo)儀于波長490 nm處測吸光度值。計算相對活細(xì)胞百分比，相對活細(xì)胞百分比=實驗組吸光度值/對照組吸光度值×100%。

1.4 細(xì)胞克隆形成率(PE)檢測 采用平板克隆形成實驗。Wnt3a誘導(dǎo)24 h，將細(xì)胞接種于12孔板中，每孔接種600個細(xì)胞。繼續(xù)培養(yǎng)8 d，先去除培養(yǎng)液，用PBS清洗3遍，甲醇固定20 min，1%亞甲基藍(lán)染色40 min，去離子水清洗兩遍，晾干。顯微鏡下計數(shù)不少于50個細(xì)胞的克隆數(shù)，計算PE。PE=克隆數(shù)/接種細(xì)胞數(shù)×100%。

1.5 細(xì)胞周期檢測 采用流式細(xì)胞術(shù)。Wnt3a誘導(dǎo)24 h，取細(xì)胞用PBS洗滌兩遍，用70%乙醇固定，4 ℃保存過夜。PBS清洗1次，將細(xì)胞調(diào)整為1×106/mL，加入碘化丙啶染色液，染色30 min。采用流式細(xì)胞術(shù)檢測細(xì)胞周期，計算各周期細(xì)胞百分比。

1.6 細(xì)胞內(nèi)ALDH1B1表達檢測 ①采用Western blotting法檢測細(xì)胞內(nèi)ALDH1B1蛋白表達。Wnt3a誘導(dǎo)48 h，根據(jù)文獻[7]進行操作，使用anti-ALDH1B1一抗及anti-GAPDH一抗，濃度為1∶200，二抗?jié)舛葹?∶1 000。用Quantity One 1-D分析軟件對蛋白質(zhì)印跡條帶進行分析。目的蛋白相對表達量=目的蛋白灰度值/內(nèi)參蛋白灰度值。②采用RT-PCR技術(shù)檢測細(xì)胞內(nèi)ALDH1B1 mRNA表達。Wnt3a誘導(dǎo)48 h，按照TRIzol reagent說明書提取細(xì)胞總RNA,將所得的RNA使用ImPron-Ⅱ逆轉(zhuǎn)錄系統(tǒng)(Roche,USA)逆轉(zhuǎn)錄。使用SYBR Green PCR master mix試劑在ABI 7500 RT-PCR儀中進行RT-PCR。ALDH1B1 正向引物:5′-CCCATTCTGAACCCAGACATC-3′;反向引物:5′-AATGACCTCCCCGGTGGTA-3′；β-actin正向引物:5′-TGGCACCCAGCACAATGAA-3′;反向引物:3′-CTAAGTCATAGTCC- GCCTAGAAGCA-5′。反應(yīng)條件：95 ℃ 5 min, 94 ℃變性30 s，60 ℃退火30 s，共進行40個循環(huán)。采用2-ΔΔCt法計算目的基因相對表達量。

2 結(jié)果

2.1 兩組細(xì)胞增殖情況比較 Wnt3a誘導(dǎo)24、48、72 h，Wnt3a組相對活細(xì)胞百分比分別為(240.63±27.34)%、(340.43±37.04)%、(410.23±45.21)%，對照組分別為(188.46±16.67)%、(236.75±26.68)%、(286.42±26.53)%；兩組各時間點活細(xì)胞百分比比較差異有統(tǒng)計學(xué)意義(P均<0.05)。

2.2 兩組PE比較 Wnt3a誘導(dǎo)7 d，Wnt3a組PE為(69.53±3.28)%，對照組為(27.36±2.57)%，兩組PE比較差異有統(tǒng)計學(xué)意義(P<0.05)。

2.3 兩組細(xì)胞周期分布情況比較 Wnt3a誘導(dǎo)24 h，Wnt3a組G1、G2期細(xì)胞百分比分別為(33.7±3.6)%、(21.2±2.5)%，對照組分別為(48.3±4.8)%、(10.8±1.4)%；兩組細(xì)胞周期分布比較差異有統(tǒng)計學(xué)意義(P均<0.05)。

2.4 兩組ALDH1B1表達比較 Wnt3a 誘導(dǎo)48 h，Wnt3a組ALDH1B1蛋白相對表達量為1.95±0.18，mRNA相對表達量為13.75±1.82，對照組分別為0.48±0.04、1.00±0.00；兩組ALDH1B1蛋白、mRNA相對表達量比較差異有統(tǒng)計學(xué)意義(P均<0.05)。

