CN114457158B - Hsa_circ_0006867作为食管癌分子靶标在制备药物和试剂盒中的应用 - Google Patents
Hsa_circ_0006867作为食管癌分子靶标在制备药物和试剂盒中的应用 Download PDFInfo
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Abstract
本发明公开了一种Hsa_circ_0006867环状RNA在制备、筛选或治疗食管鳞状细胞癌药物中的应用,其特征在于所述环状RNA在食管鳞状细胞癌癌组织表达水平显著低于癌旁组织。本发明通过制备hsa_circ_0006867过表达质粒载体,过表达慢病毒验证其食管鳞状细胞癌细胞中的功能,首次发现了过表达hsa_circ_0006867可显著抑制食管鳞状细胞癌细胞增殖、侵袭及迁移等肿瘤细胞生物学表型。因此,hsa_circ_0006867在食管鳞状细胞癌发挥抑癌作用,其可作为治疗靶点在制备、筛选或治疗食管鳞状细胞癌或抑制食管鳞状细胞癌转移药物中的应用,为食管鳞状细胞癌的干预或治疗提供一种新的治疗方法。
Description
技术领域
本发明涉及一种环状RNA Hsa_circ_0006867在制备、筛选或治疗食管鳞状细胞癌药物中的应用,属于生物医药技术领域。
背景技术
食管癌是一种具有高发病率、高死亡率及高复发率等特征的消化道肿瘤。据2020年全球癌症统计数据显示,食管癌发病率位居全球第七,死亡率位居第六位,是世界范围内威胁人类健康的高发恶性肿瘤之一。食管癌病理类型主要包括鳞状细胞癌和腺癌,在南非、东非、东亚等食管癌高发区,90%的病理类型为食管鳞状细胞癌。我国是世界上食管癌发病率和死亡率最高的国家,由于我国人口基数大,病程进展迅速等因素,我国每年食管癌新发病例和死亡病例分别占居全球的53.7%和55.7%左右,给我国带来了严重的疾病负担。随医学技术的快速发展在食管癌治疗方面虽取得一定改善,如开胸手术到微创食管癌手术,再到免疫抑制治疗等,但患者5年生存率仍低于15%。目前,对于食管鳞状细胞癌的术后高复发及高转移也仍缺少有效的控制手段。因此,阐明食管鳞状细胞癌发生发展的分子机制,寻找调控食管鳞状细胞癌发生、侵袭转移及凋亡的新靶点以及针对性的抗肿瘤药物的研发,在此基础上制定靶向干预策略有望为食管鳞状细胞癌治疗带来新的选择,对相关药物、试剂具有重要意义。
环状RNA(circular RNA,circRNA)是一类具有共价闭合结构的非线性非编码RNA,既无5’端帽子也无3’端polyA尾巴结构,其稳定性超过其他线性RNA分子,且不易被RNA核酸外切酶和RNA酶降解,从而使其具有高度的保守性、稳定性及组织特异性。近年来,circ RNA与癌症的关系已成为癌症研究领域的重点问题,数百个circ RNA已被证明在包括食管鳞状细胞癌在内的各种人类癌症中异常表达,主要在miRNA分子海绵,调控宿主基因表达,调控可变剪接等方面发挥作用,进而参与调控肿瘤细胞增殖、上皮间充质转化、肿瘤细胞侵袭和转移。随“mRNA时代”的到来,“环形RNA疗法”在治疗癌症、自身免疫病、再生医学、遗传性疾病等方面已进入研究者视线,如利用工程化手段制造出具有高效编码蛋白质潜力的环状RNA(oRNA),可在真核细胞中高效且稳定地表达蛋白质,这意味着oRNA能够让患者以更小剂量获得所需的治疗效果,可能更适用于那些需要高蛋白水平且超出线性mRNA表达水平治疗的疾病。但目前,关于circRNA在食管细胞癌变过程中表达及调控作用的研究尚不清晰,尚无明确食管鳞状细胞癌circRNA分子靶标,因此,进一步系统挖掘食管鳞状细胞癌中发挥作用的circ RNA,对于进一步了解食管鳞状细胞癌发生发展机制,探索新的食管鳞状细胞癌治疗分子靶标,推动“环形RNA疗法”进入IND(Investigational New Drug)阶段,为全世界肿瘤患者带来新的治疗药物具有重要意义。
发明内容
本发明目的是提供一种RNA Hsa_circ_0006867在制备、筛选或治疗食管鳞状细胞癌药物中的应用,主要解决食管鳞状细胞癌药物治疗单一、治疗效果不佳及肿瘤易转移等问题。
