二烯丙基二硫上調(diào)miR-22通過(guò)Wnt-1通路抑制人胃癌細(xì)胞增殖與遷移侵襲

唐云云，唐 儀，劉 芳，蘇 堅(jiān),4，夏 紅，蘇 波，曾 希，蘇 琦

(1.南華大學(xué)腫瘤研究所, 湖南省胃癌研究中心，湖南省高校腫瘤細(xì)胞與分子病理學(xué)重點(diǎn)實(shí)驗(yàn)室，湖南 衡陽(yáng) 421001；2.永州職業(yè)技術(shù)學(xué)院基礎(chǔ)醫(yī)學(xué)部，湖南 永州 425100；3.南華大學(xué)附屬湘潭醫(yī)院病理科，湖南 湘潭 411101；4南華大學(xué)附屬第二醫(yī)院病理科，湖南 衡陽(yáng) 421001)

二烯丙基二硫上調(diào)miR-22通過(guò)Wnt-1通路抑制人胃癌細(xì)胞增殖與遷移侵襲

唐云云1,2，唐 儀1,3，劉 芳1，蘇 堅(jiān)1,4，夏 紅1，蘇 波1，曾 希1，蘇 琦1

(1.南華大學(xué)腫瘤研究所, 湖南省胃癌研究中心，湖南省高校腫瘤細(xì)胞與分子病理學(xué)重點(diǎn)實(shí)驗(yàn)室，湖南 衡陽(yáng) 421001；2.永州職業(yè)技術(shù)學(xué)院基礎(chǔ)醫(yī)學(xué)部，湖南 永州 425100；3.南華大學(xué)附屬湘潭醫(yī)院病理科，湖南 湘潭 411101；4南華大學(xué)附屬第二醫(yī)院病理科，湖南 衡陽(yáng) 421001)

目的 探討二烯丙基二硫(diallyl disulfide，DADS)上調(diào)miR-22是否通過(guò)Wnt-1通路抑制人胃癌MGC803細(xì)胞增殖與遷移侵襲。方法 MTT、細(xì)胞劃痕實(shí)驗(yàn)、侵襲實(shí)驗(yàn)分別檢測(cè)DADS與miR-22對(duì)MGC803細(xì)胞增殖與遷移侵襲的影響。在線預(yù)測(cè)軟件尋找miR-22調(diào)控的靶基因，熒光素酶報(bào)告基因檢測(cè)miR-22對(duì)Wnt-1 3′UTR熒光酶活性的影響。qRT-PCR檢測(cè)Wnt-1mRNA表達(dá)變化。Western blot檢測(cè)Wnt-1、β-catenin與TCF-4蛋白表達(dá)。結(jié)果 MTT顯示，DADS 與miR-22可明顯抑制MGC803細(xì)胞增殖(P<0.05)。劃痕實(shí)驗(yàn)顯示，DADS與miR-22可明顯抑制MGC803細(xì)胞遷移，而miR-22+DADS更為明顯(P<0.05)。侵襲實(shí)驗(yàn)顯示，miR-22可抑制人胃癌MGC803細(xì)胞侵襲，而miR-22+DADS更為明顯(P<0.05)。在線預(yù)測(cè)軟件尋找miR-22調(diào)控的靶基因顯示，Wnt-1可能是miR-22的靶基因，熒光素報(bào)告基因檢測(cè)證實(shí)Wnt-1是miR-22直接調(diào)控的靶基因；qRT-PCR顯示，DADS與miR-22能下調(diào)Wnt-1 mRNA表達(dá)，而miR-22+DADS更為明顯(P<0.05)。Western blot顯示，DADS 與miR-22能下調(diào)Wnt-1、β-catenin與TCF-4蛋白表達(dá)，而miR-22+DADS尤為明顯(P<0.05)。結(jié)論 DADS可上調(diào)miR-22 通過(guò)Wnt-1通路明顯抑制MGC803細(xì)胞增殖與遷移侵襲。

