CN112691195B - Prpf8表达抑制剂在制备治疗肺癌的药物中的应用 - Google Patents
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Abstract
PRPF8表达抑制剂在制备治疗肺癌的药物中的应用，及生物技术领域，尤其涉及一种PRPF8表达抑制剂在制备治疗肺癌的药物中的应用。本发明研究发现，通过在非小细胞肺癌细胞系中敲减PRPF8可抑制细胞的生长，抑制细胞克隆形成能力，因此在非小细胞肺癌中PRPF8可作为新型的抑制剂，用于临床对非小细胞肺癌的治疗。
Description
技术领域
本发明涉及生物技术领域，尤其涉及一种PRPF8表达抑制剂在制备治疗肺癌的药物中的应用。
背景技术
肺癌是常见的恶性肿瘤，非小细胞肺癌(NSCLC)是肺癌的一种，约占80％-85％，仅次于女性乳腺癌。非小细胞肺癌的治疗方案主要以化疗为主。到目前为止，顺铂仍然是NSCLC治疗的最广泛的一线化疗药物。但是，治疗效果并不显著，根本原因是并没有从蛋白产生的源头开始寻找靶基因。
PRPF8蛋白约280kDa，是U5 snRNP剪接体装配动力学的中心。它不仅在RNA底物中与5'的分支点和3'剪切位点中的多嘧啶区域形成直接接触。同时也参与了U5和U6snRNAs组成。在人类常染色体显性遗传性视网膜炎中发现，PRPF8可产生影响剪切体组装和功能的突变，引起局部疾病，导致细胞减少。
目前，PRPF8在肺癌发生、迁移和侵袭中的功能未见相关报道。
发明内容
本发明的目的是提供一种PRPF8表达抑制剂在制备治疗肺癌的药物中的应用。
本发明PRPF8表达抑制剂在制备治疗肺癌的药物中的应用。
优选的，所述肺癌为非小细胞肺癌。
进一步的，所述PRPF8表达抑制剂包括下述至少一种：
特异性抑制PRPF8表达的化合物；
特异性干扰PRPF8表达的干扰分子；
特异性与PRPF8蛋白结合的抗体或配体。
PRPF8基因敲减试剂，所述PRPF8基因敲减试剂为特异性敲减PRPF8的基因编辑试剂。
进一步的，所述干扰分子为miRNA或siRNA。
进一步的，所述PRPF8基因敲减试剂包括一种表达载体，所述表达载体包括一种shRNA片段的DNA编码序列，所述shRNA片段的靶基因为PRPF8基因。
优选的，所述shRNA片段的序列如序列表中SEQ ID NO：1所示。
优选的，所述表达载体为质粒载体或病毒载体。
本发明还提供一种治疗肺癌的药物，包括一种PRPF8基因敲减试剂，所述PRPF8基因敲减试剂包括一种shRNA片段，所述shRNA片段的靶基因为PRPF8基因。
优选的，所述shRNA片段的序列如序列表中SEQ ID NO：1所示。
优选的，所述肺癌为非小细胞肺癌。
本发明还提供一种试剂在制备试剂盒中的应用，所述试剂用于定量检测PRPF8蛋白表达水平，所述试剂盒用于判断药物治疗肺癌的有效性。
优选的，所述肺癌为非小细胞肺癌。
本发明的有益效果：
人类PRPF8的功能缺失突变延迟了剪接体组分对前mRNA的组装，并且抑制了9％(5/57)受试基因的剪切，产生功能不同的蛋白，对疾病的发生和肿瘤的生长具有重要的作用。
本发明通过在非小细胞肺癌细胞系中敲减PRPF8可抑制细胞的生长，抑制细胞克隆形成能力，因此在非小细胞肺癌中PRPF8可作为新型的抑制剂，用于临床对非小细胞肺癌的治疗。
通过抑制该基因的表达量可抑制非小细胞肺癌的细胞生长，可成为临床上治疗非小细胞肺癌的药物。
附图说明
图1为敲降细胞系荧光鉴定结果；
图2为PRPF8敲降细胞系WB鉴定结果；
图3为KEGG富集分析结果；
图4为A549敲降PRPF8细胞生长曲线结果；
图5为A549敲降PRPF8细胞克隆形成结果；
图6为imageJ分析图5克隆形成结果；
图7为MDA-MB-468敲降PRPF8细胞生长曲线；
图8为MDA-MB-468敲降PRPF8细胞克隆形成结果；
图9为imageJ分析图8克隆形成结果。
具体实施方式
下面对本发明的实施例做详细说明，以下实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方案和具体的操作过程，但本发明的保护范围不限于下述的实施例。
实施例1、生物信息学分析PRPF8在肺癌中的表达
利用数据库对PRPF8在肺癌中的表达进行分析，通过KEGG等数据库对PRPF8进行富集分析，关注肺癌中PRPF8调控的通路，分析PRPF8在肺癌中的主要作用途径。
