CN116536429B - c8orf76在制备肺腺癌靶向药物中的应用 - Google Patents
c8orf76在制备肺腺癌靶向药物中的应用 Download PDFInfo
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Abstract
本发明属于癌症治疗技术领域，具体涉及c8orf76在制备肺腺癌靶向药物中的应用。本发明要解决的技术问题是为肺腺癌治疗提供一种新选择。本发明的技术方案是c8orf76在制备肺腺癌靶向药物中的应用，其特征在于：c8orf76的核苷酸序列如SEQ ID NO.1所示。本发明设计出能够特异性敲低c8orf76表达siRNA，以及特异性识别c8orf76的引物对，并提出其在肺腺癌靶向药物研发与预后评估中的应用。
Description
技术领域
本发明属于癌症治疗技术领域，具体涉及c8orf76在制备肺腺癌靶向药物中的应用。
背景技术
肺癌是目前最常见的人类恶性肿瘤之一，位居全球癌症死亡率之首。非小细胞肺癌是肺癌的主要病理类型，约占肺癌确诊病例的85％，其中肺腺癌约占原发肺癌40％，是最常见的非小细胞肺癌。尽管近年来新的治疗方法不断出现，但肺腺癌患者的预后仍不尽人意，5年生存率不超过20%。因此，发现新的与肺腺癌发生、发展密切相关的调控基因，对于进一步探究肺腺癌复杂的肿瘤生物学特性以及促进肺腺癌靶向治疗策略的发展具有重要意义。
小干扰RNA(Small interfering RNA，siRNA)是一种小分子RNA，通常由内切核糖核酸酶Dicer切割双链RNA而成，长度为21个核苷酸。在转染试剂的作用下，siRNA能够进入细胞并形成RNA诱导沉默复合体。该复合体能够识别含有与其互补序列的mRNA，并切割该mRNA使其降解，从而阻止该基因的转录产物翻译成蛋白。因此，siRNA常被用于研究某个基因在生理或病理进程中的作用，以及相应的靶向药物研发。为了避免siRNA脱靶效应对研究结果的不良影响，需要设计出针对靶点mRNA特异且高效的siRNA。
人c8orf76，为8号染色体开放阅读框76，位于第8q24.13号染色体上，是一种核蛋白编码基因。
发明内容
本发明要解决的技术问题是为肺腺癌治疗提供一种新选择。
本发明提供了c8orf76在制备肺腺癌靶向药物中的应用，所述c8orf76的核苷酸序列如SEQ ID NO .1所示。
本发明进一步提供了一种肺腺癌靶向药物，所述药物为敲低c8orf76表达的物质。
进一步的，所述敲低c8orf76表达的物质为靶向c8orf76的siRNA。
其中，所述siRNA的靶序列如SEQ ID NO .2所示。
具体的，针对c8orf76的siRNA为siRNA-c8orf76，其具有以下特征中的至少一项：
a、siRNA-c8orf76的正义链的核苷酸序列如SEQ ID NO .7；
b、siRNA-c8orf76的正义链的核苷酸序列如SEQ ID NO .8。
特别的，所述药物还包括药学上可接受的辅料或者辅助性成分。
本发明提供了c8orf76在制备肺腺癌预后评估的生物标志物中的应用，所述c8orf76的核苷酸序列如SEQ ID NO .1所示。
本发明还提供了检测c8orf76表达水平的物质在制备肺腺癌预后评估试剂盒中的应用， 所述c8orf76的核苷酸序列如SEQ ID NO .1所示。
具体的，所述检测c8orf76表达水平的物质为扩增c8orf76的引物。
进一步的，所述引物的核苷酸序列如SEQ ID NO .3和SEQ ID NO .4所示。
特别的，所述试剂盒还包括RNA提取试剂、PCR缓冲液、PCR酶、dNTP中的至少一种。
本发明还提供了针对c8orf76的siRNA，其靶序列如SEQ ID NO .2所示。
进一步的，针对c8orf76的siRNA为siRNA-c8orf76，其具有如下特征中的至少一项：
a、siRNA-c8orf76的正义链的核苷酸序列如SEQ ID NO .7；
b、siRNA-c8orf76的正义链的核苷酸序列如SEQ ID NO .8。
本发明的有益效果：本发明研究发现应用siRNA特异性敲低c8orf76表达水平能够显著抑制肺腺癌细胞的增殖、迁移与侵袭，并发现c8orf76高表达预示着肺腺癌患者总生存期的缩短。据此，本发明设计出能够特异性敲低c8orf76表达siRNA，以及特异性识别c8orf76的引物对，并提出其在肺腺癌靶向药物研发与预后评估中的应用。基于本发明的技术方案，为肺腺癌的治疗提供了一种新选择和新的可能性。
附图说明
