CN116694759B - Scarna12基因调节细胞增殖与存活中的应用 - Google Patents
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Abstract
SCARNA12基因调节细胞增殖与存活中的应用,属于生物技术领域。为了在snoRNAs中挖掘识别结直肠癌的生物标志物,研究SCARNA12在结直肠癌中的临床意义和生物学功能,本发明通过检测SCARNA12在CRC组织和细胞中的表达水平,分析其高表达与结直肠癌患者临床病理特征的相关性,证实了SCARNA12在CRC中的潜在诊断价值,且SCARNA12可作为CRC患者不良预后的潜在生物标志物。此外,通过分析SCARNA12过表达对结直肠癌细胞增殖和存活及对结直肠癌肿瘤生长的影响,发现SCARNA12在CRC中作为癌基因,具有成为CRC患者新的治疗靶点的临床价值。
Description
技术领域
本发明属于生物技术领域,具体涉及SCARNA12基因在结直肠癌检测试剂盒及调节结直肠癌细胞增殖与存活中的应用。
背景技术
结直肠癌(CRC)是消化系统最常见的恶性肿瘤之一,在2020年全球癌症统计数据中排名第三,也是癌症死亡的第二大原因(Sung H, Ferlay J, Siegel RL, LaversanneM, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCANEstimates of Incidence and Mortality Worldwide for 36 Cancers in 185Countries. CA Cancer J Clin. 2021;71(3):209-49;Siegel RL, Miller KD, FuchsHE, Jemal A. Cancer Statistics, 2021. CA Cancer J Clin. 2021;71(1):7-33.)。根据GLOBOCAN 2020数据推测,美国有147950人被诊断患有CRC,其中53200人死于这种疾病(Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al.Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and MortalityWorldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49.)。早期发现-定期粪便检测或结构检查筛查对于改善患者的预后至关重要。数据显示,在诊断为CRC的患者中,约有20%属于转移性结直肠癌(Kahi CJ, Boland CR, DominitzJA, Giardiello FM, Johnson DA, Kaltenbach T, et al. Colonoscopy SurveillanceAfter Colorectal Cancer Resection: Recommendations of the US Multi-SocietyTask Force on Colorectal Cancer. Gastroenterology. 2016;150(3):758-68 e11.)。据估计,早期结直肠癌患者的5年生存率为91%,但转移性结直肠癌患者的5年生存率降至15%(Wang Q, Shen X, Chen G, Du J. Drug Resistance in Colorectal Cancer: FromMechanism to Clinic. Cancers (Basel). 2022;14(12).)。目前,手术联合化疗和/或放疗是结直肠癌最常见的治疗方法。尽管CRC诊断(例如生物分子标志物)和治疗技术(例如免疫治疗和靶向治疗)有所改进,但由于该疾病早期诊断技术和患者个性化临床管理的不完善,相当部分CRC患者的预后远未令人满意。因此,需要更多有效的新型生物标志物用于CRC患者的临床诊断和预后风险分析。
CRC的癌变始于异常隐窝的出现,逐渐演变为癌前病变(息肉),并最终在10-15年的时间发展为CRC。其中,70%-90%的CRC来源于结直肠腺瘤,而10%-20%的CRC起源于锯齿状瘤(Dekker E, Tanis PJ, Vleugels JLA, Kasi PM, Wallace MB. Colorectal cancer.Lancet. 2019;394(10207):1467-80.)。虽然已经研究了很长时间,但CRC致癌的机制仍然不是很清楚。据我们所知,抑癌基因(例如APC,p53,PETN等),癌基因(例如RAS,BRAF,PIK3CA等)或错配修复基因(例如MSH2/6,PMS2/6等)的突变诱导的致癌信号通路失调在CRC病理过程中起着至关重要的作用。PI3K/AKT信号通路的异常激活在CRC致癌过程中发挥关键的作用,参与多种生物学过程的调控,包括细胞增殖、存活、迁移和机体代谢。