CN117487803A - 一种卵巢癌抑癌因子及其靶基因与应用 - Google Patents
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Abstract
本发明公开了一种卵巢癌抑癌因子及其靶基因与应用,所述卵巢癌抑癌因子为miR‑34a‑5p,其序列为SEQ ID NO:1,miR‑34a‑5p的靶基因为TRIM44。miR‑34a‑5p作为抑癌因子,可以抑制其下游靶基因TRIM44 mRNA的蛋白的表达,从而抑制卵巢癌细胞A2780和/或ES‑2的增殖、迁移和侵袭能力,从而抑制卵巢癌的生长、扩散。本发明提供的卵巢癌因子miR‑34a‑5p可以用于制备治疗卵巢癌的药物,或用于对卵巢癌的治疗进行用药指导,具备较好的临床应用价值和广阔的应用前景。
Description
技术领域
本发明属于卵巢癌治疗药物筛选技术领域,具体涉及一种卵巢癌抑癌因子及其靶基因与应用。
背景技术
卵巢癌是发生于卵巢上皮的恶性肿瘤,其发病率在女性生殖系统恶性肿瘤中位居第三,仅次于宫颈癌和子宫内膜癌。由于卵巢癌起病隐匿,早期缺乏有效的筛查方法,且癌细胞扩散迅速,75%的卵巢癌患者确诊时已是晚期。目前,卵巢癌的标准治疗方案是细胞减灭术、铂类/紫杉烷联合化疗和靶向治疗。一线治疗有效率约为80%-90%,但易复发并发展为化疗耐药,5年生存率仅35%。因此,基因靶向治疗药物成为目前卵巢癌的主要研究方向之一。
微小核苷酸(microRNA,miRNA)是一类小的、非编码RNA,其大小约为19-24个核苷酸,通过抑制蛋白质翻译和促进mRNA剪切来抑制靶基因的表达。已被证实miRNA的失调与包括癌症、心血管疾病、代谢疾病及糖尿病等多种疾病的发生及进展有关,这提示它们可被用作诊断、预后及预测的生物标记物。且越来越多的研究表明miRNA可作为潜在的致癌基因或抑癌基因,因此基于miRNA的靶向抗癌治疗也成为研究的热点。据最新报道,miR-34a家族成员miR-34a-5p在肝癌(Niu X,Wei N,Peng L,et al.miR-34a-5p plays an inhibitoryrole in hepatocellular carcinoma by regulating target gene VEGFA[J].Malays JPathol,2022,44(1):39-52.)、胃癌(Hong S,Li Q,Yang Y,et al.Silencing of LongNon-coding RNALINC01106 Represses Malignant Behaviors of Gastric Cancer Cellsby Targeting miR-34a-5p/MYCN Axis[J].Mol Biotechnol,2022,64(2):144-155.)、食管癌(Su J,Wang G,Li C,et al.Screening for differentially expressed miRNAs inAedes albopictus(Diptera:Culicidae)exposed to DENV-2and their effect onreplication of DENV-2in C6/36cells.Parasites&vectors.2019;12(1):44.)、结直肠癌(Zhao J,Lin H,Huang K.Mesenchymal Stem Cell-derived Extracellular VesiclesTransmitting MicroRNA-34a-5p Suppress Tumorigenesis of Colorectal CancerThrough c-MYC/DNMT3a/PTEN Axis[J].Mol Neurobiol,2022,59(1):47-60.)等恶性肿瘤中充当肿瘤抑制因子,抑制肿瘤细胞的增殖、侵袭、迁移及上皮间质转化。且近期研究发现,miR-34a-5p通过靶向PD-L1来调节卵巢癌细胞的顺铂耐药性(Zuo Y,Zheng W,Liu J,etal.MiR-34a-5p/PD-L1 axis regulates cisplatin chemoresistance of ovariancancer cells[J].Neoplasma,2020,67(1):93-101.)。但是对卵巢癌细胞的作用尚未可知,需要进行进一步研究。
