CN111944906A - Trpm7在制备肝癌诊疗药物中的应用 - Google Patents
Trpm7在制备肝癌诊疗药物中的应用 Download PDFInfo
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Abstract
本发明公开了TRPM7在制备肝癌诊疗药物中的应用，属于生物医学技术领域。本发明在肝癌缺氧微环境中缺氧诱导因子HIF可调控TRPM7表达的研究基础上，进一步筛选得到了有效的TRPM7 siRNA序列，从而达到有效的肝癌精准治疗靶点，提高肝癌患者的生存期和生存质量。
Description
技术领域
本发明属于生物医学技术领域，具体涉及TRPM7在制备肝癌诊疗药物中的应用。
背景技术
肝细胞癌(Hepatocellular carcinoma，HCC)是原发性肝癌的主要病理类型，约占全部肝癌的90％。HCC的主要风险因素包括乙型/丙型肝炎病毒感染、饮酒、非酒精性脂肪肝等，近年来针对这些因素的研究极大促进了患者的风险评估、预后估计和选择正确的治疗策略，但HCC转移和复发仍然难以得到控制。近年来，越来越多研究趋向于认为，肿瘤转移是肿瘤细胞自身特性及所处的肿瘤微环境相互作用的结果。然而，由于肿瘤患者个体差异以及肿瘤细胞和其微环境的复杂性等，对其认识尚有不足。与直接针对肿瘤细胞治疗手段相比，干预微环境为肿瘤治疗提供了新思路。
肿瘤微环境，即肿瘤细胞及其周围环境所组成的高度特异性、且随肿瘤发生发展而不断进化的微小生态系统。肿瘤细胞的快速增殖导致了细胞缺氧及相应缺氧诱导因子(hypoxia inducible factors,HIFs)稳定性增加，从而引起与缺氧耐受相关基因的改变，并可能导致肿瘤细胞转移增加。目前HIF-α的家族成员已知的有3种，即HIF1α、HIF2α、HIF3α。其中HIF1α是肝癌细胞对低氧环境做出适应性应答的关键因子，可以诱导多种靶基因表达，而HIF2α在肿瘤中的作用也不容忽视。
TRPM7(Transient receptor potential melastatin 7)是广泛表达的具有通道和激酶双重特性的膜蛋白，其激活参与重要的病理生理过程，比如镁离子平衡、胚胎发育、缺氧神经元死亡和细胞粘附过程，近年来研究发现TRPM7参与多种肿瘤的发生，比如乳腺癌、视网膜母细胞瘤、胃癌、鼻咽癌、胰腺癌、前列腺癌、卵巢癌和膀胱癌等，而人肝癌中的TRPM7表达情况还少有报道，TRPM7在肝癌进展中的作用及机制也尚不清楚。
发明内容
本发明的目的是提供TRPM7在制备肝癌诊断或治疗药物中的应用。
通过生物信息学分析TRPM7序列发现，TRPM7启动子区域存在低氧反应原件HRE，前期研究显示，低氧条件下，多种肝癌细胞中的TRPM7表达上调，发明人推出肝癌细胞系中TRPM7的表达受缺氧的调控，进而影响肝癌的转移及预后。
本发明在肝癌缺氧微环境中缺氧诱导因子HIF可调控TRPM7表达的研究基础上，进一步筛选有效的TRPM7 siRNA序列，从而达到有效的肝癌精准治疗靶点，提高肝癌患者的生存期和生存质量。
附图说明
图1为低氧条件(1％O2)处理肝癌细胞24h后TRPM7和HIF的表达结果。
图2为HIF1αsiRNA、HIF2αsiRNA、HIF1α抑制剂2-MeOE2处理SK-Hep1或者HepG2细胞后，TRPM7的表达结果。
图3为染色质免疫共沉淀结果。
图4为CCK-8实验和Transwell迁移实验检测TRPM7siRNA对肝癌细胞的增值和迁移的影响。
具体实施方式
下面将结合具体实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明公开了肝癌缺氧微环境中缺氧诱导因子HIF调控TRPM7表达，并进一步筛选有效的TRPM7 siRNA序列，抑制肝癌细胞的增值，包括如下过程：
1.通过qRT-PCR实验，检测低氧或CoCl2诱导肝癌细胞系中TRPM7和HIFα表达增加；
2.使用HIF1αsiRNA，HIF2αsiRNA或者HIF1α抑制剂2-MeOE2处理SK-Hep1或者HepG2细胞后，通过qRT-PCR实验，检测TRPM7 mRNA表达。
3.通过染色质免疫共沉淀(CHIP)和荧光素酶报告分析荧光素酶实验分析，HIF1α通过结合到TRPM7启动子区域的两个结合位点发挥调控作用。
4.筛选并鉴定有效的TRPM7siRNA序列；并通过CCK-8和Transwell迁移实验，观察有效TRPM7 siRNA能够抑制肝癌细胞的增值和迁移。
实施例1
一、细胞培养
肝癌细胞系SK-Hep1、SMMC-7721、HepG2、Huh7和LM3分别于37℃，在10％FBS的DMEM培养基条件下，放置在5％CO2培养箱培养。
二、RNA提取和qRT-PCR反应
1.RNA提取
(1)六孔板细胞使用PBS洗涤两遍，加入1mL TRIZOL试剂；
