CN116162621A - Dna双链序列及应用和肺腺癌细胞增殖抑制剂或在肺腺癌药物 - Google Patents

Dna双链序列及应用和肺腺癌细胞增殖抑制剂或在肺腺癌药物 Download PDF

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CN116162621A
CN116162621A CN202111410962.0A CN202111410962A CN116162621A CN 116162621 A CN116162621 A CN 116162621A CN 202111410962 A CN202111410962 A CN 202111410962A CN 116162621 A CN116162621 A CN 116162621A
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许国旺
耿鹏宇
秦望舒
罗圆媛
林志坤
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Dalian Institute of Chemical Physics of CAS
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Abstract

本发明公开了DNA双链序列及应用和肺腺癌细胞增殖抑制剂或在肺腺癌药物。本发明针对HPRT1设计了表达shRNA的DNA双链序列，并构建了包含上述DNA双链序列的慢病毒载体质粒。通过实验证实本发明的HPRT1shRNA构建物能够显著降低HPRT1mRNA和蛋白水平，敲降作用非常明显。鉴于HPRT1shRNA构建物能够显著降低肺腺癌细胞的生长，因此本发明的HPRT1shRNA构建物可作为一种肺腺癌治疗的潜在靶点。

Description

DNA双链序列及应用和肺腺癌细胞增殖抑制剂或在肺腺癌 药物
技术领域
本发明属于分子生物学领域，具体涉及作用于人HPRT1基因的shRNA质粒构建及其用途。
背景技术
肺癌死亡率位居恶性肿瘤之首，肺癌早期干预和治疗刻不容缓。非小细胞肺癌(Non-small cell lung cancer,NSCLC)约占肺癌发病率的80％，其治疗包括传统的手术和放化疗，新兴技术包括介入、靶向治疗及免疫治疗等。尽管治疗方式在持续更新，但NSCLC患者预后仍不太理想，生存率无明显改善，5年生存率不足20％。肺腺癌是NSCLC的主要类型，研究发现肺腺癌的发病率呈逐年递增趋势。因此对于肺腺癌发生发展作用机制的探索一直是广大科研工作者研究的热点问题，对于提高肺腺癌的生存率具有重要意义。
次黄嘌呤磷酸核糖基转移酶(HPRT)是嘌呤核苷酸补救合成途径中的关键酶，可催化次黄嘌呤与鸟嘌呤，使其向各自的单核苷酸转化。利用免疫组织化学法比较人类肺、乳腺、结肠和前列腺来源的正常组织和恶性肿瘤组织中HPRT的表达水平，研究者发现肿瘤组织中HPRT的表达上调，且与肺癌的临床分期较晚有关。截至目前，HPRT在肺腺癌中的生物学功能和作用机制尚无报道。如果HPRT1能成为潜在的肺腺癌治疗靶点，采用HPRT1的shRNA或抑制剂等方法敲降HPRT1的表达在临床上用于肺腺癌的治疗。
发明内容
本发明的目的旨在提供一种作用于人HPRT1基因的shRNA及其用途。
本发明提供了一种用于敲降HPRT1基因的DNA双链序列，所述的DNA双链序列包含以下序列:
上游链：
5’-CCGGCCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGGTTTTTG-3’
下游链：
5’-AATTCAAAAACCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGG-3’
本发明还提供了一种构建物，所述的构建物包含以下DNA双链序列:
上游链：
5’-CCGGCCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGGTTTTTG-3’
下游链：
5’-AATTCAAAAACCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGG-3’
所述的构建物为慢病毒载体。
本发明还提供了所述的DNA双链序列或所述的构建物在抑制肺腺癌细胞增殖中的应用。
通过实验证实本发明的HPRT1 shRNA构建物能够显著降低HPRT1 mRNA和蛋白水平，敲降作用非常明显。鉴于HPRT1 shRNA构建物能够显著降低肺腺癌细胞的生长，因此本发明的HPRT1 shRNA构建物可作为一种肺腺癌治疗的潜在靶点。
附图说明
图1：pLKO.1puro慢病毒载体图谱；
图2：HPRT1 shRNA敲降效率验证，图2A为Real time PCR结果，图2B为Westernblot结果；
图3：在肺腺癌细胞株PC9中敲降HPRT1对细胞增殖的影响。
具体实施方式
现结合实例，对本发明做进一步说明。实施例仅限于说明本发明，而非对本发明的限定。
实施例1 HPRT1 shRNA质粒构建，病毒包装及稳转细胞系的构建
(1)HPRT1敲降质粒的表达载体pLKO.1puro如图1，HPRT1 shRNA在GeneticPerturbation Platform Web Portal网站上设计，由长春库美生物科技有限公司合成，序列如下：
上游链：
5’-CCGGCCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGGTTTTTG-3’
下游链：
5’-AATTCAAAAACCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGG-3’
(2)将正反DNA片段分别加水溶解至100μM，各吸取10μl混合均匀，利用PCR仪将片段进行退火，程序如下：
95℃ 4min—85℃ 4min—80℃ 4min—75℃ 4min—70℃ 10min—65℃ 4min—60℃ 4min—55℃ 4min—50℃ 4min—45℃ 4min—40℃ 4min—37℃ 15min—30℃ 4min—25℃10min。
程序进行完即是退火后的shRNA片段。
(3)酶切载体
将pLKO.1puro载体(VT2318，miaolingbio)用AgeI酶(#FD1464，ThermoFisher)和EcoRI酶(#FD0274，ThermoFisher)于37℃双酶切2h，而后使用胶回收试剂盒(#28704，Qiagen)回收线性化载体。
(4)连接与转化
用T4连接试剂盒(C301-01，Vazyme)连接退火后的shRNA片段与线形化载体，
体系如下：
线性化pLKO.1-puro 1μl
退火后shRNA 1μl
T4 ligase 1μl
10X ligase buffer 1μl
ddH2O 6μl
室温孵育3小时后，将10μl孵育后体系加入到含有50μlDH5α化学感受态细胞的离心管中(#11802ES80，YEASEN)中，冰上孵育30分钟，42℃热激90秒后迅速冰上静止3分钟。而向离心管中加入无氨苄青霉素的LB培养基1ml，在37℃摇床中220rpm震摇1小时，而后5000rpm离心5min后弃0.9ml上清，将沉淀用剩余上清溶解后涂布在含100μg/ml氨苄霉素(A8180，Solarbio)的LB固体培养皿上，37℃倒置培养16小时。挑选2个单克隆菌株接种于3ml含100μg/ml氨苄霉素的LB培养基中培养16h，而后各取1ml菌液送长春库美生物科技有限公司测序验证插入序列正确性。
(5)病毒包装
转染前一天将293T细胞铺到10cm皿中。第二天待细胞密度达到70％时进行病毒包装。将培养基换成无血清的DMEM(MA0212,meilunbio)培养基4ml。取出2个1.5ml离心管，分别加入500μl无血清的DMEM培养基，而后依次加入pLKO.1puro质粒或pLKO.1-shHPRT1质粒12μg，包装质粒pMDlg/pRRE(VT1449，youbio)：6.5μg，pRSV-Rev(VT1445，youbio)：2.5μg，pLP/VSVG(VT1491，youbio)：3.5μg，最后加入转染试剂聚乙烯亚胺(23966，Polyscienceas)：9μl，轻轻混匀后，室温静置孵育15min，然后分别加入到细胞培养皿中。细胞在37℃，CO2体积含量5％空气的培养箱中培养6h后，更换培养基为含有10％体积血清的DMEM培养基5ml。转染72h后收集病毒原液，0.45μm滤膜过滤，-80℃保存备用。
(6)病毒侵染，稳转细胞系的构建
将肺腺癌细胞PC9细胞提前铺到二个3.5cm皿中，待细胞密度为30％时，向培养皿中分别加入1ml pLKO.1puro和1ml pLKO.1-shHPRT1病毒原液。侵染48h后，进行嘌呤霉素的筛选，筛选浓度为2ug/ml，连续筛选3天后即可获得对照组细胞系和稳定敲降HPRT1的PC9细胞系。
(7)HPRT1 shRNA敲降效率的验证
取对照组(pLKO.1-puro)PC9细胞和稳定敲降HPRT(pLKO.1-shHPRT1)PC9细胞，按照文献“Targeting KDM1A attenuates Wnt/β-catenin signaling pathwaytoeliminatesorafenib-resistant stem-like cells in hepatocellularcarcinoma”条件提取RNA和蛋白，并进行Real time PCR和Western blot实验检测HPRT1 shRNA的敲降效率。
结果：图2A Real time PCR和图2B Western blot实验结果表明pLKO.1-shHPRT1PC9细胞中的HPRT mRNA和蛋白表达水平显著低于pLKO.1-puro PC9细胞。
实施例2 HPRT shHPRT1抑制肺腺癌细胞增殖
将pLKO.1-puro PC9细胞和pLKO.1-shHPRT1 PC9细胞用胰酶消化成单个细胞后，利用细胞计数仪对其计数，而后按800个细胞/孔接种到96孔板中。接种后分别在0、1、2、3和4天利用Cell Counting Kit(CCK8，C0037，Beyotime)检测OD450的吸光度。
结果：图3 CCK8法测细胞活力检测后发现在PC9细胞中敲降HPRT1后，细胞的增殖明显受到抑制。
序列表
<110> 中国科学院大连化学物理研究所
<120> DNA双链序列及应用和肺腺癌细胞增殖抑制剂或在肺腺癌药物
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccggccaggt tatgaccttg atttactcga gtaaatcaag gtcataacct ggtttttg 58
<210> 2
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aattcaaaaa ccaggttatg accttgattt actcgagtaa atcaaggtca taacctgg 58
<210> 3
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccggccaggt tatgaccttg atttactcga gtaaatcaag gtcataacct ggtttttg 58
<210> 4
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aattcaaaaa ccaggttatg accttgattt actcgagtaa atcaaggtca taacctgg 58