二烯丙基二硫；人胃癌細(xì)胞；miR-22；Wnt通路；遷移；侵襲

胃癌是最常見的惡性腫瘤之一，發(fā)生率與死亡率分別為全球第4位與第3位[1]。據(jù)最新統(tǒng)計(jì)，胃癌在我國(guó)每年約新發(fā)67.9萬(wàn)，死亡49.8萬(wàn)人，發(fā)生率與死亡率位于第2。由于患者就診時(shí)大多已發(fā)生侵襲轉(zhuǎn)移，常規(guī)外科手術(shù)和化學(xué)藥物治療效果較差，5年生存率低于10%[2-3]。因此，開發(fā)高效低毒藥物與尋找靶點(diǎn)對(duì)防治胃癌具有重要意義。

二烯丙基二硫(diallyl disulfide，DADS)是大蒜中烯丙基硫化物的一種脂溶性的有效成分，對(duì)多種腫瘤均有明顯的抑制作用[4]。近年來(lái)，miRNAs在腫瘤中的作用引起人們高度關(guān)注。miRNAs是一類含量豐富且高度保守的非編碼內(nèi)源性小RNA分子，通過(guò)與靶基因mRNA的3′非編碼區(qū)完全或不完全結(jié)合，抑制靶基因的表達(dá)，從而調(diào)控細(xì)胞增殖、分化與凋亡等重要過(guò)程[5]。我們先前研究表明，miR-22在胃癌中表達(dá)下調(diào)與臨床分期和淋巴結(jié)轉(zhuǎn)移有關(guān)[6]。并且，DADS作用人胃癌MGC803細(xì)胞的差異miRNAs中，發(fā)現(xiàn)miR-22上調(diào)[7]。本文進(jìn)一步探討DADS上調(diào)miR-22是否通過(guò)Wnt-1通路抑制MGC803細(xì)胞遷移與侵襲。

1 材料與方法

1.1 試劑 質(zhì)粒抽提試劑盒購(gòu)自上海華瞬公司；逆轉(zhuǎn)錄試劑盒、熒光素酶活性檢測(cè)試劑盒購(gòu)自Promega公司；miR-22 mimics與Wnt-1基因3′UTR的結(jié)合位點(diǎn)序列分別由Exiqon公司與Invitrogen公司合成；qRT-PCR miRNA試劑盒購(gòu)自上海吉瑪公司；蛋白濃度測(cè)定試劑盒、ECL化學(xué)發(fā)光檢測(cè)試劑盒購(gòu)自Pierce公司；Wnt-1(sc-6280)、β-catenin (sc-7963)、TCF-4(sc-166699)、β-actin(sc-47778)抗體購(gòu)自Santa Cruz公司；RPMI 1640培養(yǎng)液購(gòu)自Hyclone公司，胎牛血清購(gòu)自四季青生物公司；轉(zhuǎn)染用Opti-MEM培養(yǎng)液購(gòu)自Invtrogen公司。

1.2 細(xì)胞培養(yǎng)與分組 人胃癌MGC803細(xì)胞由本實(shí)驗(yàn)室保存，置于含10%小牛血清的RPMI 1640培養(yǎng)基中，37℃、5% CO2、飽和濕度的培養(yǎng)箱內(nèi)傳代培養(yǎng)。采用胰酶消化預(yù)先培養(yǎng)的MGC803細(xì)胞并接種于6孔板中，按每孔2 mL鋪好，培養(yǎng)至細(xì)胞匯合度達(dá)30%～50%用于轉(zhuǎn)染。用滅菌的無(wú)酶水稀釋上述各種轉(zhuǎn)染質(zhì)粒干粉，按說(shuō)明配制成20 μmol·L-1的溶液備用。用不含血清的Opti-MEM培養(yǎng)液分別稀釋100 pmol轉(zhuǎn)染質(zhì)粒與5 μL的Lipofectamine 2000，混勻，室溫孵育5 min。將MGC803細(xì)胞分為轉(zhuǎn)染scramble對(duì)照組、scramble+DADS組、轉(zhuǎn)染miR-22 mimics組與miR-22 mimics+DADS組，轉(zhuǎn)染培養(yǎng)48 h，收獲細(xì)胞。

