CN107475369A - Hoxa9基因在制备治疗皮肤鳞状细胞癌药物中的应用 - Google Patents
Hoxa9基因在制备治疗皮肤鳞状细胞癌药物中的应用 Download PDFInfo
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Abstract
本发明公开了HOXA9基因在制备治疗皮肤鳞状细胞癌药物中的应用,属于基因治疗领域。发明人及其整个团队经过两年的实验研究,以皮肤鳞癌特征性的细胞株A431为实验对象,通过实验发现靶向上调HOXA9,可以抑制肿瘤细胞增殖、迁移和侵袭能力,并抑制肿瘤细胞糖酵解,最终改善或治疗皮肤鳞癌。本发明提供了一种能够最大程度保证器官功能完好及外型美观的基础上治疗CSCC的新方法,可以克服现有技术的不足,具有重大意义。
Description
技术领域
本发明属于基因治疗领域,具体涉及HOXA9基因在制备治疗皮肤鳞状细胞癌药物中的应用。
背景技术
皮肤鳞状细胞癌(Cutaneous squamous cell carcinoma,CSCC)是起源于表皮附属器角质形成细胞的一类恶性肿瘤,是临床上最常见的皮肤恶性肿瘤之一,发病率仅次于基底细胞癌,占皮肤恶性肿瘤的20%。在我国CSCC的高发年龄为50-60岁,多见于头、颈、手及前臂伸侧等暴露位置。CSCC病因复杂,长期紫外照射是其最大的危险因素。此外,人乳头瘤病毒(HPV)16、18和31型感染、器官移植、化学制剂等因素与其发病密切相关。虽然CSCC死亡率不高,但具有较强的转移和侵袭的能力,可以侵蚀重要的组织结构或者转移至淋巴结,相比基底细胞癌其恶性程度更高,预后极差。其转移率为2%-5%,影响转移的危险因素包括:免疫抑制,肿瘤直径>10mm或浸润深度>6mm,中低分化,神经浸润,淋巴管浸润,初次手术切除不完整及局部复发等。
目前,皮肤鳞癌确切的发病机制尚不完全清楚,部分研究指出转录因子(Transcription factor,TFs)在CSCC的发生发展过程中起到了重要的作用,如p53、NFκB、AP-1等。TFs的突变和异常表达会直接或间接导致某些甚至全部肿瘤特征的出现,包括增殖、抗凋亡、供养血管的形成、迁移和侵袭能力等。此外,UVB(Ultraviolet B)不仅可以引起肿瘤抑制因子p53的突变,同时可诱导NFκB的激活并向核内转位上调IL-6的表达和分泌导致慢性炎症的发生。
HOX基因家族编码一类TFs,180个核苷酸编码影响转录调控活动的DNA结合结构域。因自身编码含有183千碱基对的DNA片段,转录由61个氨基酸组成的同源功能结构域。其第42至50位9个氨基酸序列片段由于富含赖氨酸和精氨酸,被认为是与DNA特异性相结合的区域,对基因的表达起主控作用。因此,HOX基因表达产物是可与特异性DNA结合的转录因子。HOXA9在胚胎发育等过程中起到了重要作用,对骨骼、乳腺、泌尿生殖道、肾等器官的发育过程中起到重要作用,同时也参与正常骨髓和T细胞的分化。近几年研究表明HOX家族在肿瘤中同样也起到非常重要的作用。HOXA9作为HOX家族一员,不仅可以调控胚胎发育过程,还在多种肿瘤发生发展的不同阶段作为抑癌因子或促癌因子起到不同的作用。在急性骨髓性白血病和急性淋巴细胞白血病中出现的混合细胞系白血病预后极差并伴随HOX基因家族的异常表达,尤其是HOXA9。
然而,在实体肿瘤中,HOXA9的功能并不一致。雌激素/孕激素受体表达缺失的乳腺癌患者中HOXA9和其下游靶基因BRCA1皆显著下调(HOXA9作为抑癌因子),这一现象被认为对乳腺癌的发展起到重要作用。在非小细胞肺癌中HOXA9的下调(HOXA9作为抑癌因子)也被认为是癌症发生的一个指证,HOXA9表达上调可以明显抑制A549和HCC95细胞的侵袭。卵巢癌中,HOXA9在转录水平调控TGF-β2(HOXA9作为促癌因子),诱导成纤维细胞增殖从而形成适宜的微环境并促进肿瘤的生长。总地来说,对于HOXA9在肿瘤中所起到的功能并没有唯一的定论。HOXA9是抑癌或者促癌因子主要取决于肿瘤类型的不同。
