FMNL3基因轉(zhuǎn)染對結(jié)直腸癌SW480細(xì)胞EMT和體外侵襲能力的影響*

曾元鳳,肖移生,羅小江,胡國柱,路名芝,吳曉牧,辛洪波

(1.江西省人民醫(yī)院病理科,江西南昌 330006;2.江西中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)院形態(tài)學(xué)教研室,江西南昌,330004;3.江西省人民醫(yī)院普外科,江西南昌 330006;4.江西省人民醫(yī)院干細(xì)胞重點(diǎn)實(shí)驗(yàn)室,江西南昌 330006;5.江西省人民醫(yī)院神經(jīng)內(nèi)科,江西南昌 330006;6.南昌大學(xué)轉(zhuǎn)化醫(yī)學(xué)研究院,江西南昌, 330031)

FMNL3基因轉(zhuǎn)染對結(jié)直腸癌SW480細(xì)胞EMT和體外侵襲能力的影響*

曾元鳳1,肖移生2,羅小江3,胡國柱4,路名芝1,吳曉牧5△,辛洪波6▲

(1.江西省人民醫(yī)院病理科,江西南昌 330006;2.江西中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)院形態(tài)學(xué)教研室,江西南昌,330004;3.江西省人民醫(yī)院普外科,江西南昌 330006;4.江西省人民醫(yī)院干細(xì)胞重點(diǎn)實(shí)驗(yàn)室,江西南昌 330006;5.江西省人民醫(yī)院神經(jīng)內(nèi)科,江西南昌 330006;6.南昌大學(xué)轉(zhuǎn)化醫(yī)學(xué)研究院,江西南昌, 330031)

目的探討成蛋白樣3型蛋白(FMNL3)基因轉(zhuǎn)染對結(jié)直腸癌SW480細(xì)胞上皮-間質(zhì)轉(zhuǎn)化(EMT)和體外侵襲能力的影響?方法 將帶綠色熒光蛋白的重組FMNL3表達(dá)質(zhì)粒pEGFP-N1/FMNL3轉(zhuǎn)染至SW480細(xì)胞中(以未轉(zhuǎn)染質(zhì)粒的SW480細(xì)胞作正常對照,轉(zhuǎn)染空載質(zhì)粒的SW480細(xì)胞為陰性對照),熒光定量PCR(RT-qPCR)和 Western blot法檢測重組質(zhì)粒轉(zhuǎn)染前?后細(xì)胞FMNL3mRNA和蛋白的表達(dá)變化;在熒光顯微鏡下觀察FMNL3轉(zhuǎn)染前?后SW480細(xì)胞的形態(tài)變化;采用RT-qPCR和Western blot檢測轉(zhuǎn)染前?后SW480細(xì)胞EMT上皮標(biāo)志物E-cadherin和間質(zhì)標(biāo)志物Vimentin的表達(dá)變化;用Transwell小室實(shí)驗(yàn)檢測FMNL3基因轉(zhuǎn)染對SW480細(xì)胞的體外侵襲能力的影響?結(jié)果FMNL3轉(zhuǎn)染48h后SW480細(xì)胞中FMNL3mRNA和蛋白表達(dá)水平明顯提高(P0.01);FMNL3蛋白表達(dá)于細(xì)胞質(zhì)中,轉(zhuǎn)染FMNL3基因后SW480細(xì)胞的形態(tài)發(fā)生了明顯的改變,由典型的圓形?多邊形變成梭形并伸出多個(gè)細(xì)長突起的間充質(zhì)樣,呈現(xiàn)典型的EMT形態(tài)學(xué)改變;轉(zhuǎn)染FMNL3基因后,SW480細(xì)胞中EMT上皮標(biāo)志物E-cadherin表達(dá)下調(diào),間質(zhì)標(biāo)志物Vimentin表達(dá)上調(diào);轉(zhuǎn)染FMNL3基因后SW480細(xì)胞的體外侵襲能力顯著增強(qiáng)?結(jié)論FMNL3基因可能通過促進(jìn)結(jié)直腸癌細(xì)胞EMT而促進(jìn)其侵襲?

