劉越峰,張勇,鐘曉東,羅衛(wèi)民
(湖北醫(yī)藥學(xué)院附屬十堰市太和醫(yī)院眼科中心1、心胸外科2,湖北 十堰 442000)
DADS上調(diào)miR-200b抑制視網(wǎng)膜母細(xì)胞瘤細(xì)胞的生長(zhǎng)與侵襲
劉越峰1,張勇1,鐘曉東1,羅衛(wèi)民2
(湖北醫(yī)藥學(xué)院附屬十堰市太和醫(yī)院眼科中心1、心胸外科2,湖北 十堰 442000)
目的 探索miR-200b和二烯丙基二硫(DADS)相關(guān)的抑瘤機(jī)制,為揭示DADS抑制視網(wǎng)膜母細(xì)胞瘤生長(zhǎng)及侵襲的內(nèi)在分子機(jī)制提供理論依據(jù)。方法采用qRT-PCR分別檢測(cè)不同濃度的DADS對(duì)Y79細(xì)胞miR-200b表達(dá)的影響,DADS分別設(shè)置六種濃度:0μmol/L、25μmol/L、50μmol/L、100μmol/L、200μmol/L和400μmol/L。MTT和Transwell侵襲實(shí)驗(yàn)分別檢測(cè)DADS和miR-200b對(duì)Y79細(xì)胞生長(zhǎng)及侵襲能力的影響,將實(shí)驗(yàn)設(shè)置成五個(gè)組,即miRNA陰性對(duì)照組(瞬時(shí)轉(zhuǎn)染scramble 40 μmol/L);miR-200b組(瞬時(shí)轉(zhuǎn)染miR-200b-mimics 40 μmol/L);DADS陰性對(duì)照組(DMSO 10 μmol/L,即mock組);DADS組(DADS 200 μmol/L)和DADS+miR-200b組(瞬時(shí)共轉(zhuǎn)染miR-200b-mimics 40 μmol/L)和DADS(200 μmol/L)。結(jié)果DADS可上調(diào)Y79細(xì)胞中miR-200b的表達(dá),且其呈劑量依賴性(P<0.05);在MTT實(shí)驗(yàn)中,miR-200b組的OD值(0.549±0.057)較miRNA陰性對(duì)照組(0.737±0.135)明顯降低;DADS組的OD值(0.508±0.064)較DADS陰性對(duì)照組(0.722±0.145)明顯降低;而DADS+miR-200b組的OD值(0.362±0.081)較miRNA陰性對(duì)照組和DADS陰性對(duì)照組降低最為顯著(P<0.05),即DADS可以通過(guò)上調(diào)miR-200b抑制Y79細(xì)胞的增殖能力。在Transwell侵襲實(shí)驗(yàn)中,外源性的高表達(dá)miR-200b組(105±13)較miRNA陰性對(duì)照組(162±12)能夠顯著抑制Y79細(xì)胞的穿膜細(xì)胞數(shù),DADS組(102±13)較DADS陰性對(duì)照組(154±8)能夠顯著降低Y79的穿膜細(xì)胞數(shù),且DADS+miR-200b組(77±8),細(xì)胞穿膜細(xì)胞數(shù)較miRNA陰性對(duì)照組和DADS陰性對(duì)照組減少最顯著(P<0.05),即DADS通過(guò)上調(diào)miR-200b抑制Y79細(xì)胞侵襲。結(jié)論DADS通過(guò)上調(diào)miR-200b的表達(dá)抑制視網(wǎng)膜母細(xì)胞瘤細(xì)胞的生長(zhǎng)及侵襲。
視網(wǎng)膜母細(xì)胞瘤;MiR-200b;二烯丙基二硫;增殖;侵襲
二烯丙基二硫(Diallyl disulfide,DADS)對(duì)腫瘤的抑制作用多有報(bào)道,其為一種最初從大蒜中提取得到的天然化合物二烯丙基二硫。研究報(bào)道顯示DADS可抑制肺癌、胃癌、乳腺癌和白血病等多種惡性腫瘤[1-2]。在胃癌中,DADS可抑制細(xì)胞的生長(zhǎng)并誘導(dǎo)分化[3-4]。在結(jié)腸癌中,DADS可抑制腫瘤細(xì)胞的增殖[5]。在白血病中,DADS可阻滯細(xì)胞的細(xì)胞周期[6]。而DADS的抑癌機(jī)制與其能抑制DNA的加合物及活性氧形成、激活解毒致癌物的代謝酶、阻滯細(xì)胞周期和誘導(dǎo)腫瘤細(xì)胞凋亡等相關(guān)[7]。