发明内容
针对上述技术问题,本发明提供一种卵巢癌抑癌因子及其靶基因与应用,通过基因芯片和生物信息学筛选得到了卵巢抑癌因子miR-34a-5p及其下游靶基因TRIM44,并通过体外细胞实验证明,miR-34a-5p可以抑制TRIM44的表达,从而抑制卵巢癌细胞的增殖、迁移和侵袭能力,从而可作为一种抑制卵巢癌生长、扩散的药物来用于治疗卵巢癌。
为了实现上述目的,本发明提供一种卵巢癌抑癌因子,所述卵巢癌抑癌因子为miR-34a-5p,其序列为SEQ ID NO:1。
本发明还提供一种卵巢癌抑癌因子在制备检测卵巢癌诊断试剂盒或制备治疗卵巢癌的药物中的应用。
优选的,所述治疗卵巢癌的药物为抑制卵巢癌细胞增殖、迁移和侵袭的药物。
进一步优选的,所述卵巢癌细胞为A2780和/或ES-2。
本发明还提供一种卵巢癌抑癌因子的靶基因,所述靶基因为TRIM44。
本发明还提供一种卵巢癌抑癌因子的靶基因在制备检测卵巢癌诊断试剂盒或制备治疗卵巢癌的药物中的应用。
优选的,所述应用为卵巢癌抑癌因子的靶基因的表达抑制剂在抑制卵巢癌细胞增殖、迁移和侵袭中的应用。
进一步优选的,所述卵巢癌细胞为A2780和/或ES-2。
本发明的有益效果在于:利用miRNA基因芯片技术筛选得到了在DZNep干预下表达量最显著的几种miRNA,其中miR-34a-5p的表达量的变化显著性处于第二位,与卵巢癌细胞具有较强的相关性,然后通过生物信息学软件预测了miR-34a-5p可能调控的下游靶基因,并选择其中预测评分最高、与癌症相关性最强的TRIM44作为miR-34a-5p的下游靶基因进行研究,研究发现通过抑制miR-34a-5p的表达量,TRIM44的mRNA和蛋白的表达量显著上升,同时卵巢癌细胞A2780和ES-2的增殖、迁移和侵袭能力也显著上升,表明miR-34a-5p可以通过抑制TRIM44的表达量从而抑制卵巢癌细胞的增殖、迁移和侵袭能力,因此miR-34a-5p可以作为一种抑癌因子进行卵巢癌细胞的治疗,也可以和TRIM44一起作为卵巢癌早期诊断和监测预后的生物学标志物。
附图说明
图1为实施例2中基因芯片筛选得到的miRNA表达量图,图中A为表达量热图,B为DZNep干预卵巢癌细胞OVSAHO、A2780、SKOVS后miRNA的表达量柱状图。
图2为实施例2中通过DZNep处理后表达量变化最显著的miRNA的表达量柱状图。
图3为实施例3中miR-34a-5p的下游靶基因的预测图。
图4为实施例4中的凝胶电泳图,图中A为TRIM44 3'UTR-WT和TRIM44-3’UTR-MUT的凝胶电泳图,M为DL2000的marker,条带1和2为TRIM44 3'UTR-WT,条带3和4为TRIM44-3’UTR-MUT;B为双酶切后pYr-MirTarget质粒的凝胶电泳图,M为DL15000的Marker,条带1为双酶切后的pYr-MirTarget质粒。
图5为实施例4中的测序结果比对,图中A为pYr-MirTarget-TRIM44-3’UTR-WT的测序结果,B为pYr-MirTarget-TRIM44-3’UTR-MUT的测序结果。
图6为实施例4中双荧光素酶报告基因活性分析柱状图。
图7为实施例5中qPCR检测miR-34a-5p的抑制效率柱状图。
图8为实施例6中不同处理下TRIM44 mRNA的表达水平变化柱状图。
图9为实施例7中不同处理下TRIM44蛋白的表达变化图,图中A为TRIM44蛋白的表达水平,B为TRIM44蛋白表达水平的相对灰度值柱状图,图中*表示p<0.05,***表示p<0.001,****表示p<0.0001,n=3。
图10为实施例8中下调miR-34a-5p后各组细胞的增殖能力柱状图,图中**表示p<0.01,****表示p<0.0001,n=3。
图11为实施例9中下调miR-34a-5p后各组细胞的迁移能力图,图中A为显微镜下不同处理条件下A2780、ES-2细胞的迁移情况,B为各组细胞迁移情况统计分析柱状图,图中*表示p<0.05,***表示p<0.001,****表示p<0.0001,n=3。
图12为实施例10中下调miR-34a-5p后各组细胞的侵袭能力图,图中A为显微镜下不同处理条件下A2780、ES-2细胞的侵袭情况,B为各组细胞侵袭情况统计分析柱状图,图中*表示p<0.05,***表示p<0.001,****表示p<0.0001,n=3。
