白一禾　秦兆宇　賀福初,2,4　丁　琛,3△

(1復(fù)旦大學(xué)生物醫(yī)學(xué)研究院醫(yī)學(xué)系統(tǒng)生物學(xué)研究中心 上?！?00032; 2 北京國家蛋白質(zhì)科學(xué)中心　北京　102206;3 復(fù)旦大學(xué)生命科學(xué)學(xué)院　上海　200438; 4 蛋白質(zhì)組學(xué)國家重點實驗室,北京蛋白質(zhì)組研究中心,北京放射醫(yī)學(xué)研究所　北京　102206)

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HMG20A對肝癌細(xì)胞體外增殖與遷移的影響及其機制

白一禾1秦兆宇1賀福初1,2,4丁琛1,3△

(1復(fù)旦大學(xué)生物醫(yī)學(xué)研究院醫(yī)學(xué)系統(tǒng)生物學(xué)研究中心 上海200032;2北京國家蛋白質(zhì)科學(xué)中心北京102206;3復(fù)旦大學(xué)生命科學(xué)學(xué)院上海200438;4蛋白質(zhì)組學(xué)國家重點實驗室,北京蛋白質(zhì)組研究中心,北京放射醫(yī)學(xué)研究所北京102206)

目的研究HMG20A在肝細(xì)胞癌(hepatocellular carcinoma,HCC)發(fā)生發(fā)展和轉(zhuǎn)移中的功能與機制。方法在不同轉(zhuǎn)移能力和遺傳背景的肝癌細(xì)胞Huh7和HCCLM3中分別構(gòu)建HMG20A過表達(dá)和敲低穩(wěn)定株,通過實時定量PCR (real time quantitative PCR,qPCR) 驗證過表達(dá)和敲低該基因的效果。用CCK8試劑盒檢測HMG20A對肝癌細(xì)胞增殖能力的影響,利用Transwell小室分析HMG20A調(diào)控肝癌細(xì)胞轉(zhuǎn)移的能力。借助Western blot和qPCR分析HMG20A調(diào)控肝癌增殖和轉(zhuǎn)移的機制。結(jié)果體外實驗表明,HMG20A能夠促進(jìn)肝癌細(xì)胞的體外增殖和遷移,顯著上調(diào)促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)通路中p38 (p38 MAPK)、細(xì)胞外調(diào)節(jié)蛋白激酶(extracellular regulated protein kinase,ERK)的活性和表達(dá)水平,同時上皮間充質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)的標(biāo)志物Vimentin、抗平滑肌抗體(anti-smooth muscle antibody,alpha-SMA)、N-cadherin受到顯著的正調(diào)控,E-cadherin受到顯著負(fù)調(diào)控。結(jié)論HMG20A可能通過促進(jìn)EMT進(jìn)程和MAPK通路促進(jìn)肝癌細(xì)胞的體外增殖與遷移。

肝癌;HMG20A;轉(zhuǎn)移;促分裂源活化蛋白激酶;上皮間充質(zhì)轉(zhuǎn)化

原發(fā)性肝細(xì)胞癌 (以下簡稱肝癌)是世界上最常見的惡性腫瘤之一,在腫瘤致死原因中位居第三,僅次于肺癌和胃癌[1],我國是全球肝癌發(fā)病率最高和死亡最多的國家[2]。肝癌的發(fā)病機制非常復(fù)雜,盡管已經(jīng)確認(rèn)乙肝和丙肝病毒感染、肝硬化、大量飲酒、黃曲霉素攝入等為致病因素,但仍然缺乏對導(dǎo)致肝癌形成和進(jìn)展的分子生物學(xué)機制的確切認(rèn)識。

轉(zhuǎn)錄因子HMG20A是高遷移率蛋白家族的一員,含有一個HMG結(jié)構(gòu)域,該結(jié)構(gòu)域含有大約70個氨基酸,形成3個α-螺旋,和DNA的小溝結(jié)合。HMG20A在成熟神經(jīng)元中高表達(dá),調(diào)控許多在神經(jīng)分化中有重要作用的分子。HMG20A可以招募組蛋白甲基轉(zhuǎn)移酶MLL,導(dǎo)致組蛋白H3K4甲基化,甲基化的H3K4可通過招募各種因子,如通用轉(zhuǎn)錄因子TFⅡD、組蛋白乙?；窼AGA,參與基因轉(zhuǎn)錄激活過程,從而誘導(dǎo)神經(jīng)分化[3]。