该通路功能障碍通常由重要调节因子或结构成分的遗传改变引起。例如,PIK3CA是一种编码p110α催化亚基(四个I类PI3K之一)的基因,已在10%-30%的CRCs中发现突变(Kato S, Iida S, HiguchiT, Ishikawa T, Takagi Y, Yasuno M, et al. PIK3CA mutation is predictive ofpoor survival in patients with colorectal cancer. Int J Cancer. 2007;121(8):1771-8.)。靶向PI3K/AKT通路被认为是治疗CRC和其他癌症的一种有效的治疗方法。然而,由于PI3K/AKT通路调控网络的复杂性,大多数靶向PI3K/AKT通路的抑制剂在临床应用中存在局限性。因此,阐明其在CRC疾病过程中的潜在分子机制对于延长患者的生存至关重要。
现有文献报道了小核仁RNA(snoRNA)对PI3K-AKT途径的影响。2017年,Fang等人发现在人肝癌细胞(HCC)细胞和CRC细胞中SNORD126激活PI3K-AKT通路(Fang X, Yang D,Luo H, Wu S, Dong W, Xiao J, et al. SNORD126 promotes HCC and CRC cell growthby activating the PI3K-AKT pathway through FGFR2. J Mol Cell Biol. 2017;9(3):243-55.)。另一个案例是ACA11,由Wu等人(Wu L, Zheng J, Chen P, Liu Q, Yuan Y.Small nucleolar RNA ACA11 promotes proliferation, migration and invasion inhepatocellular carcinoma by targeting the PI3K/AKT signaling pathway. BiomedPharmacother. 2017;90:705-12.)证明。然而,小卡哈尔体特异性RNA(scaRNA)和PI3K-AKT途径之间的相互作用尚未报道。
snoRNA是一类约60-300个核苷酸的非编码RNA,主要位于核仁。ScaRNA于1984年首次被发现,是snoRNA的一种亚型(Fakan S, Leser G, Martin TE. Ultrastructuraldistribution of nuclear ribonucleoproteins as visualized byimmunocytochemistry on thin sections. J Cell Biol. 1984;98(1):358-63.)。它们共享保守的结构域C/D box和H/ACA box,并且具有相似的生物学功能(Darzacq X, Jady BE,Verheggen C, Kiss AM, Bertrand E, Kiss T. Cajal body-specific small nuclearRNAs: a novel class of 2'-O-methylation and pseudouridylation guide RNAs.EMBO J. 2002;21(11):2746-56;Kiss T. Small nucleolar RNAs: an abundant groupof noncoding RNAs with diverse cellular functions. Cell. 2002;109(2):145-8.)。SnoRNA(包括scaRNA)的典型功能是分别通过C/D box和H/ACA box引导靶向RNA(主要是核糖体RNA和小核RNA)的2'-O-甲基化和假尿苷化(Henras AK, Dez C, Henry Y. RNAstructure and function in C/D and H/ACA s(no)RNPs. Curr Opin Struct Biol.2004;14(3):335-43;Hamma T, Ferre-D'Amare AR. The box H/ACA ribonucleoproteincomplex: interplay of RNA and protein structures in post-transcriptional RNAmodification. J Biol Chem. 2010;285(2):805-9;Accardo MC, Giordano E, RiccardoS, Digilio FA, Iazzetti G, Calogero RA, et al. A computational search for boxC/D snoRNA genes in the Drosophila melanogaster genome. Bioinformatics. 