(2)加入1/5体积氯仿约200μL，颠倒混匀15s，室温放置5min；
(3)4℃，12000rpm离心15min，分为三层：上层为无色水相(RNA主要存在于此)，中间层为白色，下层为红色有机相(用于提DNA或者蛋白质)；
(4)转移上层水相(约400μL-500μL)于另一新的1.5mL离心管；
(5)加入等体积的异丙醇(约400μL-500μL)，混匀后置于-20℃冰箱1小时；
(6)4℃，12000rpm离心10min，离心管管底或侧壁可见白色小点沉淀，弃去上清；
(7)加入冰预冷的75％乙醇1mL，4℃，7500rpm离心5min，弃去上清，用移液器尽量吸掉残余液体，将离心管倒扣在滤纸上吸干水，室温放置10min；
(8)加入DEPC水20μL，55℃金属浴，8min助溶，后立即置于冰上；
(9)用Nanodrop 2000分光光度计测定RNA浓度及纯度后立即保存于-80℃冰箱中备用或者立即用于下步逆转录。
2.逆转录反应
使用逆转录试剂盒(Biorad)，20μL体系
反应条件：25℃10min，42℃30min，85℃5min，所得cDNA-20度保存备用。
3.实时定量PCR(Real-time qPCR)
(1)设计合成引物序列
引物采用Primer Premier3.0设计，由上海生工生物技术有限公司合成。
(2)反应体系
上述体系放入CFX 96TM实时PCR仪中，设定运行程序。
(3)反应体系反应数据由Bio-Rad CFX Manager软件收集，通过软件计算出样本的Ct值，以β-Actin作为内参，采用
计算TRPM7及HIF基因的相对表达量。
如图1所示，在低氧条件(1％O2)处理肝癌细胞24h后，肝癌细胞中的TRPM7和HIF表达上调。
如图2所示，使用HIF1αsiRNA、HIF2αsiRNA或者HIF1α抑制剂2-MeOE2处理SK-Hep1或者HepG2细胞后，通过qRT-PCR实验，检测得到TRPM7 mRNA表达下调。
三、染色质免疫共沉淀(CHIP)
1.人肝癌细胞SK-Hep1和Huh7细胞的准备及固定
实验分组：正常组
缺氧组：200μmol/L CoCl2处理12h，造模完毕待细胞状态稳定后交联固定。
(1)细胞固定及染色体破碎
固定：倾去完全培养基，加入1％甲醛的PBS溶液，室温孵育10min；
终止交联：加10×Glycine溶液至培养皿中，混匀后，室温静置5min；
吸去交联液，用预冷的PBS清洗细胞2次；
用细胞刮刮取收集细胞于15mL离心管中，2 000rpm，4℃，离心5min弃去上清，收集细胞；
(2)预冷PBS重悬细胞，每组细胞均分成两份(IgG组和HIF1α抗体组)，2 000rpm，4℃，离心5min弃上清，每组细胞加入100μL Lysis Buffer 1(内含1μL Halt Cocktail)，振荡混匀15s，冰上静置10min；
(3)9000g，室温离心3min，弃上清；
(4)加入100μL MNase Digestion Buffer Working Solution，重悬细胞；
(5)上述溶液中加0.25μL Micrococcal Nuclease，混匀，37℃水浴15min，每5min颠倒混匀一次；
(6)溶液中加入10μL MNase Stop Solution，混匀，冰浴5min；
(7)9000g，室温离心3min，弃上清；
(8)加入50μL Lysis Buffer 2(内含0.5Halt Cocktail)重悬，冰浴15min，每5min取出振荡混匀15s；
(9)9000g，室温离心3min，转移上清至新的1.5mL离心管中，每管50μL，此为已经消化完毕的染色体。
琼脂糖凝胶电泳分析消化效果，消化后的染色体在200-1000bp。
2.免疫沉淀
(1)将上述染色体溶液，每组细胞吸取5μL于新的1.5mL离心管，存于-20℃，作为10％Input组(同组的其他几份染色体溶液也取5μL，舍弃)；
(2)向剩余的45μL上清中加入450μL 1x IP Dilution Buffer混匀，再加入相应的抗体，如下表：
置于旋转培养器上孵育，4℃，过夜；
(3)免疫复合物的沉淀及清洗
(3-1)过夜孵育后，每管中加入60μL Protein A Agarose/SalmonSperm DNA，旋转培养器上4℃颠转2h；
(3-2)4℃静置10min后，700rpm离心1min，弃去上清；
(3-3)依次用下列溶液清洗沉淀复合物：
洗涤溶液：a.IP wash buffer 1(洗涤一次)
b.IP wash buffer 2(洗涤二次)
c.IP wash buffer 3(洗涤一次)
洗涤步骤：加入洗涤溶液，在旋转培养器上4℃颠转5min，3000g离心1min，弃去上清；
(3-4)IP wash buffer 3洗涤后，3000g再次离心1min，加入150μL IP Elutionbuffer，65℃水浴40min，每10min轻弹离心管；