還有研究顯示,在乳腺癌中敲除HMG20A后,許多與上皮間充質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)相關(guān)的基因下調(diào),HMG20A和LSD1 (lysine specific demethylase 1)參與snail1 (snail family zinc finger 1)和轉(zhuǎn)化生長因子β (transforming growth factor-β,TGF-β)調(diào)控的EMT,HMG20A缺失削弱LSD1和上皮基因啟動子的結(jié)合,導(dǎo)致細(xì)胞的轉(zhuǎn)移能力減弱[4]。

EMT是哺乳動物胚胎發(fā)育過程中的生理現(xiàn)象,它對胚胎發(fā)生和器官發(fā)育十分重要。上皮細(xì)胞是高度有序的單層細(xì)胞,細(xì)胞間的緊密連接和黏附連接的存在使上皮細(xì)胞緊密相連發(fā)揮功能,同時也限制了任意遷移的能力。與之相反,間充質(zhì)細(xì)胞則形態(tài)各異,表現(xiàn)出更多的遷移和侵襲能力[5]。這種表型上的變化被認(rèn)為參與了一些致癌通路。EMT在腫瘤的侵襲過程中起著重要的作用,促使良性腫瘤發(fā)展成轉(zhuǎn)移性腫瘤,進(jìn)而侵襲其他組織。

我們推測HMG20A可能在肝癌的發(fā)展與轉(zhuǎn)移過程中同樣發(fā)揮潛在功能,但目前尚無相關(guān)報道。本研究分別在低轉(zhuǎn)移潛能的肝癌細(xì)胞Huh7和高轉(zhuǎn)移潛能的肝癌細(xì)胞HCCLM3中過表達(dá)和干擾HMG20A,通過一系列體外實驗研究了HMG20A對肝癌細(xì)胞增殖和轉(zhuǎn)移的影響,及其可能的機制。

材 料 和 方 法

主要試劑與材料細(xì)胞株HCCLM3購自復(fù)旦大學(xué)中山醫(yī)院肝癌研究所;Huh7購自中國科學(xué)院上海生命科學(xué)研究院生物化學(xué)與細(xì)胞生物學(xué)研究所;DMEM/high Glucose (NYA0782)購自美國Thermo Scientific公司;AgeI快切酶 (FD1464)和EcoRI快切酶 (FD0274)購自美國Fermentas公司;蛋白預(yù)染Marker (SM0671)購自美國Fermentas公司;PrimeScript?RT reagent Kit (DRR037A)和SYBR?Premix Ex TaqTM (DRR041A)購自日本Takara公司;細(xì)胞增殖與活性檢測試劑 (CK04)購自日本Dojindo公司;TransZol UP (ET111-01)購自北京全式金生物技術(shù)有限公司;Transwell小室購自美國BD公司;BCA蛋白濃度測定試劑盒 (P0012)購自上海碧云天生物技術(shù)有限公司;一抗稀釋液 (P0023D)和二抗稀釋液 (P0023D)購自上海碧云天生物技術(shù)有限公司。

敲低穩(wěn)定株的構(gòu)建shRNA干擾序列由上海拓然生物科技有限公司設(shè)計,序列如下:shHMG20A-1,5′-CCGGGUGAACAGACUCGAU-CGUUCTCGAGAACGAUCGAGUCUGUUCAC-TTTTTG-3′ (Oligo F),5′-AATTCAAAAAGUG-

AACAGACUCGAUCGUUCTCGAGATTGGCG-GCTAGTTCCACTGC-3′ (Oligo R);shHMG20A-2,5′-CCGGGAAAUCACAAGGAUGUUAGCTC-

GAGCUAACAUCCUUGUGAUUUCTTTTTG-3′ (Oligo F),5′-AATTCAAAAA GAAAUCACAA-

GGAUGUUAGCTCGAGCUAACAUCCUUGUG-A-UUUC-3′ (Oligo R)。在一個10 cm細(xì)胞培養(yǎng)皿約鋪5×106個HEK293T細(xì)胞,第2天進(jìn)行轉(zhuǎn)染。取1.5 mL Ep管2支,其中一支加入10 μg pMK0.1、7.5 μg Gag-Pol、2 μg VSV-G待轉(zhuǎn)染質(zhì)粒,加500 μL Opti-MEM培養(yǎng)基混勻。另一支中加入25 μL PEI溶液和500 μL Opti-MEM培養(yǎng)基,2支Ep管的混合物混勻后室溫放置20 min。將得到的DNA-PEI復(fù)合體加入到細(xì)胞培養(yǎng)器皿中,繼續(xù)培養(yǎng)4～6 h后,更換為10 mL的新鮮完全培養(yǎng)基。培養(yǎng)36～48 h后收集上清,感染HCCLM3細(xì)胞 (30%～40%密度)。24 h后換用含有4 μg/μL Puromycin的培養(yǎng)基篩選培養(yǎng)1周,所得細(xì)胞即為靶基因穩(wěn)定干擾細(xì)胞株。