2004;20(18):3293-301.)。尽管两者有相似之处,但也存在一些差异。通常位于核仁中的snoRNA仅包含一个H/ACA盒或C/D盒,而scaRNA通常包含两个,或者在某些情况下两种功能结构域各包含一个,称为混合域scaRNA(Kiss AM, Jady BE, Darzacq X, Verheggen C,Bertrand E, Kiss T. A Cajal body-specific pseudouridylation guide RNA iscomposed of two box H/ACA snoRNA-like domains. Nucleic Acids Res. 2002;30(21):4643-9.)。此外,scaRNA包含一个卡哈尔体盒(CAB盒),将其引导到特定的细胞器卡哈尔体, scaRNA在此被组装并执行功能(Marnef A, Richard P, Pinzon N, Kiss T.Targeting vertebrate intron-encoded box C/D 2'-O-methylation guide RNAs intothe Cajal body. Nucleic Acids Res. 2014;42(10):6616-29;Richard P, Darzacq X,Bertrand E, Jady BE, Verheggen C, Kiss T. A common sequence motif determinesthe Cajal body-specific localization of box H/ACA scaRNAs. EMBO J. 2003;22(16):4283-93.)。近二十年来,越来越多的研究表明,snoRNA在多个水平上调控癌变过程,在癌症临床治疗中显示出很好的应用前景,已成为肿瘤生物学领域的研究热点。迄今为止,已经发现数种snoRNA在CRC中功能失调,如癌基因和抑癌基因(例如SNORD12C,SNORA21,SNORA24,SNORA42,SNORD44,SNORD57,SNORA71A和SNORD78等)。同样,研究人员发现scaRNA的异常表达参与了包括CRCs在内的少数癌症的进展(Zhang PF, Wu J, Wu Y, Huang W,Liu M, Dong ZR, et al. The lncRNA SCARNA2 mediates colorectal cancerchemoresistance through a conserved microRNA-342-3p target sequence. J CellPhysiol. 2019;234(7):10157-65;Ronchetti D, Todoerti K, Tuana G, Agnelli L,Mosca L, Lionetti M, et al. The expression pattern of small nucleolar andsmall Cajal body-specific RNAs characterizes distinct molecular subtypes ofmultiple myeloma. Blood Cancer J. 2012;2:e96;Ronchetti D, Mosca L, Cutrona G,Tuana G, Gentile M, Fabris S, et al. Small nucleolar RNAs as new biomarkersin chronic lymphocytic leukemia. BMC Med Genomics. 2013;6:27;Zhang S, Ding Y,Sun Z, Ge Y, Li Y, Han X, et al. Identification of Expression Pattern andClinical Significance of the Small Cajal Body-specific RNA SCARNA16 inHepatocellular Carcinoma. J Clin Transl Hepatol. 2022;10(1):104-11.),这些发现使其成为癌症的候选生物标志物或治疗靶点。然而,只有有限的研究聚焦scaRNA在癌变过程中的生物学功能,其中大多数仍然未知。
SCARNA12由其宿主基因Prohibitin 2的内含子加工而成,包含一个H/ACA盒和一个C/D盒。它是一种小核RNA,引导U5小核RNA中残基U46的假尿苷化。先前的研究表明,SCARNA12与其他非编码RNA组合可作为筛查宫颈癌的诊断标志物(Cho O, Kim DW, CheongJY. Screening Plasma Exosomal RNAs as Diagnostic Markers for Cervical Cancer:An Analysis of Patients Who Underwent Primary Chemoradiotherapy.Biomolecules. 2021;11(11).)。然而,SCARNA12在结直肠癌中的临床意义和生物学功能尚不清楚。因此,研究SCARNA12在结直肠癌中的临床意义和生物学功能可能对于结直肠癌患者的临床评估和预后分析有所帮助。