实施例2、敲降细胞系的建立
根据PRPF8序列设计合成2条shRNA，如下表所示，连入p-GIPZ和p-TRIPZ载体上(购买自美国Addgene生物技术有限公司,USA)。
表shRNA序列
实施例3、慢病毒制备
(1)293T细胞接种25cm小瓶，过夜培养后弃培养基，换2.5mL DMEM无抗培养基；
(2)将质粒pPAX2、pMD2、GIPZ-CTRL；质粒pPAX2、pMD2、GIPZ-PRPF8-1；质粒pPAX2、pMD2、GIPZ-PRPF8-4混匀后，加入250μL opti-MEM培养基，再用250μL opti-MEM培养基稀释Lipo2000，室温放置5min后，将质粒与Lipo2000混合，室温放置20min，加入293T细胞中；
(3)5％CO2培养18h后换DMEM培养基；
(4)继续培养48h后回收上清，并用0.45μM滤膜过滤；
(5)加入1/3体积的病毒浓缩液(25％PEG8000/0.75M NaCl)，混匀后4℃过夜；
(6)然后于4℃、1500g离心45min，弃上清，沉淀用1640培养基溶解，分别得到GIPZ-CTRL、GIPZ-PRPF8-1、GIPZ-PRPF8-4病毒上清。分装，-80℃保存。
实施例4、细胞转染
(1)嘌呤浓度的筛选
将A549细胞系用胰酶消化3分钟，用培养基重悬成单细胞悬液，调整细胞密度为每孔105个细胞，培养30小时后，换成含嘌呤不同浓度的培养基，终浓度分别为：0.25μg/ml、0.4μg/ml、0.6μg/ml、0.75μg/ml、1.0μg/ml，每隔24h换液一次，连续培养7天，观察到细胞死亡的浓度，即最适嘌呤霉素浓度。
(2)病毒转染
A549细胞接种6孔板，每孔细胞2×105个细胞，过夜培养；24h后弃培养基，用PBS洗一次，加入1.2mL无抗、无血清含polybrene的新鲜培养基；加入GIPZ-CTRL、GIPZ-PRPF8-1、GIPZ-PRPF8-4病毒上清；32℃2000rpm离心1h；37℃5％CO2培养5h后，每孔加入1mL完全培养基；培养24h后弃培养基，加入含puromycin(终浓度0.75μg/mL)新鲜培养基，每三天换液一次；培养5天后收细胞，提RNA。
实施例5、q-PCR鉴定
将细胞接种6孔板，每孔2×105个细胞，过夜培养后弃培养基，加入新鲜的含puromycin培养基，每天换液一次，连续3天后收细胞，提取RNA反转录成cDNA。进行q-PCR。
q-PCR反应体系如下：
实时定量PCR反应程序：预变性94℃5min，95℃10sec，60℃20sec，72℃10sec，共40个循环。
实施例6、细胞增殖检测
A549细胞6孔板每孔接种2×105个/孔，加入GIPZ-CTRL、GIPZ-PRPF8-1、GIPZ-PRPF8-4病毒，嘌呤霉素puromycin 0.75μg/mL筛选5天后，收细胞接种96孔板，每孔1×104个细胞，每个细胞系设6个重复孔，连续培养7天，每天用CCK8试剂盒测OD值。
实施例7、细胞克隆形成实验
A549细胞6孔板每孔接种2×105个/孔，加入GIPZ-CTRL、GIPZ-PRPF8-1、GIPZ-PRPF8-4病毒，嘌呤霉素puromycin筛选5天后，调整细胞数目，6孔板每孔接种500个/孔，每个细胞系3个样本重复，培养7天后，弃培养基，用PBS洗涤细胞2次后，每孔加入1mL甲醇固定细胞20min，弃甲醇PBS洗涤2次，每孔加入1mL 1％结晶紫溶液，室温染色20min后，PBS洗涤2次，ddH2O洗涤一次，凝胶成像系统拍照。
为了验证PRPF8对肺癌细胞生长的抑制作用是仅限于对肺癌细胞内的作用机制，我们采用同样的方法和同一批制备的病毒侵染乳腺癌MDA-MB-468细胞，在乳腺癌细胞中敲降PRPF8检测细胞的生长能力。
敲降细胞系荧光鉴定结果如图1所示，左侧为TRIPZ-shCTRL Doxycycline诱导72h；中间为TRIPZ-shPRPF8-1 Doxycycline诱导72h，右侧为TRIPZ-shPRPF8-4Doxycycline诱导72h。PRPF8敲降细胞系WB鉴定结果如图2所示。
检测结果说明：