1.3 MTT實(shí)驗(yàn) 用胰酶消化上述各組細(xì)胞，吹打成單細(xì)胞懸液并收集離心。取200 μL的5×103個(gè)細(xì)胞接種于96孔板中，設(shè)復(fù)孔6個(gè)。未接種細(xì)胞的孔中加入RPMI 1640培養(yǎng)液作調(diào)零孔。于37℃、5% CO2細(xì)胞培養(yǎng)箱中培養(yǎng)24～48 h，細(xì)胞匯合度達(dá)90%。接種細(xì)胞的孔中加入滅菌MTT液20 μL，繼續(xù)孵育4 h。取出培養(yǎng)板，吸棄上清液，加入150 μL DMSO溶液，低速振蕩10 min，使結(jié)晶物溶解。選擇570 nm波長(zhǎng)，酶標(biāo)儀測(cè)定各孔吸光度值，記錄結(jié)果，實(shí)驗(yàn)重復(fù)3次。

1.4 劃痕實(shí)驗(yàn) 將上述細(xì)胞接種于6孔板中，RPMI 1640培養(yǎng)基37℃、5% CO2培養(yǎng)，直至形成單層細(xì)胞，每組3個(gè)平行樣本。吸棄上層培養(yǎng)液，PBS緩沖液洗滌2次，用無(wú)菌10 μL Eppendorf Tip在細(xì)胞板上劃痕，無(wú)血清培養(yǎng)液洗3次，加入新鮮無(wú)血清培養(yǎng)基。倒置顯微鏡下觀察、測(cè)量劃痕區(qū)相對(duì)距離。實(shí)驗(yàn)重復(fù)3次。

1.5 侵襲實(shí)驗(yàn) 將基質(zhì)膠稀釋液鋪置在transwell小室中制成膜。加無(wú)血清RPMI 1640培養(yǎng)液100 μL至transwell小室的上腔，水化基質(zhì)膠20 min。取100 μL上述各組細(xì)胞稀釋液接種至小室上腔，下腔中加入含10%胎牛血清的RPMI 1640培養(yǎng)液500 μL。37℃、5% CO2的培養(yǎng)箱中培養(yǎng)48 h，取出transwell小室，用棉簽擦棄小室上層細(xì)胞，PBS液洗3遍。4%多聚甲醛固定10 min，0.1%結(jié)晶紫染色20 min，PBS液清洗，晾干。光鏡下觀察并隨機(jī)選取4個(gè)高倍視野進(jìn)行細(xì)胞計(jì)數(shù)，取平均值。實(shí)驗(yàn)重復(fù)3次。