发明内容
为了在snoRNAs中挖掘识别结直肠癌的生物标志物,研究SCARNA12在结直肠癌中的临床意义和生物学功能,本发明对SCARNA12在结直肠癌中的表达水平、SCARNA12高表达与结直肠癌患者临床病理特征的关系、及其对结直肠癌细胞增殖、存活和对结直肠癌肿瘤生长的影响进行了相关研究,具体技术方案如下:
本发明的第一个目的是提供SCARNA12基因在制备结直肠癌检测试剂盒中的应用,所述SCARNA12基因的核苷酸序列如SEQ ID No.1所示。
进一步地限定,所述试剂盒用于结直肠癌诊断和/或预后评估;与正常癌旁组织相比,所述SCARNA12基因在结直肠癌细胞或组织中高表达。
本发明的第二个目的是提供上述SCARNA12基因的引物在制备结直肠癌检测试剂盒中的应用。
进一步地限定,所述试剂盒用于结直肠癌诊断和/或预后评估;与正常癌旁组织相比,所述SCARNA12基因在结直肠癌细胞或组织中高表达。
进一步地限定,所述引物的序列为:
正向:5'-CATTTCTGGTGCTGCCCCTA-3';
反向:5'-AGATCCAAGGTTGCGCTCAG-3'。
本发明的第三个目的是提供一种结直肠癌诊断试剂盒,所述试剂盒包括SCARNA12基因的引物。
本发明的第四个目的是提供一种结直肠癌预后评估试剂盒,所述试剂盒包括SCARNA12基因的引物。
本发明的第五个目的是提供敲低SCARNA12基因或者能够抑制SCARNA12基因表达的物质在抑制结直肠癌细胞增殖,或制备用于抑制结直肠癌细胞增殖的药物中的应用,所述SCARNA12基因的核苷酸序列如SEQ ID No.1所示。
本发明的第六个目的是提供敲低SCARNA12基因或者能够抑制SCARNA12基因表达的物质在抑制结直肠癌细胞存活,或制备用于抑制结直肠癌细胞存活的药物中的应用,所述SCARNA12基因的核苷酸序列如SEQ ID No.1所示。
本发明的第七个目的是提供敲低SCARNA12基因或者能够抑制SCARNA12基因表达的物质在抑制结直肠癌肿瘤生长,或制备用于抑制结直肠癌肿瘤生长的药物中的应用,所述SCARNA12基因的核苷酸序列如SEQ ID No.1所示。
本发明的有益效果:
(1)本发明发现SCARNA12在包括CRC在内的多种癌症中高表达,且SCARNA12在CRC细胞系中的表达显著上调,该结果表明SCARNA12在CRC中的潜在诊断价值。
(2)本发明发现高表达的SCARNA12与结直肠息肉病史和CRC患者不良预后有关,结肠息肉通常被认为是CRC进展的驱动力,这表明高水平的SCARNA12是CRC患者不良预后的潜在生物标志物。
(3)本发明发现SCARNA12参与CRC细胞的增殖,存活和体内肿瘤的生长。在进一步的研究中,我们证实SCARNA12通过调节蛋白磷酸化修饰作为PI3K-AKT信号通路的激活剂。进一步表明,SCARNA12通过靶向PI3K-AKT信号通路在CRC中作为癌基因发挥作用,促进结直肠癌的进展。本发明揭示了SCARNA12作为CRC患者的预后指标或新的治疗靶点的临床价值。
附图说明
图1为CRC组织/细胞中SCARNA12基因表达水平分析结果图;其中,图1中的A为cBioPortal网站分析SCARNA12在CRC癌组织与邻近癌旁组织中表达情况的结果图;图1中的B为cBioPortal网站分析SCARNA12表达与CRC肿瘤临床分期的相关性结果图;图1中的C为40对临床CRC癌与邻近癌旁配对组织样本中SCARNA12表达分析结果图;图1中的D为40例临床CRC癌组织中SCARNA12表达分布结果图;图1中的E为正常结直肠上皮细胞系(FHC)和三种CRC细胞系(SW620、HT29、HCT116)中SCARNA12表达分析结果图;图1中的F为ROC曲线分析SCARNA12表达水平用于CRC与邻近正常组织分类的特异性和敏感性结果图;
图2为SCARNA12表达水平与CRC临床预后相关性的分析结果图;其中,图2中的A为Kaplan-Meier和log-rank检验分析SCARNA12表达水平与CRC患者总生存率的相关性结果图,图2中的B为Kaplan-Meier和log-rank检验分析SCARNA12表达水平与CRC患者无复发生存率的相关性结果图;图2中的C为单因素Cox回归模型分析SCARNA12与CRC临床预后风险的相关性结果图;图2中的D为多因素Cox回归模型分析SCARNA12与CRC临床预后风险的相关性结果图;