本发明的第一个方面提供了一种食管鳞状细胞癌分子靶标,所述分子靶标是Hsa_circ_0006867,其是一种环状RNA,位于染色体4q31.3位点的一种抑癌基因。
本发明通过对食管鳞状细胞癌患者癌组织和癌旁组织样本中circRNA表达差异性分析,确定了Hsa_circ_0006867具有作为食管鳞状细胞癌治疗分子靶标的潜能,所述分子靶标在食管鳞状细胞癌癌组织样本中的表达明显低于癌旁组织,差异具有统计学意义,其可作为食管鳞状细胞癌药物制备、筛选及治疗的分子靶标。
优选地,本发明还提供了一种检测食管组织样本Hsa_circ_0006867的方法,包括以下步骤:
1)采集食管鳞状细胞癌癌组织和癌旁组织(距癌组织>5cm),并对组织样本进行处理,提取组织RNA;
2)使用MMLV逆转录酶对步骤1)组织RNA逆转录成cDNA;
3)根据Hsa_circ_0006867接口序列设计其特异性引物对步骤2)cDNA进行RT-QPCR扩增,收集荧光,计算熔解曲线;
4)根据步骤3)所得ΔCt值,并按照ΔCt值越大,表明实际表达量越低原则,根据T检验筛选综合分析结果最优、差异表达最显著的circRNA。
本发明的第二个方面提供了一种食管鳞状细胞癌的抗癌药物,其含有Hsa_circ_0006867的促进剂,所述用途包括但不局限于制备、筛选及治疗食管鳞状细胞癌的药物。
优选地,所述促进剂在制备及筛选抑制食管鳞状细胞癌细胞增殖药物中的应用;
优选地,所述促进剂在制备及筛选抑制食管鳞状细胞癌细胞侵袭迁移药物中的应用;
优选地,所述的促进剂,载体为pLent-EF1a-circRNA-CMV-RFP-P2A-Puro(9.2Kb),hsa_circ_0006867序列连接位置于Asisl和MIuI,启动子为EF1a,荧光蛋白为RFP。
优选地,所用食管鳞状细胞癌细胞是在含100nmol/L微囊藻毒素(MC-LR)+100nmol/L甲基苄基亚硝胺(NMBzA)培养基对Het-1A进行35代染毒形成的T-Het-1A细胞。
本发明的优点和有益效果
本研究首次发现Hsa_circ_0006867在食管鳞状细胞癌及癌组织的差异表达,通过制备Hsa_circ_0006867过表达载体验证了其在食管鳞状细胞癌细胞中的功能,我们发现过表达Hsa_circ_006386则可以显著抑制食管鳞状细胞癌的增殖、侵袭及迁移,这是食管鳞状细胞癌治疗非常重要的分子靶标,为肿瘤患者的治疗提供了一种新的有效治疗手段,减少了患者的治疗成本且提高了其生存率,具有很好的药物开发前景。
下面结合附图和具体实施方式进一步详细说明,以使本发明公开的各个方面及其优势显而易见,从而更容易被理解,并非对本发明的限制。凡依照本发明公开内容进行的本领域的等同替换,均属于本发明的保护范围。
附图说明
图1为Hsa_circ_0006867的生物合成及结构示意图;
图2为Hsa_circ_0006867过表达载体结构示意图;
图3为食管鳞状细胞癌组织和癌旁组织Hsa_circ_0006867差异表达水平;
图4为Hsa_circ_0006867稳转过表达Het-1A中Hsa_circ_0006867表达水平;
图5为微囊藻毒素和亚硝胺联合染毒Hsa_circ_0006867过表达Het-1A细胞35代平板克隆实验、软琼脂克隆实验及裸鼠皮下移植瘤解剖结果图;
图6为Hsa_circ_0006867促进剂调控细胞增殖、侵袭及迁移结果图;
图7为Hsa_circ_0006867促进剂在裸鼠皮下移植瘤细胞增殖能力及转移能力结果图。
具体实施方式
下来结合实施例对本发明作进一步详细说明,可以理解为该实施例仅为解释本发明,而非对本发明加以限制,但针对本发明教导所作的任何变换或替换,均属本发明的保护范围。
实施例1:RT-QPCR检测Hsa_circ_0006867在食管鳞状细胞癌组织中的表达水平明显低于癌旁组织;
1.食管鳞状细胞癌组织样本RNA提取
1)在经东南大学医学伦理委员会批准,患者/家属知情同意后,选取2009-2012年于江苏省淮安市第一人民医院诊断为食管鳞状细胞癌的患者96例,共收集96例癌组织和对应癌旁组织;
