HMG20A對肝癌細胞體外增殖與遷移的影響及其機制

白一禾　秦兆宇　賀福初,2,4　丁　琛,3△

(1復旦大學生物醫(yī)學研究院醫(yī)學系統(tǒng)生物學研究中心 上?！?00032; 2 北京國家蛋白質科學中心　北京　102206;3 復旦大學生命科學學院　上?！?00438; 4 蛋白質組學國家重點實驗室,北京蛋白質組研究中心,北京放射醫(yī)學研究所　北京　102206)

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HMG20A對肝癌細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復旦大學生物醫(yī)學研究院醫(yī)學系統(tǒng)生物學研究中心 上海200032;2北京國家蛋白質科學中心北京102206;3復旦大學生命科學學院上海200438;4蛋白質組學國家重點實驗室,北京蛋白質組研究中心,北京放射醫(yī)學研究所北京102206)

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

肝癌;HMG20A;轉移;促分裂源活化蛋白激酶;上皮間充質轉化

原發(fā)性肝細胞癌 (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)病機制非常復雜,盡管已經(jīng)確認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,但仍然缺乏對導致肝癌形成和進展的分子生物學機制的確切認識。

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

還有研究顯示,在乳腺癌中敲除HMG20A后,許多與上皮間充質轉化(epithelial-mesenchymal transition,EMT)相關的基因下調,HMG20A和LSD1 (lysine specific demethylase 1)參與snail1 (snail family zinc finger 1)和轉化生長因子β (transforming growth factor-β,TGF-β)調控的EMT,HMG20A缺失削弱LSD1和上皮基因啟動子的結合,導致細胞的轉移能力減弱[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。上皮細胞是高度有序的單層細胞,細胞間的緊密連接和黏附連接的存在使上皮細胞緊密相連發(fā)揮功能,同時也限制了任意遷移的能力。與之相反,間充質細胞則形態(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)展成轉移性腫瘤,進而侵襲其他組織。

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

材 料 和 方 法

主要試劑與材料細胞株HCCLM3購自復旦大學中山醫(yī)院肝癌研究所;Huh7購自中國科學院上海生命科學研究院生物化學與細胞生物學研究所;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有限公司。

敲低穩(wěn)定株的構建shRNA干擾序列由上海拓然生物科技有限公司設計,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細胞培養(yǎng)皿約鋪5×106個HEK293T細胞,第2天進行轉染。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待轉染質粒,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復合體加入到細胞培養(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,所得細胞即為靶基因穩(wěn)定干擾細胞株。

過表達穩(wěn)定株的構建轉染前一天準備293T細胞,轉染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待轉染質粒,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轉染試劑。將2支離心管內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,逆轉錄以后進行qPCR反應。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,電泳后進行轉膜,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檢測細胞遷移 對構建成功的穩(wěn)定細胞株及對照組細胞進行消化并計數(shù),用無血清DMEM將細胞懸液稀釋成1×106cells/mL,每個Transwell上室加入100 μL細胞懸液,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,用棉簽檫凈上層細胞,多聚甲醛固定后結晶紫染色,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。

細胞增殖檢測 對構建成功的穩(wěn)定株進行消化計數(shù),把細胞鋪到96孔板中,每組6個副孔,每孔加2 000個細胞。鋪板后每24 h取出一板細胞進行檢測,連續(xù)4天。每孔細胞中加入10 μL CCK-8細胞增殖與毒性檢測試劑和90 μL的DMEM培養(yǎng)基,放至細胞培養(yǎng)箱內孵育2 h,用酶標儀檢測,使用450 nm濾光片,以不接種細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)計學處理 Graphpad 5.0軟件對所有的數(shù)據(jù)進行統(tǒng)計處理,差異顯著性用雙側t檢驗,P<0.05為差異有統(tǒng)計學意義。

結　　　果

HMG20A過表達和敲低效率驗證構建HMG20A過表達穩(wěn)定株Huh7,以空質粒pCDH為陰性對照,pCDH-HMGH20A為過表達HMG20A的穩(wěn)定細胞株,qPCR實驗證明pCDH-HMGH20A組能夠顯著過表達目標基因,差異具有統(tǒng)計學意義 (P<0.001)。同時,為了在HCCLM3中敲低HMG20A,設計2條特異shRNA干擾序列shHMG20A-1和shHMG20A-2,在HCCLM3中構建HMG20A敲低穩(wěn)定株,shNC1和shNC2為陰性對照,qPCR結果顯示敲低穩(wěn)定株中HMG20A的表達水平顯著低于對照組,差異具有統(tǒng)計學意義 (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過表達和敲低效果的驗證

Fig 1The effects of HMG20A overexpression and knockdown

HMG20A促進肝癌細胞的體外增殖 在Huh7中構建HMG20A過表達的穩(wěn)定株,在HCCLM3中構建敲低穩(wěn)定株,分別于鋪板后第1、2、3、4天檢測細胞的增殖情況。結果顯示過表達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)計學意義 (P<0.01),表明HMG20A能顯著促進肝癌細胞的體外增殖。