結(jié)直腸腫瘤;成蛋白樣3型蛋白;上皮-間質(zhì)轉(zhuǎn)化;侵襲

轉(zhuǎn)移是導(dǎo)致結(jié)直腸癌患者死亡的主要原因?已有研究發(fā)現(xiàn)上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)在腫瘤細(xì)胞侵襲和轉(zhuǎn)移中起重要作用[1]?因此,對結(jié)直腸癌EMT發(fā)生機(jī)制的研究,將有助于揭示結(jié)直腸癌侵襲?轉(zhuǎn)移的調(diào)控機(jī)制,并可能為結(jié)直腸癌治療靶點(diǎn)的尋找提供堅(jiān)實(shí)的理論依據(jù)?新近發(fā)現(xiàn)的成蛋白樣3型蛋白(formin-like,FMNL3)屬Diaphanous相關(guān)成蛋白(Diaphanous-related formins,DRFs)家族成員,該家族蛋白是目前發(fā)現(xiàn)的一類最有效的細(xì)胞骨架裝配和組建的關(guān)鍵調(diào)控分子,其在腫瘤轉(zhuǎn)移中的作用已引起人們的廣泛重視[2]?目前有關(guān)FMNL3的表達(dá)和功能學(xué)研究文獻(xiàn)甚少,其是否與結(jié)直腸癌EMT和侵襲?轉(zhuǎn)移相關(guān)尚少見文獻(xiàn)報(bào)道?因此,本課題將初步探討FMNL3基因轉(zhuǎn)染對結(jié)直腸癌SW480細(xì)胞EMT及體外侵襲能力的影響?

1 材料與方法

1.1 材料 (1)細(xì)胞株和質(zhì)粒:結(jié)直腸癌細(xì)胞株SW480及pEGFP-N1質(zhì)粒為江西省人民醫(yī)院中心實(shí)驗(yàn)室自存;自行構(gòu)建FMNL3重組表達(dá)質(zhì)粒 pEGFP-N1/FMNL3;Transwell小室(8μm孔徑)購自 Corning公司?(2)主要試劑:LipofectamineTM2000?各種引物購自Invitrogen公司;災(zāi)光定量PCR(RT-qPCR)試劑盒購自TOYOBO公司;蛋白提取試劑盒購自南京凱基公司;BCA蛋白定量試劑盒和化學(xué)發(fā)光液購自碧云天公司;鼠抗人FMNL3一抗購自Abnova公司,兔抗人E-cadherin和Vimentin一抗購自CST公司,羊抗兔二抗購自中杉金橋公司?

1.2 方法

1.2.1 基因轉(zhuǎn)染實(shí)驗(yàn) 自行構(gòu)建FMNL3重組表達(dá)質(zhì)粒pEGFP-N1/FMNL3,經(jīng)鑒定正確后將其轉(zhuǎn)染SW480細(xì)胞?轉(zhuǎn)染前24h將細(xì)胞接種于24孔板中,待細(xì)胞生長至90%～95%匯合時(shí)進(jìn)行基因轉(zhuǎn)染實(shí)驗(yàn):用50μL的Opti-MEM稀釋1μg重組質(zhì)粒并混勻;取2μL的LipofectamineTM2000用50μL的Opti-MEM稀釋并混勻;室溫孵育5min后將兩液混勻;室溫孵育20min后均勻加入SW480細(xì)胞培養(yǎng)孔中,培養(yǎng)6h后換完全培養(yǎng)液?實(shí)驗(yàn)組轉(zhuǎn)染FMNL3重組質(zhì)粒(SW480/FMNL3組),陰性對照組轉(zhuǎn)染空載質(zhì)粒(SW480/mock組),正常對照組未進(jìn)行質(zhì)粒轉(zhuǎn)染(SW480/NC組)?