图3为SCARNA12在体外细胞实验中对CRC细胞增殖和存活的影响结果图;其中,图3中的A为qRT-PCR检测感染过表达SCARNA12基因慢病毒的SW620细胞SCARNA12表达水平结果图;图3中的B为qRT-PCR分别检测转染靶向SCARNA12 ASO敲低序列的HCT116细胞和转染靶向SCARNA12 ASO敲低序列的HT29细胞SCARNA12表达水平的结果图;图3中的C为CCK-8法分别检测感染过表达SCARNA12基因慢病毒的SW620细胞、转染靶向SCARNA12 ASO敲低序列的HCT116细胞和转染靶向SCARNA12 ASO敲低序列的HT29细胞的增值结果图;图3中的D为感染过表达SCARNA12基因慢病毒的SW620细胞、转染靶向SCARNA12 ASO敲低序列的HCT116细胞和转染靶向SCARNA12 ASO敲低序列的HT29细胞的平板克隆实验结果图;图3中的E和图3中的F为感染过表达SCARNA12基因慢病毒的SW620细胞、转染靶向SCARNA12 ASO敲低序列的HCT116细胞和转染靶向SCARNA12 ASO敲低序列的HT29细胞凋亡分析结果图;
图4为敲低SCARNA12表达的CRC细胞转录组测序分析及验证结果图;其中,图4中的A为qRT-PCR检测SCARNA12敲低的HCT116细胞SCARNA12基因表达水平结果图;图4中的B为转录组测序筛选SCARNA12敲低的HCT116细胞中差异表达基因结果图;图4中的C为SCARNA12敲低的HCT116细胞中PI3K-AKT信号通路相关基因分析结果图;图4中的D为SCARNA12敲低的HCT116细胞中PI3K-AKT信号通路相关基因的qRT-PCR检测结果图;
图5为SCARNA12通过激活PI3K-AKT通路促进CRC细胞的增殖和存活的相关结果图;其中,图5中的A为转录组测序差异基因KEGG富集分析结果图,发现敲低SCARNA12富集PI3K-AKT通路;图5中的B为SW620细胞中过表达SCARNA12,或HCT116、HT29细胞中敲低SCARNA12后,蛋白质印迹实验检测PI3K和AKT蛋白磷酸化水平实验结果图;图5中的C为使用AKT抑制剂(MK2206)条件下,过表达SCARNA12分析AKT磷酸化水平实验结果图;图5中的D为使用MK2206条件下,过表达SCARNA12调节CRC细胞增殖实验结果图;图5中的E为使用MK2206条件下,过表达SCARNA12调节CRC细胞克隆形成实验结果图;
图6为SCARNA12在裸鼠体内促进CRC细胞皮下移植瘤生长的相关结果图;其中,图6中的A为过表达SCARNA12的SW620细胞系(LV-SCARNA12)及阴性对照细胞系(LV-NC)进行裸鼠皮下荷瘤实验,手术取瘤结果图;图6中的B为移植瘤重量分析结果图;图6中的C为根据移植瘤体积测定数据拟合肿瘤生长曲线图;图6中的D为SCARNA12基因qRT-PCR检测结果图;图6中的E为肿瘤组织石蜡切片经苏木精和伊红(H&E)和免疫组织化学(IHC)方法检测Ki67和p-AKT蛋白表达实验结果图。
具体实施方式
以下结合具体实施例和附图,对本发明作进一步的详细说明。本发明所使用的药品、试剂、仪器、设备等如无特殊说明,均可通过商业化途径购买获得,涉及的PCR扩增等分子生物学实验操作如无特殊说明,均为本领域常规实验操作或依照相应试剂的产品说明书进行。
本发明涉及的材料具体如下:
(1)临床样本
从辽宁省肿瘤医院获取40对新鲜冷冻的CRC肿瘤组织和相邻的正常粘膜组织标本。标本的使用经辽宁省肿瘤医院人体调查伦理委员会(20190970)批准,并征得每位患者的知情同意。
(2)细胞系
SW620,HCT116和HT29细胞系购自GeneChem(中国上海),人正常结肠上皮细胞(FHC)购自ATCC(马纳萨斯,弗吉尼亚州)。
(3)试剂和抗体
MK2206(一种AKT抑制剂)购自Med Chem Express,p-PI3K(#4228)、PI3K(#4292)、p-AKT(#4060)、AKT(#4691)和PARP(#9542)的一抗从Cell Signaling Technology购买,Bcl-2(#YM3041)从ImmunoWay购买,β-actin(60009-1-Ig)购自Proteintech,小鼠IgG(#5220-0341)和兔IgG(#5220-0336)的抗体从SeraCare(美国KPL)购买。
本发明涉及的研究方法具体如下:
(1)公共癌症数据库的生物信息学分析
从TCGA下载了包括548名CRC患者的数据集,实际上我们的研究包括了324名患者(缺乏准确临床信息的患者被排除)。分析SCARNA12的表达与总生存期(OS)、无复发生存期(RFS)和临床病理因素的相关性。同时,采用cBioPortal网站(http://www.cbioportal.org)数据,包括CRC肿瘤组织(n=273)和邻近正常组织(n=256),分析CRC与邻近正常组织SCARNA12表达的差异及其与CRC患者临床病理特征的相关性。
(2)细胞培养