具体实施方式
下面结合附图和具体实施例对本发明的技术方案做进一步解释说明,值得注意的是,下述实施例仅为本发明的优选实施例,而不应理解为对本发明的限制,本发明的保护范围应以权利要求记载的内容为准。本领域技术人员在没有做出创造性劳动而对本发明的技术方案做出的修改、替换均落入到本发明的保护范围之内。
实验细胞:卵巢癌细胞株A2780、ES-2均来自华中科技大学同济医学院附属协和医院,由三峡大学医学院肿瘤与免疫微环境实验室保存。
实验试剂:
双荧光素酶报告基因检测试剂盒购买于Beyotime公司;
DMEM购买于Procell公司;
I、0.25%胰酶(Trypsin-EDTA)购买于GIBCO公司;
lipofectamine 2000购买于Invitrogen公司;
Fetal Bovine Serum购买于Excell Bio公司;
Ampicillin、Streptomycin sulfate购买于Aladdin公司;
Trypsin-EDTA(0.25%)购买于Procell公司;
Trizol购买于Ambion公司;
IIQ Select RT SuperMix for qPCR、SYBR Green Master Mix、TaqPlus DNAPolymerase、DL2000 DNAMarker购买于TIANGEN公司;
T4 DNALigase购买于TRANS公司;
XhoI、Not I购买于TAKARA公司;
薄型琼脂糖凝胶DNA回收试剂盒、质粒小量制备试剂盒购买于GENERAYBiotechnology公司;
金牌超量无内毒素质粒大提试剂盒购买于CWBIO公司;
磷酸酶抑制剂、PMSF、RIPA裂解液、BCA蛋白浓度测定试剂盒、CCK-8试剂盒购买于Beyotime公司;
TEMED、SDS、溴酚蓝、甘油、甲醇、氯化钠、氯化钾、磷酸二氢钠、磷酸二氢钾、冰醋酸、吐温20均购买于国药集团化学试剂有限公司;
30%丙烯酰胺购买于Biosharp公司;
甘氨酸、Trise-Base、二硫苏糖醇(DTT)购买于Biofroxx公司;
DZNep购买于Caymen Chemical公司;
胎牛血清(FBS)购买于浙江天杭四季青生物工程有限公司;
DMEM/F12培养基、青霉素-链霉素(双抗)购买于Hyclone公司;
二甲基亚砜(DMSO)购买于美国Sigma公司;
4%多聚甲醛固定液购买于武汉赛维尔生物科技有限公司;
基质胶(Matrigel)、脱脂牛奶购买于美国BD Biosciences公司;
蛋白marker(10-180KD)购买于武汉三鹰生物技术有限公司;
蛋白marker(10-120KD)购买于金斯瑞公司;
兔多抗GAPDH购买于杭州贤至生物有限公司;
兔多抗TRIM44(50-55KD)购买于Affinity公司;
HRP标记羊抗兔二抗购买于武汉博士德生物工程有限公司;
ECL底物液购买于北京普利莱基因技术有限公司;
PVDF膜(0.45μm)购买于Millipore公司;
主要溶液与试剂的配制:
细胞培养所用试剂
1)完全培养基:89mL无血清DMEM培养基、10mL FBS及1mL双抗,混匀后4℃保存备用。
2)细胞冻存液:7mL无血清DMEM培养基、2mL FBS及1mL冻存级DMSO,混匀后4℃避光保存备用。
0.2%结晶紫溶液:0.1g结晶紫粉末溶解于50mL的4%多聚甲醛固定液中,过滤后室温避光保存备用。
RNA/DNA电泳相关试剂:
1)5×TBE缓冲液:称取24.2g Tris,5.71mL冰醋酸,吸取0.5mmol/L EDTA(pH 8.0)10mL,加入800mL双蒸水中,充分混匀后定容至1L,置于常温储存备用。
2)6×DNA上样缓冲液:称取0.025g溴酚蓝,充分溶解于10mL双蒸水中,置于4℃备用。
3)溴化乙锭溶液:称取1.0g溴化乙锭,充分溶解于100mL双蒸水中,置于4℃避光保存备用。
4)1%琼脂糖凝胶:取0.15g琼脂糖粉末,加入15mL 1×TAE缓冲液,摇匀后加热,重复数次至完全溶解,加入0.5μL溴化乙锭溶液混匀,冷却至60℃后倒入插好齿梳的胶板上,室温下冷却凝固。
引物稀释:将引物置于离心机中,3000rpm离心1min,取出后据说明书加入适量双蒸水,配制成100μmol/L的储存液,分装后置于-20℃保存备用。使用时,取5μL的储存液加入45μL的双蒸水稀释为10μmol/L。