1.6 miR-22的靶基因驗(yàn)證

1.6.1 通過(guò)在線預(yù)測(cè)軟件(Targetscan and Miranda)尋找miR-22調(diào)控的靶基因

1.6.2 熒光素酶報(bào)告基因檢測(cè) 根據(jù)Wnt-1基因3′-UTR序列與miR-22結(jié)合位點(diǎn)，設(shè)計(jì)合成引物序列， Wnt-1-wt：F: 5′-CCGCTCGAGCCCTCCCCCAAAC-3′, R: 5′-GAATGCGGCCGCCTGGGAGTGGTAAAAGG GGAGGAT-3′；Wnt-1-mut：F: 5′-CCGCTCCTCCAAGCC ATTC-3′, R: 5′-ATGCCGACTTGGCCGAAT-3′。相應(yīng)的上、下引物一起退火完成后，將其連接到含有熒光素酶報(bào)告基因的載體質(zhì)粒上。將MGC-803細(xì)胞培養(yǎng)于12 孔培養(yǎng)板，細(xì)胞分為轉(zhuǎn)染miR-22 mimics與Wnt-1-wt組、轉(zhuǎn)染scramble與Wnt-1-wt組、轉(zhuǎn)染miR-22 mimics與Wnt-1-mut組和轉(zhuǎn)染scramble與Wnt-1-mut組，48 h后收獲細(xì)胞。按照Promega公司雙熒光素酶活性檢測(cè)試劑盒操作說(shuō)明書，在單光子檢測(cè)儀檢測(cè)細(xì)胞熒光素酶的活性，計(jì)算相對(duì)熒光素酶活性=熒光素酶活性值/海腎熒光素酶活性。實(shí)驗(yàn)獨(dú)立重復(fù)3次。1.7 qRT-PCR檢測(cè) 收集上述各組細(xì)胞，采用RNA抽提試劑盒抽提細(xì)胞總RNA，逆轉(zhuǎn)錄合成cDNA。設(shè)計(jì)合成PCR引物序列：Wnt-1：F: 5′-TGGCTGGGTTTCTGCTACG-3′，R: 5′-CCCGGATTTTGGCGTATC-3′；GAPDH：F: 5′-GCTGAGAACGGGAAGCT TGT-3′, R: 5′-GCCAGGGGTGCTAAGCAG-3′。PCR擴(kuò)增反應(yīng)體系為20 μL，包括PCR primers(5 mmol·L-1)0.4 μL、RT product 2.0 μL、Taq DNA polymerase(5 U·μL-1)0.2 μL、2×SYBR Mix 10 μL、滅菌蒸餾水7.4 μL。反應(yīng)條件：95 ℃ 3 min、95 ℃ 12 s、62 ℃ 35 s、72 ℃ 30 s，共40個(gè)循環(huán)。以GAPDH為內(nèi)參，采用2-ΔΔCT法計(jì)算Wnt-1 mRNA相對(duì)表達(dá)量。

1.8 Western blot檢測(cè) 收集上述各組細(xì)胞，提取細(xì)胞總蛋白，BCA法測(cè)定蛋白濃度，每組取等量樣本進(jìn)行SDS-PAGE凝膠電泳，電泳后轉(zhuǎn)膜，封閉1 h，加一抗，4 ℃過(guò)夜，TBST洗膜，加二抗，孵育1 h，洗膜，ECL發(fā)光，X線曝光、顯影、定影。

2 結(jié)果

2.1 DADS和miR-22對(duì)MGC803細(xì)胞增殖的影響 將miR-22 mimics轉(zhuǎn)染于MGC803細(xì)胞中，轉(zhuǎn)染miRNA的無(wú)關(guān)序列scramble作為對(duì)照，并用30 mg·L-1DADS分別處理。Tab 1 MTT結(jié)果顯示，48、72、96 h后，DADS與miR-22高表達(dá)均可明顯抑制MGC803細(xì)胞增殖(P<0.05)。表明DADS與miR-22能明顯抑制MGC803細(xì)胞增殖，且miR-22可增強(qiáng)DADS的作用。

2.2 DADS和miR-22對(duì)MGC803細(xì)胞遷移的影響 Fig 1顯示，48 h后，DADS處理組、miR-22組與miR-22+DADS組傷口愈合率分別為(60.17±2.22)%、(61.57±1.54)%與(49.85±1.98)%，較對(duì)照組(91.94±2.01)%明顯減緩(P<0.01)。表明DADS與miR-22能明顯抑制MGC803細(xì)胞的遷移能力，miR-22可增強(qiáng)DADS的抑制作用。

Fig 1 Effect of DADS and miR-22 on migration of MGC803 cells(×20)