SW620和HCT16细胞使用RPMI-1640培养基培养,HT29细胞在DMEM(HyClone,美国)中培养。两种培养基均补充了10%胎牛血清(ExCell Bio,中国)和1%青霉素-链霉素(HyClone,美国)。FHC在DMEM:F-12(ATCC,美国)中培养,补充10 ng/mL霍乱毒素、0.005 mg/mL胰岛素、10 mM HEPES(终浓度为25 mM)、20 ng/mL人重组EGF(赛默飞世尔 PHG0311)、100ng/mL氢化可的松、0.005 mg/mL转铁蛋白、10%胎牛血清(GIBCO)和1%青霉素-链霉素。所有细胞在含5% CO2的37°C保湿的孵箱中孵育。
(3)RNA提取和实时荧光定量PCR(qRT-PCR)
使用TRIzol试剂(美国Sigma)从组织样品或培养细胞中提取总RNA,并通过PrimeScriptTM RT试剂盒(Takara,Shiga,Japan)反转录为cDNA。在Bio-Rad CFX96系统中使用iTaq Universal SYBR Green Supermix(BioRad,美国)和特异性引物(表1)进行QRT-PCR。U6和GAPDH被用作内参。采用2-ΔΔCt法计算基因表达量。
SCARNA12的核苷酸序列如SEQ ID NO.1所示:
5’-CAGGCTGATGAGACTAAGGCGAATGCGACTCCGTGCTCTCTGGCCCTTGGCTCCTTGTTGGGGGTGGGGACTACAGATGAGATCTGAAATCTTAGTGGTAGTACCTGAGCCATGACTCCCCACTGTAAGGCCAGATCAATAGCATTGGTGGCCTTGCCTTCATTTCTGGTGCTGCCCCTAGTTCCTGGCAGCAGCCTGCAGGGAGGCCCACAGGTGGGGTCCACGGTAGGGCTGGGCACAAGCCACCTGAGCGCAACCTTGGATCTGACA-3’
表1 用于靶向敲低SCARNA12基因的ASO序列和qRT-PCR引物序列
(4)细胞转染
SW620细胞感染过表达SCARNA12和阴性对照的慢病毒(GeneChem,中国上海),感染慢病毒的MOI为20。感染72小时后,用嘌呤霉素(2 μg/ mL)筛选过表达SCARNA12的稳定细胞株。靶向SCARNA12的反义寡核苷酸(ASO)由锐博生物(中国广州)设计和合成。ASO-SCARNA12的寡核苷酸序列为5'-CTAGTTCCTGGCAGCAGCCT-3'。根据产品说明,使用riboFECT™ CPReagent(锐博生物,中国广州)进行ASO转染。
(5)细胞增殖检测
根据制造商的说明,使用CCK8试剂盒检测细胞增殖。将细胞接种在96孔板中,每孔100 μL培养基包含2×103个细胞。对于每组,设置5个复孔。然后将10% CCK-8试剂加入每个孔中,并将板在37℃孵箱中孵育2小时。使用酶标仪(BIO-RAD,170-6750)在450 nm处检测吸光度值。
(6)克隆形成实验
将800个细胞接种在6孔板中并培养2周,每4天更换培养基。将细胞固定在甲醇中20分钟,并在室温下用Giemsa染色30分钟。以无偏方式计算每个孔中的克隆总数。
(7)细胞凋亡分析
使用Annexin V-FITC凋亡检测试剂盒(DOJINDO)对不同组的细胞进行染色。通过流式细胞术分析凋亡细胞的百分比(ACEA Bio,San Diego,California)。该实验独立进行3次。
(8)蛋白质提取和蛋白质印迹分析
细胞在含有蛋白酶抑制剂和磷酸酶抑制剂(罗氏)的RIPA缓冲液中进行裂解。蛋白质浓度通过BCA蛋白质测定试剂盒(Beyotime,P0010)进行定量。将等量的蛋白质(30 μg)通过10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离并转移到硝酸纤维素膜上。使用5%脱脂奶粉在室温下封闭2小时,并在4℃条件下将一抗孵育过夜。用TBS-吐温20(TBS-T)缓冲液洗涤膜3次,然后在室温下与辣根过氧化物酶(HRP)偶联的二抗孵育2小时。使用ECL试剂盒(赛默飞世尔科技)可视化蛋白质条带。β-actin作为内参。最后,使用Image J软件分析相对蛋白表达水平。
(9)裸鼠肿瘤异种移植实验
5周龄雄性BALB/c裸鼠(中国北京维通利华),将其分为两组(LV-NC,LV-SCARNA12,每组n=5)用于小鼠异种移植模型。将感染LV-SCARNA12或LV-NC的SW620细胞(在100 μL PBS中每个位点约5×106个细胞)皮下注射到小鼠体内。每4天用游标卡尺估计异种移植物的长度(L)和宽度(W),用公式计算异种移植体积:V=(L×W2)/2。30天后,收集皮下异种移植物,拍照并称重。所有动物实验均按照军事医学科学院伦理委员会(AMMS,中国北京)允许的伦理规定进行。
(10)苏木精和伊红(H&E)和免疫组织化学(IHC)
