CN111387143A - miRNA-203a-3p在开发抑制胰腺癌药物中的应用 - Google Patents
miRNA-203a-3p在开发抑制胰腺癌药物中的应用 Download PDFInfo
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Abstract
本发明提供了miRNA‑203a‑3p在开发抑制胰腺癌药物中的应用,1)制备用于筛选抑制胰腺癌转移药物的模型;2)制备抑制胰腺癌转移的模型;miRNA‑203a‑3p的核苷酸序列如SEQ ID NO:1所示。本发明发现miR‑203a‑3p不能抑制胰腺癌细胞增殖和肿瘤形成。但是它可以诱导胰腺癌细胞转移相关基因CERKL的负相关表达,可用于制备抑制胰腺癌转移的小鼠模型,该模型可应用于筛选抑制胰腺癌转移药物,以及用于研究原位肿瘤(胰腺癌)发展为转移肿瘤(胰腺癌)机制的工具。
Description
技术领域
本发明涉及生物技术领域,特别涉及miRNA-203a-3p在开发抑制胰腺癌药物中的应用。
背景技术
胰腺癌(Pancreatic cancer,PC)是消化道肿瘤中最常见的癌症类型之一,是导致美国癌症死亡的第四大原因,也是预后最差的恶性肿瘤之一,只有少数患者存在治愈的机会,在诊断时,大多数PDAC患者发现在手术切除之外的晚期阶段这一统计数据在过去四十年基本未变。胰腺癌患者的特点是直到晚期都缺乏临床表现,导致预后不良和高病死率。大约只有8%的胰腺肿瘤是可定位并适宜切除的。目前胰腺癌生物标记物具有相对较低的敏感性和特异性,限制了其临床应用的范围。胰腺癌的发生发展是个多基因、多步骤复杂的病理和生理过程。多种因素可导致胰腺细胞癌的发生,例如:长期高脂高糖饮食、胰腺结石机械性刺激胰管粘膜上皮促使其增生、吸烟及嗜饮用咖啡人群。另外,上皮-间质转化(EMT)是一种丢失上皮表型和获得间充质表型的过程,导致早期肿瘤细胞的播散和转移。EMT现象在胚胎发育,伤口愈合,组织再生,器官纤维化和癌症进展中起关键作用,有研究表明EMT作为肾纤维化发展中肾脏肌成纤维细胞群的直接贡献者,特别是糖尿病肾病,涉及信号分子和II型EMT途径以及特定miRNA表达的变化,抑制EMT可逆转器官纤维化。目前科学界正探索胰腺癌潜在EMT marker及其机制作用。
发明内容
本发明的一个目的是解决至少上述问题,并提供至少后面将说明的优点。
本发明还有一个目的是提供miRNA-203a-3p在开发抑制胰腺癌药物中的应用。
为了实现根据本发明的这些目的和其它优点,提供了miRNA-203a-3p在如下1)-2)中至少一种的应用,
1)制备用于筛选抑制胰腺癌转移药物的模型;
2)制备抑制胰腺癌转移的模型;
miRNA-203a-3p的核苷酸序列如SEQ ID NO:1所示。
优选的是,所述与胰腺癌相关的基因为CERKL。
优选的是,所述产品为试剂盒或药物;所述模型为动物模型,所述动物为小鼠。
如1)-6)中任一产品,包括所述的miRNA-203a-3p,
1)制备用于筛选抑制胰腺癌转移药物的模型;
2)制备抑制胰腺癌转移的模型;
miRNA-203a-3p的核苷酸序列如SEQ ID NO:1所示。
优选的是,所述与胰腺癌相关的基因为CERKL。
优选的是,所述产品为试剂盒或药物;所述模型为动物模型,所述动物为小鼠。
本发明至少包括以下有益效果:
本发明发现miR-203a-3p不能抑制胰腺癌细胞增殖和肿瘤形成,但是它可以诱导胰腺癌细胞转移相关基因CERKL的负相关表达,提高胰腺癌细胞侵袭能力,促进胰腺癌细胞在小鼠体内的迁徙和转移。可用于制备胰腺癌转移的小鼠模型,该模型可应用于筛选抑制胰腺癌转移药物。
本发明的其它优点、目标和特征将部分通过下面的说明体现,部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
附图说明
图1为胰腺癌组织中高表达miRNAs物种保守型预测结果;
图2为胰腺癌组织中低表达miRNAs物种保守型预测结果;
图3为胰腺癌组织中差异表达miRNAs与患者临床分期的相关性结果;
图4为miR-203a-3p与靶基因相关性分析;
图9为miR-203a-3p对各组Bxpc-3细胞集落形成能力的影响;
图11为显微镜下miR-203a-3p对各组Bxpc-3细胞迁移结果(100×);
