CN114689856A - Vgf基因在转移性前列腺癌治疗中的应用 - Google Patents
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Abstract
本发明属于生物医药技术领域，VGF基因在转移性前列腺癌治疗中的应用。VGF基因抑制剂在制备前列腺癌药物中的应用。本发明首次发现VGF可以促进前列腺癌的侵袭和转移，VGF在恶性程度更高的前列腺癌组织中表达更高，抑制VGF能够抑制前列腺癌的发生发展。制备VGF小分子抑制剂siRNA从而治疗前列腺癌以及抑制前列腺癌的转移，抑制PI3K/AKT信号通路。本发明为进一步研究前列腺癌的发病机制以及VGF基因的功能提供了新的思路，为前列腺癌药物的开发提供新的方向。
Description
技术领域
本发明属于生物医药技术领域，VGF基因在转移性前列腺癌治疗中的应用。
背景技术
在全球，约一半患有前列腺癌的男性发生转移性疾病，是男性最常见的癌症之一。据Cancer Statistics2022报告发布，2022年美国将有1918030例新增癌症病例，对于男性而言，仅前列腺癌就占新增癌症的27％，死亡人数仅次于肺癌。随着经济水平提高、人民生活水平改善和人均寿命延长，前列腺癌的发病率呈显著上升趋势，我国大多数前列腺癌患者在初诊时就已经出现转移的现象，一项研究表明，国内新增前列腺癌患者中，54％的患者在诊断时已发生远处转移(包括骨和腹部器官转移)，发生远处转移的患者，5年相对生存率从未转移患者的80％降至30％，无进展生存时间是未转移患者的一半。目前，雄激素剥夺疗法是转移性前列腺癌的主要治疗方法，在初期显示出优异的抗肿瘤作用，例如缓解癌症相关症状、肿瘤缩小和肿瘤相关标志物下降。然而，大多数转移性前列腺癌患者最终会产生去势抵抗并变得致命，因此探索出一些具有应用前景的针对转移性前列腺癌的治疗靶点尤为重要。
根据课题组前期研究，我们进行了转移性前列腺癌与非转移性前列腺癌的转录组测序，筛选出与前列腺癌侵袭转移相关的基因VGF(神经生长因子诱导因子)，该基因能够被神经生长因子上调，其结构组织与大鼠基因相似，翻译区和非翻译区也都与大鼠基因的序列高度相似，编码的蛋白也与分泌粒蛋白/嗜铬粒蛋白家族具有相似之处。VGF基因负责能量平衡和新陈代谢，能够促进神经元生长并防止细胞凋亡，目前已经在多种神经内分泌细胞和癌症细胞中检测到VGF基因的表达。一些研究表明，VGF基因与肺癌、胰腺癌、尿路上皮细胞癌等癌症的进展相关，然而目前为止，关于VGF基因在前列腺癌侵袭转移中的作用和分子机制还未见文献报道。
发明内容
本发明的目的在于，针对现有技术的不足，一方面提供一种以VGF基因作为前列腺癌诊断的分子标志物的用途。另一方面，提供一种以VGF为作用靶标去制备得到的对VGF 具有抑制作用的抑制剂并将其用于制备抗前列腺癌药物。
为了实现上述目的，本发明提供了如下技术方案。
一方面在于提供一种前列腺癌特异性表达的基因，用以标记前列腺癌的发生发展程度，提供VGF作为前列腺癌诊断分子标志物的用途。
另一方面在于提供一种VGF基因抑制剂，其通过抑制前列腺癌的侵袭转移，提供作为抑制前列腺癌转移药物的应用。
进一步地提供一种VGF基因抑制剂，其通过抑制PI3K/AKT信号通路，提供作为抑制PI3K/AKT信号通路药物的应用。
优选的，所述VGF基因具有核苷酸序列。
优选的，所述VGF基因抑制剂可以抑制VGF表达的siRNA序列，所述siRNA的正义链和反义链分别为：
正义链：
5’-CUUCUGCCUUCUGCUGAUCAA-3’(SEQ ID NO.1)；
反义链：
5’-UUGAUCAGCAGUUGGCAGAAG-3’(SEQ ID NO.2)。
进一步地，所述抑制剂包括但不限于：核酸分子、碳水化合物、脂类、小分子、化学药、抗体药、多肽、蛋白或干扰慢病毒。
进一步地，所述抑制剂为任何药物治疗上可接受的剂量。