過表達(dá)穩(wěn)定株的構(gòu)建轉(zhuǎn)染前一天準(zhǔn)備293T細(xì)胞,轉(zhuǎn)染1 h前換為無血清的培養(yǎng)基,取1.5 mL離心管2支,其中一支加入4 μg pCDH-HMG20A、1 μg pSD、3 μg psPAX2待轉(zhuǎn)染質(zhì)粒,加500 μL Opti-MEM培養(yǎng)基混勻,另一支加入加500 μL Opti-MEM培養(yǎng)基和25 μL PEI轉(zhuǎn)染試劑。將2支離心管內(nèi)的混合物混勻后靜置20 min,點加到培養(yǎng)皿中,4～6 h后換為正常的培養(yǎng)基。

qPCR用Trizol試劑提取總RNA,逆轉(zhuǎn)錄以后進(jìn)行qPCR反應(yīng)。HMG20A引物序列(5′-3′):上游-AGCTACACATCACTTGACACCA,下游-ATCTGCAAAAAGGGGCGGTA;Vimentin引物序列:上游-GGACAGCTAACCAACGAACGACA,下游-AAGGTCAAGACGTGCCAGAG;alpha-SMA引物序列:上游-AGAGGAACACCCCACT-CTGT,下游-GTCCAGCACAATGCCTGTTG;N-cadherin引物序列:上游-CCTGGATCGCGAGC-AGATAG,下游-TCCCTCAGGAACTGTCCCAT;E-cadherin引物序列:上游-AGGCCAAGCAGCA-TACATT,下游-GGGGGCTTCATTCACATC-CA;ERK引物序列:上游-GCCGAAGCACCATT-CAAGTT,下游-GGACCAGGGGTCAAGAACT-G;p38引物序列:上游-ATGCGTCTGACAGGA-ACACC,下游-CGCAAAGTTCATCTTCGGCA。

Western blot 待細(xì)胞長到90%的匯合度時,用SDS裂解液提取全蛋白,用BCA法測蛋白濃度,配置10%的SDS-PAGE凝膠,電泳后進(jìn)行轉(zhuǎn)膜,抗體孵育過夜,用ECL發(fā)光試劑孵育后使用Las 4000儀器掃膜獲取蛋白條帶。

Transwell檢測細(xì)胞遷移 對構(gòu)建成功的穩(wěn)定細(xì)胞株及對照組細(xì)胞進(jìn)行消化并計數(shù),用無血清DMEM將細(xì)胞懸液稀釋成1×106cells/mL,每個Transwell上室加入100 μL細(xì)胞懸液,下室加入600 μL含有30%FBS的DMEM,置于5% CO2培養(yǎng)箱培養(yǎng)12～30 h。取出Transwell小室,用棉簽檫凈上層細(xì)胞,多聚甲醛固定后結(jié)晶紫染色,于顯微鏡鏡下觀察,選取上、中、下、左、右5個視野計數(shù)并拍照。

細(xì)胞增殖檢測 對構(gòu)建成功的穩(wěn)定株進(jìn)行消化計數(shù),把細(xì)胞鋪到96孔板中,每組6個副孔,每孔加2 000個細(xì)胞。鋪板后每24 h取出一板細(xì)胞進(jìn)行檢測,連續(xù)4天。每孔細(xì)胞中加入10 μL CCK-8細(xì)胞增殖與毒性檢測試劑和90 μL的DMEM培養(yǎng)基,放至細(xì)胞培養(yǎng)箱內(nèi)孵育2 h,用酶標(biāo)儀檢測,使用450 nm濾光片,以不接種細(xì)胞的空白孔加入相同體積的檢測試劑和培養(yǎng)基為對照,保存數(shù)據(jù),繪制增殖曲線。

統(tǒng)計學(xué)處理 Graphpad 5.0軟件對所有的數(shù)據(jù)進(jìn)行統(tǒng)計處理,差異顯著性用雙側(cè)t檢驗,P<0.05為差異有統(tǒng)計學(xué)意義。

結(jié)　　　果

HMG20A過表達(dá)和敲低效率驗證構(gòu)建HMG20A過表達(dá)穩(wěn)定株Huh7,以空質(zhì)粒pCDH為陰性對照,pCDH-HMGH20A為過表達(dá)HMG20A的穩(wěn)定細(xì)胞株,qPCR實驗證明pCDH-HMGH20A組能夠顯著過表達(dá)目標(biāo)基因,差異具有統(tǒng)計學(xué)意義 (P<0.001)。同時,為了在HCCLM3中敲低HMG20A,設(shè)計2條特異shRNA干擾序列shHMG20A-1和shHMG20A-2,在HCCLM3中構(gòu)建HMG20A敲低穩(wěn)定株,shNC1和shNC2為陰性對照,qPCR結(jié)果顯示敲低穩(wěn)定株中HMG20A的表達(dá)水平顯著低于對照組,差異具有統(tǒng)計學(xué)意義 (P<0.01,圖1)。

Pvalues were calculated using an unpairedt-test (HMG20Avs.pCDH,(1)P<0.001,shHMG20Avs.shNC,(2)P<0.01).