(3-5)水浴过程中，取出10％Input组，溶解后加入150μL IP Elution buffer，6μL5M NaCl(终浓度为0.2M)和2μL Proteinase K，混匀后置于室温；
(3-6)65℃水浴结束后，向每管中加入6μL 5M NaCl和2μL Proteinase K，混匀后，连同10％Input组一起，65℃，1.5h解交联，6000g，离心1min，取上清至新的离心管中；
3.DNA样品的回收
(1)解交联结束后，向每管上清中加750μL DNA binding buffer；
(2)取500μL加入DNA回收柱中，1000g离心1min，弃去收集管废液，再将剩余的加入DNA回收柱内，1000g离心1min，弃去废液，吸附柱放回收集管内；
(3)加750μL DNA wash buffer，1000g离心1min，弃去废液，吸附柱放回收集管内；
(4)1000g再次离心1min；
(5)弃废液收集管，将回收柱放入新的离心管，向回收柱膜中央加50μL DNAElution Buffer，1000g离心1min，离心管中所收集的液体即为纯化的DNA，可进行PCR或qPCR。
4.CHIP结果验证
使用GAPDH promoter的引物(试剂盒里提供)，常规PCR验证CHIP结果的有效性，PCR体系(20μL体系)如下表：
PCR产物通过跑2％琼脂糖凝胶电泳观察条带进行分析：
从PCR产物中取5μL加入2％琼脂糖凝胶电泳的梳孔中，将加好样的琼脂糖凝胶放入装有TAE缓冲液的电泳槽中，缓冲液要覆盖胶面以上，且梳孔要放在负极方向，60V电压跑45min，化学发光仪上拍片看条带分析结果的有效性。
5.PCR检测
(1)引物设计
通过Pubmed查找目标基因人TRPM7基因的mRNA全序列，在通过UCSC(http://genome.ucsc.edu/)确定其ORF上游启动子区3000bp范围序列，每隔300bp左右，使用PrimerPremier3.0设计一对引物，共设计10对引物。
TRPM7的启动子区3000bp序列如SEQ ID NO.9所示。
10对引物序列如下：
TRPM7p1+AGTGTTTCACCATGTTGCCCAGACT(SEQ ID NO.10)
TRPM7p1-CAGTGTCTAAGAATATGTCAGGAGC(SEQ ID NO.11)
TRPM7p2+GAATATAAATATATATATTTCAAGA(SEQ ID NO.12)
TRPM7p2-CCCAGCACTTTGGGAGGCCGAGGCG(SEQ ID NO.13)
TRPM7p3+GATTACTGGCGTGATATTTTTTTAA(SEQ ID NO.14)
TRPM7p3-CATGTCTGTATTCCCAGCTACTCCA(SEQ ID NO.15)
TRPM7p4+GGGCCACTGTGCCTGACTCAATACA(SEQ ID NO.16)
TRPM7p4-GAGGCAGAAGAATCGCTTGAACCCA(SEQ ID NO.17)
TRPM7p5+CGGCCCCCCGAGTAGCTGTGATTAC(SEQ ID NO.18)
TRPM7p5-CGAATGCTTCGCTCCTTAGTTGGAT(SEQ ID NO.19)
TRPM7p6+CGTGAGTACAAAAAGACCAGAGTTA(SEQ ID NO.20)
TRPM7p6-CAATCTTCAGGTATTGTGTATTTCT(SEQ ID NO.21)
TRPM7p7+GTTCACAAGCTGGGGGAACCAGTGG(SEQ ID NO.22)
TRPM7p7-CACCATAATCAAATGAACGTATTCA(SEQ ID NO.23)
TRPM7p8+GATTTCATGGGTGCACAGGTATGTC(SEQ ID NO.24)
TRPM7p8-TATCCACCTTTCCATTTATTTTGCT(SEQ ID NO.25)
TRPM7p9+GGTACATATTACATCAAGGGAACAA(SEQ ID NO.26)
TRPM7p9-CCGCTAACTAGGCCTGGCCTGTGCC(SEQ ID NO.27)
TRPM7p10+GAATGAGCCCACACAGCAACTCCAG(SEQ ID NO.28)
TRPM7p10-GGCGCGGCGCGTGCGTCGCCGCCCG(SEQ ID NO.29)
(2)通过PCR检测结果
将上述引物逐个做常规PCR，样品分别为IgG组，HIF1α抗体组和Input组。
PCR体系(20μL)如下：
扩增条件为：95℃预变性5min；95℃变性30sec，60℃退火30sec，72℃延伸30sec，35个循环；最后72℃延伸10min。结果通过2％琼脂糖电泳验证。