2)取0.1cm3组织用剪刀剪碎,放入1.5mL EP管,加入1mL Trizol、3颗研磨钢珠,70Hz研磨10min(研磨盒提前预冷),后续按照Trizol试剂常规提取步骤进行操作;
3)取1μl RNA样品于紫外分光光度计检测其浓度和纯度,所得RNA样品A260/A280比值均在1.8-2.0之间。
2、RNA逆转录成cDNA
使用MMLV逆转录酶进行逆转录,逆转录反应体系及条件如下:
RNA | 1μg |
Random primer | 1μL |
补无酶水 | 至15μL |
70℃反应10min,冰浴2min。
上述反应混合液 | 15μL |
10mM dNTP | 1μL |
MMLV | 1μL |
补无酶水 | 至25μL |
37℃反应60min,90℃10min,4℃保持。
3、cDNA荧光定量PCR:
根据Hsa_circ_0006867结构及接头序列(结果见图1)设计其特异性引物:circ-0006867-F的核苷酸序列为GATGTTTCTCAACACGATAGCAGGAC(SEQ ID NO.2);circ-0006867-R的核苷酸序列为ATTGATCGTATCTCTGTGAACAGCCAT(SEQ ID NO.3);
反应体系:
cDNA | 1μL |
SYBR Green Mix | 5μL |
5μL | 1μL |
上、下游引物 | 各0.6μL |
补水 | 补水至10μL |
反应条件:94℃预热5min,94℃30s,60℃30s,反应40个循环,每个循环60℃时收集荧光,60℃~95℃升温,收集荧光,计算熔解曲线。
4、数据处理与分析
待检样本Hsa_circ_0006867表达水平通过其Ct值与内参β-actin Ct值法进行计算得出ΔCt,ΔCt值越大,表明实际表达量越低。统计学分析方法采用T检验进行分析,以P<0.05为差异存在统计学意义。
5、实验结果与分析
在该队列食管鳞状细胞癌患者癌组织Hsa_circ_0006867表达水平明显低于癌旁组织,差异具有统计学意义(结果见图3),表明Hsa_circ_0006867作为一个抑癌基因,可能是治疗食管鳞状细胞癌的重要分子靶标。
实施例2Hsa_circ_0006867参与亚硝胺联合藻毒素致食管癌恶性转化
1、构建Hsa_circ_0006867过表达细胞模型
在前期已构建的Hsa_circ_0006867过表达载体基础上,进行Hsa_circ_0006867过表达慢病毒包装。将3×106个293T细胞接种于直径10cm的培养皿,待细胞生长至50~70%融合率时即可用于质粒转染;取10μgHsa_circ_0006867过表达质粒、6.6μg pMDLg/pRRE质粒、2.4μg pRSV-Rev质粒以及3.6μg pMD2G质粒,加培养基稀释至100μL,吹打混匀,室温静置5~10min;取44μL/>2000,加56μL/> 培养基吹打混匀,室温静置5~10min;将上述两种混合液混合,吹打混匀,室温静置20min;将上述脂质体-质粒混合物加入5mL/>培养基吹打混匀;去除293T细胞培养基,PBS清洗两遍,将稀释的混合液全部加入293T细胞培养皿,培养六小时后更换正常含10%血清无双抗培养基培养,24小时后更换含双抗及血清的正常培养基,一定时间后收获病毒并进行病毒滴度测定。
2、Hsa_circ_0006867参与亚硝胺联合藻毒素致食管癌恶性转化
1)细胞染毒恶性转化:取Hsa_circ_0006867过表达(Hsa_circ_0006867high)及Hsa_circ_0006867过表达对照(over-NC)Het-1A细胞在含100nmol/L MC-LR+100nmol/LNMBzA培养基对其染毒35代,观察并分析Hsa_circ_0006867在恶性转化过程中发挥的作用。实验分为染毒组和对照组,对照组在培养基中加入相同剂量的DMSO溶液。
2)平板克隆实验:取对数增长期细胞,使用0.25%胰酶消化离心制备单细胞悬液,按照2×102/孔密度将细胞种入六孔板,正常培养,隔天观察细胞克隆生长情况,培养2周后将细胞取出,使用结晶紫染色,拍照计数>50个细胞的克隆,计算克隆形成率。