圖1HMG20A過表達(dá)和敲低效果的驗證

Fig 1The effects of HMG20A overexpression and knockdown

HMG20A促進(jìn)肝癌細(xì)胞的體外增殖 在Huh7中構(gòu)建HMG20A過表達(dá)的穩(wěn)定株,在HCCLM3中構(gòu)建敲低穩(wěn)定株,分別于鋪板后第1、2、3、4天檢測細(xì)胞的增殖情況。結(jié)果顯示過表達(dá)HMG20A后Huh7的生長速率顯著高于對照組 (圖2 A),而兩條干擾序列敲低HMG20A后,HCCLM3的增殖能力較對照組顯著降低 (圖2 B),差異均具有統(tǒng)計學(xué)意義 (P<0.01),表明HMG20A能顯著促進(jìn)肝癌細(xì)胞的體外增殖。

HMG20A overexpression stable cell lines (A) or knockdown stable cell lines (B) were plated onto 96-well plates.Cell proliferation was detected by using CCK8-kit assay from the first day to the fourth day.Pvalues were calculated using an unpairedt-test (P<0.01).

圖2HMG20A對肝癌細(xì)胞增殖的影響

Fig 2HMG20A promotes the proliferation of HCC cells

HMG20A促進(jìn)肝癌細(xì)胞的體外遷移 通過Transwell實驗觀察肝癌細(xì)胞轉(zhuǎn)移能力的變化,結(jié)果顯示過表達(dá)HMG20A后,Huh7的遷移能力增加 (圖3 A),而兩條干擾序列敲低HMG20A后HCCLM3的遷移能力均顯著降低 (圖3 B)。選取上、中、下、左、右5個視野統(tǒng)計計數(shù)差異 (圖3C,3D),差異均具有統(tǒng)計學(xué)意義 (HMG20Avs.pCDH,P<0.001,shHMG20Avs.shNC,P<0.01),證實HMG20A能夠顯著促進(jìn)肝癌細(xì)胞的體外遷移能力。

HMG20A上調(diào)MAPK通路分裂原激活的蛋白激酶 (mitogen activated protein kinases,MAPK)家族是非常保守的絲氨酸/蘇氨酸蛋白激酶,研究表明MAPK通路參與細(xì)胞的生長、發(fā)育、分化、凋亡等一系列細(xì)胞生理活動,可以促進(jìn)腫瘤的發(fā)生發(fā)展和轉(zhuǎn)移,幾種MAPK亞家族參與的信號轉(zhuǎn)導(dǎo)通路各有不同的功能,如ERK調(diào)控細(xì)胞生長和分化[6],JNK和p38 MAPK信號通路在炎癥和細(xì)胞凋亡等應(yīng)激反應(yīng)中發(fā)揮重要作用[7-8]。MAPK家族分子如p38、ERK、JNK可以被生長因子、高糖、炎性細(xì)胞因子、激素等刺激磷酸化激活,激活后MAPK轉(zhuǎn)移到細(xì)胞核,并磷酸化和激活許多蛋白激酶以及轉(zhuǎn)錄因子,參與細(xì)胞增殖與分化、細(xì)胞形態(tài)維持以及細(xì)胞凋亡和細(xì)胞骨架的構(gòu)建、癌癥的發(fā)生發(fā)展等多種生理過程[9]。我們在Huh7和HCCLM3中分別構(gòu)建了HMG20A過表達(dá)和敲低的穩(wěn)定株,分析HMG20A對肝癌細(xì)胞相關(guān)信號通路的影響。Western blot和qPCR的結(jié)果顯示在過表達(dá)HMG20A的穩(wěn)定株中,HMG20A能顯著促進(jìn)MAPK家族p38、ERK的活性和表達(dá)水平 (P<0.001,圖4 A、4C);同時在HMG20A敲低的穩(wěn)定株中,p38、ERK的活性和表達(dá)水平受到明顯的抑制 (P<0.01,圖4 B、4D)。表明HMG20A可能通過促進(jìn)MAPK通路,影響肝癌的發(fā)展。