Tab 1 Effect of DADS and miR-22 on proliferation in MGC803 cells

*P<0.05vscontrol group

2.3 DADS和miR-22對(duì)MGC803細(xì)胞侵襲的影響 Fig 2顯示，48 h后，DADS處理組、miR-22組與miR-2+DADS組穿過(guò)基質(zhì)膠的細(xì)胞數(shù)分別為(137±11)、(120±9)與(70±10)，較對(duì)照組(253±16)明顯減少(P<0.01)。表明DADS與miR-22高表達(dá)能明顯抑制MGC-803細(xì)胞的侵襲能力，且miR-22可增強(qiáng)DADS的抑制作用。

Fig 2 Effect of DADS and miR-22 on invasion in MGC803 cells(×10)

A:Control;B:DADS;C:miR-22;D:miR-22+DADS

2.4 Wnt-1是miR-22的靶基因

2.4.1 miR-22候選靶基因的篩選 為進(jìn)一步明確miR-22調(diào)控胃癌生物學(xué)行為的作用機(jī)制，運(yùn)用生物信息學(xué)方法，通過(guò)在線預(yù)測(cè)軟件(Targetscan and Miranda)尋找miR-22相關(guān)蛋白編碼的靶基因。經(jīng)在線軟件交叉預(yù)測(cè)后，發(fā)現(xiàn)miR-22有500多個(gè)靶基因，miR-22具有Wnt-1的3′UTR的結(jié)合位點(diǎn)，Wnt-1可能是miR-22的靶基因(Fig 3)。

Fig 3 Putative binding sites of miR-22 and 3′UTRs of Wnt-1

2.4.2 熒光素酶報(bào)告基因驗(yàn)證Wnt-1是miR-22直接調(diào)控的靶基因 為證實(shí)miR-22作用預(yù)測(cè)的結(jié)合位點(diǎn)導(dǎo)致熒光素酶活性發(fā)生改變，設(shè)計(jì)Wnt-1 3′UTR缺失的miR-22結(jié)合位點(diǎn)的突變序列和野生序列插入報(bào)告質(zhì)粒中。胃癌MGC803細(xì)胞分別轉(zhuǎn)染miR-22 mimics、Wt-miR-22/Wnt-1和Mut-miR-22/Wnt-1重組質(zhì)粒。熒光素酶報(bào)告基因檢測(cè)發(fā)現(xiàn)(Tab 2)，Mut-miR-22/Wnt-1熒光素酶活性為(1.033±0.128)，較對(duì)照組(1.075±0.062)差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)。但Wt-miR-22/Wnt-1熒光素酶活性為(0.522±0.318)，較對(duì)照組(1.081±0.984)明顯下降(P<0.05)。表明Wnt-1是miR-22直接的靶基因。

2.5 DADS與miR-22對(duì)MGC803細(xì)胞Wnt-1表達(dá)的影響 Fig 4 qRT-PCR結(jié)果顯示，DADS組和miR-22組Wnt-1 mRNA的表達(dá)水平2-△△CT分別為(0.721±0.034)與(0.745±0.038)較對(duì)照組2-△△CT(1.066±0.714)明顯下降，并以miR-22+DADS組2-△△CT(0.495±0.029)下降尤為明顯(P<0.05)。Fig 5 Western blot結(jié)果顯示，DADS處理與miR-22高表達(dá)的MGC803細(xì)胞 Wnt-1蛋白表達(dá)水平較對(duì)照組分別下調(diào)31.84%與28.77%，并且，miR-22+DADS下調(diào)尤為明顯(48.9%)(P<0.05)。

Tab 2 Effect of miR-22 on luciferase activity

*P<0.05vsscramble group

Fig 4 Effect of DADS and miR-22 on Wnt-1 mRNA expression in MGC803 cells

1:Scramble;2:Scramble+DADS;3:miR-22;4:miR-22+DADS.*P<0.05vsscramble;#P<0.05vsmiR-22.