图1为本发明应用肿瘤与癌症基因组图谱项目（TCGA）数据库分析c8orf76高表达与低表达的肺腺癌患者之间总生存期之间的差异示意图，用对数秩检验（Log-rank检验）两组患者之间预后差异是否具有统计学意义。
图2为本发明siRNA特异性敲低A549细胞中c8orf76表达水平示意图，用T检验（Student’s t检验）验证组间差异的统计学意义；CK，空白对照组；NC，阴性对照组；siRNA，敲低A549细胞中c8orf76表达组。
图3为本发明特异性敲低c8orf76表达的siRNA转染至A549细胞，应用CCK-8实验检测c8orf76敲低对A549细胞增殖能力的影响示意图。
图4为本发明siRNA特异性敲低A549细胞中c8orf76表达水平后，应用划痕实验及侵袭实验检测c8orf76表达下调对A549细胞迁移与侵袭能力的影响示意图。
具体实施方式
基于肿瘤与癌症基因组图谱项目（TCGA）公开数据库中的数据，发明人从中筛选了大量患者的信息，分析了c8orf76的表达水平在肺腺癌患者体内中的差异。发现与c8orf76低表达的肺腺癌患者相比，c8orf76高表达的肺腺癌患者的总生存期明显缩短。因此可见，c8orf76能够用于评估肺腺癌患者总生存期。
为了进一步研究c8orf76在肺腺癌中能发挥的作用，发明人分析了c8orf76的序列，从诸多的候选区域选择了合适序列作为靶序列，设计了针对c8orf76的siRNA。将设计好的siRNA-c8orf76进行了体外转染实验。通过CCK-8实验验证c8orf76 mRNA敲低会降低A549细胞的增殖能力。应用划痕实验及侵袭实验证明了c8orf76 mRNA表达下调，降低了A549细胞迁移与侵袭能力。
下面结合实施例对本发明的技术方案作进一步描述，但是本发明的保护范围并不仅限于此。实施例中涉及的试剂与材料，若无特殊说明，均为普通市售产品。
人源肺腺癌细胞系A549细胞购自中国科学院典型培养物保藏委员会细胞库。
实施例1 应用TCGA数据库分析肺腺癌患者肿瘤组织中c8orf76表达水平对其预后的影响
在TCGA网站(https://portal.gdc.cancer.gov/)中下载502例肺腺癌患者的预后信息，按照c8orf76表达水平中位数将肺腺癌患者分为高、低表达两组，应用乘积极限法（Kaplan-Meier法）分析两组患者总生存期的差异，c8orf76低表达组共251例肺腺癌患者，其中位生存期45.6个月；c8orf76高表达组包含251例肺腺癌患者，其中位生存期58.3个月，Log-rank 检验显示组间生存差异具有统计学意义。具体分析结果如图1所示，与c8orf76低表达的肺腺癌患者相比，c8orf76高表达的肺腺癌患者的总生存期明显缩短。因此，c8orf76能够用于评估肺腺癌患者总生存期。
实施例2用siRNA-c8orf76特异性干扰A549细胞中c8orf76表达水平
(1)针对c8orf76的编码区域，设计能够特异结合并诱导其剪切降解的siRNAc8orf76，其靶向序列为5’-acctggcctacagacgacaagagta-3’(SEQ ID NO .2)，siRNA-c8orf76（正义链SEQ ID NO .7：5’ accuggccuacagacgacaagagua 3’，反义链SEQ ID NO.8：5’ uacucuugucgucuguaggccaggu 3’） 和阴性对照siRNA-NC（正义链SEQ ID NO .9：5’accgcuacagacgacaagauggua 3’，反义链SEQ ID NO .10：5’ uaccaucuugucgucuguaggcggu3’）均均由上海和元生物公司合成。
SEQ ID NO .1 c8orf761
gcttcctcgt tgcccccgcc gcgggcgcga gatggattcc gggtgctggt tgttcggcggcgagttcgag gactcggtgt tcgaggagag gccggagcgg cggtcaggac cgcccgcgtc ctactgcgccaagctctgcg agccgcagtg gttttatgaa gaaacagaaa gcagtgatga tgttgaagtg ctgactctcaagaaattcaa aggagacctg gcctacagac gacaagagta tcagaaagca ctgcaggagt attccagtatctctgaaaaa ttgtcatcaa ccaattttgc catgaaaagg gatgtccagg aaggtcaggc tcggtgtctggctcacctgg gtaggcatat ggaggcgctg gagattgctg caaacttgga aaataaagca