CN113995841A - Mettl3/alkbh5/eno1调控轴作为靶位点在制备治疗肺腺癌药物中的应用 - Google Patents
Mettl3/alkbh5/eno1调控轴作为靶位点在制备治疗肺腺癌药物中的应用 Download PDFInfo
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Abstract
本发明涉及METTL3/ALKBH5/ENO1调控轴作为靶位点在制备治疗肺腺癌药物中的应用。本发明首次证实肺腺癌中“METTL3/ALKBH5/ENO1”调控轴的存在,该调控轴是肺腺癌治疗领域的一种非常有价值的潜在靶点,有助于通过多个节点的干预彻底打破促进肿瘤发生发展的恶性循环,更有效抑制肿瘤进展,此外METTL3/ALKBH5/ENO1调控轴还可作为临床上预测肺腺癌患者的分子标志物。本发明还证实了甲基化抑制剂DAA,糖酵解抑制剂2DG、ENOblock可作为安全高效的肺腺癌治疗药物。
Description
技术领域
本发明涉及生物医药技术领域,具体地说,涉及METTL3/ALKBH5/ENO1调控轴作为靶位点在制备治疗肺腺癌药物中的应用。
背景技术
甲基转移酶3(methyltransferase-like 3,METTL3)是m6A甲基转移酶复合体中的关键蛋白,能够催化RNA上的腺嘌呤(A)第6位氮原子发生甲基化。METTL3介导的m6A修饰与RNA的剪接、出核、降解和翻译都有着密切联系,并且在肿瘤的发生发展中起着重要作用。
AlkB同源蛋白5(Alk B homologue 5,ALKBH5)是一种m6A去甲基化酶,其可通过影响m6A修饰水平参与调节底物RNA的稳定性、翻译效率以及选择性剪接等。目前研究表明ALKBH5在绝大多数恶性肿瘤中均通过影响底物m6A修饰水平来激活多种信号通路以促进肿瘤进展。
α-烯醇化酶(alpha-enolase,ENO1)是烯醇化酶家族的3种同功酶之一,是糖酵解过程中的重要限速酶,其催化2-磷酸甘油酸脱水成磷酸烯醇式丙酮酸,并产生ATP。近年来,研究发现ENO1在多种类型肿瘤中存在显著的差异表达,并在肿瘤的发生、发展过程中起到重要作用。
然而,目前的研究证实以上蛋白酶在不同组织、不同细胞的不同生理或病理状态下,甚至在不同的细胞亚定位均有不同的作用途径,也显示出不同的功能。因此并不能明确它们与肿瘤发生、发展的确切关系。
肺癌主要分为非小细胞肺癌(NSCLC,约85%)和小细胞肺癌(SCLC,约15%)。NSCLC根据组织病理学类型又可分为腺癌(lung adenocarcinoma)、鳞状细胞癌、腺鳞癌、大细胞癌及肉瘤样癌等亚型。肺腺癌发病率逐年增长,已经成为NSCLC中最常见的亚型,几乎占全部肺癌的50%,且总体生存率较低。
目前未见肺腺癌中存在“METTL3/ALKBH5/ENO1”调控轴的报道,而该调控轴的发现将为肺腺癌诊断和治疗提供一种非常有价值的潜在靶点,通过从多个节点干预将彻底打破促进肿瘤发生发展的恶性循环,更有效抑制肿瘤进展。
发明内容
本发明的目的是针对现有技术中的不足,提供METTL3/ALKBH5/ENO1调控轴作为靶位点在制备治疗肺腺癌药物中的应用。
第一方面,本发明提供了METTL3基因或蛋白的抑制剂在制备治疗肺腺癌的药物中的应用。
优选地,所述抑制剂选自生物大分子或化合物小分子。所述生物大分子为以METTL3蛋白或其转录本为靶序列、且能够抑制METTL3蛋白表达或基因转录的小干扰RNA、dsRNA、shRNA、微小RNA、反义核酸;或能表达或形成所述小干扰RNA、dsRNA、微小RNA、反义核酸的构建物。
第二方面,本发明提供了ALKBH5基因或蛋白或它们的促进剂在制备治疗肺腺癌的药物中的应用。
优选地,所述促进剂选自生物大分子或化合物小分子。所述生物大分子为包含编码ALKBH5的多聚核苷酸及与之操作性相连的表达调控序列的表达载体。
第三方面,本发明提供了ENO1基因或蛋白的抑制剂在制备治疗肺腺癌的药物中的应用。
优选地,所述抑制剂选自生物大分子或化合物小分子。所述生物大分子为以ENO1蛋白或其转录本为靶序列、且能够抑制ENO1蛋白表达或基因转录的小干扰RNA、dsRNA、shRNA、微小RNA、反义核酸;或能表达或形成所述小干扰RNA、dsRNA、微小RNA、反义核酸的构建物。