Western blot相关试剂:
1)蛋白裂解液:将RIPA蛋白裂解液、磷酸蛋白酶抑制剂A液、磷酸蛋白酶抑制剂B液和PMSF按照100:1:1:1比例混合,现配现用。
2)30%Acr-Bis溶液:称取58.0g Acrylamide和2.0g Bis-acrylamide,加入150mL的双蒸水,充分溶解后定容至200mL,4℃避光保存。
3)1.5M Tris-HCl:18.17g Tris-base充分溶解于80mL的双蒸水中,调节pH值至8.8后定容至100mL,4℃避光保存。
4)1.0M Tris-HCl:称取12.1g Tris-base,充分溶解于80mL的双蒸水中,调节pH值至6.8后定容至100mL,置于4℃避光保存。
5)10%SDS:称取10g SDS,充分溶解于100mL的双蒸水中,室温保存。
6)10%过硫酸胺溶液:称取0.1g APS,充分溶解于1mL的双蒸水中,4℃避光保存备用。
7)5×SDS-PAGE电泳缓冲液:取15.15g Tris-base和94.0g甘氨酸,溶解于800mL的双蒸水中,随后加入5g SDS粉末,完全溶解后定容至1L,置于4℃保存,使用时加入稀释至1×使用。
8)5×SDS-PAGE转膜缓冲液:称取15.15g Tris-base和72g甘氨酸,溶解于600mL的双蒸水中,定容至800mL后4℃保存备用,使用时稀释至1×,再加入四分之一体积的甲醇,现配现用。
9)1×TBST Buffer:称取12.1g的Tris-base和44g的NaCl,充分溶解于800mL的双蒸水中,随后加入2.5mL吐温20,混匀后调节pH至7.4,最后加入双蒸水定容至5L,置于常温保存。
10)5%牛奶封闭液:称取2.5g脱脂奶粉,充分溶解于50mL TBST中,定容至50mL后使用。
实施例1卵巢癌细胞培养
一、细胞复苏:
1)将T25培养瓶、15mL离心管、1mL移液枪及枪头置于无菌超净台中,紫外线照射30min;
2)取无菌离心管,加入2mL完全培养基备用;
3)将含有ES-2或A2780细胞冻存管从液氮中取出,快速放入37℃水浴锅中,轻摇使其溶解;
4)用移液器将解冻的细胞液迅速转移到加入完全培养基的无菌离心管中,配平后置于低速离心机中,转速1000rmp,离心5min;
5)离心结束后,消毒离心管,在无菌超净台中吸去上清液,加入1mL完全培养基,吹打重悬;
6)在T25培养瓶中加入3mL完全培养基,用加样枪将上述吹打混匀后的细胞悬液移入培养瓶中,摇晃使细胞分布均匀,置于37℃、5%CO2的细胞培养箱中培养,在显微镜下观察细胞密度及生长状态,在细胞密度达90%-100%时完成复苏,可进行细胞传代。
二、细胞传代
1)T25培养瓶、15mL离心管、1mL移液枪、枪头及PBS置于无菌超净台中,紫外线照射30min;
2)用75%酒精消毒后,将步骤一细胞密度达90%-100%的细胞培养瓶放入超净台内,弃去旧培养基,加入PBS清洗3次,弃去PBS,加入0.25%胰酶1mL,轻晃培养瓶后放入细胞培养箱消化1min;
3)显微镜下观察,可看到细胞相互分离变圆,即消化完成;加入完全培养基终止消化,轻轻吹打细胞,将细胞悬液移入15mL离心管中,室温800rpm离心3min;
4)准备2-3个细胞培养瓶,加入3mL完全培养基备用;
5)离心完毕后,消毒离心管,在超净台中弃去上清,加入完全培养基重悬细胞;将细胞悬液按比例移入到准备好的培养瓶中,轻晃培养瓶后放入细胞培养箱中培养,密切观察细胞生长状态,待细胞密度达到80%-90%时,得到传代后含有A2780细胞或ES-2细胞的细胞培养瓶。
三、细胞冻存
1)将1mL冻存管、15mL离心管、移液枪、枪头及PBS置于无菌超净台中,紫外线照射30min;
2)用75%酒精喷洒消毒后,将步骤二中得到的细胞培养瓶放入超净台内,弃去旧培养基,加入PBS清洗细胞3次,弃去PBS,加入0.25%胰酶1mL,轻晃培养瓶后放入细胞培养箱消化1min;
3)显微镜下观察,可看到细胞相互分离变圆,即消化完成,然后加入完全培养基终止消化,轻轻吹打细胞,将细胞悬液移入15mL离心管中,室温800rpm离心3min;
4)离心完毕后,消毒离心管,在超净台中弃去上清,加入1mL冻存液吹打均匀后移入冻存管中,标明时间和细胞类别;