2.6 DADS與miR-22對(duì)β-catenin與TCF-4表達(dá)的影響 Western blot結(jié)果顯示(Fig 6、7)，DADS處理與miR-22高表達(dá)的MGC803細(xì)胞β-catenin與TCF-4蛋白表達(dá)水平較對(duì)照組分別下調(diào)，且miR-22+DADS下調(diào)尤為明顯(P<0.05)。表明DADS上調(diào)miR-22通過(guò)Wnt-1信號(hào)下調(diào)β-catenin與TCF-4。

3 討論

研究證實(shí)，miRNA在腫瘤中通過(guò)調(diào)控抑癌基因或癌基因的表達(dá)，表現(xiàn)為類似癌基因或抑癌基因樣作用[8]。大量研究表明，miR-22在多種腫瘤中表達(dá)下調(diào)，可抑制腫瘤細(xì)胞遷移侵襲，表現(xiàn)為抑癌基因，其作用機(jī)制與靶向調(diào)控多種靶基因有關(guān)[9-19]。研究顯示，miR-22通過(guò)靶向TIAM1抑制結(jié)腸癌HCT-116細(xì)胞增殖與遷移侵襲[9]。ATP citrate lyase(ACLY)在肺癌、前列腺癌、子宮頸癌、骨肉瘤等中表達(dá)上調(diào)，而miR-22表達(dá)下調(diào)，ACLY與miR-22表達(dá)呈負(fù)相關(guān)，且miR-22可直接靶向轉(zhuǎn)錄后調(diào)控ACLY抑制腫瘤生存與轉(zhuǎn)移[10]。miR-22在胃癌中表達(dá)較正常組織明顯下調(diào)，與淋巴結(jié)轉(zhuǎn)移、遠(yuǎn)距離轉(zhuǎn)移、臨床分期和患者生存率下降有關(guān)。而miR-22高表達(dá)可通過(guò)靶向Sp1或MMP-14、Snail，抑制胃癌細(xì)胞遷移與侵襲[11-13]。Xu等[14]研究顯示，miR-22通過(guò)靶向CDK6、MDM2、LEF1、MYB、FOS等基因在卵巢癌細(xì)胞遷移與侵襲中起著重要作用。miR-22在腎細(xì)胞癌表達(dá)下調(diào)與組織學(xué)類型、腫瘤分期和淋巴結(jié)轉(zhuǎn)移有關(guān)，其通過(guò)直接靶向SIRT1，抑制腎細(xì)胞癌細(xì)胞增殖與遷移侵襲。miR-22高表達(dá)體外可抑制腎細(xì)胞癌細(xì)胞增殖、遷移侵襲與誘導(dǎo)凋亡，體內(nèi)可抑制移植瘤生長(zhǎng)?？苫罨痯53及其下游靶點(diǎn)p21與PUMA，裂解CASP3與PARP，抑制EMT[15]。

Fig 5 Effect of DADS and miR-22 on Wnt-1 protein expression in MGC803 cells

1: Scramble; 2: Scramble+DADS; 3: miR-22; 4: miR-22+DADS.*P<0.05vsscramble;#P<0.05vsmiR-22.

Fig 6 Effect of DADS and miR-22 on β-catenin protein expression in MGC803 cells

1: Scramble; 2: Scramble+DADS; 3: miR-22; 4: miR-22+DADS.*P<0.05vsscramble;#P<0.05vsmiR-22;△P<0.05vsDADS.

Fig 7 Effect of DADS and miR-22 on TCF-4 protein expression in MGC803 cells

1: Scramble; 2: Scramble+DADS; 3: miR-22; 4: miR-22+DADS.*P<0.05vsscramble;#P<0.05vsmiR-22;△P<0.05vsDADS.