3)软琼脂克隆实验:配制100mL 1.2%和0.7%的琼脂、含20%血清及2×抗生素的DMEM培养基;按1:1的比例混合1.2%琼脂和配制好的2×培养基,将1.5mL混合液迅速加入六孔板,室温静置至胶凝固备用;取对数期生长细胞,0.25%胰酶消化后用2×培养基调整浓度至8×102/mL,将细胞悬液与0.7%琼脂溶液混匀,迅速滴加在配好的下层胶上,每孔加入1mL,待上层胶凝固,将细胞放入含5% CO2培养箱37℃培养3周;间隔三天补加200μL含10% FBS培养基;隔天观察克隆生长情况,培养结束后在显微镜下观察计数克隆形成情况,计数>50个细胞的克隆,计算克隆形成率。
4)裸鼠皮下成瘤实验:选用3-4周龄,SPF级,BALB/c Nude CRLJ雄性裸鼠。取指数增长期Hsa_circ_0006867high、Over-NC组Het-1A-T种板,制备细胞密度为4×107/mL单细胞悬液,加入等体积的Matrigel胶;每只裸鼠左腋皮下注射200μL细胞悬液;每日观察接种后皮下肿瘤生长情况,待出现肿瘤后,每隔3天测量一次,瘤体生成两周后停止饲养,采用颈椎脱臼法处死,取出皮下移植瘤测量肿瘤大小并拍照记录。
5)结果显示:Hsa_circ_0006867稳转过表达细胞中Hsa_circ_0006867表达显著升高,表达是对照细胞的254倍(图4),染毒35代时Hsa_circ_0006867过表达细胞克隆数显著低于对照细胞(图5A),Hsa_circ_0006867过表达染毒细胞形成克隆数目显著低于对照(图5B),对解剖下来的移植瘤进行体积比较,结果显示Hsa_circ_0006867过表达细胞移植瘤体积显著低于对照(图5C)。提示Hsa_circ_0006867作为一抑癌基因在食管癌发生过程中发挥至关重要的作用。
实施例3过表达Hsa_circ_0006867对食管鳞状细胞癌细胞生物表型影响
1)Hsa_circ_0006867过表达稳转细胞模型构建:使用Hsa_circ_0006867过表达质粒及干扰对照慢病毒转染Het-1A-T,使用嘌呤筛选获取Hsa_circ_0006867high、Over-NC细胞株。
2)细胞增殖能力检测:以1×104/孔将细胞接种于96孔板,待细胞长至50%融合度时进行EdU孵育2h,后续步骤按照BeyoClick EdU细胞增殖检测试剂盒说明书进行;最后使用PBS清洗两遍,每次5min;弃掉清洗PBS,每孔加入50μL新的PBS,显微镜下观察拍照;每孔随机选取5个视野拍摄,计算绿色细胞(增殖细胞)、蓝色细胞(总细胞)数目。
3)细胞侵袭能力检测:取对数增长期细胞进行铺板,待细胞融合度70%,将-20℃Matrigel基质胶放于4℃冰箱溶解,另将Transwell小室放入4℃冰箱预冷;按照1:9的比例使用无血清培养基稀释基质胶,将稀释好的基质胶快速加入小室,每个小室加50μL;将小室放入37℃培养箱中加热1h,使基质胶凝固,取出小室,吸去上层未凝固液体;消化收集细胞,使用无血清培养基制备单细胞悬液,按照5×105/孔,将细胞加入小室内,下室加入600μL含50% FBS的完全培养基;培养24h后,取出小室,用棉签擦去未穿膜的细胞及基质胶,甲醇固定风干后使用0.1%结晶紫进行染色;显微镜下观察,每个小室随机选取5个视野拍照,统计穿膜细胞数。
4)细胞迁移能力检测:使用8μm孔径Transwell小室进行细胞迁移能力检测,取指数增长期细胞,经胰酶消化制备单细胞悬液,每个小室接种5×104细胞,小室上室加无血清培养基补足200μL,下室加入600μL含20%FBS完全培养基。常规培养24h后取出小室,使用枪头吸掉小室残余的液体,用棉签转动擦掉小室内侧未穿过的细胞,使用甲醇浸泡10min,取出小室,待膜风干后,使用0.1%结晶紫染液染色,常温染色15min,使用PBS淋洗,自然风干后放回24孔板。显微镜下观察,每个小室随机选取5个视野拍照,统计穿膜细胞数。
5)裸鼠移植瘤中Hsa_circ_0006867功能分析:于实施例2第4步)获得的裸鼠皮下移植瘤切片,采用免疫组化常规操作步骤进行,所述抗体为裸鼠皮下移植瘤增殖相关基因PCNA抗体,裸鼠皮下移植瘤迁移相关基因E-cadherin抗体。