HMG20A可能促進(jìn)肝癌細(xì)胞EMT進(jìn)程 EMT參與了胚胎發(fā)生與器官發(fā)育、腫瘤轉(zhuǎn)移等多種生理病理過程,標(biāo)志EMT過程的分子標(biāo)志物有很多種,主要包括細(xì)胞表面標(biāo)志物 (如E-cadherin、MMP)[10]、轉(zhuǎn)錄因子 (如Snail、Twist)[11]、細(xì)胞外基質(zhì)蛋白 (如層粘連蛋白、纖維連接蛋白)[12]、細(xì)胞支架標(biāo)志物 (如Vimentin、alpha-SMA)4類[13]。在Huh7中構(gòu)建HMG20A過表達(dá)的穩(wěn)定株,通過檢測EMT相關(guān)標(biāo)記蛋白表達(dá)水平的變化,分析HMG20A對EMT乃至肝癌細(xì)胞轉(zhuǎn)移能力的影響。結(jié)果表明過表達(dá)HMG20A能提高Vimentin、N-cadherin和alpha-SMA的表達(dá)水平,并抑制E-cadherin的表達(dá) (圖5A～C),差異具有統(tǒng)計學(xué)意義 (P<0.001)。構(gòu)建HMG20A敲低穩(wěn)定株HCCLM3,E-cadherin的表達(dá)水平上調(diào) (圖5 D),Vimentin、alpha-SMA和N-cadherin的表達(dá)水平下調(diào) (圖5 E),差異具有統(tǒng)計學(xué)意義 (P<0.01)。上述結(jié)果提示HMG20A可能促進(jìn)了EMT進(jìn)程。

HMG20A promotes cell migration in Transwell assays.A:HMG20A overexpression stable cell lines were constructed in Huh7 cells;B:HMG20A knockdown stable cell lines were constructed in HCCLM3 cells.Transwell inserts were displaced 12 h (A) or 30 h (B) later.At this time the microporous membrane were stained by using crystal violet,phase-contrast pictures of the membrane at different locations were taken.C&D:Counted the cell numbers of 5 visions of the membranes,Pvalues were calculated using an unpairedt-test (HMG20Avs.pCDH,(1)P<0.001,shHMG20Avs.shNC,(2)P<0.01).

圖3HMG20A對肝癌細(xì)胞遷移能力的影響

Fig 3HMG20A promotes the migration of HCC cells

討　　　論

前期研究顯示,HMG20A是一個在腦中表達(dá)水平較高的轉(zhuǎn)錄因子,在神經(jīng)分化中有重要作用。HMG20A/iBRAF招募甲基轉(zhuǎn)移酶MLL甲基化H3K4,進(jìn)而調(diào)控神經(jīng)分化特異性基因[3],MLL蛋白在基因調(diào)控、細(xì)胞增殖、生長分化等生理功能中發(fā)揮著重要作用,與腫瘤密切相關(guān)[14]。

還有研究表明HMG20B/BRAF35激活抑癌基因REST,HMG20B的抑制因子HMG20A能夠抑制REST,從而激活被REST抑制的神經(jīng)分化相關(guān)的基因[15]。REST缺失導(dǎo)致腫瘤的發(fā)生,REST參與細(xì)胞凋亡、可能參與細(xì)胞復(fù)制、血管形成等,對基因轉(zhuǎn)錄起負(fù)調(diào)控作用[16]。

Total proteins and RNA were extracted from HMG20A overexpression and knockdown stable cell lines.Levels of MAPKs such as ERK and p38 proteins were determined by Western blot (A&B),and levels of transcripts were determined by qPCR (C&D).Pvalues were calculated using an unpairedt-test (HMG20Avs.pCDH,(1)P<0.001,shHMG20Avs.shNC,(2)P<0.01).

圖4HMG20A激活MAPK通路并上調(diào)MAPKs

Fig 4HMG20A activates the MAPK pathway and upregulates the expression levels of MAPKs

Rivero等[4]發(fā)現(xiàn)在RPE1細(xì)胞中HMG20A和LSD1對于調(diào)控間充質(zhì)表型是必需的,敲減HMG20A或者LSD1會上調(diào)上皮表型標(biāo)志物如CXADR、CLDN12、KRT18和CDH4。敲減HMG20A會削弱過表達(dá)Snail1導(dǎo)致的CXADR和CLDN12的下調(diào),說明Snail1通過HMG20A抑制CXADR和CLDN12。

