SCUBE2過表達(dá)通過Wnt/β-catenin抑制結(jié)直腸癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化

王 麗， 張 寧， 張龍江， 席作武

(河南省中醫(yī)院肛腸科，河南 鄭州 450002)

SCUBE2過表達(dá)通過Wnt/β-catenin抑制結(jié)直腸癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化

王 麗， 張 寧△， 張龍江， 席作武

(河南省中醫(yī)院肛腸科，河南 鄭州 450002)

目的： 研究信號肽-CUB-EGF結(jié)構(gòu)域蛋白2(SCUBE2)對結(jié)直腸癌細(xì)胞上皮-間質(zhì)轉(zhuǎn)化(EMT)的影響及可能的分子機(jī)制。方法:首先，用real-time PCR與Western blot法檢測人結(jié)直腸癌HCT116細(xì)胞和正常人結(jié)直腸上皮FHC細(xì)胞SCUBE2的表達(dá)；HCT116細(xì)胞轉(zhuǎn)染GV144-SCUBE2質(zhì)粒過表達(dá)SCUBE2后，MTT、Transwell和流式細(xì)胞術(shù)分別檢測其對細(xì)胞活力、遷移和凋亡的影響。轉(zhuǎn)染GV144-SCUBE2質(zhì)粒6 h后加入10 μg/L重組轉(zhuǎn)化生長因子(TGF)-β1蛋白繼續(xù)培養(yǎng)48 h，real-time PCR或者Western blot法檢測EMT標(biāo)志物[E-鈣黏蛋白(E-cadhe-rin)、波形蛋白(vimentin)和Snail]、β-catenin、c-Myc和細(xì)胞周期蛋白(cyclin)D1的表達(dá)。隨后，在HCT116細(xì)胞中使用Wnt/β-catenin通路激活劑氯化鋰(LiCl)或抑制劑XAV93920并加入TGF-β1且過表達(dá)SCUBE2，檢測Wnt/β-catenin通路激活或阻斷后對HCT116細(xì)胞EMT的影響。結(jié)果:HCT116細(xì)胞中SCUBE2的表達(dá)明顯低于FHC細(xì)胞；過表達(dá)SCUBE2能夠抑制HCT116細(xì)胞的活力與遷移，但是對其凋亡影響不大；SCUBE2增加了E-cadherin的表達(dá)，降低了TGF-β1誘導(dǎo)的vimentin、Snail、β-catenin、c-Myc和cyclin D1的表達(dá)，XAV93920增強(qiáng)了SCUBE2對EMT的影響，而LiCl阻礙SCUBE2對EMT的影響。結(jié)論:過表達(dá)SCUBE2能夠抑制結(jié)直腸癌細(xì)胞的生長與遷移，抑制其EMT的發(fā)生，這一過程可能是通過抑制Wnt/β-catenin信號通路發(fā)揮作用的。

SCUBE2； 結(jié)直腸癌； 上皮-間質(zhì)轉(zhuǎn)化； Wnt/β-catenin信號通路

結(jié)直腸癌是全球最常見的惡性腫瘤之一，在男性中居于第2位，在女性中居于第3位[1]。在結(jié)直腸癌中，上皮-間質(zhì)轉(zhuǎn)化(epithelial-to-mesenchymal transition, EMT)會導(dǎo)致基底膜缺失，且與不良預(yù)后有關(guān)[2]。信號肽-CUB-EGF結(jié)構(gòu)域蛋白2 [signal peptide-CUB (complement C1r/C1s, Uegf and Bmp1)-EGF (epidermal growth factor) domain-containing protein 2, SCUBE2]是SCUBE家族中一個分泌型且與膜相關(guān)的多結(jié)構(gòu)域糖蛋白[3]。近年來的研究顯示，SCUBE2在乳腺癌中是一個新的腫瘤抑制劑，它能夠通過逆轉(zhuǎn)EMT來抑制乳腺癌細(xì)胞的遷移和侵襲[4]，且它也是乳腺癌的一個重要預(yù)后標(biāo)志物[5]。最新的研究顯示SCUBE2在結(jié)直腸癌組織中表達(dá)降低，且與臨床表現(xiàn)密切相關(guān)[6]，但是SCUBE2對結(jié)直腸癌細(xì)胞EMT的作用及機(jī)制還不清楚。本研究通過在結(jié)直腸癌細(xì)胞中過表達(dá)SCUBE2，探究其對腫瘤細(xì)胞生長、遷移和凋亡的影響，并分析SCUBE2對轉(zhuǎn)化生長因子(transforming growth factor, TGF)-β1誘導(dǎo)的EMT的影響及其作用機(jī)制。