MicroRNAs(miRNAs)是一種內(nèi)源性的單鏈非編碼RNA分子,通過(guò)與其靶基因3′-UTR(3′-非編碼區(qū))的完全或不完全配對(duì),miRNA能使翻譯抑制或者使靶mRNA降解,并轉(zhuǎn)錄后水平上對(duì)靶基因的表達(dá)進(jìn)行調(diào)控[8]。在腫瘤的發(fā)生及發(fā)展過(guò)程中,miRNA可對(duì)細(xì)胞的增殖、分裂、分化及調(diào)亡等重要的生物學(xué)過(guò)程進(jìn)行調(diào)控,發(fā)揮抑制或促進(jìn)腫瘤進(jìn)程的作用。據(jù)報(bào)道,miR-200b在多種細(xì)胞,組織或?qū)嶓w腫瘤中表達(dá)下調(diào),并與多種腫瘤的生長(zhǎng)增殖或復(fù)發(fā)轉(zhuǎn)移等相關(guān),如乳腺癌[9]、胃癌[10]和膠質(zhì)瘤[11]等。
視網(wǎng)膜母細(xì)胞瘤是原發(fā)于視網(wǎng)膜的惡性腫瘤,常見(jiàn)于兒童的眼部腫瘤。患者按類型可分為非遺傳型和遺傳型兩種,非遺傳型常見(jiàn)于單側(cè)發(fā)病,無(wú)遺傳性;遺傳型常見(jiàn)于雙眼發(fā)病,有陽(yáng)性家族史,發(fā)病迅速,易轉(zhuǎn)移惡化導(dǎo)致死亡[12]。目前對(duì)miR-200b在視網(wǎng)膜母細(xì)胞瘤組織和細(xì)胞中的表達(dá)情況報(bào)道較少,DADS在視網(wǎng)膜母細(xì)胞瘤中的抑制作用和內(nèi)在機(jī)制的研究亦不深入。本研究從miR-200b的角度揭示DADS抑制視網(wǎng)膜母細(xì)胞瘤生長(zhǎng)及轉(zhuǎn)移的分子機(jī)制,為視網(wǎng)膜母細(xì)胞瘤的治療提供新的思路。
1.1 主要材料 人視網(wǎng)膜母細(xì)胞瘤Y79細(xì)胞;Lipofectamine 2000購(gòu)于美國(guó)Invitrogen公司;MTT試劑盒購(gòu)于美國(guó)Sigma公司;Transwell小室購(gòu)于美國(guó)BD公司;miR-200b mimics、miR-200b inhibitor和scramble購(gòu)于丹麥Exiqon公司;RNA抽提試劑盒購(gòu)于美國(guó)Applied Biosystems公司;TIMP3和β-actin抗體購(gòu)于美國(guó)Santa Cruz公司。
1.2 qRT-PCR 適量人視網(wǎng)膜母細(xì)胞瘤標(biāo)本和癌旁正常組織,抽提組織中總RNA,逆轉(zhuǎn)錄合成cDNA,并保存于-80°C中備用。采用六種濃度的DADS(0μmol/L、25μmol/L、50μmol/L、100μmol/L、200μmol/L和400μmol/L)分別處理Y79細(xì)胞,48 h后收集每組細(xì)胞,提取總RNA。miR-200b(正向)5′-CGCAGCTACATCTGGCTACTG-3′,miR-200b(反向) 5′-GTGCAGGGTCCGAGGT-3′;U6(正向)5′-GCGCG TCGTGAAGCGTTC-3′,U6(反向)5′-GTGCAGGGTC CGAGGT-3′。PCR體外擴(kuò)增反應(yīng)體系為20 μl,包括Taq DNA polymerase 0.2 μl(5 U/μl),PCR primers 0.4 μl (5 μmol/L),2×SYBR Mix 10 μl,RT product 2.0 μl及滅菌蒸餾水7.4 μl。循環(huán)體系為:96°C 3min,96°C 15 s,65°C、35 s,73°C、30 s,共計(jì)38個(gè)循環(huán),以U6為內(nèi)參,測(cè)得的miR-200b相對(duì)表達(dá)量用2-ΔΔCT法來(lái)進(jìn)行分析。
