CN112342295A - 一种检测人结直肠癌的肿瘤标志物及其应用 - Google Patents
一种检测人结直肠癌的肿瘤标志物及其应用 Download PDFInfo
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Abstract
本发明公开了一种检测人结直肠癌的肿瘤标志物及其应用,属于生物检测技术领域。本发明提供一种诊断结直肠癌的肿瘤标志物N6‑甲基腺嘌呤识别酶YTHDF3及其应用方法,拟提高临床上结直肠诊断的阳性率及特异性,从而做到结直肠癌早期诊断及早期治疗,提高患者预后生存率。
Description
技术领域
本发明涉及一种检测人结直肠癌的肿瘤标志物及其应用,属于生物检测技术领域。
背景技术
结直肠癌(Colorectal cancer,CRC)是导致全球癌症患者死亡人数最多的癌症类型之一,近年来,结直肠癌的发病率和致死率在中国仍居高不下。纤维肠镜下取病灶组织做病理活检是结直肠癌诊断的金标准,目前结直肠癌的诊断指标包括癌胚抗原(CEA)、CA199、CA125、P53等。但是癌胚抗原(CEA)、CA199、CA125、P53等存在假阳性率高,特异性低等缺点,因此,如何寻找结直肠癌精准诊断及复发转移的有效指标,提高患者的生存率,降低死亡率是亟需解决的关键问题。
发明内容
为解决上述技术问题,本发明提供一种诊断结直肠癌的新型肿瘤标志物及其应用方法,拟提高临床上结直肠诊断的阳性率及特异性,从而做到结直肠癌早期诊断及早期治疗,提高患者预后生存率。
本发明的第一个目的是提供一种检测人结直肠癌的肿瘤标志物,所述的标志物为N6-甲基腺嘌呤(m6A)识别酶YTHDF3。
进一步地,所述的N6-甲基腺嘌呤(m6A)识别酶YTHDF3(NCBI,Gene ID:253943)的氨基酸序列如SEQ ID NO.1所示。
本发明的第二个目的是提供一种检测人结直肠癌的引物组和抗体的组合,所述的引物组和抗体的组合可定量检测肿瘤组织中N6-甲基腺嘌呤识别酶YTHDF3的表达量。
进一步地,所述的引物组中正向引物如SEQ ID NO.2所示,反向引物如SEQ IDNO.3所示。
进一步地,所述的抗体可特异性识别并结合所述的N6-甲基腺嘌呤识别酶YTHDF3。
进一步地,所述的抗体识别肽段的氨基酸序列如SEQ ID NO.8所示。
本发明的第三个目的是提供一种检测人结直肠癌的试剂盒,所述的试剂盒包含所述的检测人结直肠癌的引物组和抗体的组合。
本发明的第四个目的是提供所述的检测人结直肠癌的肿瘤标志物在制备检测人结直肠癌的产品中的应用。
本发明的有益效果是:
本发明提供一种诊断结直肠癌的新型肿瘤标志物及其应用方法,拟提高临床上结直肠诊断的阳性率及特异性,从而做到结直肠癌早期诊断及早期治疗,提高患者预后生存率。
附图说明
图1为本发明的技术路线;
图2为免疫组化实验证实YTHDF3在结直肠癌中表达较癌旁组织显著增高(A-B),且高表达YTHDF3的结直肠癌患者(n=94)相比低表达YTHDF3的结直肠癌患者(n=89)预后较差(C);
图3为qRT-PCR实验证实YTHDF3在8株不同结直肠癌细胞株均有表达;
图4为CCK8细胞增殖实验证实稳定沉默YTHDF3抑制结直肠癌细胞系体外增殖能力(A);稳定过表达YTHDF3促进结直肠癌细胞系体外增殖能力(B);
图5为Transwell实验证实稳定沉默YTHDF3抑制结直肠癌细胞系体外侵袭能力(A);稳定过表达YTHDF3促进结直肠癌细胞系体外侵袭能力(B);
图6为裸鼠皮下瘤实验证实稳定过表达YTHDF3促进结直肠癌肿瘤体内增殖能力(A);稳定沉默YTHDF3抑制结直肠癌肿瘤体内增殖能力(B)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1:
收集了200例有完整随访信息的结直肠癌患者肿瘤组织石蜡标本,通过免疫组化证实了一种N6-甲基腺嘌呤(m6A)识别酶YTHDF3在结直肠癌肿瘤组织中的表达较癌旁组织显著上调,并且高表达YTHDF3的结直肠癌患者预后较差;进一步通过结直肠癌细胞功能学实验及裸鼠结直肠癌皮下瘤成瘤实验模型证实了稳定过表达YTHDF3促进结直肠癌体内增殖能力,稳定敲除YTHDF3的结直肠癌细胞体内外增殖速度减慢。技术路线如图1所示。