6)结果显示:在Het-1A-T中Hsa_circ_0006867过表达抑制细胞增殖(图6A),迁移能力(图6B)和细胞侵袭(图6C)。使用免疫组化检测皮下移植瘤中PCNA表达,结果显示Hsa_circ_0006867过表达细胞中PCNA表达显著低于对照(图7A),细胞中E-cadherin表达显著高于对照(图7B),提示Hsa_circ_0006867在食管癌进展过程具有重要地位,且Hsa_circ_0006867促进剂可以显著抑制肿瘤进展,其可作为药物研发及食管癌治疗中新的分子靶标。
在本说明书的描述中,参考术语“一个实施例”、“示例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。
序列表
<120> Hsa_circ_0006867作为食管癌分子靶标在制备药物和试剂盒中的应用
<141> 2022-01-19
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 450
<212> DNA/RNA
<213> Homo sapiens
<400> 1
atcttggcat atacagaagg gctgcatgga aaatggctgt tcacagagat acgatcaatc 60
ttttctcgtc gttatctttt gcaaaataca gccctggaga tctttatggc aaacagagtt 120
gctgtgatgt tcaacttccc agaccctgca acagtaaaga aagtggttaa ctatctacct 180
cgtgttggcg ttggaacaag ttttggattg cctcaaacca gacgtatttc attagctagt 240
ccacgtcagc tttttaaggc ttctaatatg acccagcgat ggcaacacag agagatatct 300
aattttgagt acttgatgtt tctcaacacg atagcaggac ggagttataa tgacttaaat 360
cagtatccag tgtttccttg ggtcatcact aattatgaat cagaagaact ggatcttacc 420
ttgcccacca acttcagaga tttgtccaag 450
<210> 2
<211> 25
<212> DNA/RNA
<213> 人工合成()
<400> 2
gatgtttctc aacacgatag caggac 26
<210> 3
<211> 27
<212> DNA/RNA
<213> 人工合成()
<400> 3
attgatcgta tctctgtgaa cagccat 27
Claims (4)
1.用于检测环状RNA hsa_circ_0006867的试剂在筛选预防、缓解和/或治疗食管鳞状细胞癌药物中的应用,hsa_circ_0006867核苷酸序列为SEQ ID NO.1所示,作为食管鳞状细胞癌的分子靶标。
2.根据权利要求1所述的应用,其特征在于,分子靶标的特异性引物包括hsa_circ_0006867-F和hsa_circ_0006867-R,所述hsa_circ_0006867-F的核苷酸序列为SEQ IDNO.2;hsa_circ_0006867-R的核苷酸序列为SEQ ID NO.3。
3.一种hsa_circ_0006867促进剂在制备用于预防、缓解和/或治疗食管鳞状细胞癌药物中的应用,所述hsa_circ_0006867的核苷酸序列为SEQ ID NO.1所示,所述促进剂为:Hsa_circ_0006867过表达质粒。
4.根据权利要求3所述的应用,其特征在于,所述促进剂载体为pLent-EF1a-circRNA-CMV-RFP-P2A-Puro,9.2Kb,hsa_circ_0006867序列连接位置于Asisl和MIuI,启动子为EF1a,荧光蛋白为RFP。
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