1.2.2 RT-qPCR及 Western blot法檢測重組質(zhì)粒轉(zhuǎn)染細(xì)胞后FMNL3的表達(dá) 轉(zhuǎn)染后48h收集細(xì)胞,用TRIZol試劑按說明書提取細(xì)胞總RNA,然后進(jìn)行RT-qPCR反應(yīng)?采用Prime 5.0自行設(shè)計(jì)并由Invitrogen公司合成,FMNL3的RT-qPCR檢測引物如下,上游引物:5′-TCC GAT TCA TTC GTT CTT AC-3′,下游引物:5′CCG CCT CAA CTC TGC TAT T-3′,產(chǎn)物長度173bp;GAPDH 內(nèi)參引物如下,上游引物:5′-ACG GAT TTG GTC GTA TTG GGC G-3′,下游引物:5′-CTC CTG GAA GAT GGT GAT GG-3′,產(chǎn)物長度138bp?應(yīng)用IBM7500定量PCR儀進(jìn)行RT-qPCR實(shí)驗(yàn),反應(yīng)條件為95℃預(yù)變性30s?95℃變性5s?55～60℃退火30s,45個(gè)循環(huán)?定量的方法參照文獻(xiàn)[3],用Folds=2-△△Ct表示實(shí)驗(yàn)組與對照組目的基因表達(dá)的倍比關(guān)系,公式如下△△Ct=(Ct目的基因-CtGAPDH)實(shí)驗(yàn)組-(Ct目的基因-CtGAPDH)對照組?實(shí)驗(yàn)重復(fù)3次,計(jì)算平均值?用南京凱基公司的蛋白提取試劑盒抽提各組細(xì)胞總蛋白,BCA法定量蛋白?取細(xì)胞總蛋白進(jìn)行十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE),轉(zhuǎn)到PVDF膜,5%脫脂奶粉室溫封閉1h,FMNL3-抗(1∶500)4℃孵育過夜,二抗(1∶500)室溫1h,用化學(xué)發(fā)光液在暗室內(nèi)顯影?曝光成像?

1.2.3 FMNL3基因轉(zhuǎn)染前?后SW480細(xì)胞形態(tài)檢測 因重組質(zhì)粒含有編碼綠色熒光蛋白EGFP的基因,轉(zhuǎn)染細(xì)胞后能發(fā)出綠色熒光,轉(zhuǎn)染48h后可在倒置熒光顯微鏡下觀察SW480細(xì)胞的形態(tài)學(xué)變化?

1.2.4 FMNL3基因轉(zhuǎn)染前?后SW480細(xì)胞的體外侵襲能力變化 實(shí)驗(yàn)前將Transwell小室濾膜按每孔50μL包被Matrigel膠并在培養(yǎng)箱中聚合2h,消化細(xì)胞并用無血清的RPMI-1640培養(yǎng)液重懸,調(diào)整細(xì)胞濃度至1×105/mL后,將細(xì)胞接種于上室,下室用常規(guī)培養(yǎng)基作趨化因子,培養(yǎng)24h后取出濾膜,4%多聚甲醛固定?Gimsa染色,光鏡下隨機(jī)挑取5個(gè)視野計(jì)數(shù)穿膜細(xì)胞數(shù),取其均值代表浸潤力量值?

1.2.5 RT-qPCR及 Western blot法檢測細(xì)胞中EMT上皮及間質(zhì)標(biāo)志物的表達(dá) 方法步驟同1.2.2,其中 Anti-E-Caherin和Anti-Vimentin均1∶1 000?

1.3 統(tǒng)計(jì)學(xué)處理 采用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行分析,計(jì)量資料用±s表示,RT-qPCR?Western blot及體外侵襲實(shí)驗(yàn)結(jié)果均采用單因素方差分析?以P0.05為差異有統(tǒng)計(jì)學(xué)意義?

2 結(jié) 果

2.1 重組表達(dá)質(zhì)粒pEGFP-N1/FMNL3轉(zhuǎn)染SW480細(xì)胞后FMNL3表達(dá)水平 FMNL3重組表達(dá)質(zhì)粒pEGFP-N1/FMNL3經(jīng)雙酶切及測序鑒定證實(shí)序列完全正確,說明FMNL3表達(dá)載體構(gòu)建成功?轉(zhuǎn)染SW480細(xì)胞48h后FMNL3mRNA表達(dá)水平提高約53.47倍(P0.01),蛋白表達(dá)約8倍(圖1),說明FMNL3已經(jīng)成功轉(zhuǎn)染入SW480細(xì)胞中?

圖1 3組SW480細(xì)胞中E-Cadherin和Vimentin蛋白表達(dá)水平比較

2.2 FMNL3基因轉(zhuǎn)染前?后SW480細(xì)胞形態(tài)觀察 倒置熒光顯微鏡下觀察FMNL3轉(zhuǎn)染前?后SW480細(xì)胞的形態(tài),結(jié)果顯示,FMNL3蛋白表達(dá)于細(xì)胞質(zhì);FMNL3轉(zhuǎn)染后SW480細(xì)胞形態(tài)變化顯著,由典型的圓形?多邊形變成梭形并伸出多個(gè)突起的間充質(zhì)細(xì)胞樣(圖2),呈現(xiàn)出典型的EMT形態(tài)學(xué)改變?