5)将冻存管放入细胞冻存盒于-80℃超低温冰箱中梯度降温,24h后转入液氮罐长期保存。
四、细胞计数
1)取状态良好、对数生长期的细胞,经消化、离心后,加入1mL培养基制备成单细胞悬液;
2)吸取10μL单细胞悬液,滴加在盖片边缘,使悬液充满盖片和计数板之间,静置30s;
3)将计数板置于显微镜下,计数四个大格的细胞数量,若存在压线,只记录左侧和上方,每毫升细胞数=四大格细胞总数/4×104×稀释倍数。
实施例2利用基因芯片实验来筛选DZNep可能作用的miRNA
为了研究在卵巢癌细胞里面DZNep作用显著改变的miRNA的功能,我们选取三种生长状态良好的卵巢癌细胞,分别为A2780、SKOV3及OVSAHO细胞,每种细胞分为两组,一组作为空白对照,另外一组待密度适中,对数生长期的单层培养细胞时,用5μM的DZNep进行干预,并换用5%胎牛血清的培养液加入到细胞中培养,持续作用96h后,用0.05%胰蛋白酶消化并用移液枪轻轻地吹打悬浊液,使其成单个细胞,1000rpm低速离心收取细胞沉淀,加入1mL Trizol液后置于干冰箱送至上海市启因生物科技有限公司进行基因芯片筛选。
结果如图1-2所示,通过基因芯片筛选了80个miRNA,其中在卵巢癌细胞中DZNep显著改变了miR-4448、miR-663a、miR-34a-5p和miR-4454的表达水平,其中最显著的为miR-4448,其次为miR-34a-5p,其序列为SEQ ID NO:1。
由于在使用OVSAHO细胞进行后续细胞迁移及侵袭实验时,穿膜细胞数过少,无法进行统计分析,因此替换卵巢癌细胞株ES-2进行研究。
实施例3miR-34a-5p靶基因的预测
将实施例2中获取的miR-34a-5p的序列,将其提交至targetscan网站上预测下游靶基因,结果如图3所示,miR-34a-5p与TRIM44的结构序列存在高度互补区域,确定TRIM44为miR-34a-5p下游靶基因之一,且经文献调查发现,TRIM44在多种恶性肿瘤中呈异常表达状态,并且与肿瘤的发生发展及预后相关(Luo Q,Lin H,Ye X,et al.Trim44 facilitatesthe migration and invasion of human lung cancer cells via the NF-kappaBsignaling pathway[J].Int J Clin Oncol,2015,20(3):508-517.Kashimoto K,KomatsuS,Ichikawa D,et al.Overexpression of TRIM44 contributes to malignant outcomein gastric carcinoma[J].Cancer Sci,2012,103(11):2021-2026.),但是在卵巢癌细胞中的作用尚不明确,因此选择TRIM44进行后续研究。
实施例4实验验证miR-34a-5p与TRIM44靶标关系
一、构建双荧光素酶载体
(1)UTR序列对比:通过miRanda、TargetScan对比miR-34a-5p与TRIM44的3’UTR结构序列,发现二者结构序列存在高度互补区域;其中TRIM44的3’UTR的序列为SEQ ID NO:2
(2)设计3’UTR的WT和MUT引物:为了验证miR-34a-5p作用于TRIM44的3’UTR,针对TRIM44的3’UTR结构序列设计了WT(野生型)和MUT(结合位点突变型)的PCR引物,并进行PCR扩增、提纯获得TRIM44 3'UTR-WT和TRIM44-3’UTR-MUT的PCR片段(图4A),其中PCR引物序列见表1,PCR反应体系见表2,PCR反应条件见表3:
表1PCR引物序列
表2PCR反应体系
表3PCR反应程序
(3)pYr-MirTarget空载质粒的准备:将大提质粒后获得的pYr-MirTarget质粒利用Xho I和Not I进行双酶切,酶切体系见表4,然后将酶切体系混合均匀后置于37℃下酶切6h,然后进行琼脂糖凝胶电泳(图4B),最后经切胶回收得到纯化后的pYr-MirTarget线性载体;
表4双酶切体系
(4)目的DNA片段和载体的连接:利用T4 DNA Ligase将步骤(2)获得的目的DNA片段与步骤(3)获得的pYr-MirTarget线性载体进行连接,获得重组pYr-MirTarget载体:pYr-MirTarget-TRIM44-3’UTR-WT和pYr-MirTarget-TRIM44-3’UTR-MUT报告质粒,按照表5所述连接反应体系进行配置,混匀后4℃下连接过夜;