图13为显微镜下各组Panc-1细胞迁移结果(100×);
图15为网站预测miR-203a-3p与CERKL为负性相关。
具体实施方式
下面结合附图对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
应当理解,本文所使用的诸如“具有”、“包含”以及“包括”术语并不排除一个或多个其它元件或其组合的存在或添加。
实施例1
1.1差异表达miRNAs的筛选
登录Google Chrome浏览器,输入网址:https://xenabrowser.net/datapages/,登录TCGA数据库,查找胰腺癌相关数据包进行下载,使用pheatmap程序包分析,对差异miRNAs采用R语言edgeR软件包提取,挑选正常胰腺组织(normal Mean)与胰腺癌患者组织(Tumor Mean)差异倍数(Fold Change)大于2倍,即logFC>2,且P<0.05,作为差异表达有统计学意义的miRNAs。
1.2分析差异表达miRNAs保守性
对筛选出的hsa-miR-192-5p(氨基酸序列如SEQ ID NO:2),hsa-miR-203a-3p和hsa-miR-451a(氨基酸序列如SEQ ID NO:3)进行保守性分析,使用miRBase和TargetScan网站查询其保守性,发现高表达组中hsa-miR-192-5p、hsa-miR-203a-3p基因序列具有物种保守性,选为差异表达的miRNAs。低表达组中hsa-miR-451a基因序列具有物种保守性,选为胰腺癌组织中差异表达的miRNAs。
1.3分析差异表达miRNAs患者的生存曲线,临床分期
利用Kaplan-Meier分析胰腺癌组织中低表达组与高表达组患者的总体生存率,进一步采用Graphpad6.0制作Kaplan-Meier生存分析图。TCGA分析出基因表达数据和临床数据相结合,找到肿瘤分期诊断信息,制作差异表达的miRNA箱式图与TNM分期散点图(stageⅰ、stageⅱ、stageⅲ、stageⅳ)。患者生存曲线具有统计学意义(P<0.05)的miRNAs进一步分析它们在胰腺癌组织中的差异表达和临床分期。
1.4相关靶基因预测
运用DIANA TOOLS v5.0生物信息网站预测高表达组差异显著的hsa-miR-192-5p、hsa-miR-203a-3p和低表达组差异显著的hsa-miR-451a的靶基因,并按与靶基因结合程度评分,筛选具有较高评分排名的靶基因,每个miRNA至少预测10个靶基因。
1.5分析靶基因生存曲线及其与miRNAs相关性分析
接下来运用数据库分析靶基因在胰腺癌中的生存曲线,排除已经验证过的靶基因和无原始数据的靶基因,对具有统计学意义的靶基因(P<0.05)继续分析它们与显著表达miRNAs之间的相关性。
1.6应用实时荧光定量PCR验证正常胰腺上皮HPNE细胞和胰腺癌Bxpc-3细胞、Aspc-1细胞、Panc-1细胞中mRNA的表达量
使用正常胰腺上皮HPNE细胞作为对照,在胰腺癌细胞系Bxpc-3、Aspc-1、Panc-1中检测miR-192-5p、miR-203a-3p、hsa-miR-451a mRNA表达水平,细胞接种于6孔板,48h后按照Trizol RNA标准,利用试剂盒提取4株细胞总RNA,整个操作在冰上进行。
具体操作步骤如下:
1)接种细胞:转染前两天,取在对数生长期的细胞系,生理盐水冲洗两遍,加入1ml0.25%胰蛋白酶(含0.02%EDTA)培养瓶中进行消化,吹打成单个悬浮细胞,牛鲍式计数板计数。将细胞按照5×105个/孔,体积为2ml接种于6孔板,放置于37℃,5%CO2恒温培养箱中过夜。转染前细胞密度到达板底面积的70%-90%即可。检测miRNA在胰腺癌细胞中表达不需要对细胞系转染,直接提取对数生长期细胞的RNA。
2)细胞转染:用于检测过表达miR-203a-3p和抑制细胞内源性miR-203a-3p后胰腺癌细胞中miR-203a-3p表达量转染液的制备。
(1)分别在两个无菌的1.5ml EP管配制A液和B液(以下为转染1孔细胞所用的剂量):
A液:用无血清培养基Opti-MEM稀释miR-203a-3p mimic和NC-mimic各4μl或miR-203a-3p inhibitor和NC-inhibitor各10μl,终量为250μl,轻轻吹打混匀,瞬时离心30s。