由此我們推測HMG20A在肝癌發(fā)展和轉(zhuǎn)移中可能發(fā)揮潛在功能。為了驗證HMG20A在肝癌中的功能,我們在肝癌細(xì)胞HCCLM3和Huh7中分別構(gòu)建HMG20A敲低和過表達(dá)穩(wěn)定株,發(fā)現(xiàn)HMG20A能顯著促進(jìn)肝癌細(xì)胞的體外增殖和遷移。

為了分析其功能可能的機制,我們通過qPCR和Western blot的方法,篩選了HMG20A可能影響的信號通路。發(fā)現(xiàn)其可上調(diào)MAPK通路中的p38、ERK的活性和表達(dá)水平。

MAPK是細(xì)胞內(nèi)的一類絲氨酸/蘇氨酸蛋白激酶,是細(xì)胞信號傳導(dǎo)的重要途徑之一。在未受刺激的細(xì)胞內(nèi),MAPK處于靜止?fàn)顟B(tài),當(dāng)受到高糖、生長因子、激素和炎性細(xì)胞因子等因素刺激后,MAPK被磷酸化激活,進(jìn)而定位到細(xì)胞核中,調(diào)控一系列轉(zhuǎn)錄因子、蛋白激酶等靶基因。研究表明MAPK是胰島素和胰島素樣生長因子(insulin-like growth factors-1,IGF-1)信號通路的下游分子,在很多癌癥,如乳腺癌、結(jié)腸癌和前列腺癌中起著促進(jìn)增殖的作用[17-19]。MAPK還可以通過磷酸化一些細(xì)胞核內(nèi)的轉(zhuǎn)錄因子,如c-Myc,c-Jun等,參與細(xì)胞增殖和分化[20-21]。我們的研究提示HMG20A可能通過促進(jìn)MAPK途徑中ERK和p38的活性和表達(dá)水平促進(jìn)肝癌細(xì)胞的增殖。

此外,p38是MAPK一個重要的亞族,能夠接受細(xì)胞外的刺激,并磷酸化下游蛋白發(fā)揮其功能。在原發(fā)性腹膜間皮細(xì)胞中,p38與Twist1的磷酸化有關(guān),能夠增加Twist1的蛋白穩(wěn)定性,促進(jìn)EMT進(jìn)程和腫瘤的浸潤[22]。在原腸胚形成中,E-鈣黏素蛋白的表達(dá)受到p38 MAPK 的負(fù)調(diào)控[23]。持續(xù)活化的ERK能夠促進(jìn)細(xì)胞增殖以及細(xì)胞的惡性轉(zhuǎn)化,已經(jīng)有研究表明ERK能夠直接或者間接上調(diào)Vimentin的表達(dá),從而促進(jìn)EMT的發(fā)生[24]。在胰腺癌中,Collagen I能夠激活JNK1,上調(diào)N-cadherin 的表達(dá),加速EMT進(jìn)程[25]。

A-C:Overexpression of HMG20A improved the EMT associated markers including Vimentin,alpha-SMA and N-cadherin ((1)P<0.001).Futhermore,it inhibited the expression of E-cadherin ((1)P<0.001).D:E-cadherin was promoted with the knock down of HMG20A ((2)P<0.01).E:EMTassociated markersVimentin,alpha-SMA and N-cadherin mRNA expression levels were determined by qPCR.They were inhibited with the knock down of HMG20A ((2)P<0.01).Pvalues were calculated using an unpairedt-test.

圖5過表達(dá)和敲低HMG20A后EMT相關(guān)標(biāo)記蛋白的變化

Fig 5Overexpression and knockdown of HMG20A regulate the EMT associated markers

我們通過qPCR和Western blot的方法,發(fā)現(xiàn)HMG20A能上調(diào)EMT標(biāo)志物Vimentin、alpha-SMA和N-cadherin的表達(dá),并抑制E-cadherin的表達(dá)水平。結(jié)果提示HMG20A可能促進(jìn)了EMT進(jìn)程。

EMT在腫瘤的轉(zhuǎn)移過程中起著重要作用,在EMT過程中,細(xì)胞功能發(fā)生變化,上皮細(xì)胞失去其上皮特征而獲得了間充質(zhì)細(xì)胞特征,使得腫瘤細(xì)胞離開原發(fā)部位,遷移到鄰近組織,其主要的上皮標(biāo)記物如角蛋白、E-鈣黏素等表達(dá)下調(diào),同時波形蛋白、N-鈣黏素等間質(zhì)標(biāo)記物表達(dá)上調(diào)。癌癥相關(guān)的許多通路已經(jīng)成為EMT的重要調(diào)控信號,其中許多通路能夠共同配合引發(fā)EMT[26]。