第四方面,本发明提供了下述a)、b)和c)中的至少2种的组合在制备治疗肺腺癌的药物中的应用:
a)METTL3基因或蛋白的抑制剂;
b)ALKBH5基因或蛋白或它们的促进剂;
c)ENO1基因或蛋白的抑制剂。
第五方面,本发明提供了甲基化抑制剂DAA在制备治疗肺腺癌的药物中的应用。
第六方面,本发明提供了糖酵解抑制剂2DG在制备治疗肺腺癌的药物中的应用。
第七方面,本发明提供了糖酵解抑制剂ENOblock在制备治疗肺腺癌的药物中的应用。
本发明优点在于:
1、本发明研究证实,在肺腺癌中,高表达的METTL3和低表达的ALKBH5,促使ENO1的蛋白表达,最终通过增强糖酵解的作用,促进肺癌细胞的不断增殖。首次证实肺腺癌中“METTL3/ALKBH5/ENO1”调控轴的存在,是肺腺癌治疗领域的一种非常有价值的潜在靶点,有助于通过多个节点的干预彻底打破促进肿瘤发生发展的恶性循环,更有效抑制肿瘤进展。此外,METTL3/ALKBH5/ENO1调控轴还可作为临床上预测肺腺癌患者的分子标志物。
2、本发明证实了甲基化抑制剂DAA,糖酵解抑制剂2DG、ENOblock能显著抑制肺腺癌皮下移植瘤和人源肿瘤异种移植模型生长,而对小鼠体重没有影响,提示甲基化抑制剂DAA,糖酵解抑制剂2DG、ENOblock可作为有效的肺腺癌治疗药物,且副作用较小。
附图说明
图1.ELISA和WB结果显示METTL3在肺腺癌组织高表达,ALKBH5在肺腺癌组织低表达(A-B);WB结果显示METTL3在肺腺癌细胞中高表达,ALKBH5在肺腺癌细胞中低表达(C)。
图2.ELISA和IHC结果及IHC评分显示ENO1在肺腺癌组织中高表达(A-B)。
图3.相关性分析结果显示肺腺癌组织中METTL3与ENO1的表达呈正相关关系(A),ALKBH5与ENO1的表达呈负相关关系(B)。
图4.代谢组结果显示ENO1对于高表达METTL3同时敲除ALKBH5所造成的糖酵解异常激活是必须的(A-C)。(D)H1975细胞中高表达ALKBH5同时敲除METTL3可削弱过表达ENO1所造成的细胞异常增殖。(E)H1299细胞中敲除ENO1可明显削弱高表达METTL3同时敲除ALKBH5所造成的细胞增殖。
图5.肺腺癌皮下移植瘤(CDX)结果显示甲基化抑制剂DAA和糖酵解抑制剂2DG,ENOblock能显著抑制CDX肿瘤细胞生长(A-B),对小鼠体重没有影响(C);人源肿瘤异种移植模型(PDX)结果显示甲基化抑制剂DAA和糖酵解抑制剂2DG,ENOblock能显著抑制PDX肿瘤细胞生长(D-E),对小鼠体重没有影响(F)。
图6.肺腺癌皮下移植瘤(CDX)结果显示高表达ALKBH5同时敲除METTL3相比于仅高表达ALKBH5或仅敲除METTL3显著抑制了移植瘤的生长。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1
一、实验方法
1.酶联免疫吸附实验(ELISA)检测组织中METTL3、ALKBH5和ENO1蛋白的含量
(1)ELISA试剂盒从冰箱取出,室温平衡60分钟;
(2)设置标准品孔和样本孔,标准品孔各加不同浓度的标准品50μL;
(3)待测样本孔(组织裂解液及细胞裂解液)先加待测样本10μL,再加样本稀释液40μL;
(4)随后标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μL,用封板膜封住反应孔,37℃水浴锅或恒温箱温育60min;
(5)弃去液体,吸水纸上拍干,每孔加满洗涤液,静置1min,甩去洗涤液,吸水纸上拍干,如此重复洗板5次(也可用洗板机洗板,每孔注入洗液350μL,浸泡1min,洗板5次);
(6)每孔加入底物A、B各50μL,37℃避光孵育15min;
(7)每孔加入终止液50μL,15min内,在450nm波长处测定各孔的OD值;
(8)根据标准孔OD值和浓度绘制标准曲线,算出待测样本孔的浓度。
2.免疫印迹WB检测蛋白的表达情况
2.1蛋白样品制备
(1)吸去培养基,PBS洗涤2次;
(2)6孔板中每孔加入200μl胰酶消化约3min,加入1ml/孔完全培养基终止消化;
(3)用枪轻轻将细胞吹下,吸入EP管,1000rpm,离心5min;
(4)吸弃上清,PBS洗涤1次,1000rpm,离心5min;