accaacacagaccatttaac cacggtactc tacctccagc ttgctatttg ttcaagtttg cagaacttgg agaaaacaattttctgcctg cagaaactga tttctttgca tccttttaat ccttggaact ggggcaaatt ggcagaggcttacctgaatc tggggccagc tctttcagca gcacttgcgt catctcagaa acagcacagt ttcacctcaagtgacaaaac tatcaaatcc ttctttccac actcaggaaa agactgtctt ttgtgttttc ctgaaaccttgcctgagagc tctttatttt ctgtggaagc gaatagcagt aatagccaga aaaatgagaa agctctgacaaatatccaaa actgtatggc agaaaagaga gaaacagtgt tgatagagac tcagctgaaa gcatgtgcctcttttatacg aaccaggctt ctgcttcagt ttacccaacc tcagcaaaca tcgtttgctt tggagaggaacttaaggact cagcaggaaa ttgaagataa aatgaaaggg ttcagcttca aagaagacac tttgctgttgatagctgagg ttatgggaga agatatccca gaaaaaataa aagatgaagt tcacccagag gtgaagtgtgttggctccgt agccctgact gccttggtga ctgtatcctc agaagaattt gaagacaagt ggttcagaaagatcaaagac catttctgtc catttgaaaa tcagttccat acagagatac aaatcttggc ttagtgggttataaaaaaca aaaccacaaa tatcttgtac tgtattaatt gtccttgttt acttcagaca ggatccattgctaatcatgg agtataaatg attatttatg ttttataaaa ctggcttctg tctcaaatga
(2)选取A549细胞，待其生长密度达到80％时，将siRNA-c8orf76 和阴性对照siRNA-NC分别转染至A549细胞中，所用转染试剂为Lipo8000™转染试剂（碧云天公司），转染48小时后，收集细胞并提取RNA，所用试剂盒为Trizol提取试剂盒(invitrogen公司)。A549细胞扩培。待细胞长满后调整细胞浓度为5×105细胞/孔，接种到六孔板内进行培养，使第二天细胞密度能达到约80%。在进行下述转染步骤前，把培养有细胞的六孔板每孔换成2mL新鲜培养液(含有血清和抗生素的完全培养液)。对于待转染的六孔板中每一个孔的细胞，加入125μL不含抗生素和血清的DMEM培养液，加入100 pmol的siRNA-c8orf76 和阴性对照siRNA-NC，并用枪轻轻吹打混匀；再加入4μL Lipo8000™转染试剂，用枪轻轻吹打混匀，配制完成后，室温存放6h内稳定。按照六孔板每孔125μL Lipo8000™转染试剂-siRNA混合物的用量，均匀滴加到整个孔内，随后轻轻混匀。继续培养48h后，收集细胞并提取总RNA，所用试剂盒为Trizol提取试剂盒(invitrogen公司)。
（3）以步骤(2)所提RNA为模板先去除基因组DNA，再进行逆转录合成cDNA产物；以cDNA产物为模板，应用SEQ ID NO .3和SEQ ID NO .4所示引物对进行荧光定量PCR，所用试剂盒为SYBRGreen PCR试剂盒（Thermo公司），PCR仪器为ABI公司荧光定量PCR仪器。c8orf76相对表达水平的计算公式为2-ΔΔCT ；
其中，扩增c8orf76引物对的核苷酸序列如下：上游引物：5’ aattggcagaggcttacc3’(SEQ ID NO .3)；下游引物：5’ gctattcgcttccacaga 3’(SEQ ID NO .4)。
Gapdh表达水平为内参，引物对的核苷酸序列如下：上游引物：5’ggagcgagatccctccaaaat 3’ (SEQ ID NO .5)，下游引物：5’ggctgttgtcatacttctcatgg 3’(SEQ ID NO .6)。
Real time-PCR反应体系，根据Real time-PCR反应体系配制反应液。在PCR反应管中分别加入ddH2O、SybrGreen qPCR Master Mix、正向引物、反向引物、cDNA模板，充分混匀。