材 料 和 方 法

1 材料

人結(jié)腸癌細(xì)胞HCT116及正常人結(jié)直腸上皮FHC細(xì)胞來源于ATCC；DMEM培養(yǎng)基、胎牛血清、青霉素/鏈霉素雙抗溶液和胰酶均購自Gibco；NanoFectin轉(zhuǎn)染試劑購自上海依科賽生物制品有限公司；氯化鋰(lithium chloride，LiCl)購自Sigma；MTT及細(xì)胞毒性試劑盒購自碧云天生物技術(shù)公司；Annexin V-FITC/PI細(xì)胞凋亡試劑盒購自BD；TRIzol購自Invitrogen; SuperQuickRT cDNA第一鏈合成試劑盒與UltraSYBR Mixture均購自北京康為世紀(jì)公司；重組人TGF-β1蛋白、SCUBE2抗體、E-鈣黏蛋白(E-cadherin)抗體、波形蛋白(vimentin)抗體、Snail抗體和β-catenin抗體均購自Abcam;細(xì)胞周期蛋白(cyclin)D1 和c-Myc 購自Cell Signaling Technology；XAV93920購自Selleck Chemicals；BCA法蛋白定量試劑盒購自上海捷瑞生物工程有限公司。

2 方法

2.1 細(xì)胞培養(yǎng)和轉(zhuǎn)染 HCT116和FHC細(xì)胞均培養(yǎng)在含有10%胎牛血清、1×105U/L青霉素和100 mg/L鏈霉素的DMEM完全培養(yǎng)基中，置于37 ℃、5% CO2培養(yǎng)箱中常規(guī)培養(yǎng)。將對數(shù)期的HCT116細(xì)胞胰酶消化傳代，接種到特定的細(xì)胞培養(yǎng)板中。待細(xì)胞融合度達(dá)到60%時，按照轉(zhuǎn)染試劑說明書操作，分別轉(zhuǎn)染GV144和GV144-SCUBE2質(zhì)粒。轉(zhuǎn)染6 h后加入10 μg/L TGF-β1繼續(xù)培養(yǎng)48 h進(jìn)行后續(xù)檢測，或者加入TGF-β1培養(yǎng)24 h后加入20 mmol/L LiCl或10 μmol/L XAV93920再常規(guī)培養(yǎng)24 h。

2.2 細(xì)胞活力分析 采用MTT法檢測過表達(dá)SCUBE2對HCT116細(xì)胞活力的影響。將轉(zhuǎn)染24 h的HCT116細(xì)胞按照每孔1×103個的密度接種到96孔板中，繼續(xù)培養(yǎng)24 h、48 h和72 h后，按照MTT試劑盒說明書操作檢測各孔在570 nm處的吸光度值。

2.3 細(xì)胞遷移的檢測 利用Transwell法檢測過表達(dá)SCUBE2對HCT116細(xì)胞遷移的影響。將轉(zhuǎn)染24 h的細(xì)胞在無血清的培養(yǎng)中培養(yǎng)12 h，然后將200 μL含有5×104個細(xì)胞的無血清培養(yǎng)基加入到Transwell上室，下室加入500 μL含有10%胎牛血清的DMEM培養(yǎng)基，繼續(xù)培養(yǎng)48 h后，取出小室，4%多聚甲醛固定30 min，用棉簽輕輕擦掉上層未遷移細(xì)胞，0.1%結(jié)晶紫染色20 min，PBS沖洗后，顯微鏡下隨機(jī)選取5個視野計(jì)數(shù)穿膜細(xì)胞數(shù)，實(shí)驗(yàn)重復(fù)3次，取平均值。