Claims (6)

1.一种用于敲降人HPRT1基因的DNA双链序列，其特征在于，所述的DNA序列包含以下序列：
上游链：
5’-CCGGCCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGGTTTTTG-3’
下游链：
5’-AATTCAAAAACCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGG-3’。
2.一种构建物，其特征在于，所述的构建物包含以下DNA双链序列:
上游链：
5’-CCGGCCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGGTTTTTG-3’
下游链：
5’-AATTCAAAAACCAGGTTATGACCTTGATTTACTCGAGTAAATCAAGGTCATAACCTGG-3’。
3.根据权利要求2所述的构建物，其特征在于，所述的构建物为慢病毒载体。
4.根据权利要求3所述的构建物，其特征在于，所述的慢病毒载体为pLKO.1puro。
5.权利要求1所述的DNA双链序列或权利要求2所述的构建物中的一种或二种以上在抑制肺腺癌细胞增殖中的应用或在制备肺腺癌药物中的应用。
6.一种肺腺癌细胞增殖抑制剂或在肺腺癌药物，以权利要求1所述的DNA双链序列或权利要求2所述的构建物中的一种或二种以上活性成份。
CN202111410962.0A 2021-11-25 2021-11-25 Dna双链序列及应用和肺腺癌细胞增殖抑制剂或在肺腺癌药物 Pending CN116162621A (zh)

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