CN116790753B - 联合靶向USP21与G3BP1 mRNA在治疗人食管鳞状细胞癌中的应用 - Google Patents
联合靶向USP21与G3BP1 mRNA在治疗人食管鳞状细胞癌中的应用 Download PDFInfo
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Abstract
本发明涉及联合靶向USP21与G3BP1mRNA在治疗人食管鳞状细胞癌中的应用。所述USP21基因的mRNA核苷酸序列如SEQ ID NO.1所示，所述G3BP1基因的mRNA核苷酸序列如SEQ ID NO.2所示。以本发明提供的USP21基因和/或G3BP1基因作为靶点，干扰抑制USP21基因和/或G3BP1基因的mRNA表达水平，可以有效抑制食管鳞状细胞癌细胞增殖、迁移与侵袭，并且设计了特异性敲低USP21基因与G3BP1基因mRNA表达水平的si‑USP21和si‑G3BP1。本发明还发现使用si‑USP21和si‑G3BP1的混合物si‑UG同时敲低USP21和G3BP1mRNA表达水平对USP21基因与G3BP1基因的抑制效果达到最佳，抑制食管鳞状细胞癌增殖与转移的效果最好，可应用于食管鳞状细胞癌靶向治疗药物的研发与制备。
Description
技术领域
本发明涉及联合靶向USP21与G3BP1 mRNA在治疗人食管鳞状细胞癌中的应用，属于生物医学技术领域。
背景技术
食管鳞状细胞癌是食管癌最常见的病理类型，占我国食管癌病例的90％以上。食管癌的传统治疗方式主要以手术、化疗及放疗为主。近年来，靶向与免疫治疗的发展与临床应用将食管癌的治疗推向了个体化精准诊疗时代。已应用于临床的食管癌治疗靶点包括HER2、EGFR、VEGFR、MET等，相应的靶向药物为食管癌患者带来了切实的预后提高，但食管癌的高肿瘤异质性经常导致单一靶向治疗方案的失败。因此，探究驱动食管癌进展的新的调控通路，并同时干预通路中的关键靶点，对于提高食管癌靶向治疗效果以及降低复发风险都具有重要意义。
RNA干扰技术是实现特异性敲低细胞内靶基因表达水平的常用手段之一。该技术的原理是通过细胞内的RNA诱导的沉默复合体降解目的基因的mRNA，而沉默复合体识别目的基因mRNA则需要在siRNA的引导下完成。因此，设计出高效特异性的siRNA是RNA干扰技术的关键所在。应用siRNA特异性有效敲低目的基因的表达水平对于研究该基因在癌细胞恶性表型中的生物学功能极为重要，也将为相关靶向药物的研发提供重要理论依据。
发明内容
针对现有技术的不足，联合靶向USP21与G3BP1 mRNA在治疗人食管鳞状细胞癌中的应用。
本发明的技术方案如下：
USP21基因和/或G3BP1基因的mRNA作为药物靶点在制备治疗食管鳞状细胞癌药物中的应用。
根据本发明优选的，所述USP21基因的mRNA核苷酸序列如SEQ ID NO.1所示，所述G3BP1基因的mRNA核苷酸序列如SEQ ID NO.2所示。
根据本发明优选的，所述治疗食管鳞状细胞癌药物以USP21基因和/或G3BP1基因作为作用靶点，能够高效特异性干扰抑制USP21基因和/或G3BP1基因的表达。
根据本发明优选的，所述治疗食管鳞状细胞癌药物为siRNA、shRNA、microRNA或USP21基因、G3BP1基因化学抑制剂。
进一步优选的，所述siRNA为si-USP21、si-G3BP1或si-UG；
所述si-USP21的核苷酸序列如SEQ ID NO.3所示，所述si-G3BP1的核苷酸序列如SEQ ID NO.4所示；
所述si-UG为si-USP21和si-G3BP1按照1:1的摩尔比混合的混合物。
一种治疗食管鳞状细胞癌药物，所述治疗食管鳞状细胞癌药物包含USP21基因和/或G3BP1基因抑制剂。
有益效果：
1、以本发明提供的USP21基因和/或G3BP1基因作为靶点，干扰抑制USP21基因和/或G3BP1基因的mRNA表达水平，可以有效抑制食管鳞状细胞癌细胞增殖、迁移与侵袭。本发明设计了通过敲低低USP21基因与G3BP1基因mRNA的表达水平来干扰抑制USP21基因和/或G3BP1基因的表达，成功抑制了食管鳞状细胞癌细胞增殖、迁移与侵袭，因此可以将USP21基因和/或G3BP1基因作为治疗靶点制备治疗食管鳞状细胞癌药物。