2.4 細(xì)胞凋亡的檢測 采用Annexin V-FITC/PI雙染進(jìn)行流式細(xì)胞術(shù)檢測過表達(dá)SCUBE2對HCT116細(xì)胞凋亡的影響。用不含EDTA的胰酶消化并收集轉(zhuǎn)染48 h的細(xì)胞，預(yù)冷的PBS洗滌細(xì)胞2次，按照Annexin V-FITC/PI細(xì)胞凋亡試劑盒說明書操作，用FACSCalibur流式細(xì)胞儀檢測細(xì)胞的凋亡情況。實(shí)驗(yàn)重復(fù)3次，凋亡細(xì)胞包括凋亡早期和凋亡晚期細(xì)胞。

2.5 Real-time PCR實(shí)驗(yàn) 按照TRIzol說明書操作，提取細(xì)胞的總RNA，取2 μg總RNA進(jìn)行逆轉(zhuǎn)錄，然后按照UltraSYBR Mixture說明書加樣后進(jìn)行Real-time PCR。以GAPDH為內(nèi)參照，按照2-ΔΔCt的方法計(jì)算目的基因的相對表達(dá)量，引物序列見表1。

表1 引物序列

2.6 Western blot實(shí)驗(yàn) RIPA裂解液將細(xì)胞充分裂解后，4 ℃、12 000×g離心15 min收集上清，即為提取細(xì)胞的總蛋白。BCA試劑盒測定蛋白質(zhì)濃度后，將蛋白樣品沸水浴煮沸5 min，每孔取相同質(zhì)量的總蛋白上樣進(jìn)行SDS-PAGE分離蛋白，然后將蛋白轉(zhuǎn)移至聚偏二氟乙烯(polyvinylidene difluoride，PVDF)膜上，5%脫脂奶粉室溫封閉2 h，加入 I 抗 [SCUBE2(1∶500稀釋)、E-cadherin(1∶800稀釋)、vimentin(1∶1 000稀釋)、Snail(1∶1 000稀釋)、β-catenin(1∶8 000稀釋)、c-Myc(1∶1 000稀釋)和cyclin D1(1∶1 000稀釋)] 4 ℃過夜孵育，TBST洗膜3次后加入HRP標(biāo)記的 II 抗室溫孵育45 min，再用TBST洗膜后加入新鮮配置的ECL發(fā)光液，將膜充分覆蓋后拍照進(jìn)行灰度值分析。

3 統(tǒng)計(jì)學(xué)處理

采用SPSS 13.0軟件進(jìn)行統(tǒng)計(jì)分析，計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，兩組間比較采用t檢驗(yàn)，多組間比較采用單因素方差分析。P<0.05表明差異具有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 人結(jié)腸癌細(xì)胞中SCUBE2的表達(dá)降低

Real-time和Western blot實(shí)驗(yàn)檢測人結(jié)腸癌細(xì)胞HCT116和正常人結(jié)直腸上皮FHC細(xì)胞中SCUBE2的表達(dá)，發(fā)現(xiàn)與FHC細(xì)胞相比，HCT116細(xì)胞中SCUBE2的表達(dá)在mRNA和蛋白水平均明顯降低(P<0.05)，見圖1。

Figure 1.The expression of SCUBE2 at mRNA (A) and protein (B) levels in different colorectal cells were detected by real-time PCR and Western blot. Mean±SD.n=3.**P<0.01vsFHC group.

圖1 不同結(jié)直腸細(xì)胞中SCUBE2的mRNA和蛋白表達(dá)

2 過表達(dá)SCUBE2抑制HCT116細(xì)胞的活力

HCT116細(xì)胞轉(zhuǎn)染GV144 或GV144-SCUBE2質(zhì)粒48 h后，Western blot法檢測SCUBE2的表達(dá)結(jié)果顯示，與GV144組相比，GV144-SCUBE2組中SCUBE2的表達(dá)明顯升高(P<0.05)。轉(zhuǎn)染24 h、48 h和72 h后分別用MTT法檢測細(xì)胞的活力，與GV144組相比，GV144-SCUBE2組中HCT116細(xì)胞的活力明顯降低(P<0.05)，見圖2。

Figure 2.The effect of SCUBE2 over-expression on the viability of HCT116 cells. A: Western blot analysis for SCUBE2 expression; B: MTT assay for cell viability. Mean±SD.n=3.**P<0.01vsGV144 group.