表5连接反应体系
(5)将步骤(4)中得到的连接产物全部转化到E.coli DH5α感受态细胞中,然后挑取转化后的连接产物单克隆扩大培养,小提质粒,菌落PCR检测并将鉴定正确(图5)菌液测序,将测序正确的菌液保存。
二、双荧光素酶报告基因实验
(1)细胞转染:
1)取处于对数期,生长状态良好的细胞A2780,消化、离心并调整细胞浓度至每孔1×105个,铺入12孔板中,置于培养箱中培养过夜;
2)转染前2h,用无血清培养基换液;
3)提前准备2个1.5mL EP管,向其中一个EP管中加入50μL无血清OPTI-MEM培养基、2.5μL小片段和1μg质粒,混匀后静置5min;另一个EP管中加入50μL无血清OPTI-MEM培养基和2.5μLLipofectamineTM 2000,混匀后静置5min。
4)将LipofectamineTM 2000-培养基混合物滴加至小片段和质粒-培养基混合物中,混匀后静置20min;
5)待上述混合物静置时,取出提前铺好细胞的12孔板,按照如下实验分组分别对A2780细胞进行重组质粒和小片段RNA的共转染,转染结束后轻晃孔板后置于培养箱中继续培养;
wt UTR+miR-34a-5p NC:转染pYr-MirTarget-TRIM44-3’UTR-WT质粒和microRNAmimics NC;
wt UTR+miR-34a-5p mimics:转染pYr-MirTarget-TRIM44-3’UTR-WT质粒和hsa-miR-34a-5pmimics;
mut UTR+miR-34a-5p NC:转染pYr-MirTarget-TRIM44-3’UTR-MUT质粒和microRNA mimics NC;
mut UTR+miR-34a-5p mimics:转染pYr-MirTarget-TRIM44-3’UTR-MUT质粒和hsa-miR-34a-5pmimics;
miR-34a-5p NC;转染pYr-MirTarget空质粒和microRNA mimics NC;
miR-34a-5p mimics:转染pYr-MirTarget空质粒和hsa-miR-34a-5p mimics;
其中microRNA mimics NC为miR-34a-5p过表达的阴性对照,序列为SEQ ID NO:7,hsa-miR-34a-5p mimics为miR-34a-5p的过表达,其序列为SEQ ID NO:8;
6)6h后换液,在完全培养基条件下继续培养。
7)按照实验设计,继续培养48h获得转染后的细胞。
(2)双荧光素酶检测:
1)取出步骤(1)中含有转染后细胞的培养板,弃去培养基,用PBS小心清洗2遍,每孔加入200uL报告基因细胞裂解液;
2)充分裂解后,15000rpm离心3min,取上清用于测定;
3)提前融化萤火虫荧光素酶检测试剂和Renilla荧光素酶检测缓冲液,按照每个样品100μL的量,按照1:100的比例加入Renill荧光素酶检测底物配制成Renilla荧光素酶检测工作液;
4)取待测样品100μL,首先加入100μL萤火虫荧光素酶检测试剂,混匀后测定RLU;再加入100μL Renilla荧光素酶检测工作液,混匀后测定RLU;
5)以萤火虫荧光素酶作为内参,用Renilla荧光素酶测定得到的RLU值除以萤火虫荧光素酶测定得到的RLU值。
结果如图6所示:在A2780细胞中,在共转染的6个组中,hsa-miR-34a-5p mimics的三个组中,只有wt UTR+miR-34a-5p mimics与wt UTR+miR-34a-5p NC相比荧光素酶的活性显著降低,而其他四个组中两两相比荧光素酶活性均没有发生明显变化,表明当使用hsa-miR-34a-5p mimics过表达miR-34a-5p时,pYr-MirTarget-TRIM44-3’UTR-WT的荧光素酶活性显著降低,而pYr-MirTarget-TRIM44-3’UTR-MUT以及pYr-MirTarget的荧光素酶活性无明显变化,表明miR-34a-5p通过与TRIM44 3'UTR结合来调控TRIM44的表达,即TRIM44是miR-34a-5p的下游靶点。
实施例5hsa-miR-34a-5p inhibitor抑制效果的确定