(5)吸弃上清,加入蛋白裂解液,50~100μl/孔,具体视细胞量而定,4℃裂解40min~1h或置于-80℃保存;
(6)4℃12000G离心10min(提前开离心机预冷),上清为目标蛋白,沉淀为细胞碎片,吸取100μl上清至EP管中,置于冰上备用。
2.2制作标准曲线,BCA法蛋白定量
使用预先配好的标准品(c=0.5μg/μl),取96孔板,按如下浓度梯度布孔加样:
标准品(μl) | 灭菌的ddH<sub>2</sub>O(μl) | 浓度(μg/μl) | 待测OD值(562nm处) |
0 | 20 | 0 | |
1 | 19 | 0.025 | |
2 | 18 | 0.05 | |
4 | 16 | 0.1 | |
8 | 12 | 0.2 | |
12 | 8 | 0.3 | |
16 | 4 | 0.4 | |
20 | 0 | 0.5 | |
待测样品2μl | 18μl | 待测 |
加好后,每孔加入200μl的AB液,BCA试剂A液:B液=50:1,轻轻振荡混匀后,37℃培养30min,酶标仪测吸光度(562nm处),在Excel中制作标准曲线,得出方程,从而计算蛋白浓度。
2.3温箱孵育过程中,每100μl样品中加入20μl的6×蛋白上样缓冲液(即样品:缓冲液=5:1),100℃水浴10min,变性蛋白样品。
2.4根据蛋白浓度计算上样量,注意此前的稀释倍数(测标曲时稀释了10倍,加蛋白上样缓冲液又进行了稀释5/6),并在EP管上标明20μg/30μg体系及所测的蛋白浓度,便于以后再用,置于-80℃或-20℃保存。
2.5根据待测蛋白的分子量配制合适浓度的凝胶,安装好电泳装置,倒入电泳缓冲液,拔去梳子,最左边孔加入Marker 5μl,上蛋白样品,接通电源80V电泳30min,待蛋白样品跑至分离胶,换为120V电泳。
2.6根据待测蛋白的分子量和Marker切取相应的胶,剪取相应的NC膜,并用圆珠笔在一角作好标记,将胶、滤纸和其它转膜所需的物品在转膜缓冲液中浸湿,制作转膜“三明治”,即黑色板-海绵-厚滤纸-凝胶-膜-厚滤纸-海绵-白色板,冰上200mA转膜,100KD以下转膜1h,100KD以上转膜2h。多个蛋白分子量相差过大时需分开转膜。
2.7取出NC膜,5%脱脂奶粉摇床封闭90min,回收封闭液。
2.8加入一抗,4℃摇床过夜或室温摇床1h~2h,摇床速度不要过快。回收一抗,标明使用次数。推荐内参要与检测的目的蛋白的分子量最好相差5KD以上。
2.9洗涤一抗,PBST洗4次,每次5min,摇床最大速率摇。
2.10在湿暗盒中孵二抗,一般每个上样孔滴100μl,注意看清一抗是兔抗还是鼠抗而选择相应的二抗,二抗按1:2000稀释,即0.5μl的二抗加1ml的封闭液。(先滴加二抗,将膜有蛋白的一面覆盖在二抗上,使充分接触)
2.11PBST洗涤4次,每次5min。
2.12配制ECL显色剂,A液和B液各500μl,混匀,将膜放在EP手套上,用100μl枪头滴加显色剂至膜的表面,曝光。
3.组织芯片(TMA)免疫组化(IHC)及IHC评分检测组织中ENO1的表达水平
3.1水化石蜡切片:
1)按顺序放置切片:二甲苯中放置15min,100%酒精中放置5min,95%酒精中放置5min,70%酒精中放置5min,最后在清水中放置5min;
2)用PBS清洗一次后浸泡5min。
3.2抗原重恢复:
1)将切片放置在片架上,并将片架放入10mM柠檬酸钠溶液中(储存液为100mM,pH6.0),沸水中蒸煮2h,或根据组织类型和要求减少或增加蒸煮时间;
2)取出片架,自然冷却至室温,用清水清洗3次,每次5min,之后浸泡于PBS至少5min。
3.3封闭内源性过氧化酶:
1)将切片置于90ml甲醇/10ml 30%H2O2中室温浸泡15-20min(不可超过30min);
2)PBS清洗3次,每次5min;
3)用卫生纸吸去多余的PBS,并用PAP笔在组织周围画圈。
3.4封闭、一抗孵育过夜:
1)将封闭液(5%BSA+1%goat serum+0.1%Tween 20)加置组织上,封闭至少1h;
2)用封闭液稀释一抗;
3)吸去组织上的封闭液,并将稀释的一抗加置组织上,4℃过夜。
3.5二抗、DAB显色
1)洗去一抗,一抗可反复使用多次;