如图3所示，HIF1α通过结合到TRPM7启动子区域的两个结合位点发挥调控作用。
四、针对TRPM7基因序列，通过网站(http://sidirect2.rnai.jp/design.cgi或者http://biodev.extra.cea.fr/DSIR/selectEP.php)在线设计设计三对TRPM7siRNA，合成相应的寡核苷酸，然后瞬时转染到肝癌细胞系中。
1.TRPM7siRNA设计
M7siRNA-1:5’-GGAGUAAGCAUGCAUAAAU-3’(SEQ ID NO.30)
M7siRNA-2:5’-CAGGGCAUCUUUAUAUUAU-3’(SEQ ID NO.31)
M7siRNA-3:5’-GGCAUAACCAUUUCAUUAA-3’(SEQ ID NO.32)。
2.转染TRPM7siRNA到肝癌细胞系SK-Hep1中
(1)六孔板中培养细胞24h后，使用lipo2000转染TRPM7siRNA至细胞中。
(2)6小时换液，24小时后提取细胞mRNA进行qPCR实验，48小时后提取细胞蛋白进行WB实验及CCK-8功能实验验证。
3.验证有效的TRPM7siRNA序列
使用qRT-PCR和WB实验，发现M7siRNA-1抑制TRPM7mRNA和蛋白最佳，因此鉴定M7siRNA-1为最有效的序列(图4A)。
3.使用上述敲除成功的细胞系进行CCK-8实验，检测M7siRNA对肝癌细胞增殖能力的影响。
(1)在96孔板中接种105个细胞的100μL细胞悬液，在培养箱中预培养24小时。
(2)培养24h、48h、72h和96h后加入10μL CCK-8溶液(注意不能产生气泡)。
(3)将培养板在培养箱内孵育1-4小时。
(4)用酶标仪测定细胞在450nm处的吸光度。
结果如图4B所示，M7siRNA-1能够沉默肝癌细胞TRPM7的表达，从而抑制癌细胞增殖。
4.使用上述敲除成功的细胞系进行Transwell实验，检测M7siRNA对肝癌细胞增殖能力的影响。
(1)所有细胞培养试剂和Transwell chamber放在37℃温育；
(2)待测细胞培养至对数生长期，消化细胞，用PBS和无血清培养基先后洗涤一次，用无血清培养基悬浮细胞，计数，调整浓度为2×105/mL；
(3)在下室(即24孔板底部)加入600－800μl含10％血清的培养基，上室加入100－150μl细胞悬液，继续在孵箱培养24小时；
(4)用镊子小心取出chamber，吸干上室液体，移到预先加入约800μl甲醇的孔中，室温固定30分钟；
(5)取出chamber，吸干上室固定液，移到预先加入约800μl Giemsa染液的孔中，室温染色15－30分钟；
(6)轻轻用清水冲洗浸泡数次，取出chamber，吸去上室液体，用湿棉棒小心擦去上室底部膜表面上的细胞；
(7)用小镊子小心揭下膜，底面朝上晾干，移至载玻片上用中性树胶封片；
(8)显微镜下取9个随机视野计数，统计结果。
结果如图4C所示，M7siRNA-1能够沉默肝癌细胞TRPM7的表达，从而抑制癌细胞增殖。
上述实验数据，应用统计学软件GraphPad Prism 6.0分析所获得的实验数据，首先对各组数据进行正态性检验，对于计量资料间的两两比较采用t检验，生存率差异比较采用Keplan-Meier法检验，多组数据资料之间比较采用单因素方差分析，数据用均数±标准差(mean±SEM)表示，P<0.05为差异有统计学意义。
序列表
<110> 南通大学
<120> TRPM7在制备肝癌诊疗药物中的应用
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gctctttcgc ccaggccgga ctgcagtggc gctatctcag ctcactgcaa gctccgcctc 420
ccgggttcac gccattctgc tgcctcagcc tcccgagtag ctgggactac aggcgcccgc 480
cactgcgccc ggctaatttt ttgtattttt agtagagacg gggtttcaca gtattagcca 540
ggatggtctc gatctcctga ccttgtgatc tgcccgcctc ggcctcccaa agtgctggga 600
ttactggcgt gatatttttt taagagacgt ggtcttgcta cgtagtccag gcttgagtgc 660
ggtggctata cacaggcact atccaactac tgatcagcag gaattctgac ctgctcaatt 720
tctgtcctgg gttgattcac ccctccttag gcaatctggt ggtcccttgc tcccaggaag 780
tcactacgtt gatgctgaac ttagcacagc cacccaatcc gcatagcaca ctacagccca 840