圖2 過表達(dá)SCUBE2對HCT116細(xì)胞活力的影響

3 過表達(dá)SCUBE2抑制HCT116細(xì)胞的遷移

Transwell實(shí)驗(yàn)的檢測結(jié)果顯示，與GV144組相比，GV144-SCUBE2組的遷移細(xì)胞數(shù)目明顯降低，差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，見圖3。

4 過表達(dá)SCUBE2對HCT116細(xì)胞的凋亡無影響

流式細(xì)胞術(shù)檢測細(xì)胞凋亡結(jié)果顯示，與GV144組相比，GV144-SCUBE2組中細(xì)胞的凋亡率稍微增加，但是差異無統(tǒng)計(jì)學(xué)顯著性，見圖4。

Figure 3.The effects of SCUBE2 over-expression on the number of migratory HCT116 cells. Mean±SD.n=3.**P<0．01vsGV144 group.

圖3 過表達(dá)SCUBE2對HCT166細(xì)胞遷移數(shù)目的影響

5 過表達(dá)SCUBE2對HCT116細(xì)胞EMT的影響

Real-time PCR和Western blot實(shí)驗(yàn)檢測E-cadherin、vimentin和Snail的表達(dá)。與對照組相比，TGF-β1組中E-cadherin的mRNA和蛋白表達(dá)均降低(P<0.05)；vimentin和Snail的mRNA和蛋白表達(dá)均明顯增加(P<0.05)；與TGF-β1組相比，TGF-β1+SCUBE2組中E-cadherin的mRNA和蛋白表達(dá)均增加(P<0.05)，vimentin和Snail的mRNA和蛋白表達(dá)均降低(P<0.05)，見圖5。

6 SCUBE2通過Wnt/β-catenin信號通路抑制EMT進(jìn)程

Western blot實(shí)驗(yàn)檢測HCT116細(xì)胞中β-catenin、c-Myc和cyclin D1的蛋白表達(dá)。與對照組相比，TGF-β1組中三者的表達(dá)均明顯增加(P<0.05)；與TGF-β1組相比，TGF-β1+SCUBE2組中三者的表達(dá)均明顯降低(P<0.05)，見圖6A。

Figure 4.The effects of SCUBE2 over-expression on the apoptosis of HCT116 cells. Mean±SD.n=3.

圖4 過表達(dá)SCUBE2對HCT116細(xì)胞凋亡的影響

Figure 5.The effects of SCUBE2 over-expression on the mRNA (A) and protein (B) expression of EMT markers. Mean±SD.n=3.**P<0．01vscontrol group;##P<0．01vsTGF-β1 group.

圖5 過表達(dá)SCUBE2對HCT116細(xì)胞EMT標(biāo)志物表達(dá)的影響

另外，HCT116細(xì)胞轉(zhuǎn)染GV144-SCUBE2質(zhì)粒6 h后加入TGF-β1蛋白(10 μg/L)，培養(yǎng)24 h后加入LiCl(Wnt/β-catenin通路激活劑，20 mmol/L)或XAV93920 (Wnt/β-catenin通路抑制劑，10 μmol/L)繼續(xù)培養(yǎng)24 h。Western blot法檢測c-Myc、E-cadhe-rin、vimentin和Snail的表達(dá)。與TGF-β1+SCUBE2組相比，TGF-β1+SCUBE2+XAV93920組的E-cadherin表達(dá)明顯增加，c-Myc、vimentin和Snail的表達(dá)明顯降低(P<0.05)；與TGF-β1+SCUBE2組相比，TGF-β1+SCUBE2+LiCl組中E-cadherin的表達(dá)明顯降低，c-Myc、vimentin和Snail的表達(dá)明顯增加(P<0.05)，見圖6B。

Figure 6.SCUBE2 inhibited EMT through Wnt/β-catenin signaling pathway. A: Western blot analysis for the expression of β-catenin, c-Myc, and cyclin D1; B: the expression of c-Myc and EMT markers in the HCT116 cells treated with 20 mmol/L LiCl or 10 μmol/L XAV93920 combined with SCUBE2 expression. Mean±SD.n=3.**P<0.01vscontrol;##P<0.01vsTGF-β1 group;△P<0.05,△△P<0.01vsTGF-β1+SCUBE2 group.

