CN113699233B - Troap在制备肾细胞癌预后产品及治疗药物中的用途 - Google Patents
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Abstract
本发明属于生物医药领域,涉及TROAP在制备肾细胞癌预后产品及治疗药物中的用途。本发明通过实验发现并证实TROAP基因可促进肾癌细胞增殖、迁移和侵袭,同时抑制凋亡,是肾癌进展非常重要的标记物。基于此,本发明提出将TROAP基因作为筛选出的分子标记物,通过检测TROAP的表达水平来判断肾细胞癌患者的预后情况,通过预后模型指导临床治疗,这对评估患者预后以及指导患者的个体化治疗具有重要的实际应用价值。同时,本发明提出将ROAP基因作为针对性的开发药物作用靶点,为肾透明细胞癌,特别是转移性透明细胞癌提供新的抑制剂及药物开发靶点,提高对这类患者的早期预警能力及后续治疗效果。
Description
技术领域
本发明涉及生物医药领域,具体涉及TROAP在制备肾细胞癌预后产品及治疗药物中的用途。
背景技术
肾细胞癌(Renal cell carcinoma,RCC),简称肾癌,是泌尿系统常见的恶性肿瘤之一,占成人恶性肿瘤的2%-3%,其中肾透明细胞癌约占80%(clear cell renal cellcarcinoma, ccRCC)[1]。目前,手术是肾癌主要的治疗方式,但较多患者在确诊肾癌时已经发生远处转移,其五年生存率不足20%。局限性肾癌患者接受手术治疗后仍有20%-40%的复发率 [2]。此外,肾癌对放疗和化疗均不敏感,晚期肾癌患者使用分子靶向药物后易出现耐药,降低治疗效果。
肾癌的发病机制复杂,其发生受到众多因素影响,其中基因表达异常在肾癌发生发展中起到重要作用[3,4]。越来越多的证据表明RNA及蛋白具有重要的生物学功能,如携带遗传信息及调控疾病的发生进程等[5]。申请人曾参与非编码RNA相关研究[6],关注该领域研究进展。且近期研究证实,肾透明细胞癌的预后与蛋白相关标志物密切相关[7,8]。
但目前这些研究尚处于基础阶段,尚未发现有效的蛋白相关标志物,且研究样本量少,研究病例有较大的异质性,未与实际临床治疗情况及预后相结合,未在较大标本病人中得到验证,也未将蛋白相关标志物用于临床预后模型的建立与完善。
TROAP又名tastin,与滋养素(trophinin)相互作用,是一种细胞质蛋白,由778个氨基酸残基组成,含有蛋白激酶的潜在磷酸化位点[9,10]。根据既往研究综合来看,TROAP高表达在卵巢癌、乳腺癌、结直肠癌和肝癌预示更差预后和疾病进展,扮演促进肿瘤进展的角色[11-14]。同样在前列腺癌中,敲低TROAP表达可抑制细胞增殖和引起细胞周期阻滞[15]。
虽然有多项研究表明TROAP参与肿瘤的发生和发展,但迄今其在肾细胞癌中的作用尚未见文献报道,TROAP是否可作为标志物用于肾细胞癌的诊断和预后仍缺乏相应研究支持;此外,TROAP对肾细胞癌的效应中发挥作用及相应的分子机制仍不明确。
发明内容
本发明的目的在于克服现有技术的不足,针对目前肾细胞癌诊断和预后缺乏有效的指标的现状,研究肾细胞癌特异性生物标志物,及与恶性进展的分子机制,指导患者临床的个体化治疗。具体地,本发明筛选出TROAP作为肾癌特异性生物标志物,提出TROAP 在制备肾细胞癌预后产品及治疗药物中的用途,通过预后模型指导临床的个体化治疗;本发明还为肾透明细胞癌提供了抑制剂及药物开发靶点,提高对肾细胞癌患者的早期预警能力及后续治疗效果。
为实现上述目的,本发明采用如下的技术方案:
第一方面,本发明提出了TROAP在制备用于检测肾细胞癌的预后产品中的用途。
在具体的实施方案中,所述预后产品以TROAP基因为特异性生物标志物,通过检测受试者样本中的TROAP基因表达水平来判断肾细胞癌患者的预后情况。
在具体的实施方案中,所述预后产品中含有检测TROAP基因的检测试剂及对照品;通过测序平台检测TROAP基因的表达水平。
在具体的实施方案中,所述肾细胞癌是原发于泌尿系统的肿瘤。
在具体的实施方案中,所述受试者样本是肾细胞癌组织样本。
在具体的实施方案中,所述肾细胞癌组织样本是通过活检、穿刺或外科切除的方式获得。
在具体的实施方案中,所述受试者是人或哺乳动物。
第二方面,本发明提出了TROAP在制备肾细胞癌抑制剂或治疗药物中的用途。
在具体的实施方案中,所述抑制剂或治疗药物是以TROAP基因为靶点的抑制剂或药物。
在具体的实施方案中,所述抑制剂或治疗药物通过敲低TROAP基因来抑制肾癌细胞的增殖、迁移和侵袭,并增加凋亡。
术语说明:
预后:在本说明书中,预后是指预测某种疾病的可能病程和结局。预后既包括判断疾病的特定后果(如康复、某种症状、体征、并发症),也包括提供时间线索(如预测某段时间内发生某种结局的可能性)。当从疾病演进过程的角度考虑时,预后包括例如缓解率、复发率、病残率。当从疾病终极状态的角度考虑时,预后包括例如治愈率、生存率、病死率。当从预后时间考虑时,预后包括例如近期病死率、远期病死率。