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對肝癌細胞增殖的影響

Fig 2HMG20A promotes the proliferation of HCC cells

HMG20A促進肝癌細胞的體外遷移 通過Transwell實驗觀察肝癌細胞轉移能力的變化,結果顯示過表達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)計學意義 (HMG20Avs.pCDH,P<0.001,shHMG20Avs.shNC,P<0.01),證實HMG20A能夠顯著促進肝癌細胞的體外遷移能力。

HMG20A上調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通路參與細胞的生長、發(fā)育、分化、凋亡等一系列細胞生理活動,可以促進腫瘤的發(fā)生發(fā)展和轉移,幾種MAPK亞家族參與的信號轉導通路各有不同的功能,如ERK調控細胞生長和分化[6],JNK和p38 MAPK信號通路在炎癥和細胞凋亡等應激反應中發(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、炎性細胞因子、激素等刺激磷酸化激活,激活后MAPK轉移到細胞核,并磷酸化和激活許多蛋白激酶以及轉錄因子,參與細胞增殖與分化、細胞形態(tài)維持以及細胞凋亡和細胞骨架的構建、癌癥的發(fā)生發(fā)展等多種生理過程[9]。我們在Huh7和HCCLM3中分別構建了HMG20A過表達和敲低的穩(wěn)定株,分析HMG20A對肝癌細胞相關信號通路的影響。Western blot和qPCR的結果顯示在過表達HMG20A的穩(wěn)定株中,HMG20A能顯著促進MAPK家族p38、ERK的活性和表達水平 (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MAPK通路,影響肝癌的發(fā)展。

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

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是一個在腦中表達水平較高的轉錄因子,在神經(jīng)分化中有重要作用。HMG20A/iBRAF招募甲基轉移酶MLL甲基化H3K4,進而調控神經(jīng)分化特異性基因[3],MLL蛋白在基因調控、細胞增殖、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[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)分化相關的基因[15]。REST缺失導致腫瘤的發(fā)生,REST參與細胞凋亡、可能參與細胞復制、血管形成等,對基因轉錄起負調控作用[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MAPKs

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

Rivero等[4]發(fā)現(xiàn)在RPE1細胞中HMG20A和LSD1對于調控間充質表型是必需的,敲減HMG20A或者LSD1會上調上皮表型標志物如CXADR、CLDN12、KRT18和CDH4。敲減HMG20A會削弱過表達Snail1導致的CXADR和CLDN12的下調,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,我們在肝癌細胞HCCLM3和Huh7中分別構建HMG20A敲低和過表達穩(wěn)定株,發(fā)現(xiàn)HMG20A能顯著促進肝癌細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MAPK通路中的p38、ERK的活性和表達水平。

MAPK是細胞內的一類絲氨酸/蘇氨酸蛋白激酶,是細胞信號傳導的重要途徑之一。在未受刺激的細胞內,MAPK處于靜止狀態(tài),當受到高糖、生長因子、激素和炎性細胞因子等因素刺激后,MAPK被磷酸化激活,進而定位到細胞核中,調控一系列轉錄因子、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、結腸癌和前列腺癌中起著促進增殖的作用[17-19]。MAPK還可以通過磷酸化一些細胞核內的轉錄因子,如c-Myc,c-Jun等,參與細胞增殖和分化[20-21]。我們的研究提示HMG20A可能通過促進MAPK途徑中ERK和p38的活性和表達水平促進肝癌細胞的增殖。

此外,p38是MAPK一個重要的亞族,能夠接受細胞外的刺激,并磷酸化下游蛋白發(fā)揮其功能。在原發(fā)性腹膜間皮細胞中,p38與Twist1的磷酸化有關,能夠增加Twist1的蛋白穩(wěn)定性,促進EMT進程和腫瘤的浸潤[22]。在原腸胚形成中,E-鈣黏素蛋白的表達受到p38 MAPK 的負調控[23]。持續(xù)活化的ERK能夠促進細胞增殖以及細胞的惡性轉化,已經(jīng)有研究表明ERK能夠直接或者間接上調Vimentin的表達,從而促進EMT的發(fā)生[24]。在胰腺癌中,Collagen I能夠激活JNK1,上調N-cadherin 的表達,加速EMT進程[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過表達和敲低HMG20A后EMT相關標記蛋白的變化

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EMT標志物Vimentin、alpha-SMA和N-cadherin的表達,并抑制E-cadherin的表達水平。結果提示HMG20A可能促進了EMT進程。

EMT在腫瘤的轉移過程中起著重要作用,在EMT過程中,細胞功能發(fā)生變化,上皮細胞失去其上皮特征而獲得了間充質細胞特征,使得腫瘤細胞離開原發(fā)部位,遷移到鄰近組織,其主要的上皮標記物如角蛋白、E-鈣黏素等表達下調,同時波形蛋白、N-鈣黏素等間質標記物表達上調。癌癥相關的許多通路已經(jīng)成為EMT的重要調控信號,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MAPK通路中的p38、ERK的活性和表達水平,提高肝癌細胞的體外增殖能力;還發(fā)現(xiàn)該基因能夠上調EMT標志物Vimentin、alpha-SMA和N-cadherin的表達,并抑制E-cadherin的表達水平,提示其可能參與了促進細胞EMT進程,導致肝癌細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。

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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)

國家重點基礎研究發(fā)展計劃 (2013CB910802);國家高技術發(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).