圖6 SCUBE2通過Wnt/β-catenin信號通路抑制EMT進(jìn)程

討 論

SCUBE2蛋白包含1個N端信號肽序列、9個EGF樣重復(fù)序列、1個沉默區(qū)、3個富含半胱氨酸的重復(fù)序列和1個C端CUB結(jié)構(gòu)域[3]。它主要表達(dá)于血管內(nèi)皮細(xì)胞和一些非內(nèi)皮類型的細(xì)胞中，如成纖維細(xì)胞、腎間質(zhì)細(xì)胞和乳腺導(dǎo)管上皮細(xì)胞[5, 7]。SCUBE2在乳腺癌中發(fā)揮關(guān)鍵作用，它能結(jié)合并拮抗骨形態(tài)發(fā)生蛋白的活性，抑制β-catenin通路從而抑制乳腺癌細(xì)胞生長[3, 5]；而且還能夠逆轉(zhuǎn)EMT過程從而抑制乳腺癌細(xì)胞的遷移和侵襲[4]。Parris等[8]的研究發(fā)現(xiàn)，SCUBE2的表達(dá)也與口腔鱗狀細(xì)胞癌的臨床病理特征相關(guān)，如癌周炎性浸潤、頸部淋巴結(jié)轉(zhuǎn)移和腫瘤大小等。最近的研究發(fā)現(xiàn)，SCUBE2與結(jié)直腸癌的疾病進(jìn)程和預(yù)后相關(guān)[6]，但是目前其相關(guān)機(jī)制還沒有報(bào)道。我們結(jié)果中發(fā)現(xiàn)與正常結(jié)直腸上皮FHC細(xì)胞相比，HCT116結(jié)腸癌細(xì)胞中SCUBE2的表達(dá)明顯降低。

EMT是上皮細(xì)胞在特定的生理或病理?xiàng)l件下向間充質(zhì)細(xì)胞轉(zhuǎn)化的現(xiàn)象，是腫瘤細(xì)胞獲得遷移和侵襲能力的分子基礎(chǔ)[9]。本研究中過表達(dá)SCUBE2能夠抑制HCT116細(xì)胞的活力與遷移，這表明SCUBE2可能與其EMT進(jìn)程相關(guān)。EMT過程伴隨著上皮表型黏附分子E-cadherin的減少或缺失，間質(zhì)表型vimentin 和Snail表達(dá)增加[10]。在EMT相關(guān)的信號通路中，TGF-β信號通路被認(rèn)為在EMT過程中發(fā)揮關(guān)鍵作用[11]，TGF-β能激活上皮細(xì)胞向間充質(zhì)轉(zhuǎn)化[12]。本研究中TGF-β1刺激后，E-cadherin的表達(dá)降低，vimentin 和Snail表達(dá)增加，表明TGF-β1成功誘導(dǎo)HCT116細(xì)胞EMT過程。隨后本研究檢測SCUBE2過表達(dá)對TGF-β1誘導(dǎo)的EMT標(biāo)志物的影響發(fā)現(xiàn)，SCUBE2能夠增加E-cadherin的表達(dá)，降低Vimentin 和Snail的表達(dá)，這表明過表達(dá)SCUBE2基因可以抑制TGF-β1誘導(dǎo)的EMT進(jìn)程。