综上所述,无论作为癌基因还是抑癌基因,HOXA9对肿瘤的发生发展确实起到重要作用。但目前对于HOXA9在实体肿瘤,尤其是CSCC中的作用尚无详尽的机制研究。
近年来,随着CSCC发病率的不断上升以及患者对术后生活质量的要求逐渐提高,如何选择更加优化的治疗方案,最大限度地保留病灶的外观及功能,是目前治疗CSCC的首要难题。CSCC治疗目前首选手术切除,直径小于2.0cm的低风险CSCC病例一般切除范围为距离肿瘤边缘至少4.0mm的组织。而对于T2期及以上直径大于2.0cm的CSCC病例切除范围则扩大至距离肿瘤边缘至少6.0mm的组织。当CSCC具有以下高危因素时:鳞癌细胞中低分化或未分化;肿瘤侵袭皮下组织;肿瘤发生部位在耳、眼睑、唇、头皮及鼻等部位时,应扩大手术切除范围,而不是仅局限于切除原始病发灶。然而,扩大手术范围也不能彻底清除肿瘤细胞,手术治疗CSCC的复发率仍处于不低的水平。直径小于2.0cm的CSCC病例手术切除后复发率为41.7%,即使选择显微手术的方法也只能将复发率降为25.2%;低分化的CSCC术后复发率为53.6%。鉴于传统治疗方法的手术需扩大切除范围至肿瘤周围组织,很可能造成表皮及皮下组织功能受损并影响器官外表的美观,同时此方法较高的复发率也使得寻找一种能够最大程度保证器官功能完好及外型美观的基础上治疗CSCC的新方法势在必行。
发明内容
本发明的目的在于提供HOXA9基因在制备治疗皮肤鳞状细胞癌药物中的应用。
本发明所采取的技术方案是:
HOXA9基因和/或其编码蛋白在制备治疗实体瘤的药物中的应用。
优选的,实体瘤包括皮肤癌、肺癌、宫颈癌。
优选的,皮肤癌为皮肤鳞状细胞癌。
HOXA9基因和/或其编码蛋白作为皮肤癌的辅助诊断和/或预后判断的敏感性检测新靶点的应用。
优选的,皮肤癌为皮肤鳞状细胞癌。
一种用于皮肤癌诊断和/或预后判断的试剂盒,该试剂盒含有特异性扩增HOXA9基因DNA链和/或其cDNA链的PCR引物,或抗HOXA9蛋白的抗体。
优选的,皮肤癌为皮肤鳞状细胞癌。
用于治疗皮肤癌的药物组合物,该药物组合物含有HOXA9基因和/或其编码的蛋白,以及医学上可接受的药用辅料。
优选的,皮肤癌为皮肤鳞状细胞癌。
特异性扩增HOXA9基因DNA链和/或其cDNA链的PCR引物,或抗HOXA9蛋白的抗体在制备皮肤癌诊断和/或预后判断试剂盒中的应用。
本发明的有益效果是:
申请人及其整个团队经过两年的实验研究,以皮肤鳞癌特征性的细胞株A431为实验对象,通过实验发现靶向上调HOXA9基因的表达,可以抑制肿瘤细胞糖酵解,并抑制肿瘤细胞增殖、迁移和侵袭能力,最终改善或治疗皮肤鳞癌。并进一步扩大发明的使用范围,以肺癌特征性的细胞株A549和宫颈癌特征性的细胞株Hela为实验对象,申请人及团队通过实验发现靶向上调HOXA9基因的表达,可以抑制肿瘤细胞糖酵解,最终达到改善或治疗该类实体肿瘤的目的。
异常的糖代谢是肿瘤细胞的一大特征,由于肿瘤细胞需要进行快速增殖扩增肿瘤体积,这一过程需要大量的底物用以合成蛋白质等大分子产物以及大量的能量作为支持,故而肿瘤细胞更加偏好采用糖酵解的方式代替氧化磷酸化完成肿瘤的快速增长。在本发明研究中发现,通过上调皮肤鳞癌细胞中HOXA9的表达水平,抑制糖酵解,减少了肿瘤细胞的合成代谢,从而在真正意义上抑制其快速增长,达到治疗肿瘤的目的。
抑制肿瘤细胞的增殖可以抑制其增长,而肿瘤细胞的迁移和侵袭能力是恶性肿瘤的浸润和转移主要指标。在本发明研究中发现,通过上调HOXA9表达抑制皮肤鳞癌细胞的迁移和侵袭能力能够减弱其向周围组织和远端器官的浸润和转移,从而降低肿瘤的恶性程度,降低肿瘤复发几率,提高患者的生存率。