1.3 MTT法 實(shí)驗(yàn)共分五組:(1)miRNA陰性對(duì)照組(瞬時(shí)轉(zhuǎn)染scramble 40 μmol/L);(2)miR-200b組(瞬時(shí)轉(zhuǎn)染miR-200b-mimics 40 μmol/L);(3)DADS陰性對(duì)照組(DMSO 10 μmol/L)(即mock組);(4)DADS組(DADS 200 μmol/L);(5)DADS+miR-200b組(瞬時(shí)共轉(zhuǎn)染miR-200b-mimics 40 μmol/L)和DADS(200 μ mol/L),將轉(zhuǎn)染后的Y79細(xì)胞接種于96孔板,每組設(shè)置5個(gè)復(fù)孔,細(xì)胞培養(yǎng)臨近飽和時(shí),在每孔加入滅菌MTT液20 μl(5 mg/ml),在孵育4 h后取出,向每孔中加入150 μl DMSO,低速振蕩10 min,隨后選擇570 nm波長(zhǎng),于酶標(biāo)儀上測(cè)定各孔的吸光值并記錄結(jié)果,每組實(shí)驗(yàn)重復(fù)3次。
1.4 Transwell侵襲實(shí)驗(yàn) 細(xì)胞分組同MTT法,鋪適量基質(zhì)膠稀釋液到Transwell小室,過(guò)夜成膜,取100 μl(1×105cells/ml)細(xì)胞稀釋液接種于Transwell小室的上腔,下腔中加入500 μl含10%胎牛血清(FBS)的RPMI-1640培養(yǎng)液,放置于37℃、5%CO2細(xì)胞培養(yǎng)箱中,36 h后取出,將小室上層細(xì)胞用棉簽擦棄,磷酸鹽緩沖液(PBS)輕洗,4%多聚甲醛固定,結(jié)晶祡染色,PBS輕洗,倒置并晾干。光學(xué)顯微鏡下觀察,攝像,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,每組重復(fù)3次。
1.5 統(tǒng)計(jì)學(xué)方法 應(yīng)用SPSS16.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(±s)表示,兩兩間比較采用t檢驗(yàn),以P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。
2.1 DADS對(duì)Y79細(xì)胞miR-200b表達(dá)的影響 采用六種濃度的DADS(0μmol/L、25μmol/L、50μmol/L、100μmol/L、200μmol/L和400μmol/L)分別處理Y79細(xì)胞,48 h后收集每組細(xì)胞,提取總RNA。qRT-PCR結(jié)果顯示:與對(duì)照組(DADS 0μmol/L)視網(wǎng)膜母細(xì)胞瘤細(xì)胞Y79中miR-200b的表達(dá)量(0.758±0.125)比較,隨DADS劑量的增加miR-200b的表達(dá)量逐漸上調(diào)。DADS濃度不同(25μmol/L、50μmol/L、100μmol/L、200μmol/L和400μmol/L)時(shí),miR-200b的表達(dá)量分別為(0.811±0.275)、(1.573±0.101)、(1.956±0.273)、(2.341±0.218)和(2.824±0.242),差異具統(tǒng)計(jì)學(xué)意義(P<0.05),即DADS可上調(diào)Y79細(xì)胞中miR-200b的表達(dá),且其呈劑量依賴性。
2.2 DADS通過(guò)上調(diào)miR-200b抑制Y79細(xì)胞增殖 實(shí)驗(yàn)分組如方法中所述,在轉(zhuǎn)染48 h后,各組分別與其對(duì)應(yīng)陰性對(duì)照組比較。MTT實(shí)驗(yàn)結(jié)果顯示miR-200b組的OD值(0.549±0.057)較miRNA陰性對(duì)照組(0.737±0.135)明顯降低;DADS組的OD值(0.508±0.064)較DADS陰性對(duì)照組(0.722±0.145)明顯降低;而DADS+miR-200b組的OD值(0.362±0.081)較miRNA陰性對(duì)照組(0.737±0.135)和DADS陰性對(duì)照組(0.722±0.145)降低最為顯著。差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05),即DADS可以通過(guò)上調(diào)miR-200b抑制Y79細(xì)胞的增殖能力。