3 討論

近年來，對結(jié)腸癌分子機制的研究已經(jīng)取得了顯著進展。然而，目前為止，結(jié)腸癌依然是高度致死性的癌癥之一[1]。研究表明，在結(jié)腸癌的發(fā)病過程中，Wnt/β-catenin信號通路出現(xiàn)異常，包括c-Myc和CyclinD1等在內(nèi)Wnt/β-catenin信號依賴性分子轉(zhuǎn)錄失調(diào)，被認(rèn)為是導(dǎo)致結(jié)腸癌發(fā)生的重要原因[5]。有催化活性的ALDH已被認(rèn)定為多種腫瘤和腫瘤干細(xì)胞的生物標(biāo)志物[5,6]。研究表明，在正常人類結(jié)腸，ALDH1B1僅在隱窩基底部及干細(xì)胞中表達。然而，該蛋白也在人結(jié)腸腺癌中高度表達[6,7]。有研究結(jié)果表明，ALDH1B1在結(jié)腸癌細(xì)胞的基因表達譜與Wnt/β-catenin信號活性具有某種同步性，因此，ALDH1B1被認(rèn)為是結(jié)腸癌發(fā)生的原因之一[8]。Wnt3a是Wnt信號蛋白的重要成員，能促進結(jié)腸癌腫瘤組織血管形成[3]。然而，Wnt3a與ALDH1B1的相互作用依然未知。本研究著重探討了Wnt3a對結(jié)腸癌細(xì)胞SW480的細(xì)胞增殖調(diào)控作用并進一步探討了Wnt3a對ALDH1B1的基因表達調(diào)控效應(yīng)。

ALDH1B1的生物學(xué)功能是通過代謝視黃醛而產(chǎn)生視黃酸(RA)[9]。RA作為一種維生素A類衍生物，是細(xì)胞增殖發(fā)育的必需成分[10]。RA能結(jié)合細(xì)胞視黃酸結(jié)合蛋白(CARBPⅡ)和脂肪酸結(jié)合蛋白5(FABP5)[11]。RA誘導(dǎo)CARBPⅡ和FABP5介導(dǎo)的視黃酸受體(RAR)或PPARβ/δ激活[12]。RAR的激活能刺激細(xì)胞分化，并抑制細(xì)胞增殖[13]。然而，PPARβ/δ的激活對PI3K/Akt介導(dǎo)的腫瘤生長有促進效應(yīng)[14]。FABP5在結(jié)腸癌細(xì)胞中高表達，因此，推測RA能通過誘導(dǎo)FABP5激活PPARβ/δ從而促進細(xì)胞增殖、抗凋亡及促進腫瘤生長[15,16]。根據(jù)本研究的結(jié)果發(fā)現(xiàn)，Wnt3a促進ALDH1B1的基因表達，后者又可能通過激活PPARβ/δ，進而最終促進PI3K/Akt介導(dǎo)的腫瘤生長。

本研究利用Wnt3a蛋白誘導(dǎo)結(jié)腸癌細(xì)胞SW480，通過MTT活細(xì)胞計數(shù)、克隆形成實驗及細(xì)胞周期分析發(fā)現(xiàn)，Wnt3a能顯著促進SW480細(xì)胞生長、克隆形成及促使SW480細(xì)胞進入G2期。同時發(fā)現(xiàn)，Wnt3a能上調(diào)SW480細(xì)胞內(nèi)ALDH1B1表達。ALDH1B1是一種重要的結(jié)腸癌細(xì)胞增殖促進因子[6]，Wnt3a通過促進ALDH1B1的表達，從而促進結(jié)腸癌細(xì)胞的增殖。

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Effect of Wnt3a on cell proliferation of colon cancer and its mechanism

MASonglin,ZHANGHeng,LIAOYusheng,XUDan,WUJie

(WuhanCentralHospitalAffiliatedtoHuazhongUniversityofScienceandTechnology,Wuhan430014,China)

Objective To investigate the potential role of Wnt3a in the regulation of colon cancer cell proliferation and its regulatory mechanism. Methods Colon carcinoma cell line SW480 was randomly divided into two groups, the control group treated with DMEM, and the Wnt3a group treated with 100 ng/mL Wnt3a. The proliferation ability was measured by MTT. The relative percentage of living cells was also counted. Cell clone formation rate (PE) was measured by plate clone formation assay. Cell cycle was analyzed by flow cytometry. RT-PCR was used to measure the expression level of ALDH1B1 mRNA. The expression level of aldehyde dehydrogenase (ALDH)1B1 protein was measured by Western blotting. Results After 24, 48 and 72 h of induction by Wnt3a, the relative percentage of living cells in the Wnt3a group was significantly higher than that of the control group (allP<0.05). After 7 d of induction by Wnt3a, the PE of the Wnt3a group was significantly higher than that of the control group (P<0.05). The cell percentage of G1phase in the Wnt3a group was significantly lower, and the cell percentage of G2phase was significantly higher than that of the control group after 24-hour treatment of Wnt3a (allP<0.05). After 48-hour treatment with Wnt3a, the expression level of ALDH1B1 protein and mRNA in the Wnt3a group was significantly higher than that of the control group (allP<0.05).Conclusion Wnt3a promotes colon cancer cell proliferation by inducing the high expression of ALDH1B1.

colonic neoplasms; SW480 cells; Wnt3a; aldehyde dehydrogenase1B1; cell proliferation

馬松林(1979-)，男，主治醫(yī)師，碩士，主要研究方向為消化道腫瘤。E-mail:msl1002@126.com

簡介：吳杰(1958-)，男，主任醫(yī)師，碩士生導(dǎo)師，主要研究方向為消化道腫瘤。E-mail:47343977@qq.com

10.3969/j.issn.1002-266X.2016.37.006

R735.35

A

1002-266X(2016)37-0018-03

2016-03-12)