本发明首次提出了一种能够最大程度保证器官功能完好及外型美观的基础上治疗CSCC的新方法,就是通过HOXA9基因和/或其编码蛋白应用于治疗皮肤鳞状细胞癌,可以克服现有技术的不足,具有重大意义。
附图说明
图1是HOXA9在CSCC细胞系和临床样本中的表达;以人永生化角质形成细胞HaCaT为对照,HOXA9在CSCC细胞系中的mRNA和蛋白水平的表达;
图2是HOXA9对A431细胞增殖的影响;A表示HOXA9过表达模型建立成功;B过表达HOXA9后检测0h、24h、48h和72h A431细胞增殖能力情况;
图3是HOXA9对A431细胞迁移和侵袭的影响;A为过表达HOXA9后48h Transwell检测A431细胞迁移能力情况(右侧为统计图);B为过表达HOXA9后48h Transwell检测A431细胞侵袭能力情况(右侧为统计图);
图4是HOXA9对A431细胞凋亡的影响;
图5是体内实验验证HOXA9对肿瘤生长的抑制作用;
图6是HOXA9对A431细胞糖酵解的影响;
图7是HOXA9抑制皮肤鳞状细胞癌(CSCC)机制。
具体实施方式
下面结合具体实验,进一步阐述本发明内容。下列实验中未注明具体条件的实验方法,通常按照常规条件,如,《分子克隆实验指南》(第三版)中所述的条件,或按照制造厂商所建议的条件。
实验中所涉及到的细胞系分别为人永生化角质形成细胞HaCaT,人皮肤鳞癌细胞株A431、HSC-1和HSC-5。
实验1 HOXA9在皮肤鳞癌细胞系中呈低表达
采用qPCR和western blot方法检测HOXA9在正常细胞系和皮肤鳞癌细胞系的表达情况与正常皮肤组织和皮肤鳞癌组织中的表达情况。
以人永生化角质形成细胞HaCaT为对照,采用qPCR和western blot方法在皮肤鳞癌细胞株中检测HOXA9mRNA和蛋白(GAPDH为内参)表达水平,结果见图1。图1中A结果显示HOXA9在A431、HSC-1、HSC-5中呈低表达(*p<0.05,**p<0.01,***p<0.001)。采用免疫组化的方法,检测皮肤鳞癌组织中HOXA9的表达情况,图1中B结果显示相比正常皮肤组织,HOXA9在皮肤鳞癌中表达较低。
实验2过表达HOXA9显著抑制皮肤鳞癌细胞增殖
采用lipo2000向A431细胞中转染HOXA9过表达质粒和空载质粒,在A431细胞系中建立HOXA9过表达模型,并采用qPCR和western blot方法检测HOXA9的表达(GAPDH为内参,NC(negative control)为空白对照),从而验证过表达模型的效率。使用CCK-8方法检测HOXA9过表达模型中A431细胞的增殖情况。结果见图2。
图2中A结果显示A431细胞转染HOXA9后HOXA9蛋白表达水平上升。使用CCK-8方法检测HOXA9过表达模型中A431细胞的增殖情况,图2中B结果显示A431细胞转染HOXA9细胞后细胞的增殖能力减弱(*p<0.05,**p<0.01,***p<0.001)。
实验3过表达HOXA9显著抑制皮肤鳞癌细胞迁移及侵袭
使用Transwell小室检测HOXA9过表达模型中A431细胞的迁移和侵袭能力。采用lipo2000向A431细胞中转染HOXA9过表达质粒和空载体质粒,将处理后的A431细胞转种于Transwell小室中,小室中加入无血清培养基,培养板中加入完全培养基,12-16h后用4%甲醛固定细胞,经甲醇透化后使用结晶紫染色观察A431细胞迁移情况,结果见图3。