将石蜡包埋的异种移植肿瘤组织切成3 μm切片。通过H&E和IHC染色观察肿瘤的组织学分析。首先用二甲苯对石蜡包埋的载玻片进行脱蜡,然后用一系列分级的醇脱水,用H&E染色进行组织学分析。使用的抗体是:Ki-67(Abcam,UK;Cat. No. ab15580,1:2000)andp-AKT(ABclonal,China;Cat. No. A11016,1:100)。使用3,3'-二氨基联苯胺四盐酸盐(DAB)进行显色反应。
(11)统计分析
所有实验均进行三次独立重复实验。数据以平均值±标准差(SD)表示。使用Kaplan-Meier with the log-rank test来分析总体生存数据。采用Chi-square (χ2)test分析结直肠癌患者SCARNA12表达与临床病理参数的相关性。Cox比例风险回归模型用于确定与总生存期相关的独立预后因素。采用The two-tailed Student’s T test分析不同组间统计学意义。所有统计分析均由SPSS 26.0和GraphPad Prism 9.0软件完成。P值为<0.05被认为具有统计学意义。
实施例1:SCARNA12在临床CRC组织中表达上调
(2)SNORA24在临床CRC组织中表达上调
为了研究CRC中SCARNA12的表达水平,我们采用cBioPortal网站(http://www.cbioportal.org)的数据来分析CRC与邻近正常组织之间SCARNA12表达的差异及其与肿瘤临床分期的相关性,该数据包括CRC肿瘤组织(n=273)和邻近正常组织(n=256)。统计分析显示,与邻近正常组织相比,CRC组织中SCARNA12表达显著上调(图1中的A),但是与CRC的TNM分期无显著相关性(图1中的B)。此外,使用qRT-PCR在40对CRC组织和配对的相邻正常组织中测定SCARNA12的表达水平(图1中的C)。与正常组织相比,90%(36/40)的CRC组织中的SCARNA12表达水平显著增加(图1中的D)。一致地,SCARNA12在CRC细胞系(SW620,HCT116和HT29)中的表达也显著高于人正常结肠上皮细胞(FHC)(图1中的E)。受试者工作特征曲线(ROC)分析显示,SCARNA12表达可区分CRC与正常黏膜组织(AUC=0.8838,95%CI:0.80-0.96,P<0.001),表明SCARNA12可用于CRC的诊断(图1中的F)。
实施例2:SCARNA12的高表达与结直肠癌患者临床病理特征相关且能预测结直肠癌患者的不良预后
我们假设SCARNA12的高表达与CRC患者的临床病理特征或预后有关。为了解决这个问题,用χ2检验对来自TCGA数据库的CRC患者进行了分析。结果表明,SCARNA12的高表达与结肠息肉病史显著相关(P<0.001)(表2)。Kaplan-Meier和log-rank检验表明,与低表达SCARNA12的患者相比,高表达SCARNA12的CRC患者的总生存期更短,复发率更高(图2中的A,图2中的B)。此外,为了分析SCARNA12的预后价值,采用单因素和多因素Cox比例风险模型分析结直肠癌患者总生存期的预后因素。结果显示,年龄、远端转移和SCARNA12表达是CRC患者总生存期的独立预后因素(图2中的C和图2中的D)。这些发现表明,SCARNA12可能在CRC的进展中发挥重要作用。
表2 SCARNA12表达水平与结直肠癌患者临床病理指标的相关性
注:SCARNA12表达的截止阈值为本队列中所有患者的中位数;*P<0.05
实施例3:SCARNA12在体外促进结直肠癌细胞增殖和存活
为了研究SCARNA12的潜在生物学功能,我们在SW620细胞中感染了过表达SCARNA12的慢病毒,因为SW620细胞的SCARNA12内源性表达水平较低。同时,HCT116和HT29细胞转染靶向SCARNA12的ASO敲低序列,因为这两种细胞系中SCARNA12表达水平较高。通过qRT-PCR检测SCARNA12的表达水平。与对照组相比,SCARNA12的基因水平在感染过表达该基因慢病毒的SW620细胞中显著上调(图3中的A)。然而,在转染了ASO寡核苷酸序列的HCT116和HT29细胞中,SCARNA12被显著敲低(图3中的B)。CCK-8和克隆形成实验表明,SCARNA12过表达显著促进了CRC细胞的增殖和克隆形成;相反,SCARNA12敲低导致相反的结果(图3中的C和图3中的D)。流式细胞术分析显示,SCARNA12的过表达可抑制细胞凋亡;相反,敲低SCARNA12显著诱导细胞凋亡(图3的E)。蛋白质印记结果表明,SCARNA12过表达上调了Bcl-2的表达,而PARP剪接体的表达下调;反之,敲低SCARNA12后结果相反(图3中的F)。这些结果表明,SCARNA12在体外影响结直肠癌的增殖和存活。
实施例4:SCARNA12通过激活PI3K-AKT通路增强CRC细胞的增殖和存活