B液:配制前先把脂质体Lipofectamine 2000轻轻混匀,然后用Opti-MEM稀释4μl的Lipofectamine 2000,轻轻混匀,瞬时离心30s,终量为250μl,室温静置5min。
将B液加入A液中,用枪头轻轻吹打4-5次,瞬时离心15s,室温静置15-20min(不超过20min)。
(2)转染准备:接近15-17min时,取出培养板,吸净原培养液,用0.9%生理盐水清洗2次,将上述混合转染液按500μl/孔缓缓滴入对应孔中,每孔补加1.5ml无血清培养基Opti-MEM,晃动培养板使试剂充分混匀,然后置于37℃、5%CO2
恒温培养箱中培养4-6h。
(3)换液:转染4-6h后换液,吸净转染液,每孔加入不含抗生素的10%FBS RPM-1640培养基,继续置于培养箱中培养48h。
3)Trizol法提取细胞RNA:
(1)吸净原培养液,生理盐水清洗两遍后每孔加入500μl Trizol,轻摇培养板,使Trizol与细胞充分接触,冰浴10min使细胞充***解,吹打细胞完全脱落,然后转入1.5ml无酶EP管中,加入液体总体积五分之一的氯仿(4℃预冷),上下震荡混匀15s,冰置5min。
(2)4℃,12000rpm,离心15min,离心后溶液分为三层,上层水样层为RNA,吸取100-200μl RNA至新无酶EP管内(注意不要接触到中间蛋白层)。
(3)按1:1比例加入预冷的异丙醇,上下混匀10s,冰浴10min。
(4)4℃,12000rpm,离心10min,离心后可见白色RNA沉淀物,小心弃去上清液。
(5)洗涤:加入预冷的75%冰乙醇(由无水乙醇与DEPC水按照3:1比例配制)DEPC水配制的乙醇溶液500μl,轻轻弹使沉淀飘起,4℃,7500rpm,离心5min。
(6)弃去上清,敞开盖晾置10min,每管加10-20μl DEPC水溶解沉淀。
(7)mRNA纯度检测:分别留取1μl用于定量,利用超微量分光光度计检测提取RNA在260nm~280nm处的吸光度值和mRNA浓度,OD260/OD280在1.8~2.0之间方可使用。剩余RNA保存于-80℃冰箱备用。
4)制备cDNA:以提取的总RNA为模板,miRNA第一链cDNA合成试剂盒(染料法)进行逆转录。PCR反应体系如下:
瞬时离心后设置PCR仪反应条件:37℃60分钟,85℃5分钟,4℃保存。将合成的cDNA产物进行实时荧光定量PCR或者放﹣20℃冰箱保存。
5)实时荧光定量PCR检测miRNA的mRNA表达水平
qRT-PCR引物通过邮件联系方式由上海吉马基因生物有限公司负责设计与合成,U6上下引物和miRNA上下游引物序列(SEQ ID NO:4~SEQ ID NO:11)如下:
miR-192-5p-FO:5’-CTGCTGCTGCTGACCTATGAAT-3’
miR-192-5p-RE:5’-CAGTTCCTCAGCAGATGTTGGTAT-3’
miR-203a-3p-FO:5’-CCGCTCGTGAAATGTTTAGG-3’
miR-203a-3p-RE:5’-CAGAGCAGGGTCCGAGGTA-3’
miR-451a-FO:5’-CAGAGCAGGGTCCGAGGTA-3’
miR-451a-RE:5’-CAGAGCAGGGTCCGAGGTA-3’
U6 snRNA-FO:5’-CGCTTCGGCAGCACATATAC-3’
U6 snRNA-RE:5’-TTCACGAATTTGCGTGTCATC-3’
使用时将获得的逆转录产物反应液稀释50倍后作为模板,建立20ul逆转录反应体系,反应体系如下:
加样完成后,瞬时离心15s,置于qRT-PCR仪中。设置反应体系:95℃预变性3min;95℃变性12s;60℃退火40s;72℃延伸30s,循环40次,每组设置2个复孔。
实时荧光定量PCR检测miR-192-5p、miR-203a-3p、hsa-miR-451a mRNA的表达水平,以U6作为阳性对照基因校正PCR模板的拷贝数,基因相对表达量采用2-ΔΔCt计算,计算公式为:ΔCt=Ct目的基因-Ct对照基因,基因的相对表达量=2–ΔΔCt。
1.7qRT-PCR分别检测NC-mimic、miR-203a-3p mimic、NC-inhibitor、miR-203a-3pinhibtor的mRNA的转染效率;