由此,我們發(fā)現(xiàn)HMG20A可能通過上調(diào)MAPK通路中的p38、ERK的活性和表達(dá)水平,提高肝癌細(xì)胞的體外增殖能力;還發(fā)現(xiàn)該基因能夠上調(diào)EMT標(biāo)志物Vimentin、alpha-SMA和N-cadherin的表達(dá),并抑制E-cadherin的表達(dá)水平,提示其可能參與了促進(jìn)細(xì)胞EMT進(jìn)程,導(dǎo)致肝癌細(xì)胞體外遷移能力的增加。而其如何通過下游靶基因參與這一過程,尚待后續(xù)研究揭示。

[1]FERLAY J,SOERJOMATARAM I,DIKSHIT R,etal.Cancer incidence and mortality worldwide:sources,methods and major patterns in GLOBOCAN 2012[J].IntJCancer,2015,136 (5):E359-E386.

[2]JEMAL A,BRAY F,CENTER MM,etal.Global cancer statistics[J].CACancerJClin,2011,61 (2):69-90.

[3]WYNDER C,HAKIMI MA,EPSTEIN JA,etal.Recruitment of MLL by HMG-domain protein iBRAF promotes neural differentiation[J].NatCellBiol,2005,7 (11):1113-1117.

[4]RIVERO S,CEBALLOS-CHAVEZ M,BHATTACHARYA SS,etal.HMG20A is required for SNAI1-mediated epithelial to mesenchymal transition[J].Oncogene,2015,34 (41):5264-5276.

[5]NAKAYA Y,SHENG G.EMT in developmental morphogenesis[J].CancerLett,2013,341 (1):9-15.

[6]LEE SK,JANG HJ,LEE HJ,etal.p38 and ERK MAP kinase mediates iron chelator-induced apoptosis andsuppressed differentiation of immortalized and malignant human oral keratinocytes[J].LifeSci,2006,79 (15):1419-1427.

[7]ADHIKARY G,SUN Y,PEARLMAN E.C-Jun NH2 terminal kinase (JNK) is an essential mediator of Toll-like receptor 2-induced corneal inflammation[J].JLeukocBiol,2008,83 (4):991-997.

[8]NICK JA,YOUNG SK,BROWN KK,etal.Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation[J].JImmunol,2000,164 (4):2151-2159.

[9]PLOTNIKOV A,ZEHORAI E,PROCACCIA S,etal.The MAPK cascades:signaling components,nuclear roles and mechanisms of nuclear translocation[J].BiochimBiophysActa,2011,1813 (9):1619-1633.

[10]VEVERIS-LOWE TL,LAWRENCE MG,COLLARD RL,etal.Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells[J].EndocrRelatCancer,2005,12 (3):631-643.

[11]ZHENG M,JIANG YP,CHEN W,etal.Snail and Slug collaborate on EMT and tumor metastasis through miR-101-mediated EZH2 axis in oral tongue squamous cell carcinoma[J].Oncotarget,2015,6 (9):6797-6810.

[12]NAKAYA Y,SUKOWATIE W,WU Y,etal.RhoA and microtubule dynamics control cell-basement membrane interaction in EMT during gastrulation[J].NatCellBiol,2008,10 (7):765-775.

[13]VUORILUOTO K,HAUGEN H,KIVILUOTO S,etal.Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer[J].Oncogene,2011,30 (12):1436-1448.

[14]VAN DER LINDEN MH,WILLEKES M,VAN ROON E,etal.MLL fusion-driven activation of CDK6 potentiates proliferation in MLL-rearranged infant ALL[J].CellCycle,2014,13 (5):834-844.

[15]ARTEGIANI B,LABBAYE C,SFERRA A,etal.The interaction with HMG20a/b proteins suggests a potential role for beta-dystrobrevin in neuronal differentiation[J].JBiolChem,2010,285 (32):24740-24750.

[16]CHO E,MOON SM,PARK BR,etal.NRSF/REST regulates the mTOR signaling pathway in oral cancer cells[J].OncolRep,2015,33 (3):1459-1464.

[17]ZHU C,QI X,CHEN Y,etal.PI3K/Akt and MAPK/ERK1/2 signaling pathways are involved in IGF-1-induced VEGF-C upregulation in breast cancer[J].JCancerResClinOncol,2011,137 (11):1587-1594.

[18]HART LS,DOLLOFF NG,DICKER DT,etal.Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab[J].CellCycle,2011,10 (14):2331-2338.

[19]IMAMURAY,SAKAMOTOS,ENDO T,etal.FOXA1 promotes tumor progression in prostate cancer via the insulin-like growth factor binding protein 3 pathway[J].PLoSOne,2012,7 (8):e42456.