1、图3为KEGG富集分析结果，通过KEGG对肺癌中PRPF8表达的分析得出，在肺癌中PRPF8与多条通路密切相关，其中包括PI3K-Akt信号通路、顺铂耐药、小细胞肺癌、同源重组等等，在这些调控通路中PI3K-Akt信号通路最为显著，PI3K-Akt信号通路是典型的细胞生长调控通路，调节细胞生长通路，因此提示PRPF8与肺癌细胞的生长有关。
2、为了研究PRPF8在非小细胞肺癌中的作用，我们在A549细胞中建立了PRPF8敲降细胞系。p-TRIPZ-shPRPF8-1、p-TRIPZ-shPRPF8-4、p-RIPZ-shCTRL慢病毒转染A549细胞，嘌呤霉素puromycin 0.75μg/mL筛选5天后，收取细胞提RNA，qPCR检测A549-TRIPZ-PRPF8-1、A549-TRIPZ-PRPF8-4、A549-TRIPZ-CTRL细胞系中PRPF8mRNA表达降低了1.5和1.63倍。
3、敲降PRPF8抑制A549细胞生长
为了验证PRPF8在肺癌细胞生长中的作用，我们在A549细胞构建的敲降细胞系中用CCK-8试剂盒检测细胞的增殖，用1％结晶紫染色观察细胞的克隆形成能力，结果显示A549细胞敲降PRPF8后，细胞增殖能力受到抑制，A549细胞敲降PRPF8后细胞的克隆形成能力下降(p<0.05)，凋亡细胞增多，细胞生长变缓慢。
A549敲降PRPF8细胞生长曲线如图4所示，图4中■表示CTRL，●表示PRPF8-1，▲表示PRPF8-4。在A549细胞中沉默PRPF8显著降低了细胞克隆形成的能力。
A549敲降PRPF8细胞克隆形成结果如图5所示，在A549细胞中敲降PRPF8后细胞克隆形成能力降低。
imageJ分析克隆形成结果如图6所示，敲降PRPF8后shPRPF8细胞克隆形成能力显著降低(p<0.05)。
4、乳腺癌细胞系中敲降PRPF8实验
MDA-MB-468敲降PRPF8细胞生长曲线如图7所示，图7中◆表示CTRL，■表示PRPF8-1，▲表示PRPF8-4。MDA-MB-468敲降PRPF8细胞克隆形成结果如图8所示。imageJ分析克隆形成结果如图9所示。
MDA-MB-468乳腺癌细胞系中敲降PRPF8后，监测5天细胞生长曲线，结果显示与对照组相比，PRPF8敲降组细胞生长无差异(p>0.05)，细胞克隆形成试验中，敲降组细胞克隆形成能力降低不显著(p>0.05)，因此证明PRPF8调控细胞生长的能力是在肺癌中特有的，在其他癌症中不具有同样的调控能力。每种癌症因为发病器官不同，产生的机制不同，癌细胞内的调控途径也不同，因此这种调控具有器官异质性，肿瘤异质性。
5、为了进一步证明PRPF8在肺癌中调控细胞生长的能力是特有性的，我们利用数据库对乳腺癌中PRPF8的表达与富集进行分析，结果发现PRPF8与多条通路密切相关，其中PRPF8与9条通路具有显著相关性(p-value<0.05),这9条通路涉及核糖体、氧化应激反应、帕金森综合征、心肌收缩、亨延顿综合征、老年痴呆症、p450的代谢以及蛋白酶体等9方面的调控，但与细胞生长调控无关。
以上得出的实验结果证实，在非小细胞肺癌中敲除PRPF8可抑制癌细胞的生长，因此针对PRPF8合成的抑制剂能高效的抑制体内癌细胞的生长，降低肿瘤细胞的生存，是临床有效的治疗药物。
Claims (6)
1.PRPF8表达抑制剂在制备治疗肺癌的药物中的应用；所述肺癌为非小细胞肺癌A549。
2.根据权利要求1所述的应用，其特征在于所述PRPF8表达抑制剂包括下述至少一种：
特异性抑制PRPF8表达的化合物；
特异性干扰PRPF8表达的干扰分子；
特异性与PRPF8蛋白结合的抗体或配体；
PRPF8基因敲减试剂，所述PRPF8基因敲减试剂为特异性敲减PRPF8的基因编辑试剂。
3.根据权利要求2所述的应用，其特征在于所述PRPF8基因敲减试剂包括一种表达载体，所述表达载体包括shRNA片段的DNA编码序列，所述shRNA片段的靶基因为PRPF8基因。
4.根据权利要求3所述的应用，其特征在于所述shRNA片段的序列如序列 PRPF8-1 和序列 PRPF8-4 所示。
5.根据权利要求3所述的应用，其特征在于所述表达载体为质粒载体或病毒载体。
6.试剂在制备试剂盒中的应用，其特征在于所述试剂用于定量检测PRPF8蛋白表达水平，所述试剂盒用于判断药物治疗肺癌的有效性，所述肺癌为非小细胞肺癌A549。
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