2、本发明还发现使用si-USP21和si-G3BP1的混合物si-UG同时特异性敲低USP21和G3BP1 mRNA表达水平，相比于应用同剂量si-USP21和si-G3BP1单独敲低USP21和G3BP1mRNA表达水平，对于食管鳞状细胞癌细胞增殖、迁移与侵袭能力的抑制作用更为显著，说明同时敲低USP21和G3BP1 mRNA表达水平对USP21基因与G3BP1基因的抑制效果达到最佳，抑制食管鳞状细胞癌增殖与转移的效果最好，可应用于食管鳞状细胞癌靶向治疗药物的研发与制备。
附图说明
图1为应用si-UG，将其转染至Eca-109细胞中，应用逆转录-实时荧光定量PCR检测USP21 mRNA与G3BP1 mRNA水平，应用Student’s t检验明确组间差异的统计学意义。
图2为应用si-UG，将其转染至Eca-109细胞中，使用相同剂量的si-USP21，si-G3BP1或者si-NC分别转染至Eca-109细胞中，应用CCK-8实验检测Eca-109细胞增殖能力的变化，应用双向方差检验明确组间差异的统计学意义。
图3为选取Eca-109细胞，分别转染si-UG、si-USP21、si-G3BP1和si-NC，每种siRNA所用转染剂量相同，应用Transwell实验评估Eca-109细胞的迁移与侵袭能力，应用单向方差检验计算组间差异的统计学意义。
具体实施方式
下面结合具体实验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。
人源食管鳞状细胞癌细胞系Eca-109细胞，在上海富衡生物科技有限公司有售。
实施例1
发明人前期研究发现，食管鳞状细胞癌肿瘤组织中USP21水平相比于临近正常食管黏膜显著上调，且提示患者不良预后。在机制探究过程中，发明人还发现G3BP1是USP21的去泛素化底物，并证明USP21通过稳定G3BP1蛋白水平在食管鳞状细胞癌中发挥促癌作用。因此，发明人认为联合靶向USP21与G3BP1在治疗人食管鳞状细胞癌中将发挥重要作用。
实施例2
根据USP21基因和G3BP1基因的mRNA核苷酸序列，设计了特异性敲低USP21与G3BP1mRNA表达水平的si-USP21、si-G3BP1，所述si-USP21的核苷酸序列如SEQ ID NO.3所示，所述si-G3BP1的核苷酸序列如SEQ ID NO.4所示，然后将si-USP21和si-G3BP1按照1:1的摩尔比混合得到si-UG。将相同剂量的si-UG与si-NC分别转染至Eca-109细胞，应用逆转录-实时荧光定量PCR检测USP21与G3BP1 mRNA mRNA水平，具体结果如图1所示。
由图1可知，转染si-UG能够有效敲低Eca-109细胞中USP21与G3BP1 mRNA水平，干扰抑制USP21基因和G3BP1基因的表达。
具体实施过程如下：
(1)选取Eca-109细胞，并培养于6孔板中，待细胞生长密度达到60％，应用转染试剂Lipofectamine RNAiMAX Reagent(Life technologies，13778-150)将相同剂量(体积3μL，浓度10μM)的si-UG与si-NC分别转染至Eca-109细胞；
(2)转染后48小时，收集细胞，应用RNA-Quick Purification Kit(上海奕杉，RN001)试剂盒提取RNA，应用琼脂糖凝胶电泳评估所提RNA完整性，并应用NanoDrop2000检测所提RNA浓度；
(3)应用逆转录试剂盒LunaScriptTMRT SuperMix Kit(NEB,E3010)以上述所提RNA为模板，合成cDNA产物，使用PowerGreen Master Mix(Thermo FisherScientific,4367659)试剂盒，并在QuantStudioTM 5System(Thermo Fisher Scientific)PCR仪器上进行荧光定量PCR，以ACTB mRNA水平作为内参，按照2-ΔΔCT方法计算USP21与G3BP1 mRNA相对表达水平。
实施例3
选取Eca-109细胞，分别转染si-UG，si-USP21、si-G3BP1和si-NC，应用CCK-8实验检测上述siRNA对Eca-109细胞增殖能力的影响，具体结果如图2所示。