2)用PBS清洗3次,每次5min;
3)用封闭液稀释二抗(生物素标记,一般1:1000稀释),室温孵育1h;
4)用PBS清洗切片3次,每次5min;
5)配制ABC试剂(含亲和素):在2.5ml PBS中加入一滴试剂A,混匀,再加入一滴试剂B,混匀,室温静置30min;
6)将ABC试剂加至组织切片上,室温孵育30min;
7)PBS清洗3次,每次5min;
8)准备DAB试剂:2.5ml蒸馏水中加入1滴DAB Buffer,混匀,再加入2滴DAB,混匀,最后加入一滴H2O2,混匀后待用;
9)将DAB试剂加至组织切片上,检测DAB反应,不要过度反应,适当反应后将切片浸浴于清水中以终止反应。
3.6反染切片
1)将切片置入苏木精4.5min;
2)清水中浸3次;
3)换掉清水,再浸5次;
4)将切片放入bluing试剂中1min;
5)清水中浸10次;
6)100%酒精中浸20次;
7)二甲苯中浸15次;
8)二甲苯中浸泡15-20min;
9)用盖玻片封片。
免疫组化评分(IHC score)用染色指数表示(0-12),即染色强度和染色区域的乘积。染色强度的分数被定为:阴性0分;弱1分;中等2分;强阳性3分。染色区域阳性细胞的频率被定义为:少于5%,0分;5%-25%,1分;26%-50%,2分;51%-75%,3分;大于75%,4分。0到7是低表达,8到12是高表达。
4.人源肿瘤异种移植模型(Patient-Derived tumor Xenograft,PDX)模型构建及药物处理
取新鲜病人肿瘤组织,剪切成小块(1mm2左右),一部分组织收蛋白,WB检测组织中METTL3、ALKBH5和ENO1的表达量;另外一部分小块组织接种到4-6周龄Balb/c裸鼠皮下,过程如下:
【实验材料】
眼科剪、眼科镊、1ml注射针、1.5ml离心管、培养皿、吸管、酒精、生理盐水、细胞培养液、4-6周龄Balb/c裸鼠。
【操作步骤】
(1)病人原代肿瘤组织,需在手术1-2小时之内进行处理,取出后,将其浸泡于含有1%的FBS和3%双抗的PBS缓冲液中。
(2)用含有1%的FBS和3%双抗的PBS缓冲液清洗3遍,弃上清液;对组织进行修剪,将所有外周非肿瘤及坏死肿瘤组织剔除,将组织块剪成组织小块(约2×2×2mm3),装于1.5ml EP管中。
(3)向EP管中加入1:1的PBS和BD MatrigelTM基质基底膜(康宁),每管加入100μl,备用。
(4)在无菌超净台中,对小鼠腹腔注射水合氯醛(10%的水合氯醛0.1ml/20g),以麻醉小鼠。
(5)用70%乙醇擦拭小鼠右下背部(小鼠左侧卧),用眼科剪将下皮剪开一小口(约3cm),并分离出一个小口袋状空间。
(6)用尖头镊子夹着肿瘤小块伸到开口深处,然后缓慢松开镊子。
(7)用伤口夹固定伤口,并在切口滴一滴100×双抗溶液防止伤口感染。
(8)术后观察,一般状态观察:术后保持观察直至小鼠完全苏醒,确定小鼠有无死亡现象出现。苏醒小鼠,每日观察其精神、饮食、排便和活动情况,并密切注意小鼠胸腔、纵隔、心包、胸水等肺癌细胞浸润和转移情况及小鼠死亡现象的出现,并作相应的记录。每两天称量小鼠体重一次,并作相应的记录。
(9)肿瘤生长情况检查:每日扪查移植瘤生长情况,自接种后4天起,每隔3-4天称量体重并用游标卡尺测量移植瘤体的长径(L)、短径(W)1次,取每例各小鼠肿瘤长短径平均值,按公式V=1/2(L×W2)计算移植瘤平均体积。并绘制肿瘤生长变化曲线。
(10)选择稳定传代后的第三代PDX小鼠,于接种10天开始进行药物腹腔注射,腹腔注射DMSO,DAA,2DG或ENO1抑制剂ENOblock,2天一次,每次剂量DAA(50mg/kg),2DG(1000mg/kg),ENOblock(20mg/kg)。每10天监测记录一次肿瘤大小,连续注射30天,30天的时候处死裸鼠,取出瘤体,拍照记录。
5.高表达ALKBH5同时敲除METTL3的H1975细胞的制备
(1)将ALKBH5表达序列(SEQ ID No:1)插入到过表达载体空载PLVX-Puro(货号BV20-pLVX,品牌BioVision Technology,Shanghai,Co.,Ltd.)的XhoI和BamHI酶切位点之间,得到ALKBH5过表达慢病毒载体。