Wnt/β-catenin通路在人類腫瘤中常被激活，它與腫瘤細(xì)胞EMT進(jìn)程密切相關(guān)[13-14]，c-Myc和cyclin D1是該通路的2個下游蛋白[15]。本研究進(jìn)一步檢測過表達(dá)SCUBE2與HCT116細(xì)胞的EMT機(jī)制發(fā)現(xiàn)，增高SCUBE2的表達(dá)能夠降低β-catenin、c-Myc和cyclin D1的表達(dá)，這表明SCUBE2抑制了Wnt/β-catenin通路的激活。糖原合成酶激酶3β(glycogen synthase kinase-3β，GSK3β)是一個絲氨酸/蘇氨酸蛋白激酶，是Wnt信號通路中一個重要的負(fù)調(diào)控因子，能夠磷酸化通路中的蛋白使其降解[16]。LiCl 是GSK3β的抑制劑，能夠激活Wnt/β-catenin通路[17]。本研究使用LiCl和Wnt/β-catenin通路抑制劑XAV93920，進(jìn)一步探究SCUBE2是否是通過Wnt/β-catenin通路抑制HCT116細(xì)胞的EMT進(jìn)程。LiCl能夠拮抗過表達(dá)SCUBE2引起的E-cadherin上調(diào)以及vimentin和Snail下調(diào)；XAV93920能夠增強(qiáng)過表達(dá)SCUBE2對TGF-β1介導(dǎo)的HCT116細(xì)胞EMT的影響。這些結(jié)果表明過表達(dá)SCUBE2是通過Wnt/β-catenin通路抑制結(jié)直腸癌細(xì)胞的EMT進(jìn)程。

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(責(zé)任編輯： 林白霜， 羅 森)

Over-expression of SCUBE2 suppresses epithelial-mesenchymal transition through Wnt/β-catenin signaling pathway in colorectal cancer cells

WANG Li, ZHANG Ning, ZHANG Long-jiang, XI Zuo-wu

(AnorectalDepartment,HenanProvinceHospitalofTCM,Zhengzhou450002,China.E-mail:wenyinxian029@163.com)

AIM: To study the effect of SCUBE2 on epithelial-mesenchymal transition (EMT) in colorectal cancer cells and its mechanism. METHODS: The expression of SCUBE2 in human colorectal cancer cell line HCT116 and normal colonic cell line FHC was detected by real-time PCR and Western blot. HCT116 cells were transfected with GV144-SCUBE2 to over-express SCUBE2, and then the cell viability, migration, and apoptosis were determined by MTT assay, Transwell assay and flow cytometry, respectively. The expression of EMT markers (E-cadherin, vimentin, and Snail), β-catenin, c-Myc and cyclin D1 in the HCT116 cells was analyzed by real-time PCR or Western blot after transfection with GV144-SCUBE2 for 6 h, followed by the stimulation of 10 μg/L recombinant TGF-β1 protein for 48 h. Additionally, the EMT process of HCT116 cells, which were stimulated by TGF-β1, over-expressed SCUBE2, and treated with Wnt/β-catenin pathway activator lithium chloride (LiCl) or inhibitor XAV93920, was analyzed by Western blot. RESULTS: Compared with FHC cells, the expression of SCUBE2 in the HCT116 cells was significantly decreased. The viability and migration ability of the HCT116 cells were suppressed by SCUBE2 over-expression, but the apoptosis was not markedly changed. Elevated expression of SCUBE2 increased E-cadherin expression, and decreased the expression of vimentin, Snail, β-catenin, c-Myc and cyclin D1 induced by TGF-β1. Treatment with LiCl blocked but treatment with XAV93920 enhanced the effects of SCUBE2 on EMT. CONCLUSION: Over-expression of SCUBE2 may inhibit the cell growth and migration, and suppress EMT through Wnt/β-catenin signaling pathway.

SCUBE2; Colorectal cancer; Epithelial-mesenchymal transition; Wnt/β-catenin signaling pathway

1000- 4718(2016)12- 2245- 06

2016- 06- 06

2016- 09- 18

R735.3； R730.23

A

10.3969/j.issn.1000- 4718.2016.12.020

雜志網(wǎng)址： http://www.cjpp.net

△通訊作者 Tel: 0371-53312157; E-mail: wenyinxian029@163.com