由图2可知，转染si-UG相比于单独转染相同剂量的si-USP21、si-G3BP1显著削弱Eca-109细胞的增殖能力。并且还发现使用si-USP21、si-G3BP1的混合物si-UG同时特异性敲低USP21与G3BP1 mRNA表达水平，相比于应用同剂量si-USP21、si-G3BP1单独敲低USP21与G3BP1mRNA表达水平，对于Eca-109细胞增殖能力的抑制作用更为显著，说明同时敲低USP21与G3BP1 mRNA表达水平对USP21与G3BP1基因的抑制效果达到最佳，抑制食管鳞状细胞癌增殖的效果最好。
具体实施过程如下：
(1)选取Eca-109细胞，分别转染相同剂量(体积3μL，浓度10μM)的si-UG、si-USP21、si-G3BP1或者si-NC，具体方法详见实施例2，24小时后收集转染细胞并转移至96孔板中(2,000个/孔)，共分4组，每组包括si-UG、si-USP21、si-G3BP1、si-NC 4种转染细胞，每种细胞5个复孔；
(2)约2-4小时后细胞贴壁，选择任意一组细胞并加入CCK-8试剂(TargetMol,C0005)(10μL/孔)，37℃孵箱中放置1小时，应用酶标仪中测量450nm处的吸光度值(起始种植细胞的吸光度值)；
(3)每隔24小时在剩余组里任选一组，重复上述测量，方法同上，以此计算各组内不同转染细胞在1、2、3天后的细胞活力。
实施例4
选取Eca-109细胞，分别转染相同剂量的si-UG、si-USP21、si-G3BP1或si-NC，应用Transwell实验评估细胞迁移与侵袭能力，具体结果如图3所示。
由图3可知，相同转染剂量下，si-UG相比于单独si-USP21、si-G3BP1显著抑制Eca-109细胞的迁移与侵袭能力。并且还发现使用si-USP21、si-G3BP1的混合物si-UG同时特异性敲低USP21与G3BP1 mRNA表达水平，相比于应用同剂量si-USP21、si-G3BP1单独敲低USP21与G3BP1 mRNA表达水平，对于Eca-109细胞迁移与侵袭能力的抑制作用更为显著，单独敲低USP21与G3BP1 mRNA表达水平抑制Eca-109细胞迁移与侵袭的效果叠加后也不如转染si-UG后的Eca-109细胞。说明同时敲低USP21与G3BP1 mRNA表达水平对USP21基因与G3BP1基因的抑制效果达到最佳，抑制食管鳞状细胞癌迁移与侵袭的效果最好。
具体实施过程如下：
(1)选取Eca-109细胞，分别转染相同剂量(体积3μL，浓度10μM)的si-UG、si-USP21、si-G3BP1或si-NC，方法如实施例2所述；
(2)应用Transwell小室(Corning，3422)进行实验，未铺基质胶用于检测细胞迁移能力，预先铺基质胶以用于检测细胞侵袭能力，将transwell小室放入24孔板中(每孔预先加入600μL完全培养基)；
(3)收集转染细胞离心，使用无血清培养基重悬，将细胞悬液转移至transwell小室内(2万个细胞/200μL)，置于37℃、5％ CO2的孵箱中培养；
(4)48小时后取出transwell小室并清洗干净，使用4％多聚甲醛于室温条件下固定细胞15分钟，并使用0.1％结晶紫染色过夜，使用棉签刮除transwell小室内面未发生迁移、侵袭的细胞，应用倒置显微镜观察transwell小室外侧面的迁移与侵袭细胞。
Claims (3)
1.特异性干扰USP21基因的si-RNA和特异性干扰G3BP1基因的si-RNA在制备靶向治疗食管鳞状细胞癌药物中的应用；
其中，所述特异性干扰USP21基因的si-RNA的核苷酸序列如SEQ ID NO.3所示，所述特异性干扰G3BP1基因的si-RNA的核苷酸序列如SEQ ID NO.4所示。
2.如权利要求1所述的应用，其特征在于，所述药物中特异性干扰USP21基因的si-RNA和特异性干扰G3BP1基因的si-RNA的摩尔比为1:1。
3.一种治疗食管鳞状细胞癌药物，其特征在于，所述治疗食管鳞状细胞癌药物包含特异性干扰USP21基因的si-RNA和特异性干扰G3BP1基因的si-RNA；
其中，所述特异性干扰USP21基因的si-RNA的核苷酸序列如SEQ ID NO.3所示，所述特异性干扰G3BP1基因的si-RNA的核苷酸序列如SEQ ID NO.4所示。
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