2.3 DADS通過(guò)上調(diào)miR-200b抑制Y79細(xì)胞侵襲 實(shí)驗(yàn)分組如方法中所述,在轉(zhuǎn)染48 h后,各組分別與其對(duì)應(yīng)陰性對(duì)照組比較。Transwell侵襲實(shí)驗(yàn)結(jié)果顯示,轉(zhuǎn)染miR-200b組的Y79的穿膜細(xì)胞數(shù)(105±13)較miRNA陰性對(duì)照組(162±12)明顯降低;DADS組的細(xì)胞穿膜數(shù)(102±13)較DADS陰性對(duì)照組(154±8)明顯減少;且DADS+miR-200b組的細(xì)胞穿膜數(shù)(77±8)較miRNA陰性對(duì)照組(162±12)和DADS陰性對(duì)照組(154±8)減少最顯著。差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05),即DADS通過(guò)上調(diào)miR-200b抑制Y79細(xì)胞侵襲。
視網(wǎng)膜母細(xì)胞瘤常發(fā)病于五歲以下的兒童,常常由于患者年齡過(guò)小,自我辨別能力較弱,大多患兒就診時(shí)實(shí)際已發(fā)生侵襲或轉(zhuǎn)移,此時(shí)的治療效果常常是有限的,甚至?xí)?dǎo)致死亡[13]。臨床上常用的治療方法有眼球摘除手術(shù)、放射或化學(xué)治療、光凝固治療等[14]。傳統(tǒng)治療方法除可能存在較大毒副反應(yīng),治療效果也常常是有限的,且眼球摘除手術(shù)造成的視力永久性失去是無(wú)法恢復(fù)的。因此,針對(duì)視網(wǎng)膜母細(xì)胞瘤的新的抗癌藥物的研發(fā)與探索也成為了當(dāng)務(wù)之急。
DADS對(duì)胃癌、乳腺癌及結(jié)腸癌等多種腫瘤具有抑制作用[3-5]。本研究筆者從miR-200b的層面來(lái)探討DADS的抑瘤機(jī)制。首先我們用不同濃度的DADS處理視網(wǎng)膜母細(xì)胞瘤細(xì)胞Y79,提取總RNA后檢測(cè)miR-200b的表達(dá)量,結(jié)果顯示隨著DADS劑量的增加,miR-200b的表達(dá)量呈梯度式上調(diào)。說(shuō)明DADS可上調(diào)Y79細(xì)胞中miR-200b的表達(dá)水平且與劑量呈依賴性關(guān)系。隨后,我們采用MTT實(shí)驗(yàn)探索Y79細(xì)胞的增殖能力,DADS處理后,Y79細(xì)胞的增殖能力明顯降低,且miR-200b和DADS共同處理后細(xì)胞的增殖能力降低最為顯著,說(shuō)明DADS能夠通過(guò)上調(diào)調(diào)miR-200b從而抑制Y79細(xì)胞的增殖。最后,我們也采用Transwell侵襲實(shí)驗(yàn)來(lái)檢測(cè)Y79的穿膜能力,DADS處理后細(xì)胞穿膜數(shù)明顯減少,miR-200b和DADS共同處理后Y79的穿膜數(shù)減少最顯著,說(shuō)明DADS能夠通過(guò)上調(diào)miR-200b來(lái)抑制Y79細(xì)胞的侵襲。證實(shí)DADS可通過(guò)上調(diào)miR-200b的表達(dá)水平抑制視網(wǎng)膜母細(xì)胞瘤細(xì)胞的生長(zhǎng)及侵襲。
本次研究從miRNA-200b層面來(lái)探討DADS在視網(wǎng)膜母細(xì)胞瘤中的抑瘤機(jī)制,證實(shí)DADS可通過(guò)上調(diào)miR-200b的表達(dá)抑制視網(wǎng)膜母細(xì)胞瘤的生長(zhǎng)及侵襲,即DADS可潛在的靶向miR-200b治療視網(wǎng)膜母細(xì)胞瘤,為視網(wǎng)膜母細(xì)胞瘤的防治提供了新的思路,也進(jìn)一步闡明了miR-200b和DADS在視網(wǎng)膜母細(xì)胞瘤可能的生物學(xué)功能。
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Suppression of diallyl disulfide on the proliferation and invasion of retinoblastoma cells by up-regulating miR-200b.