gaactgggct cacatgagcc ttctgcctca acctctggag tagctgggaa tacagacatg 900
ggccactgtg cctgactcaa tacatatttt ttgaatgaat aaattcattc tccagaattg 960
attgctgatt gaaagtcagc atctgtgaat ttcaaggctg gttgatcact tgaaaattaa 1020
ggaatttatt ttgattagtt caattaagga taaatcctgt tccccatttc ttttcttttt 1080
cttttttttt tttttttttt gagacgaagt cctgctcttg tcccccaggc tggagtgaga 1140
tggcgcgatc tcggctcact gcaacctctg cctcctgggt tcaagcgatt cttctgcctc 1200
ggccccccga gtagctgtga ttacaggcgc ctgccaccat gcccagctaa tttttgtatt 1260
tttagtagag acggggtttc accgtgttgg ccaggctggt ctagaactcc tgacctcagg 1320
tgatccaccc gcctcggcct cccaaagtgc tgggattaca agcatgagcc accgcgccca 1380
cccaatcctg ttccccattt ctaactctaa attttccaat ttaccaaaga gttctggcaa 1440
gtgccaagga gctcatcttg tttcatttct acaccatcca actaaggagc gaagcattcg 1500
tgagtacaaa aagaccagag ttacaaagtt ctaacgaaac aaagcactct aatctcattt 1560
tattttgatt ttccagtttg tctgcctcta ccttctagat tataaactcc ctacaggcag 1620
gaaatgtgta ggcccaactg aaatccagaa gataaacatt tattgactgc atgcataaat 1680
tagtcaagat gccttcccca aggctgacaa tttagtgtga cacatatatt aaaacatttt 1740
taagtcttgg ttctgctgga cctgccagat ggggaagaaa tacacaatac ctgaagattg 1800
ttcacaagct gggggaacca gtgggtggat tagttttttt tctgagagaa gatcctactt 1860
cagtgaattg tgggtcataa tgtttgagag gcaataatta tgctgaatga aagaagttag 1920
acggaaaaaa aaggtgcgtg ctgtatgatt cccgtatata aaactttagc atgtgcaaac 1980
ttatctgtac tgaaagtaga tctgtagttg ataagggacg tgatggagag ggacgagatg 2040
aagggattat gaaggggggg gaggaaactt agaggtgaat acgttcattt gattatggtg 2100
atttcatggg tgcacaggta tgtcaaaact tatcaaattg gacatttaaa tacgtgcagt 2160
ttgtcatgtc aattatttta aaataaagtc gtttttaaaa aagagataag acagtcagat 2220
ccctgagggt cagattgagg aactctcgaa gacttcagga gcagaaagat gttaaagcag 2280
ttttaagaaa attaaactga ttgtggtctt cccagtagat aaggaataac atttagtaaa 2340
ccattgctaa tagccaaacc ttggcatgtg tgacagcaaa ataaatggaa aggtggatag 2400
gtacatatta catcaaggga acaacccagt ttttggtgca gaaggtgtgg tgtatgcgtg 2460
tatgtgggtg agtatctctg gagggaaaaa gacagtccca tagttaaaca aagaagaata 2520
tttaccggca gcaacaggag aggctagaga aagaacatga ttggggtaaa aagatgattc 2580