眾所周知，Wnt信號(hào)通路在腫瘤發(fā)生與侵襲轉(zhuǎn)移中起著重要作用。當(dāng)胞外Wnt蛋白增加時(shí)，Wnt與Frizzled結(jié)合，激活Dsh/Dvl蛋白，抑制GSK3β/APC/Axin復(fù)合物對(duì)β-catenin的降解，而促進(jìn)β-catenin入核，與轉(zhuǎn)錄因子Tcf/Lef結(jié)合，引起靶基因的轉(zhuǎn)錄，導(dǎo)致腫瘤發(fā)生。研究發(fā)現(xiàn)，Wnt信號(hào)通路與miRNAs之間存在交叉對(duì)話，miRNA可調(diào)控Wnt信號(hào)通路，如miR-122、miR-148a、miR-22、miR-200b、miR-185-3p、miR-324-3p、miR-26a、 miR-487b、miR-329、miR-410、miR-374b等可靶向抑制Wnt通路[16]。Liang等[17]研究顯示，miR-22可負(fù)調(diào)控Wnt/β-catenin信號(hào)通路，抑制β-catenin表達(dá)。近年來(lái)，研發(fā)天然植物有效成分防治腫瘤已成為新策略。研究表明，miR-22在腫瘤發(fā)生發(fā)展中可抑制腫瘤干細(xì)胞(CSC)表型與功能。姜黃素、大豆異黃酮、茶多酚、白藜蘆醇、維生素D等天然植物有效因子通過(guò)靶向CSC相關(guān)基因，上調(diào)miR-22，抑制腫瘤增殖、遷移、侵襲與轉(zhuǎn)移[18]。我們研究顯示，DADS可上調(diào)miR-22靶向Wnt-1通路，抑制人胃癌細(xì)胞增殖與誘導(dǎo)凋亡[7]。

我們前期工作證明，DADS可體內(nèi)外抑制MGC803細(xì)胞的增殖，其機(jī)制與激活p38與抑制ERK通路，調(diào)節(jié)ATR/Chk1/Cdc25C/cyclin B1，上調(diào)組蛋白乙?；cp21，G2/M阻滯等有關(guān)。并且，DADS可通過(guò)Rac1-Pak1/Rock1通路下調(diào)LIMK1、MMP-9，上調(diào)TIMP-3，抑制人胃癌細(xì)胞EMT與侵襲[19-22]。

本研究在前期研究的基礎(chǔ)上，進(jìn)一步確定DADS上調(diào)miR-22是否通過(guò)Wnt-1通路抑制MGC803細(xì)胞遷移與侵襲。結(jié)果表明，DADS與miR-22均可明顯抑制MGC803細(xì)胞增殖。DADS處理組、miR-22組與miR-2+DADS組遷移能力較對(duì)照組明顯降低，并且穿過(guò)基質(zhì)膠的細(xì)胞數(shù)較對(duì)照組明顯減少，表明DADS與miR-22能明顯抑制MGC-803細(xì)胞的遷移侵襲能力，且miR-22可增強(qiáng)DADS的抑制作用。靶基因預(yù)測(cè)發(fā)現(xiàn)，miR-22具有Wnt-1的3′UTR的結(jié)合位點(diǎn)，Wnt-1可能是miR-22的靶基因。熒光素酶報(bào)告基因檢測(cè)顯示，Mut-miR-22/Wnt-1熒光素酶活性較對(duì)照組差異無(wú)顯著性，而Wt-miR-22/Wnt-1熒光素酶活性較對(duì)照組明顯下降，確定Wnt-1是miR-22直接的靶基因。qRT-PCR驗(yàn)證顯示，DADS組和miR-22組Wnt-1 mRNA表達(dá)較對(duì)照組明顯下降，并以miR-22+DADS組更為明顯。Western blot顯示，DADS組與miR-22組Wnt-1蛋白表達(dá)較對(duì)照組明顯下調(diào)，并且miR-22+DADS下調(diào)尤為明顯。另外，DADS組與miR-22組β-catenin與TCF-4蛋白表達(dá)較對(duì)照組明顯下調(diào)，且miR-22+ DADS下調(diào)尤為明顯。上述結(jié)果證明，DADS可上調(diào)miR-22，通過(guò)Wnt-1通路下調(diào)β-catenin與TCF-4，抑制人胃癌細(xì)胞增殖與遷移侵襲。