图3中A结果显示HOXA9过表达后与对照组相比,A431迁移细胞数显著减少,差异具有统计学意义(*p<0.05,**p<0.01,***p<0.001),说明HOXA9可使皮肤鳞癌细胞迁移能力降低。同样地,采用matrigel包被的Transwell小室用上述实验方法检测过表达HOXA9对皮肤鳞癌细胞侵袭能力的影响,图3中B显示HOXA9过表达后与对照组相比,A431侵袭细胞数显著减少,差异有统计学意义(*p<0.05,**p<0.01,***p<0.001),说明HOXA9可使皮肤鳞癌细胞侵袭能力降低。
实验4过表达HOXA9显著促进皮肤鳞癌细胞凋亡
用AnnexinV/PI双染法检测HOXA9过表达对皮肤鳞癌细胞凋亡的影响,实验采用全式金公司AnnexinV-FITC/PI细胞凋亡试剂盒(货号:FA101)并通过流式细胞术检测过表达HOXA9模型中A431细胞的凋亡情况,结果见图4。
图4显示,HOXA9过表达后与对照组相比,A431侵袭细胞数显著减少,差异有统计学意义(*p<0.05,**p<0.01,***p<0.001),说明HOXA9可诱导皮肤鳞癌细胞的凋亡。
实验5裸鼠皮下成瘤实验证实HOXA9可抑制皮肤鳞癌肿瘤的生长
采用裸鼠皮下成瘤实验进一步证实HOXA9对皮肤鳞癌的抑制作用。采用A431细胞皮下注射于免疫缺陷裸鼠(BALB/C-nu/nu,4–5周龄)建立肿瘤模型,并进行HOXA9过表达或对照处理,观察肿瘤的生长情况,在不同的时间点测量和记录裸鼠肿瘤体积大小,最后取实体肿瘤组织,结果见图5。
图5结果显示HOXA9过表达后与对照组相比,裸鼠肿瘤生长减慢,体积明显减小,差异有统计学意义(*p<0.05,**p<0.01,***p<0.001),说明HOXA9可抑制皮肤鳞癌细胞的裸鼠皮下肿瘤的生长,进一步证实HOXA9对皮肤鳞癌细胞的抑制作用。
实验6过表达HOXA9显著抑制皮肤鳞癌细胞糖酵解
采用seahorse细胞外能量代谢检测系统检测了HOXA9过表达模型中A431细胞的糖酵解能力变化情况,结果见图6。
图6中A结果显示HOXA9过表达后与对照组相比,A431细胞的氧消耗速率(OCR)明显增加,说明HOXA9能够促进皮肤鳞癌细胞的氧化磷酸化。图6中B结果显示:与对照组相比,过表达HOXA9处理A431细胞的胞外产酸能力(ECAR)明显减少,说明HOXA9能够抑制皮肤鳞癌细胞的糖酵解,并抑制氧化磷酸化。
综合以上实验可以得出:HOXA9通过抑制肿瘤细胞增殖、迁移和侵袭,促进凋亡,并抑制糖酵解,从而抑制皮肤鳞癌,机制通路如图7所示。
可以预见,通过过表达HOXA9,可以抑制影响肿瘤细胞增殖、转移和侵袭,促进凋亡,并抑制糖酵解,最终改善或治疗皮肤鳞癌。
Claims (10)
1.HOXA9基因和/或其编码蛋白在制备治疗实体瘤的药物中的应用。
2.根据权利要求1所述的所述的应用,其特征在于:实体瘤包括皮肤癌、肺癌、宫颈癌。
3.根据权利要求1或2所述的所述的应用,其特征在于:皮肤癌为皮肤鳞状细胞癌。
4.HOXA9基因和/或其编码蛋白作为皮肤癌的辅助诊断和/或预后判断的敏感性检测新靶点的应用。
5.根据权利要求4所述的应用,其特征在于:皮肤癌为皮肤鳞状细胞癌。
6.一种用于皮肤癌诊断和/或预后判断的试剂盒,其特征在于:该试剂盒含有特异性扩增HOXA9基因DNA链和/或其cDNA链的PCR引物,或抗HOXA9蛋白的抗体。
7.根据权利要求6所述的试剂盒,其特征在于:皮肤癌为皮肤鳞状细胞癌。
8.用于治疗皮肤癌的药物组合物,其特征在于:该药物组合物含有HOXA9基因和/或其编码的蛋白,以及医学上可接受的药用辅料。
9.根据权利要求8所述的药物组合物,其特征在于:皮肤癌为皮肤鳞状细胞癌。
10.特异性扩增HOXA9基因DNA链和/或其cDNA链的PCR引物,或抗HOXA9蛋白的抗体在制备皮肤癌诊断和/或预后判断试剂盒中的应用。
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