LIU Yue-feng1,ZHANG Yong1,ZHONG Xiao-dong1,LUO Wei-min2.Department of Ophthalmology1, Department of Cardiothoracic Surgery2,the Affiliated Hospital of Hubei University of Medicine,Shiyan 442000,Hubei, CHINA
Objective To explore the internal molecular mechanisms related to miR-200b for the suppressing effect of diallyl disulfide(DADS)on the proliferation and invasion of retinoblastoma cells.MethodsThe expression of miR-200b regulated by different dose of DADS(0μmol/L,25μmol/L,50μmol/L,100μmol/L,200μmol/L and 400μmol/L)in Y79 cells was detected by quantitative real time polymerase chain reaction(qRT-PCR).The proliferation and invasion ability of Y79 cells in vitro regulated by miR-200b and DADS were assessed by MTT and Transwell invasion assays,with five groups set up∶miRNA-negative control group(transient transfection with scramble 40 μmol/L);miR-200b group(transient transfection with miR-200b-mimics 40 μmol/L);DADS-negative control group(mock group,DMSO 40 μmol/L);DADS group(DADS 200 μmol/L)and DADS+miR-200b group(transient transfection with both miR-200b-mimics 40 μmol/L and DADS 200 μmol/L).ResultsmiR-200b expression was increased with the increase of the doses of DADS,in a dose-dependent manner(P<0.05).For MTT,the OD value was significantly lower in miR-200b group than miRNA-negative control group[(0.549±0.057)vs(0.737±0.135)],in DADS group than DADS-negative control group[(0.508±0.064)vs(0.722±0.145)],and the OD value in DADS+miR-200b group was the lowest[(0.362±0.081)].The differences were all statistically significant(P<0.05).The results indicate that DADS could inhibit the proliferation capacity of Y79 cells by increasing the expression of miR-200b.In Transwell invasion assays,the number of cells penetrating membrane was significantly less in miR-200b group than miRNA-negative control group[(105±13)vs(162±12)],in DADS group than DADS-negative control group[(102±13)vs(154±8)],and the number in DADS+miR-200b group was the lowest[(77±8)].The differences were all statistically significant (P<0.05).The results indicate that DADS could inhibit the invasion capacity of Y79 cells by increasing the expression of miR-200b.ConclusionDADS can suppress the proliferation and invasion of human retinoblastoma cells by up-regulation of miR-200b.
Retinoblastoma;MiR-200b;Diallyl disulfide(DADS);Proliferation;Invasion
R739.7
A
1003—6350(2016)03—0351—03
2015-09-11)
湖北省教育廳科學(xué)研究計(jì)劃指導(dǎo)性項(xiàng)目(編號(hào):B2015477);湖北省十堰市科技課題(編號(hào):14Y40)
羅衛(wèi)民。E-mail:luoweimin0803@sina.com