本发明通过公开数据库分析，发现并验证VGF在前列腺癌中高表达，并与前列腺癌的恶性程度高度相关，通过构建VGF siRNA、shRNA来抑制细胞中VGF的表达，通过 VGF靶向抑制剂抑制前列腺癌细胞的侵袭和转移，进一步地抑制PI3K/AKT信号通路。
与现有技术比，本发明有益效果如下。
本发明首次发现VGF在前列腺癌中高表达，其在恶性程度更高的前列腺癌中表达更高，通过本发明提供的抑制剂能够明显抑制前列腺癌的侵袭和转移，同时能够下调PI3K/AKT信号通路。因此，VGF可以作为前列腺癌新的治疗靶点。为进一步研究前列腺癌的发病机制以及VGF基因的功能提供了新的思路，为前列腺癌药物的开发提供新的方向。
附图说明
图1：图A为TCGA数据库中VGF在正常前列腺组织与前列腺癌组织中的表达情况；图B为TCGA数据库在VGF在前列腺癌不同恶性程度组织中的表达情况；
图2：图A为VGF在前列腺增生组织与前列腺癌组织中蛋白的表达情况；图B为VGF在正常前列腺细胞(RWPE-1)以及前列腺癌细胞(C4-2、22Rv1、PC3)中mRNA的表达情况；图C为VGF在正常前列腺细胞(RWPE-1)以及前列腺癌细胞(C4-2、22Rv1、PC3)中蛋白的表达情况；
图3：图A-B为VGF抑制剂对VGF的mRNA和蛋白的抑制效果；
图4：图A-B为抑制VGF后细胞侵袭迁移能力变化的情况；图C为抑制VGF后细胞中EMT相关标志物蛋白的表达情况；
图5：图A为抑制VGF后PI3K/AKT通路相关蛋白的表达情况；图B为加入AKT激活剂SC79后PI3K/AKT通路相关蛋白的表达情况。
具体实施方式
下面将结合具体实施例对本发明做进一步说明，本发明还可以通过其它不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的前提下进行各种修饰或改变。
VGF在转移性前列腺癌中的用途，本发明通过广泛而深入的研究，发现VGF在前列腺癌组织及细胞中均呈现高表达，接着本发明发现抑制VGF抑制了前列腺癌细胞的侵袭和转移，进一步地，本发明发现抑制VGF可以下调PI3K/AKT信号通路。
下述实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。实施例中涉及的试剂及药品，若无特殊说明，均为普通市售产品。
实施例1.TCGA数据库探究VGF在前列腺癌中表达水平。
通过UALCAN癌症数据库查询TCGA中VGF的表达情况，如图1A-1B所示。
结果显示VGF在前列腺癌中高表达，并与其恶性程度相关。
实施例2.前列腺癌组织及细胞验证。
提取前列腺增生患者、前列腺癌患者组织中的蛋白，通过Western blot检测VGF在组织中的表达量，如图2A所示。
提取前列腺细胞中的RNA及蛋白，通过实时荧光定量PCR和Western blot检测VGF在细胞中的表达量，如图2B-2C所示。
1.Western blot
(1)裂解组织和细胞；
(2)BCA蛋白定量确定上样量及制备蛋白样品；
(3)SDS-聚丙烯酰胺凝胶电泳及转膜；
(4)封闭；
(5)一抗孵育4℃过夜；
(6)室温二抗孵育1h；
(7)ECL发光检测。
2.实时荧光定量PCR
(1)引物序列如下：
VGF上游引物：5’-GGAACTGCGAGATTTCAGTCC-3’
VGF下游引物：5’-GTGCGGGTTTCCGTCTCTG-3’
β-Actin上游引物：5’-GGGACCTGACTGACTACCTC-3’
β-Actin下游引物：5’-ACGAGACCACCTTCAACTCCAC-3’
(2)常规处理细胞48h，且汇合度达到90％后,用Trizol法提取细胞的总RNA,逆转录为 cDNA后用SYBR Green进行实时荧光定量PCR(以β-Actin为内参)；
(3)反应条件如下：95℃预变性3min；95℃变性10s；59℃退火30s；72℃延伸20s；共39 个循环。