扩增条件：94℃ 10 min，(94℃ 20秒, 55℃ 20秒, 72℃ 20秒) 40个循环。
上述试剂均来自逆转录试剂盒(Thermo公司)与 SYBRGreen PCR试剂盒(Thermo公司)。
荧光定量PCR的结果如图2所示，siRNA-c8orf76能够有效地特异性敲低A549细胞中c8orf76表达水平。
实施例3 应用CCK-8实验检测敲低c8orf76表达对A549细胞增殖能力的影响
(1)将siRNA-c8orf76和siRNA-NC分别转染至A549细胞中，具体方法见实施例1，转染后24小时，收集细胞，将细胞种于96孔板中，每孔含2500个细胞，每个细胞设置5个复孔，重复4组；
(2)在细胞铺板后2～4小时，加入CCK-8试剂(TargetMol，C0005)10μL，避光条件下37℃孵箱中孵育1小时，震荡后使用酶标仪测量450nm的吸光度值，此为起始读数；
(3)在首次测量细胞吸光度值后的24、48和72小时分别向六孔板中加入CCK-8试剂(TargetMol，C0005)，每孔10μL，简单震荡，使用酶标仪测量测量并记录上述孔板中450nm的吸光度值，以及计算细胞生长1天，2天，3天后的数量。
具体结果如图3所示。 由图3可知，应用siRNA-c8orf76特异性敲低c8orf76表达水平能够显著抑制肺腺癌细胞A549的增殖能力。
实施例4 检测c8orf76表达下调对A549细胞迁移与侵袭能力的影响
（1）选取A549细胞，分别转染siRNA-c8orf76和siRNA-NC，具体方法见实施例1，转染后24小时，收集细胞，离心保留细胞沉淀；
（2）细胞迁移：先用记号笔在3.5cm皿背后用直尺比着，均匀地划横线，横线大约隔0.5~1cm；将已转染好的各组细胞用0.25%胰酶分别消化计数约为5×105个细胞，接种于3.5cm皿，37℃、5%CO2饱和湿度条件培养过夜 ；待细胞密度达到90%左右，铺满3.5 cm皿底部，用200 μL的枪头比着直尺，尽量垂直于背后的横线划痕，枪头要垂直，不要倾斜；用PBS洗细胞3次，去除划下的细胞；按照实验分组，在37 ℃、5% CO2条件下培养；48h显微镜下拍照观察细胞迁移情况。
（3）细胞侵袭：将细胞调整细胞密度1为2×105细胞/mL、密度2为1×105细胞/mL；
人工基底膜制备：取出-20 ℃保存的基质膜，将其在4 ℃下过夜解冻，以无血清DMEM培养液按照体积3︰1稀释基质膜；将Boyden小室放置到24孔培养板，形成上下两室。将制备的人工基底膜100 μL加入每个Boyden小室的上室，37 ℃孵育2~6 h使其呈凝胶状；于上室加入300 μL无血清细胞悬液，下室内加入600 μL的含有血清的培养基后，37 ℃，5%CO2条件下培养48h；取出小室，吸弃上室液体，用棉签仔细擦净膜上未侵袭的细胞以及人工基底膜胶，37 ℃ 预温的PBS液漂洗两次，用冰预冷的4%多聚甲醛固定30 min，结晶紫染色10 min；小心将聚碳酯膜自上室基底切取下来，封片后在显微镜下计数浸润到小室背面的细胞，光镜随机视野拍照。
由图4可知，应用siRNA-c8orf76特异性敲低c8orf76表达水平能够显著抑制A549细胞的迁移（4A）与侵袭（4B）。
以上所述实施例仅为本发明优选的具体实施方案，并不用来限制本发明，凡是在本发明精神和原则之内，所作的任何修改、等同替换以及改进等，均应包含在本发明的保护范围之内。
Claims (4)
1.c8orf76 的siRNA在制备肺腺癌靶向药物中的应用，其特征在于：c8orf76 的核苷酸序列如SEQ ID NO .1 所示。
2.根据权利要求1 所述应用，其特征在于：所述siRNA 的靶序列如SEQ ID NO .2 所示。
3.根据权利要求1 所述应用， 其特征在于： 针对c8orf76 的siRNA 为siRNA-c8orf76，其具有以下特征中的至少一项：
a、siRNA-c8orf76 的正义链的核苷酸序列如SEQ ID NO .7；
b、siRNA-c8orf76 的反义链的核苷酸序列如SEQ ID NO .8。
4.检测c8orf76 表达水平的物质在制备肺腺癌预后评估试剂盒中的应用，其特征在于：所述c8orf76 的核苷酸序列如SEQ ID NO .1 所示；所述检测c8orf76 表达水平的物质为扩增c8orf76 的引物，引物的核苷酸序列如SEQ ID NO .3 和SEQ ID NO .4 所示。
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