2.3 EMT上皮及間質(zhì)標(biāo)志物的表達(dá)變化 RT-qPCR及Western blot法檢測結(jié)果顯示,與SW480/NC組比較,SW480/FMNL3組細(xì)胞E-cadherin mRNA表達(dá)水平下調(diào)55%,而Vimentin的mRNA表達(dá)上調(diào)15.47倍,其蛋白表達(dá)變化趨勢與mRNA水平的表達(dá)變化一致?

圖2 倒置熒光顯微鏡下觀察3組SW480細(xì)胞的形態(tài)學(xué)改變(×200)

圖3 顯微鏡下觀察3組SW480細(xì)胞的體外侵襲能力(×200)

2.4 FMNL3基因轉(zhuǎn)染前?后SW480細(xì)胞的體外侵襲能力變化 Transwell體外侵襲實(shí)驗(yàn)觀察到,48h后各組細(xì)胞均已穿出微 孔 濾 膜 (圖 3?4),SW480/NC 組?SW480/mock 組?SW480/FMNL3組顯微鏡下計(jì)數(shù)穿膜細(xì)胞數(shù)分別為(125.78±20.12)個(gè)?(125.46±16.59)個(gè)?(164.78±23.37)個(gè),FMNL3轉(zhuǎn)染后SW480細(xì)胞的體外侵襲能力顯著增強(qiáng)(P0.05)?

圖4 3組SW480細(xì)胞的體外侵襲能力變化統(tǒng)計(jì)圖形

3 討 論

FMNL3基因編碼產(chǎn)物屬DRFs家族成員,該家族是一類細(xì)胞中普遍存在?高度保守的多功能蛋白家族,通過其保守的FH2結(jié)構(gòu)域促進(jìn)肌動蛋白單體聚合并加快肌動蛋白纖維延伸,是目前發(fā)現(xiàn)的細(xì)胞中最有效的肌動蛋白成核因子之一[4];作為細(xì)胞骨架組建和裝配的關(guān)鍵調(diào)控分子,其參與了眾多以肌動蛋白為基礎(chǔ)的生命過程,包括細(xì)胞質(zhì)分裂?細(xì)胞極性和運(yùn)動?偽足形成等[5-6]?目前研究已經(jīng)發(fā)現(xiàn),該家族與Rho細(xì)胞運(yùn)動信號通路相關(guān),通過調(diào)控細(xì)胞骨架如絲狀偽足?板層偽足等的形成而影響腫瘤細(xì)胞的運(yùn)動?侵襲和轉(zhuǎn)移能力[2]?

FMNL3是新近發(fā)現(xiàn)的基因[7],位于染色體12q13?目前有關(guān)其表達(dá)和功能學(xué)研究文獻(xiàn)甚少,生物信息學(xué)分析顯示其在脾臟?卵巢纖維瘤?橫紋肌肉瘤?胃癌中表達(dá)[7];有研究發(fā)現(xiàn)在體內(nèi)高運(yùn)動能力的黑色素細(xì)胞亞群中存在FMNL3的高表達(dá)[8];FMNL3調(diào)控肌動蛋白的成核和延伸[9],與血管形成過程中內(nèi)皮細(xì)胞的細(xì)胞骨架重組有關(guān)[10],并能促進(jìn)細(xì)胞骨架絲狀偽足的形成[11],FMNL3依賴RhoC誘導(dǎo)前列腺癌細(xì)胞遷移[12-13];但FMNL3是否與結(jié)直腸癌EMT相關(guān)目前還少見文獻(xiàn)報(bào)道?本課題前期研究中用免疫組織化學(xué)和RT-qPCR法檢測結(jié)直腸癌組織和多個(gè)細(xì)胞系中FMNL3?EMT上皮和間質(zhì)標(biāo)志物的表達(dá)發(fā)現(xiàn),FMNL3在結(jié)直腸癌原發(fā)灶組織中的表達(dá)高于對應(yīng)癌旁正常組織,在淋巴結(jié)轉(zhuǎn)移灶組織中的表達(dá)高于其原發(fā)灶組織[14];同時(shí)發(fā)現(xiàn)FMNL3的表達(dá)與EMT上皮標(biāo)志物的表達(dá)呈負(fù)相關(guān),而與間質(zhì)標(biāo)志物呈正相關(guān);FMNL3及間質(zhì)標(biāo)記物Vimentin在高轉(zhuǎn)移潛能細(xì)胞系(SW620和LOVO)中的表達(dá)高于其在低轉(zhuǎn)移潛能細(xì)胞系(SW480和HT29)中的表達(dá),而上皮標(biāo)記物的表達(dá)趨勢正好相反[15];這提示FMNL3可能與結(jié)直腸癌EMT相關(guān)?在本研究中也發(fā)現(xiàn),轉(zhuǎn)染FMNL3基因后SW480細(xì)胞的形態(tài)發(fā)生明顯變化,由圓形或多邊形變?yōu)樗笮尾⑸斐龆鄠€(gè)細(xì)長突起的間充質(zhì)樣,呈現(xiàn)典型的EMT形態(tài)學(xué)改變;并且不論mRNA水平還是蛋白水平,與SW480/Mock組細(xì)胞相比,SW480/FMNL3組細(xì)胞中上皮標(biāo)志物E-cadherin表達(dá)均降低,間質(zhì)標(biāo)志物Vimentin表達(dá)均升高,這進(jìn)一步說明FMNL3可能促進(jìn)結(jié)直腸癌細(xì)胞EMT,與本課題的前期研究結(jié)果相吻合;同時(shí)還發(fā)現(xiàn),轉(zhuǎn)染FMNL3基因后SW480細(xì)胞的體外侵襲能力顯著增強(qiáng),說明在體外FMNL3能夠促進(jìn)結(jié)直腸癌細(xì)胞侵襲?綜合以上研究結(jié)果,本研究認(rèn)為FMNL3可能通過促進(jìn)結(jié)直腸癌細(xì)胞EMT而促進(jìn)結(jié)直腸癌侵襲,但具體的分子機(jī)制還有待進(jìn)一步深入研究?