[20]STEPNIAK E,RICCI R,EFERL R,etal.c-Jun/AP-1 controls liver regeneration by repressing p53/p21 and p38 MAPK activity[J].GenesDev,2006,20 (16):2306-2314.

[21]XU W,GU J,REN Q,etal.NFATC1 promotes cell growth and tumorigenesis in ovarian cancer up-regulating c-Myc through ERK1/2/p38 MAPK signal pathway[J].TumourBiol,2016,37(4):493-500.

[22]STRIPPOLI R,BENEDICTO I,FORONDA M,etal.p38 maintains E-cadherin expression by modulating TAK1-NF-kappa B during epithelial-to-mesenchymal transition[J].JCellSci,2010,123 (Pt 24):4321-4331.

[23]ZOHN IE,LI YQ,SKOLNIK EY,etal.p38 and a p38-interacting protein are critical for downregulation of E-cadherin during mouse gastrulation[J].Cell,2006,125 (5):957-969.

[24]JAVLE MM,GIBBS JF,IWATA KK,etal.Epithelial-mesenchymal transition (EMT) and activated extracellular signal-regulated kinase (p-Erk) in surgically resected pancreatic cancer[J].AnnSurgOncol,2007,14 (12):3527-3533.

[25]SHINTANI Y,HOLLINGSWORTH MA,Wheelock MJ,etal.Collagen I promotes metastasis in pancreatic cancer by activating c-Jun NH (2)-terminal kinase 1 and up-regulating N-cadherin expression[J].CancerRes,2006,66 (24):11745-11753.

[26]LIN LC,HSU SL,WU CL,etal.TGFbeta can stimulate the p (38)/beta-catenin/PPARgamma signaling pathway to promote the EMT,invasion and migration of non-small cell lung cancer (H460 cells)[J].ClinExpMetastasis,2014,31 (8):881-895.

E-mail:crickding@163.com

Effect and mechanism of HMG20A on the proliferation and migration of hepatocellular carcinoma cells

BAI Yi-he1,QIN Zhao-yu1,HE Fu-chu1,2,4,DING Chen1,3 △

(1CenterofMedicalSystemsBiology,InstitutesofBiomedicalSciences,FudanUniversity,Shanghai200032,China;2NationalCenterforProteinScience,Beijing102206,China;3SchoolofLifeSciences,FudanUniversity,Shanghai200438;4StateKeyLaboratoryofProteomics,BeijingProteomeResearchCenter,BeijingInstituteofRadiationMedicine,Beijing102206,China)

ObjectiveTo study the mechanism of HMG20A in the development and metastasis of hepatocellular carcinoma(HCC).MethodsWe constructed HMG20A overexpression and knock-down stable cell lines in Huh7 and HCCLM3 which had different metastatic abilities and genetic backgrounds.The over expression and knock down effects of HMG20A were analyzed by real time quantitative PCR (qPCR).The effect of HMG20A on the proliferation of HCC cell lines was detected by cell counting kit-8 assay.The Transwell analysis was proceeded to assess the effect of HMG20A on metastasis of HCC cells.We investigated the mechanism of HMG20A in proliferation and metastasis of HCC cells using qPCR analysis and Western blot.ResultsTheinvitroexperiments suggested that HMG20A could promote the proliferation and metastasis of HCC.It also showed that HMG20A could upregulate the activity and expression levels of mitogen-activated protein kinases (MAPKs) such as p38 and extracellular regulated protein kinase (ERK).Futhermore,it could regulate the expression levels of the biomarkers of epithelial-mesenchymal transition (EMT) such as Vimentin,anti-smooth muscle antibody (alpha-SMA) and N-cadherin positively and E-cadherin negatively.Conclusions HMG20A may promote the proliferation and migration of HCC cellsinvitroby promoting the EMT process and MAPK pathway.

liver cancer;HMG20A;metastasis;mitogen-activated protein kinase;epithelial-mesenchymal transition

Q291,R735

Adoi: 10.3969/j.issn.1672-8467.2016.04.002

2016-03-10;編輯:王蔚)

國家重點基礎(chǔ)研究發(fā)展計劃 (2013CB910802);國家高技術(shù)發(fā)展研究計劃 (2015AA020108);國家國際科技合作專項 (2014DFB30020);十二五科技部重大專項計劃(2012ZX10002012-006)

* This work was supported by the National Basic Research Program of China (2013CB910802),the National High Technology Research and Development Program of China (2015AA020108),the International S&T Cooperation Program of China (2014DFB30020),and the Major Projects of the Ministry of Science and Technology in the 12thFive-Year (2012ZX10002012-006).