敲低:在本说明书中,敲低是指通过降解具有同源序列靶基因的mRNA,达到阻止基因表达的作用。利用双链小RNA高效、特异性降解细胞内同源mRNA,从而阻断体内靶基因的表达,使细胞出现靶基因缺失的表型。它不同于基因敲除使目标基因永久性的表达沉默,而是通过降解具有同源序列靶基因的mRNA,达到阻止基因表达的作用。
表达:在本说明书中,表达是基因经过转录、翻译、产生有生物活性的蛋白质的整个过程。高表达是指转录或翻译出数量很多的最终产物,而低表达是转录或翻译出数量很少的最终产物。
靶点:在本说明书中,靶点是指药物在体内的作用结合位点。现代新药研究与开发的关键首先是寻找、确定和制备药物筛选靶,包括基因位点、受体、酶、离子通道、核酸等生物大分子。
本发明的显著效果在于:
本发明通过回顾性和前瞻性研究,首次发现并证实TROAP可促进肾癌细胞增殖、迁移和侵袭,同时抑制凋亡,是肾癌进展非常重要的标记物。因此,TROAP可通过预后模型指导临床的个体化治疗,对评估患者预后以及指导患者的个体化治疗具有重要的实际应用价值。TROAP还可能成为针对性的开发药物作用的潜在靶点,用来作为筛选的分子标记物,为肾透明细胞癌,特别是转移性透明细胞癌提供新的抑制剂及药物开发靶点,提高对这类患者的早期预警能力及后续治疗效果。
附图说明
图1是免疫组化验证TROAP在肾透明细胞癌组织中的表达图。
图2是TROAP敲减验证图。
图3是MTT细胞生长实验图。
图4是细胞凋亡实验图。
图5是划痕愈合实验图。
图6是基质胶Transwell侵袭实验图。
图7是裸鼠皮下成瘤生长实验图。
具体实施方式
以下结合附图,通过实施例进一步说明本发明,但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是,应当理解的是,这些仅仅是示例性的,并不意图限制本发明,与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1,
TROAP与肾癌相关性实验。
通过免疫组化检测20对肾癌及对应肾正常组织中TROAP的蛋白表达水平情况,结果显示TROAP在肾癌组织显著高表达;随后对肾癌分级进行亚组分析,提示随着级别上升,癌组织TROAP表达量亦呈增加趋势,结果有统计学差异(图1),结果提示TROAP 可能与肾癌发生发展密切相关。
参见图1.免疫组化验证TROAP在肾透明细胞癌组织中的表达。
免疫组化(IHS)结果显示:
A.B.C.TROAP在肾癌组织中高表达,统计具有显著差异(P<0.001);
D-H.肾癌组织临床分级越高,TROAP表达量则越高,统计具有显著差异(P<0.001)。
实施例2.
体内外实验表明敲低TROAP能够抑制肾癌细胞的增殖、迁移和侵袭。
因TROAP与肾癌临床密切相关,进一步探讨其在肾癌进展中发挥的功能。选定常用肾癌细胞系786-0及ACHN,采用慢病毒干扰技术敲低TROAP(图2),功能实验包括体外层面的MTT细胞增殖、细胞凋亡、细胞划痕愈合和Transwell侵袭。敲低TROAP能显著抑制786-0及ACHN细胞生长(图3);敲低TROAP显著增加786-0及ACHN细胞的凋亡占比(图4);敲低TROAP显著降低786-0及ACHN细胞划痕愈合的能力(图5);敲低TROAP显著抑制786-0及ACHN细胞的侵袭能力(图6)。
参见图2.TROAP敲减验证。
A-B:786-0细胞RT-PCR与Westernblot验证慢病毒敲减TROAP效率;
C-D:ACHN细胞RT-PCR与Westernblot验证慢病毒敲减TROAP效率。
参见图3.MTT细胞生长实验。干扰TROAP后显著影响786-0和ACHN细胞的生长,被显著抑制。
参见图4.细胞凋亡实验。干扰TROAP后显著增加786-0和ACHN细胞凋亡比率,说明TROAP敲减后能抑制细胞的增殖。
参见图5.划痕愈合实验。荧光显微镜下,干扰TROAP后显著减低786-0和ACHN细胞的自我修复愈合能力。
参见图6.基质胶Transwell侵袭实验。干扰TROAP后显著减低786-0和ACHN细胞侵袭能力。
实施例3.
体内裸鼠皮下成瘤实验。
本发明进行体内裸鼠皮下成瘤实验,啰嗦皮下接种ACHN细胞,结果显示TROAP敲低组瘤体生长缓慢,最后瘤体体积明显小于对照组(图7)。
参见图7.裸鼠皮下成瘤生长实验。干扰TROAP后皮下瘤生长被抑制。
A:10对裸鼠及其皮下瘤。
B:裸鼠皮下瘤生长曲线,显示TROAP干扰组皮下瘤生长受到显著抑制。
C:裸鼠皮下瘤4周后平均体积,显示TROAP干扰组体积明显低于对照组。
综合以上研究,本发明认为TROAP可促进肾癌细胞增殖、迁移和侵袭,同时抑制凋亡,是肾癌进展非常重要的标记物。因此,研究TROAP在肾癌进展中的作用机制以及针对性的开发药物作用靶点有着重要临床价值。
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Claims (1)
1.敲低TROAP基因表达水平的试剂在制备肾透明细胞癌治疗药物中的用途。
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