结果显示VGF在前列腺癌患者的组织中高表达，正常前列腺细胞RWPE-1低表达，前列腺癌细胞C4-2、22Rv1、PC3均有表达，其中，22Rv1细胞表达最高。
实施例3.VGF抑制剂的制备。
根据VGF核苷酸序列，设计VGF的siRNA和shRNA，如SEQ ID NO.1和SEQ ID NO.2所示。
实施例4.VGF抑制剂的应用。
1.transwell实验
(1)用无血清培养液制备单细胞悬液，细胞密度调整为3×105个/ml；
(2)取400μl加入上室中；在下室即24孔板内加入600μl含20％FBS的培养液；
(3)将24孔板置于37℃、5％CO2细胞培养箱内，培养24小时；
(4)待培养时间结束后，吸尽上室内液体，小心取出上室，用蘸有PBS的湿棉签檫去膜上面未穿过膜的细胞；
(5)自然风干后，向小室内加入无水乙醇，37℃固定20min；
(6)弃尽固定液，结晶紫染色15-20min，清水冲洗；
(7)小室自然风干，将小室置于载玻片上在显微镜下观察、计数。
2.划痕实验
(1)制备单细胞悬液，传代于6孔板中，置于细胞培养箱内培养；
(2)细胞贴壁后(细胞融合度80％)，用灭菌的中号枪头人工划痕(十字)；
(3)弃去孔中培养基；PBS洗两次，弃去悬浮细胞；
(4)加2mL无血清或1％血清培养基；
(5)镜下观察划痕效果，采集不同时间点的白光图片(0h，48h)；
3.Westernblot实验
结果显示，利用已构建好的VGF稳定敲低细胞系进行侵袭迁移实验，证明敲低VGF抑制了前列腺癌细胞的侵袭和迁移能力。
实施例5.VGF对PI3K/AKT信号通路的影响。
Western blot检测敲低VGF后，以及加入AKT通路激活剂SC79后PI3K/AKT信号通路相关蛋白水平。
结果显示，用已构建好的VGF稳定敲低细胞系进行Western blot，证明VGF能够影响AKT的磷酸化水平。
Claims (10)
1.VGF基因在标记前列腺癌转移中的应用。
2.如权利1要求所述的VGF基因在标记前列腺癌转移中的应用，其特征在于，所述VGF基因待检测组织样本中的表达情况是标记前列腺癌转移的指标。
3.如权力1要求所述的VGF基因在标记前列腺癌转移中的应用，其特征在于，所述的VGF基因在待检测组织样本中的表达情况越高，前列腺癌转移的概率越高。
4.VGF基因抑制剂在制备抗前列腺癌药物中的应用。
5.如权利要求4所述的VGF基因抑制剂在制备抗前列腺癌药物中的应用，其特征在于，所述的VGF基因抑制剂为核酸分子，本发明所述核酸分子包括但不限于：双链RNA(dsRNA)、短发夹RNA(shRNA)、反义寡核苷酸(ASO)、小干扰RNA(siRNA)。
6.如权利要求4所述的VGF基因抑制剂在制备抗前列腺癌药物中的应用，其特征在于，所述的VGF基因抑制剂的载体包括逆转录病毒、腺相关病毒、慢病毒、腺病毒、脂质体、囊泡中的任意一种。
7.如权利要求4所述的VGF基因抑制剂在制备抗前列腺癌药物中的应用，其特征在于，所述的VGF基因抑制剂为能够抑制VGF表达的siRNA序列，所述siRNA的正义链和反义链分别为：
正义链：
5’-CUUCUGCCUUCUGCUGAUCAA-3’(SEQ ID NO.1)；
反义链：
5’-UUGAUCAGCAGUUGGCAGAAG-3’(SEQ ID NO.2)。
8.如权利要求4所述的VGF基因抑制剂在制备抗前列腺癌药物中的应用，其特征在于，所述的VGF基因抑制剂是以VGF为作用靶标制备得到的对VGF具有抑制作用的抑制剂。
9.如权利要求4所述的VGF基因抑制剂在制备抗前列腺癌药物中的应用，其特征在于，所述的药物是抑制前列腺癌侵袭转移的药物。
10.如权利要求4所述的VGF基因抑制剂在制备抗前列腺癌药物中的应用，其特征在于，所述的药物针对PI3K/AKT信号通路。
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