分别在Bxpc-3和Panc-1细胞中转染NC-mimic、miR-203a-3p mimic、NC-inhibitor、miR-203a-3p inhibtor,检测各组mRNA的表达水平。将细胞按照5×105个/孔,体积为2ml悬液接种于6孔细胞培养板中,放置于37℃,5%CO2恒温培养箱过夜。转染后48h用Trizol法提取总RNA;以提取的总RNA为模板制备cDNA进行实时荧光定量PCR实验,具体操作参照1.6步骤进行。数据分析按照Comparative Delta Ct相对定量方法,U6用作内源对照以使基因水平标准化,计算公式:ΔCt=Ct目的基因-Ct对照基因,基因的相对表达量=2–ΔΔCt。
1.8CCK-8测定miR-203a-3p对Bxpc-3和Panc-1细胞增殖能力的影响
1)细胞转染:按照1.6的步骤进行转染。
2)细胞增殖实验利用Cell Counting Kit-8(CCK8)试剂测定增殖率。将1000个细胞每孔密度接种到96孔板中,使用Opti-MEM和Lipofectamine2000将miR-203a-3p mimic,NC-mimic,miR-203a-3p inhibitor和NC-inhibitor分别转染Bxpc-3和Panc-1细胞,在细胞培养箱中孵育0h,24h,48h和72h后,用CCK-8标记细胞,4h后使用酶标仪测定细胞在450nm波长处的OD值,每组重复三次。
3)数据处理:对各组细胞吸光值进行平均值和标准差计算分析,使用Prism 7软件制作折线图。
1.9细胞集落形成实验检测miR-203a-3p对胰腺癌细胞集落形成能力的影响
1)Bxpc-3和Panc-1细胞接种于6孔培养板,置于37℃,5%CO2恒温中培养48h,转染过表达质粒miR-203a-3p及其对照NC-mimic;miR-203a-3p抑制物miR-203a-3p inhibitor及其对照NC-inhibitor于胰腺癌细胞Bxpc-3和Panc-1细胞中,转染方法与1.6节步骤相似,细胞接种数量和转染试剂剂量按照培养板底面积计算。
2)细胞转染48h后,吸出原培养液,生理盐水清洗2次,每孔加入300μl胰酶消化,加入RPM-1640培养基2ml/孔,吹打细胞,形成均匀的单细胞悬液。使用细胞计数板进行计数,按1000个/孔细胞转移至6孔板中,每组设2个复孔,置于培养箱中继续培养。
3)培养期间每2-3天更换一次新鲜培养液,从接种的第14天开始计数,显微镜下观察细胞克隆形成情况,选取细胞数≥50个的集落作为计数对象,吸净每孔培养液,生理盐水冲洗两遍,结晶紫染色法对克隆染色5min,再用生理盐水冲洗2-3遍,拍照保存实验结果。
4)数据处理:计算每组细胞形成的集落数的平均值和标准差,计算细胞集落形成率=(细胞集落数/初始接种细胞数)×100%。
1.10Transwell实验检测miR-203a-3p对胰腺癌细胞迁移和侵袭能力的影响
1)细胞被接种于6孔细胞培养板,培养24h后,转染过表达质粒组miR-203a-3p和其对照组NC-mimic或miR-203a-3p inhibitor组和其对照NC inhibitor组于胰腺癌细胞Bxpc-3和Panc-1细胞,转染方法参照1.6节步骤,细胞接种数量和转染试剂用量按照培养板底面积比例计算。
2)迁移实验:细胞转染48h后,用生理盐水清洗2次,300μl/孔胰酶消化转染的细胞,用不含胎牛血清的RPM-1640培养基吹打细胞成悬液状态,调整细胞密度为2×104,在Transwell上层小室加入200μl细胞悬液,下层小室加入800μl含20%胎牛血清的RPM-1640培养基,每组设置2个复孔,于培养箱中连续培养24h。
3)侵袭实验:细胞转染48h后,全程冰上操作配制基质胶(Metrigel胶),浓度为1mg/ml,上层小室每孔加入50μl,轻轻拍走气泡,使胶平铺于上层小室底部。接着将其放于37℃,5%CO2恒温箱中固化15min。期间配制无胎牛血清的RPM-1640培养基和含20%胎牛血清的RPM-1640培养基,培养板生理盐水清洗2次,300μl/孔胰酶消化转染细胞,用不含胎牛血清的RPM-1640培养基吹打细胞成悬液状态,调整细胞密度为2×104/孔。吸取200μl细胞悬液移至铺好胶的Transwell上层小室,下层小室加入800μl含20%胎牛血清的RPM-1640培养基,每组设置2个复孔,于培养箱中连续培养24h。