(2)将METTL3-sgRNA序列(SEQ ID No:2)克隆至LentiCrisprV2(Addgene,#98290)慢病毒载体的两个BsmBI酶切位点之间,得到METTL3敲除载体。
(3)转染
1)第一天,将1.5×106HEK-293T细胞铺于6孔板。
2)第二天,按下列配方配制A液和B液:
A液:375μl无血清无双抗DMEM中加入如下质粒,一共为4μg;
B液:375μl无血清无双抗DMEM中加入10μl Lipofectamine 2000(2.5μl/μg DNA);
A液和B液在室温下孵育5min;将A和B液混合均匀,并在室温下孵育20min后加入293T细胞;6小时后用2ml正常培养基(含血清和双抗)替代转染培养基。
3)第三天,换液后24h后用一次性无菌针筒吸取293T细胞中含有病毒颗粒的培养基,并用0.45μM的滤器过滤,过滤后的病毒液可立即使用或冻存于-80冰箱。
(4)感染靶细胞
1)需要感染的靶细胞按照要求在感染前一天铺于六孔板内;
2)按体积加入1×Polybrene(0.4mg/ml 100×),混匀后室温孵育5min后直接加入靶细胞;
3)感染24h后,用含有puromycin(2mg/ml 1000×)的培养基替代感染培养基,根据细胞对puromycin的敏感性筛选若干天。
二、实验结果
1、METTL3在肺腺癌组织和细胞中高表达,ALKBH5在肺腺癌组织和细胞中低表达
由图1可见,ELISA和WB结果显示METTL3在肺腺癌组织高表达,ALKBH5在肺腺癌组织低表达(A-B);WB结果显示METTL3在肺腺癌细胞中高表达,ALKBH5在肺腺癌细胞中低表达(C)。
2.ENO1在肺腺癌组织中高表达
由图2可见,ELISA和IHC结果及IHC评分显示ENO1在肺腺癌组织中高表达(A-B)。
3.METTL3与ENO1在肺腺癌组织中呈正相关关系,ALKBH5与ENO1在肺腺癌组织中呈负相关关系
由图3可见,相关性分子结果显示肺腺癌组织中METTL3与ENO1的表达呈正相关关系(A),ALKBH5与ENO1的表达呈负相关关系(B)。
4.ENO1高表达刺激糖酵解产生能量促进肿瘤增殖
由图4可见,代谢组结果显示ENO1对于高表达METTL3同时敲除ALKBH5所造成的糖酵解异常激活是必须的(A-C)。(D)H1975细胞中高表达ALKBH5同时敲除METTL3可削弱过表达ENO1所造成的细胞异常增殖。(E)H1299细胞中敲除ENO1可明显削弱高表达METTL3同时敲除ALKBH5所造成的细胞增殖。
5.甲基化抑制剂和糖酵解抑制剂能显著抑制肺腺癌皮下移植瘤(CDX),人源肿瘤异种移植模型(PDX)肿瘤的生长
由图5可见,肺腺癌皮下移植瘤(CDX)结果显示甲基化抑制剂DAA(3-Deazaadenosine)和糖酵解抑制剂2DG(2-Deoxy-D-glucose),ENOblock(AP-III-a4)能显著抑制CDX肿瘤细胞生长(A-B),对小鼠体重没有影响(C);人源肿瘤异种移植模型(PDX)结果显示甲基化抑制剂DAA和糖酵解抑制剂2DG,ENOblock能显著抑制PDX肿瘤细胞生长(D-E),对小鼠体重没有影响(F)。
6.高表达ALKBH5同时敲除METTL3相比于仅高表达ALKBH5或仅敲除METTL3显著抑制了腺癌皮下移植瘤(CDX)的生长
由图6可见,肺腺癌皮下移植瘤(CDX)结果显示高表达ALKBH5同时敲除METTL3相比于仅高表达ALKBH5或仅敲除METTL3显著抑制了移植瘤的生长,抑瘤率高达90%以上。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 上海市胸科医院
<120> METTL3/ALKBH5/ENO1调控轴作为靶位点在制备治疗肺腺癌药物中的应用
<130> /
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1185
<212> DNA
<213> 人工序列
<400> 1
atggcggccg ccagcggcta cacggacctg cgtgagaagc tcaagtccat gacgtcccgg 60
gacaactata aggcgggcag ccgggaggcc gccgccgctg ccgcagccgc cgtagccgcc 120