为了探究miR-34a-5p对TRIM44表达情况的影响,设计了miR-34a-5p抑制子hsa-miR-34a-5pinhibitor与阴性对照MicroRNA inhibitor NC,通过将其转染至卵巢癌细胞A2780和ES-2细胞中,通过qPCR检测的hsa-miR-34a-5p inhibitor的抑制效果,具体步骤如下:
(1)根据miR-34a-5p设计并合成转染序列hsa-miR-34a-5p inhibitor(序列为SEQID NO:16)和MicroRNA inhibitor NC(序列为SEQ ID NO:17),将合成的序列样品置于4℃、1000rpm下离心2min,然后根据说明书加入适量的DEPC水,震荡溶解,得到转染样品;
(2)提前准备一个6孔板,取状态良好,对数生长期的细胞进行消化、离心和计数,控制细胞密度为1×105/mL,接种于6孔板上,每孔2mL;将6孔板置于培养箱中继续培养,待细胞密度为80%时进行转染
(3)提前准备2个1.5mL EP管,向其中一个EP管中加入235μL无血清OPTI-MEM培养基及5μL LipofectamineTM 2000,混匀后静置5min;另一个EP管加入250μL无血清OPTI-MEM及10μL hsa-miR-34a-5p inhibitor/MicroRNA inhibitor NC,混匀后静置5min,分别得到miR-34a-5pinhibitor组和inhibitor NC组;
(4)将LipofectamineTM 2000-培养基混合物滴加至siRNA-培养基混合物中,混匀后静置20min;
(5)待上述混合物静置时,取出提前铺好细胞的6孔板弃去培养基,用PBS清洗2遍后加入预热的1500μL新鲜无血清培养基,再加入上述混合物使每孔体积为2mL,轻晃6孔板后继续培养;
(6)6h后换液,在完全培养基条件下继续培养24h,取出转染后的细胞分别提取hsa-miR-34a-5p inhibitor组和inhibitor NC组细胞的RNA,逆转录后进行qPCR检测,逆转录及qPCR所用的引物如表6所示,逆转录采用5×HiScript II Select qRT SuperMix II,qPCR采用SYBR Green Master Mix:
表6逆转录及qPCR引物序列
结果如图7所示,与inhibitor NC组相比,在卵巢癌细胞A2780和ES-2中分别转入hsa-miR-34a-5p inhibitor后,miR-34a-5p的表达量显著降低,表明hsa-miR-34a-5pinhibitor对miR-34a-5p的表达抑制效果显著,可以继续进行后续实验。
实施例6下调miR-34a-5p对TRIM44 mRNA表达的影响
取实施例5中miR-34a-5p inhibitor组和inhibitor NC组的卵巢癌细胞,提取细胞的RNA用qPCR检测TRIM44 mRNA的表达,具体分组如下:
inhibitor NC:用含DMSO完全培养基培养的转染了MicroRNA inhibitor NC的卵巢癌细胞,
miR-34a-5p inhibitor:用含DMSO完全培养基培养的转染了hsa-miR-34a-5pinhibitor的卵巢癌细胞。
结果如图8所示,与inhibitor NC相比,转染了hsa-miR-34a-5p inhibitor的卵巢癌细胞中的TRIM44 mRNA表达水平明显升高,这表明,下调miR-34a-5p减弱了对TRIM44表达的抑制作用。
实施例7miR-34a-5p对TRIM44蛋白表达的影响
为了进一步验证miR-34a-5p对TRIM44表达情况的影响,取实施例5中miR-34a-5pinhibitor组和inhibitor NC组的卵巢癌细胞,用Western Blot实验检测各组细胞TRIM44蛋白的表达。
结果图表9所示,与inhibitor NC相比,miR-34a-5p inhibitor组中TRIM44蛋白表达水平明显升高,这表明,下调miR-34a-5p减弱了DZNep对TRIM44表达的抑制作用。
实施例8miR-34a-5p对卵巢癌细胞增殖能力的影响