4)次日,用镊子取出上层小室,吸净上层小室中的培养液,生理盐水轻轻冲掉上层小室残余的细胞,棉签轻拭上室小室聚碳酸酯微膜水分,使用1%多聚甲醛溶液固定酯膜细胞30min,0.2%结晶紫溶液染色5min,生理盐水脱色3-4遍。在100倍显微镜下随机选取5个视野进行拍照,计算穿膜的细胞数平均值和标准差,计算细胞迁移率和侵袭率。细胞迁移率=[(实验组迁移细胞数-对照组迁移细胞数)/对照组迁移细胞数]×100%。细胞侵袭率=[(实验组侵袭细胞数-对照组侵袭细胞数)/对照组侵袭细胞数]×100%。
1.11荧光报告载体实验验证二者靶定关系
为了鉴定miR-203a-3p与CERKL mRNA 3'UTR之间的直接靶关系,使用双荧光素酶报告分析***(Promega,Madison,WI)进行荧光强度的测定。用pcDNA3/pri-miR-203a-3p和CERKL野生体3'UTR或者突变体3'UTR共同转染Bxpc-3和Panc-1细胞(pcDNA3/EGFP、pcDNA3/EGFP-3’UTR和pcDNA3/EGFP-3’UTR-mut各1ug)。通过与对照组(不包含miR-203a-3p作用位点的EGFP报告载体即空白对照组,包含特异性突变EGFP报告载体即阴性对照组)进行比较,转染48h后裂解细胞,使用荧光化学发光仪SpectraMax Gemini EM对红色荧光蛋白RFP的表达强度(激发光558nm,发射光583nm)和绿色荧光蛋白EGFP(报告分子)的表达强度(激发光488nm,发射光507nm)进行测定。
实验结果
TCGA数据库筛选差异表达miRNAsmiRNA差异分析使用pheatmap R语言包https:// cran.r-project.org/web/packages/pheatmap/)。
挑选正常胰腺组织(normal Mean)与胰腺癌患者组织(Tumor Mean)差异倍数(Fold Change)大于2倍,即logFC>2,且P<0.05,作为有统计学意义的miRNAs。
与正常胰腺组织相比较,胰腺癌患者组织中高表达组miRNAs 9个,低表达组miRNAs9个,差异表达均有统计学意义(P<0.05)(如表1-2所示)。
表1胰腺癌患者组织高表达组主要失调的miRNAs(9个)
表2胰腺癌患者组织低表达组主要失调的miRNAs(9个)
差异表达miRNAs物种保守性预测结果
应用miRBase和TargetScan网站查询miRNA的保守性,发现高表达组中hsa-miR-192-5p、hsa-miR-194-5p、hsa-miR-203a-3p、hsa-miR-203a-5p、hsa-miR-592基因序列具有物种保守性(图1),选为分析胰腺癌组织中差异表达的miRNAs。高表达组中hsa-miR-192-3p、hsa-miR-215、hsa-miR-4724、hsa-miR-6514没有物种保守性,hsa-miR-196a-1、hsa-miR-196a-2、hsa-miR-203a-5p在TCGA中无原始数据,不作为研究对象,对具有广泛保守性的miRNAs进一步分析其生存曲线。
同样应用miRBase和TargetScan网站查询低表达组hsa-miR-142-5p、hsa-miR-150-5p、hsa-miR-206、hsa-miR-451a基因序列具有物种保守性(图2),选为分析胰腺癌组织中差异表达的miRNAs。低表达组中hsa-miR-142-3p、hsa-miR-150-3p、hsa-miR-1537-3p、hsa-miR-1537-5p、hsa-miR-3191-3p、hsa-miR-3191-5p、hsa-miR-4665-3p、hsa-miR-4722-3p、hsa-miR-4722-5p、hsa-miR-6502-3p没有物种保守性,不作为研究对象,对具有广泛保守性的miRNAs进一步分析其生存曲线。
实施例2
物种保守性差异表达miRNAs生存曲线和临床分期
分析胰腺癌组织中差异表达并且物种保守的miRNAs,其表达水平与患者生存相关,结果提示胰腺癌患者组织中高表达组hsa-miR-192-5p、hsa-miR-203a-3p及低表达组hsa-miR-451a的表达水平与患者生存率相关,结果具有统计学意义(P<0.05)。