tgcattttgg acgaggagag agtaacattt aaagcctcta ctcgctctga agcacacaga 2640
gtgtgttgcg acactcgagt ttgctggagc gagtcggcac aggccaggcc tagttagcgg 2700
aatgagccca cacagcaact ccagactgga tccgcaagcc ttgaacctta accctcaacc 2760
tccgattccc caagttccac tctcgtagct gaggctgaag tttgtctaag tcccgcctag 2820
gtacccaacg gagatacggc gtgaacaccc caccactttc tgggaaccca gaggcggtcg 2880
gcgtggcggc gcgaacctgc ccagcgcgtg cgcgcggcgc gcagggaggc ggcctggaat 2940
cgagggagcg cgcgcccttc acgtgacccg acccgcgggc ggcgacgcac gcgccgcgcc 3000
<210> 10
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
agtgtttcac catgttgccc agact 25
<210> 11
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cagtgtctaa gaatatgtca ggagc 25
<210> 12
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gaatataaat atatatattt caaga 25
<210> 13
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cccagcactt tgggaggccg aggcg 25
<210> 14
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gattactggc gtgatatttt tttaa 25
<210> 15
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
catgtctgta ttcccagcta ctcca 25
<210> 16
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gggccactgt gcctgactca ataca 25
<210> 17
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gaggcagaag aatcgcttga accca 25
<210> 18
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cggccccccg agtagctgtg attac 25
<210> 19
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cgaatgcttc gctccttagt tggat 25
<210> 20
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
cgtgagtaca aaaagaccag agtta 25
<210> 21
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
caatcttcag gtattgtgta tttct 25
<210> 22
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gttcacaagc tgggggaacc agtgg 25
<210> 23
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
caccataatc aaatgaacgt attca 25
<210> 24
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gatttcatgg gtgcacaggt atgtc 25
<210> 25
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tatccacctt tccatttatt ttgct 25
<210> 26
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ggtacatatt acatcaaggg aacaa 25