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Influence of FMNL3 gene transfection on epithelial-mesenchymal transition and in vitro invasion potential of colorectal carcinoma SW480 cells*

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(1.,,, 330006,;2.,,,, 330004,;3.,,, 330006,;4.,,, 330006,;5.,,, 330006,;6.,,, 330031,)

ObjectiveTo investigate the impact of FMNL3gene transfection on the epithelial-mesenchymal transition(EMT)and the in vitro invasive potential of colorectal carcinoma SW480cells.MethodsThe recombination FMNL3expression plasmid with EGFP was transfected into SW480cells(SW480cells without transfected plasmid as the normal control group and SW480cells with empty vector as the negative control group).The changes of FMNL3mRNA and protein expression were detected by real-time PCR and Western blot after transfection of 48hwith recombination FMNL3expression plasmid.The changes of SW480cell morphology were detected by the fluorescence microscope after FMNL3transfection.The changes of EMT epithelial and mesenchymal markers in SW480cells were detected by real-time PCR and Western blot after FMNL3transfection.The Transwell chamber invasion assay in vitro was used to investigate the effects of FMNL3gene trasfection on the invasive potential of SW480cells.ResultsThe levels of FMNL3mRNA and the protein expression were significantly increased at 48hafter transfection with recombination FMNL3expression plasmid(P0.01);the FMNL3protein was expressed within cytoplasma,the morphology of SW480cells after FMNL3transfection showed significantly changes from round and polygon to spindle and stretched out the multiple long and thin prominences as mesenchyma,which appeared the typical EMT change;after FMNL3gene transfection,the expression of EMT epithelial marker E-cadherin was down-regulated and the expression of mesenchymal marker Vimentin was up-regulated in SW480 cells;the in vitro invasive potential of SW480cells was remarkably enhanced after FMNL3transfection.ConclusionFMNL3gene may promote its invasion by promoting EMT of colorectal carcinoma.

colorectal cancer;FMNL3;epithelial-mesenchymal transition;invasion

* 基金項(xiàng)目:國家自然科學(xué)基金資助項(xiàng)目(81201972?81260327)?

曾元鳳(1974-),主治醫(yī)師,博士后在站工作人員,主要從事結(jié)直腸癌轉(zhuǎn)移的分子機(jī)制研究?△

,Tel:(0791)8895633;E-mail:wuxm@163.com?▲通訊作者,Tel:(0791)83827168;E-mail:hongboxin@yahoo.com?

10.3969/j.issn.1671-8348.2014.12.018

A

1671-8348(2014)12-1460-03

2013-10-20

2013-12-05)

論著?臨床研究