胰腺癌患者高表达组生存率具有统计学意义(P<0.05)的miRNAs有hsa-miR-192-5p、hsa-miR-203a-3p。低表达组患者生存率具有统计意义(P<0.05)的miRNAs有hsa-miR-451a进一步分析它们在胰腺癌组织中临床分期(图3)。
实施例3
靶基因结果的预测
利用DIANA TOOLS v5.0生物信息网站预测高表达组差异显著的hsa-miR-192-5p、hsa-miR-203a-3p和低表达组差异显著hsa-miR-451a的靶基因,并按与靶基因结合程度评分,筛选具有较高评分的靶基因。
接下来分析miR-203a-3p候选靶基因与胰腺癌患者生存相关性,结果提示表达水平与患者生存相关且具有统计学意义(P<0.05)的候选靶基因为ZNF148、TMEM69、ABCE1、CERKL、TSHR、MBNL2、HNRNPL、CCDC50。分析它们与miR-203a-3p的相关性,分析后发现ZNF148、TMEM69、ABCE1、MBNL2、HNRNPL、CCDC50与miR-203a-3p是正相关(r值分别为0.07115、0.1145、0.06799、0.04999、0.2795、0.2765);CERKL、TSHR与miR-203a-3p是负相关(r值分别为-0.4603、-0.2229)(图4)。miRNA与靶基因正相关的研究价值小,故本课题仅研究二者呈负相关的靶关系。
实施例3
qRT-PCR验证mRNAs在HPNE细胞、Bxpc-3细胞、Aspc-1细胞、Panc-1细胞中mRNA表达水平结果
应用qRT-PCR检测在正常胰腺上皮细胞HPNE和胰腺癌细胞Bxpc-3、Aspc-1、Panc-1中hsa-miR-192-5p、hsa-miR-203a-3p、hsa-miR-451a的表达水平。
①用qRT-PCR方法验证高表达组hsa-miR-203a-3p在正常胰腺上皮细胞HPNE和胰腺癌细胞Bxpc-3、Aspc-1、Panc-1中的mRNA表达水平。
qRT-PCR结果显示在正常胰腺上皮细胞HPNE和胰腺癌细胞Bxpc-3、Aspc-1、Panc-1中,hsa-miR-203a-3p mRNA表达水平升高,相对表达量分别是对照组的7.115倍、7.714倍和6.154倍(图7),差异具有统计学意义(P<0.05)。
实施例4
qRT-PCR检测NC-mimic、miR-203a-3p mimic、NC-inhibitor、miR-203a-3pinhibitor转染效率结果
实验分为miR-203a-3p mimic组、对照NC-mimic组和miR-203a-3p inhibitor组、NC-inhibitor对照组,在Bxpc-3、Panc-1细胞中分别转染4组。qRT-PCR检测各组细胞中miR-203a-3p mRNA表达水平。结果显示(图9a、图10a),相比于对照组,过表达miR-203a-3p后miR-203a-3p组mRNA表达水平高升高,相对表达量分别是对照组的26.234倍和27.947倍,差异具有统计学意义(P<0.05),提示转染miR-203a-3p mimic可有效升高miR-203a-3p的表达。而封闭内源性miR-203a-3p的功能后miR-203a-3p mRNA表达水平又低于对照组(图9b、图10b),相对表达量分别是对照组的0.414倍和0.534倍,差异具有统计学意义(P<0.05),提示转染miR-203a-3p inhibitor可有效降低miR-203a-3p的表达,差异具有统计学意义(P<0.05)。
实施例5
miR-203a-3p促进胰腺癌Bxpc-3、Panc-1细胞的增殖能力
实验分为miR-203a-3p mimic组、对照NC-mimic组和miR-203a-3p inhibitor组、NC-inhibitor对照组。采取CCK-8实验检测各组在Bxpc-3、Panc-1细胞的增殖情况,结果显示miR-203a-3p mimic促进了Bxpc-3、Panc-1细胞的增殖(P<0.05),而miR-203a-3pinhibitor抑制了Bxpc-3、Panc-1细胞的增殖,差异均具有统计学意义(P<0.05),(如图8a和8b,如表3-4所示)。