实施例2:
免疫组化(IHC):常规染色步骤:二甲苯Ⅰ脱蜡15min,二甲苯Ⅱ脱蜡15min,二甲苯Ⅲ脱蜡15min,100%乙醇Ⅰ水化5min,100%乙醇Ⅱ水化5min,80%乙醇水化5min,蒸馏水1min,苏木精液染色5min,1%盐酸乙醇1-3s,流水洗去苏木精液15min,0.5%伊红液染色1-3min,蒸馏水冲洗1-2s,80%乙醇脱水1min,95%乙醇Ⅰ脱水1min,95%乙醇Ⅱ脱水1min,无水乙醇脱水1min,二甲苯透明2min,中性树胶封片,晾干,显微镜下观察。对于IHC,标本复水后加入0.3%的过氧化氢甲醇溶液作用30min以消除内源性过氧化物酶的影响,然后使用PBS清洗3遍,每次5分钟;山羊血清封闭液作用30min,加入相应浓度的特异性一抗(Anti-YTHDF3:特异性识别肽段的氨基酸序列为:GEAAWSTAGDQPMPYLTTYGQMSNGEHHYI PDGVFSQPGALGNTPPFLGQ(SEQ ID NO.8))孵育过夜。第二天复温后使用PBS清洗3遍,加入HRP(辣根过氧化物酶)标记的二抗在37度孵育30min后加入PBS清洗3次,每次5min;加入显色液,进行免疫组织化学显色,在显微镜下终止染色。使用苏木素复染,梯度脱水,封片。显微镜下观察,如果染色呈现棕褐色则为阳性结果;如染色呈现蓝色,则为阴性结果。评分标准:通过两位有经验的病理科医生进行双盲评分。评估者结合染色阳性强度和染色面积综合评分。染色评分=染色阳性强度×染色面积。(染色强度:0,代表阴性;1,代表弱阳性;2,代表中等阳性;3,代表强阳性;染色面积:0,代表0%;1,代表1-25%;2,代表26-50%;3,代表51-75%;4,代表76-100%)截断值定义:若评分高于中位值则该例肿瘤组织YTHDF3高表达,若评分低于中位值则该例肿瘤组织YTHDF3低表达。
结果如图2所示,免疫组化实验发现YTHDF3在所有结直肠癌患者肿瘤组织中均有表达,并且YTHDF3在患者结直肠癌组织中的表达较癌旁正常肠上皮组织显著增高(A-B),图(C)利用Kaplan-Meier曲线模型分析结直肠癌患者生存时间的差异,结果显示高表达YTHDF3的结直肠癌患者(n=94)相比低表达YTHDF3的结直肠癌患者(n=89)预后较差。以上结果证实使用Anti-YTHDF3抗体能特异性地检测结直肠癌肿瘤组织,YTHDF3可以作为一种结直肠癌诊断的新型肿瘤标志物。
实施例3:
qRT-PCR:(1)使用TRIzol提取结直肠癌细胞总RNA;(2)使用RNA逆转录试剂盒使RNA逆转录为cDNA;(3)进行定量PCR的检测。
(1)使用TRIzol提取结直肠癌细胞总RNA:收集细胞沉淀,加入1ml的TRIzol涡旋混匀;加入0.2ml氯仿,剧烈振荡15秒,室温放置3分钟。2-8℃10000g离心15分钟。样品分为三层:底层为黄色有机相,上层为无色水相和一个中间层。RNA主要在水相中,吸取水层。用异丙醇沉淀水相中的RNA。每使用1ml TRIzol加入0.5ml异丙醇,室温放置10分钟。2-8℃10000g离心10分钟,移去上清。用75%乙醇洗涤RNA沉淀,2-8℃10000g离心5分钟,弃上清。室温干燥RNA沉淀,溶于DEPC水。
(2)使用RNA逆转录试剂盒使RNA逆转录为cDNA;使用TaKaRa逆转录试剂盒(codeNo.RR037A)使总RNA逆转为cDNA。
(3)进行定量PCR的检测:利用YTHDF3特异性引物(Forward:TCAGAGTAACAGCTATCCACCA(SEQ ID NO.2);Reverse:GGTTGTCAGATATGGCATAGGCT(SEQ IDNO.3))进行qRT-PCR实验,检测其在结直肠癌中的表达水平。具体步骤详见TaKaRa SYBRPremix Ex Taq(Code No.RR420L)使用说明书。
结果如图3所示,qRT-PCR实验证实YTHDF3在8株不同结直肠癌细胞株均有表达。
实施例4:
CCK8:在96孔板中接种细胞悬液(100ul/孔,共1000个细胞)。将培养板放在培养箱预培养(在37℃,5%CO2的条件下)。每孔加入10ul的CCK8溶液。将培养板在培养箱内孵育2小时。用酶标仪测定在570nm处的吸光度。
稳定沉默YTHDF3是采用小干扰RNA包装为慢病毒载体,小干扰RNA序列如下:
si-YTHDF3-1:(Forward:GGUGGAUUUCACCAGUUAAUG(SEQ ID NO.4);Reverse:UUAACUGGUGAAAUCCACCAA(SEQ ID NO.5))
si-YTHDF3-2:(Forward:AGAUGGUGUAUUUAGUCAACC(SEQ ID NO.6);Reverse:UUGACUAAAUACACCAUCUGG(SEQ ID NO.7))
结果如图4所示,CCK8细胞增殖实验证实稳定沉默YTHDF3抑制结直肠癌细胞系体外增殖能力(A);稳定过表达YTHDF3促进结直肠癌细胞系体外增殖能力(B)。
实施例5:
Transwell:应用Transwell小室法测定细胞迁移能力的变化,Transwell小室内室中加300ul无血清的DMEM培养液,首先置于培养箱中孵育1-2h,使基质胶层亲水。胰酶消化生长状态良好的细胞,用PBS洗涤细胞两次,最后用无血清的DMEM培养液重悬并调整细胞数为(0.5-1.0)×106个/ml。加入500ul含10%小牛血清的培养液于外室中,加300ul细胞悬液于内室中,于37℃、5%CO2孵箱中培养24h-48h。取出小室,倒掉培养液,用棉签擦去未穿过ECMatrix膜的细胞。将小室侵入细胞染色液中染色20min,用蒸馏水小心冲洗数次,空气中风干。显微镜观察计数,随机选取3个高倍视野计数穿过ECMatrix膜的细胞数。
结果如图5所示,Transwell实验证实稳定沉默YTHDF3抑制结直肠癌细胞系体外侵袭能力(A);稳定过表达YTHDF3促进结直肠癌细胞系体外侵袭能力(B)。
实施例6:
裸鼠皮下瘤能力:生长状态良好的培养细胞,用0.25%胰酶消化成单细胞悬液,用无血清的培养基洗两次后,再用无血清的培养基重悬细胞,将处理组及对照组细胞,均以5×106个细胞/只接种在裸鼠左右腋下,每组各10只。用LT-9MACIMSYSPULS整体荧光体视镜连续观察细胞在体内生长情况,并用游标卡尺测量肿瘤体积(体积=长×宽×高/2)。
结果如图6所示,裸鼠皮下瘤实验证实稳定过表达YTHDF3促进结直肠癌肿瘤体内增殖能力(A);稳定沉默YTHDF3抑制结直肠癌肿瘤体内增殖能力(B)。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
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Claims (8)
1.一种检测人结直肠癌的肿瘤标志物,其特征在于,所述的标志物为N6-甲基腺嘌呤识别酶YTHDF3。
2.根据权利要求1所述的肿瘤标志物,其特征在于,所述的N6-甲基腺嘌呤识别酶YTHDF3的氨基酸序列如SEQ ID NO.1所示。
3.一种检测人结直肠癌的引物组和抗体的组合,其特征在于,所述的引物组和抗体的组合可定量检测肿瘤组织中N6-甲基腺嘌呤识别酶YTHDF3的表达量。
4.根据权利要求3所述的检测人结直肠癌的引物组和抗体的组合,其特征在于,所述的引物组中正向引物如SEQ ID NO.2所示,反向引物如SEQ ID NO.3所示。
5.根据权利要求3所述的检测人结直肠癌的引物组和抗体的组合,其特征在于,所述的抗体可特异性识别并结合所述的N6-甲基腺嘌呤识别酶YTHDF3。
6.根据权利要求5所述的检测人结直肠癌的引物组和抗体的组合,其特征在于,所述的抗体识别肽段的氨基酸序列如SEQ ID NO.8所示。
7.一种检测人结直肠癌的试剂盒,其特征在于,所述的试剂盒包含权利要求3所述的检测人结直肠癌的引物组和抗体的组合。
8.一种权利要求1所述的检测人结直肠癌的肿瘤标志物在制备检测人结直肠癌的产品中的应用。
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