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Up-regulation of miR-22 through Wnt pathway suppresses proliferation, migration and invasion in human gastric MGC803 cells by DADS

TANG Yun-yun1,2, TANG Yi1,3, LIU Fang1, SU Jian1,4, XIA Hong1, SU Bo1, ZENG Xi1, SU Qi1
(1.CancerResearchInstitute,CenterforGastricCancerResearchofHunanProvince,KeyLabofCancerCellularandMolecularPathologyofHunanProvincialUniversity,UniversityofSouthChina,HengyangHunan421001,China;2.DeptofBasicMedicine,YongzhouVocationalTechnicalCollege,YongzhouHunan425100,China; 3.DeptofPathology,theAffiliatedXiangtanHospital,UniversityofSouthChina,XiangtanHunan411101,China;4.DeptofPathology,theSecondAffiliatedHospital,UniversityofSouthChina,HengyangHunan421001,China)

Aim To investigate the up-regulation of miR-22 through Wnt pathway inhibits the proliferation, migration and invasion in human gastric MGC803 cells induced by diallyl disulfide(DADS).Methods The effects of proliferation, migration, and invasion of gastric cancer cells were evaluated by MTT, wound-healing and invasion assays. Online prediction software was applied to search the target gene of miR-22. Luciferase report gene assay was used to assess the target genes Wnt-1 of miR-22. The expressions of Wnt-1, β-catenin and TCF-4 were tested by qRT-PCR and Western blot, respectively.Results MTT showed that DADS and miR-22 notably decreased the proliferation compared with control group(P<0.05). Wound-healing assay showed that DADS and miR-22 could significantly inhibit the migration of MGC803 cells compared with the control group, especially in miR-22+DADS(P<0.05). Invasion assay showed that DADS and miR-22 could markedly inhibit the invasion of MGC803 cells compared with the control group, especially in miR-22+DADS(P<0.05). Online prediction software to search the target gene exhibited that Wnt-1 may be a target gene of miR-22. Luciferase report gene assay disclosed that Wnt-1 was identified as a direct target of miR-22. qRT-PCR showed that the expression of Wnt-1 mRNA was respectively down-regulated by DADS and miR-22 compared with control group, especially in miR-22+DADS(P<0.05). Western blot exhibited that DADS and miR-22 obviously suppressed the expressions of Wnt-1, β-catenin and TCF-4 proteins, especially in miR-22+DADS(P<0.05).Conclusion Up-regulation of miR-22 through Wnt pathway can remarkably suppress the proliferation, migration and invasion in MGC803 cells by DADS.

diallyl disulfide; human gastric cancer cells; miR-22; Wnt pathway; migration; invasion

2017-04-15,

2017-05-12

國(guó)家自然科學(xué)基金資助項(xiàng)目(No 81374013, 81102854, 31100935)

唐云云(1984-)，女，碩士，助教，研究方向：胃癌發(fā)生與防治的分子機(jī)制，E-mail: 176024235@qq.com； 唐 儀(1983-)，女，碩士，主治醫(yī)師，研究方向：胃癌發(fā)生與防治的分子機(jī)制，并列第一作者，E-mail: tangyi9921@126.com； 蘇 琦(1945-)，男，教授，博士生導(dǎo)師，研究方向：胃癌發(fā)生與防治的分子機(jī)制，通訊作者，E-mail: suqi1945@163.com

時(shí)間：2017-7-7 11:05 網(wǎng)絡(luò)出版地址：http://kns.cnki.net/kcms/detail/34.1086.R.20170707.1104.040.html

10.3969/j.issn.1001-1978.2017.08.020

A

1001-1978(2017)08-1141-07

R329.24；R394.2；R735.2；R977.3；R977.6