注:*:P<0.05,**:P<0.01,与NC组相比;
注:*:P<0.05,**:P<0.01,与NC组相比;
实施例6
miR-203a-3p促进胰腺癌细胞集落形成能力
通过胰腺癌细胞Bxpc-3、Panc-1集落形成实验观察各组细胞的集落形成情况,实验分为miR-203a-3p mimic组、对照组、miR-203a-3p inhibitor组及NC inhibitor组。如图9和图10,结果显示在Bxpc-3和Panc-1中,与NC mimic对照组相比,过表达miR-203a-3p组细胞集落数明显增加,差异具有统计学意义(P<0.05);与NC inhibitor对照组相比,在Bxpc-3和Panc-1中抑制细胞内源性miR-203a-3p功能后,细胞集落形成数明显减少,差异具有统计学意义(P<0.05),这些结果表明miR-203a-3p的过表达可以促进Bxpc-3和Panc-1癌细胞集落形成能力。
实施例7
miR-203a-3p促进胰腺癌细胞迁移和侵袭的能力
为了证实miR-203a-3p影响胰腺癌细胞迁移和侵袭能力的变化,在Bxpc-3和Panc-1细胞中按照如下分组进行实验:NC-mimic、miR-203a-3p mimic、NC-inhibitor、miR-203a-3p inhibitor四个组,采用Transwell实验观察胰腺癌细胞迁移侵和袭情况。结果显示(图11、图12、图13和图14),与对照促进组相比,过表达miR-203a-3p可以促进Bxpc-3和Panc-1细胞迁移和侵袭能力,差异具有统计学意义(P<0.05);与对照抑制组相比,抑制miR-203a-3p表达后,细胞迁移和侵袭数目明显减少,差异具有统计学意义(P<0.05)。这些结果表明miR-203a-3p过表达可以促进胰腺癌细胞Bxpc-3和Panc-1迁移和侵袭的能力。
实施例8
CERKL为miRNA-203a-3p的相关靶基因
通过生物信息学网站(DIANATOOLS、TargetScan、StarBase)预测miR-203a-3p可能靶基因,三个网站均预测出CERKL是miR-203a-3p的相关靶基因。并且miR-203a-3p与CERKL之间存在高度保守的互补序列,检索发现CERKL 3’UTR在1499-1506位点处与miRNA-203a-3p存在互补序列,二者结合度评分为98分,提示CERKL是miR-203a-3p的下游靶基因(图15),二者呈负相关(r值为-0.4603)。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的图例。
<110>华北理工大学
<120>CN20BD007A--miRNA-203a-3p在促进胰腺癌细胞增殖和迁移中的应用
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<212>RNA
<213>人工序列
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Claims (6)
1.miRNA-203a-3p在如下1)-2)中至少一种的应用,其特征在于,
1)制备用于筛选抑制胰腺癌转移药物的模型;
2)制备抑制胰腺癌转移的模型;
miRNA-203a-3p的核苷酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的应用,其特征在于,所述与胰腺癌相关的基因为CERKL。
3.如权利要求1所述的应用,其特征在于,所述产品为试剂盒或药物;所述模型为动物模型,所述动物为小鼠。
4.如1)-2)中任一产品,其特征在于,包括如权利要求1所述的miRNA-203a-3p,
1)制备用于筛选抑制胰腺癌转移药物的模型;
2)制备抑制胰腺癌转移的模型;
miRNA-203a-3p的核苷酸序列如SEQ ID NO:1所示。
5.如权利要求4所述的产品,其特征在于,所述与胰腺癌相关的基因为CERKL。
6.如权利要求4所述的产品,其特征在于,所述产品为试剂盒或药物;所述模型为动物模型,所述动物为小鼠。
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