<210> 27
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ccgctaacta ggcctggcct gtgcc 25
<210> 28
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gaatgagccc acacagcaac tccag 25
<210> 29
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
ggcgcggcgc gtgcgtcgcc gcccg 25
<210> 30
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 30
ggaguaagca ugcauaaau 19
<210> 31
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 31
cagggcaucu uuauauuau 19
<210> 32
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 32
ggcauaacca uuucauuaa 19
Claims (4)
1.TRPM7在制备肝癌诊断试剂中的应用。
2.TRPM7抑制剂在制备治疗或预防肝癌的药物中的应用。
3.TRPM7抑制剂在制备抑制肝癌细胞增殖的药物中的应用。
4.根据权利要求2或3所述的应用，其特征在于：所述TRPM7抑制剂为siRNA ，该siRNA的序列为：5’-GGAGUAAGCAUGCAUAAAU-3’。
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Citations (4)
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|---|---|---|---|---|
| CN102690866A (zh) * | 2011-03-23 | 2012-09-26 | 上海人类基因组研究中心 | Trpm7基因及其表达产物的应用 |
| CN105078936A (zh) * | 2015-08-27 | 2015-11-25 | 云南中医学院 | 4-甲氧基苯甲醇在制备trpm7抑制剂类药物中的用途 |
| CN111349706A (zh) * | 2020-03-20 | 2020-06-30 | 青岛思拓新源细胞医学有限公司 | 一种基因抑制剂在制备治疗肝癌药物中的应用 |
| CN111500734A (zh) * | 2020-05-28 | 2020-08-07 | 南通大学 | 一种肝癌诊断标志物及其应用 |
-
2020
- 2020-08-24 CN CN202010856654.XA patent/CN111944906A/zh active Pending
Patent Citations (4)
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|---|---|---|---|---|
| CN102690866A (zh) * | 2011-03-23 | 2012-09-26 | 上海人类基因组研究中心 | Trpm7基因及其表达产物的应用 |
| CN105078936A (zh) * | 2015-08-27 | 2015-11-25 | 云南中医学院 | 4-甲氧基苯甲醇在制备trpm7抑制剂类药物中的用途 |
| CN111349706A (zh) * | 2020-03-20 | 2020-06-30 | 青岛思拓新源细胞医学有限公司 | 一种基因抑制剂在制备治疗肝癌药物中的应用 |
| CN111500734A (zh) * | 2020-05-28 | 2020-08-07 | 南通大学 | 一种肝癌诊断标志物及其应用 |
Non-Patent Citations (3)
| Title |
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| SANDRA VORINGER ET AL.: "Inhibition of TRPM7 blocks MRTF/SRF-dependent transcriptional and tumorigenic activity", 《ONCOGENE》 * |
| 崔志华等: "TRPM7通过STAT3途径参与IL-6诱导的肝细胞增殖", 《安徽医科大学学报》 * |
| 李立等: "TRPM7在胆管癌组织中的表达及其与预后的关系", 《重庆医学》 * |
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