为了进一步阐明SCARNA12在CRC中的潜在分子机制,我们使用SCARNA12敲低的HCT116细胞进行转录组测序。qRT-PCR检测揭示了SCARNA12的敲低效果(图4中的A)。使用log2倍数变化(FC)>1或log2倍数变化(FC)<-1,以p值<0.05作为临界值,共鉴定出1515个差异表达基因,其中1065个基因显著上调,450个基因下调(图4中的B)。我们进行了KEGG信号通路富集分析,发现与肿瘤进展密切相关的PI3K-AKT信号通路明显富集(图5中的A)。这些发现表明,SCARNA12可能是PI3K-AKT信号通路中的关键调节因子。为了证实这些发现,对参与PI3K-AKT信号通路的几个基因绘制了热图(图4中的C),并通过qRT-PCR进行了验证。如图4中的D所示,在HCT116细胞中,敲低SCARNA12显著降低了EGFR、CCND1、FGF9和PIK3R3基因的基础表达水平。
PI3K-AKT通路通常通过特定位点的磷酸化在癌症中异常激活,从而导致不受控制的细胞增殖和存活。为了证明SCARNA12对CRC中PI3K-AKT信号通路的影响,我们使用蛋白质印迹实验进行进一步分析。结果表明,在SW620细胞中的过表达SCARNA12分别提高了PI3K和AKT在Tyr458和Ser473位点的磷酸化水平,HCT116和HT29细胞中敲低SCARNA12降低了PI3K和AKT的磷酸化水平。需要注意的是,SCARNA12水平的改变对PI3K和AKT的总蛋白水平没有影响(图5中的B)。为了进一步确定SCARNA12通过激活PI3K-AKT通路促进CRC细胞增殖和克隆形成,用AKT抑制剂(MK-2206)处理过表达SCARNA12的SW620细胞。值得注意的是,MK2206显著抑制了p-AKT的水平(图5中的C)。此外,CCK-8和克隆形成实验结果显示,由SCARNA12促进的SW620细胞的增殖和存活被MK-2206逆转(图5中的D和图5中的E)。我们的结果表明,SCARNA12加速了CRC的细胞增殖和克隆形成,至少部分是通过激活PI3K-AKT信号通路实现的。敲低SNORA24表达诱导CRC细胞凋亡、抑制细胞存活。
实施例5:SCARNA12在体内促进CRC异种移植物的生长
为了进一步分析SCARNA12过表达在体内对CRC细胞生长的影响,我们使用免疫缺陷小鼠建立了异种移植肿瘤模型。将稳定转染LV-SCARNA12或对照LV-NC(每只小鼠5×106个细胞)的SW620细胞系注射到裸鼠皮下。如图6中的A和图6中的B所示,裸鼠移植肿瘤的大小和重量因SCARNA12的过表达而显着增加,移植肿瘤的体积也显着增加(图6中的C)。qRT-PCR的结果表明,感染LV-SCARNA12的异种移植肿瘤中SCARNA12的表达显着上调(图6中的D)。此外,IHC结果显示,在表达LV-SCARNA12的异种移植肿瘤组织中,Ki67和p-AKT的蛋白质水平显着升高(图6中的E)。上述结果表明,SCARNA12的过表达促进了体内肿瘤生长并激活了PI3K-AKT信号通路,这与我们在体外实验中的发现一致。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.检测SCARNA12基因的试剂在制备结直肠癌检测试剂盒中的应用,所述SCARNA12基因的核苷酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的应用,其特征在于,所述试剂盒用于结直肠癌诊断和/或预后评估;与正常癌旁组织相比,所述SCARNA12基因在结直肠癌细胞或组织中高表达。
3.扩增权利要求1中所述SCARNA12基因的引物在制备结直肠癌检测试剂盒中的应用。
4.根据权利要求3所述的应用,其特征在于,所述试剂盒用于结直肠癌诊断和/或预后评估;与正常癌旁组织相比,所述SCARNA12基因在结直肠癌细胞或组织中高表达。
5.根据权利要求3所述的应用,其特征在于,所述引物的序列为:
正向:5'-CATTTCTGGTGCTGCCCCTA-3';
反向:5'-AGATCCAAGGTTGCGCTCAG-3'。
6.敲低SCARNA12基因的物质在制备用于抑制结直肠癌细胞增殖的药物中的应用,其特征在于,所述SCARNA12基因的核苷酸序列如SEQ ID No.1所示,所述敲低SCARNA12基因的物质为用于靶向敲低SCARNA12基因的ASO序列,所述ASO序列如SEQ ID No.2所示。
7.敲低SCARNA12基因的物质在制备用于抑制结直肠癌细胞存活的药物中的应用,其特征在于,所述SCARNA12基因的核苷酸序列如SEQ ID No.1所示,所述敲低SCARNA12基因的物质为用于靶向敲低SCARNA12基因的ASO序列,所述ASO序列如SEQ ID No.2所示。
8.敲低SCARNA12基因的物质在制备用于抑制结直肠癌肿瘤生长的药物中的应用,其特征在于,所述SCARNA12基因的核苷酸序列如SEQ ID No.1所示,所述敲低SCARNA12基因的物质为用于靶向敲低SCARNA12基因的ASO序列,所述ASO序列如SEQ ID No.2所示。
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