取实施例5中miR-34a-5p inhibitor组和inhibitor NC组的卵巢癌细胞A2780和ES-2,在每个培养孔中加入10μL CCK-8溶液(注意避光),置于细胞培养箱中继续培养4h,然后置于酶标仪检测450nm波长处各孔的OD值,计算每组的细胞增殖率。
结果如图10所示,与inhibitor NC组相比,miR-34a-5p inhibitor组细胞的增殖能力增强,表明下调miR-34a-5p增强了卵巢癌细胞的增殖能力。
实施例9miR-34a-5p对卵巢癌细胞迁移能力的影响
1)取出实施例5中miR-34a-5p inhibitor组和inhibitor NC组的细胞培养板,消化、离心和计数细胞,用无血清培养基将细胞密度调整为2×105个/mL;
2)提前准备24孔板和孔径大小为8μm的聚碳酸酯膜小室,在孔板内每孔加入600μL的完全培养基,小室加入100μL的细胞悬液,倾斜孔板再将小室放入孔板中,注意不要产生气泡,继续培养;
3)培养24h后,取出小室,放入新的24孔板中,PBS清洗2遍,每孔加入0.2%结晶紫溶液700μL,固定染色15min;
4)弃去结晶紫溶液,PBS清洗2遍,用棉签小心擦去小室上层细胞,晾干后置于显微镜下观察,随机挑选3个视野拍照并统计细胞数量,检测细胞的迁移能力。
结果如图11所示,与inhibitor NC组相比,miR-34a-5p inhibitor组细胞的迁移能力增强;表明下调miR-34a-5p增强了卵巢癌细胞的迁移能力。
实施例10miR-34a-5p对卵巢癌细胞侵袭能力的影响
1)将Matrigel基质胶置于4℃过夜融化,用预冷的无血清培养基以1mg/mL的浓度稀释基质胶,每个小室内加入100μL稀释后的基质胶,置于培养箱2h;
2)取出实施例5中miR-34a-5p inhibitor组和inhibitor NC组的细胞培养板,消化、离心和计数细胞,用无血清培养基将细胞密度调整为2×105个/mL;
3)提前准备24孔板,取出加有基质胶的小室,在孔板内每孔加入600μL的完全培养基,小室内加入100μL的细胞悬液,倾斜孔板再将小室放入孔板中,注意不要产生气泡,继续培养;
4)培养24h后,取出小室放入新的24孔板中,PBS清洗2遍,每孔加入0.2%结晶紫溶液700μL,固定染色15min;
5)弃去结晶紫溶液,PBS清洗2遍,用棉签小心擦去小室上层细胞,晾干后置于显微镜下观察,随机挑选3个视野拍照并统计细胞数量,检测细胞的侵袭能力。
结果如图12所示,与inhibitor NC组相比,miR-34a-5p inhibitor组细胞的侵袭能力增强;表明下调miR-34a-5p增强了卵巢癌细胞的侵袭能力。
综上表明,通过生物信息学软件预测到miR-34a-5p调控的下游靶基因TRIM44,并且在卵巢癌细胞A2780和ES-2中验证了miR-34a-5p和TRIM44的靶向关系,并且发现当下调miR-34a-5p时,TRIM44的mRNA和蛋白表达量均升高,而卵巢癌细胞的增殖、迁移、侵袭能力也增强,表明miR-34a-5的表达量与卵巢癌细胞的能力呈负相关,TRIM44的表达量与卵巢癌细胞的能力呈正相关,miR-34a-5和TRIM44的表达量呈负相关,说明miR-34a-5可以作为一种抑癌因子用于卵巢癌的早期诊断和监测预后的生物学标志物,而TRIM44也可以作为一种调控卵巢癌细胞能力的基因进行进一步研究。
Claims (8)
1.一种卵巢癌抑癌因子,其特征在于:所述卵巢癌抑癌因子为miR-34a-5p,其序列为SEQ ID NO:1。
2.一种如权利要求1所述的卵巢癌抑癌因子在制备检测卵巢癌诊断试剂盒或制备治疗卵巢癌的药物中的应用。
3.根据权利要求2所述的应用,其特征在于:所述治疗卵巢癌的药物为抑制卵巢癌细胞增殖、迁移和侵袭的药物。
4.根据权利要求3所述的应用,其特征在于:所述卵巢癌细胞为A2780和/或ES-2。
5.一种如权利要求1所述的卵巢癌抑癌因子的靶基因,其特征在于:所述靶基因为TRIM44。
6.一种如权利要求5所述的卵巢癌抑癌因子的靶基因在制备检测卵巢癌诊断试剂盒或制备治疗卵巢癌的药物中的应用。
7.根据权利要求6所述的应用,其特征在于:所述应用为卵巢癌抑癌因子的靶基因的表达抑制剂在抑制卵巢癌细胞增殖、迁移和侵袭中的应用。
8.根据权利要求7所述的应用,其特征在于:所述卵巢癌细胞为A2780和/或ES-2。
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