gcagccgcag ccgccgctgc cgccgaacct taccctgtgt ccggggccaa gcgcaagtat 180
caggaggact cggaccccga gcgcagcgac tatgaggagc agcagctgca gaaggaggag 240
gaggcgcgca aggtgaagag cggcatccgc cagatgcgcc tcttcagcca ggacgagtgc 300
gccaagatcg aggcccgcat tgacgaggtg gtgtcccgcg ctgagaaggg cctgtacaac 360
gagcacacgg tggaccgggc cccactgcgc aacaagtact tcttcggcga aggctacact 420
tacggcgccc agctgcagaa gcgcgggccc ggccaggagc gcctctaccc gccgggcgac 480
gtggacgaga tccccgagtg ggtgcaccag ctggtgatcc aaaagctggt ggagcaccgc 540
gtcatccccg agggcttcgt caacagcgcc gtcatcaacg actaccagcc cggcggctgc 600
atcgtgtctc acgtggaccc catccacatc ttcgagcgcc ccatcgtgtc cgtgtccttc 660
tttagcgact ctgcgctgtg cttcggctgc aagttccagt tcaagcctat tcgggtgtcg 720
gaaccagtgc tttccctgcc ggtgcgcagg ggaagcgtga ctgtgctcag tggatatgct 780
gctgatgaaa tcactcactg catacggcct caggacatca aggagcgccg agcagtcatc 840
atcctcagga agacaagatt agatgcaccc cggttggaaa caaagtccct gagcagctcc 900
gtgttaccac ccagctatgc ttcagatcgc ctgtcaggaa acaacaggga ccctgctctg 960
aaacccaagc ggtcccaccg caaggcagac cctgatgctg cccacaggcc acggatcctg 1020
gagatggaca aggaagagaa ccggcgctcg gtgctgctgc ccacacaccg gcggaggggt 1080
agcttcagct ctgagaacta ctggcgcaag tcatacgagt cctcagagga ctgctctgag 1140
gcagcaggca gccctgcccg aaaggtgaag atgcggcggc actga 1185
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<400> 2
atatcacaac agatccactg 20
Claims (10)
1.METTL3基因或蛋白的抑制剂在制备治疗肺腺癌的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述抑制剂选自生物大分子或化合物小分子。
3.ALKBH5基因或蛋白或它们的促进剂在制备治疗肺腺癌的药物中的应用。
4.根据权利要求3所述的应用,其特征在于,所述促进剂选自生物大分子或化合物小分子。
5.ENO1基因或蛋白的抑制剂在制备治疗肺腺癌的药物中的应用。
6.根据权利要求5所述的应用,其特征在于,所述抑制剂选自生物大分子或化合物小分子。
7.下述a)、b)和c)中的至少2种的组合在制备治疗肺腺癌的药物中的应用:
a)METTL3基因或蛋白的抑制剂;
b)ALKBH5基因或蛋白或它们的促进剂;
c)ENO1基因或蛋白的抑制剂。
8.甲基化抑制剂DAA在制备治疗肺腺癌的药物中的应用。
9.糖酵解抑制剂2DG在制备治疗肺腺癌的药物中的应用。
10.糖酵解抑制剂ENOblock在制备治疗肺腺癌的药物中的应用。
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