siRNA IRAK1基因转染人子宫内膜样腺癌HEC-1-B和JEC细胞对裸鼠成瘤的影响
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摘要
目的探讨siRNA IRAK1基因转染人子宫内膜样腺癌细胞对裸鼠成瘤的影响。方法体外培养子宫内膜样腺癌细胞系HEC-1-B和JEC,随机分为空白对照组(Con组)、阴性对照组(siNC组)和siRNA IRAK1组,siNC组和siRNA IRAK1组分别转染空载质粒慢病毒、siRNA IRAK1慢病毒。采用Western blotting法检测各组IRAK1蛋白表达情况。将体外转染siRNA IRAK1慢病毒、siNC空载质粒慢病毒的人子宫内膜样腺癌细胞系HEC-1-B和JEC接种到裸鼠皮下,制备皮下移植瘤模型,观察各组小鼠状态及移植瘤的生长情况,50 d后处死裸鼠,剥取移植瘤观察其体积大小,并用Western blot检测各组移植瘤组织IRAK1的表达。结果与对照组比较,siRNA-IRAK1组在HEC-1-B和JEC中均有效地下调了IRAK1的表达,差异有统计学意义(P<0.01);稳定转染siRNA IRAK1或siNC的HEC-1-B和JEC悬液,皮下注射裸鼠,24只裸鼠成瘤率为100%。与siNC组比较,稳定转染siRNA IRAK的HEC-1-B和JEC组裸鼠精神及活动能力良好,未见明显活动异常,体重未见进行性下降;第50天平均体重分别为(16.3±3.5)、(16.6±4.1)g,高于siNC组的(13.1±4.2)、(13.6±3.8)g,差异有统计学意义(P<0.05)。50 d后观察siRNA-IRAK1组裸鼠成瘤的体积明显小于对照组,差异有统计学意义(P<0.01);与siNC组比较,siRNA-IRAK1组肿瘤的生长速度慢,差异有统计学意义(P<0.01)。Western blot结果显示,与siNC组比较,siRNA IRAK1处理的HEC-1-B和JEC组小鼠肿瘤组织中IRAK1蛋白表达水平分别降低59.3%和64.5%,差异有统计学意义(P<0.05)。结论抑制IRAK1表达能降低子宫内膜样腺癌细胞增殖速度,抑制裸鼠移植瘤生长。
Objective To investigate the effect of siRNA IRAKI gene transfected human endometrioid adenocarcinoma HEC-1-B and JEC cells on tumor formation in nude mice. Methods Endometrial adenocarcinoma cell lines HEC-1-B and JEC cultured in vitro were randomly divided into the blank control group, negative control group, and siRNA-IRAK1 group.Recombinant lentivirus virus vectors(NC-siRNA and IRAK1-siRNA) were constructed and were respectively transfected into negative control group and siRNA-IRAK1 group.The expression levels of IRAK1 protein were measured by Western blotting. Human endometrial adenocarcinoma cell lines HEC-1-B and JEC transfected with lentiviral vectors expressing siRNA IRAK1, or empty lentiviral vectors were transplanted subcutaneously into nude mice to generate tumor xenografts,respectively. The status of mice and the growth of the xenografts in each group of mice was observed. At 50 d after transplantation, the mice were sacrificed and their xenografts were isolated. The size of the xenografts was measured and the expressions of IRAK1 were determined by Western blot. Results Compared with the control group, the expression of IRAK1 in siRNA-IRAK1 group was effectively down-regulated in both HEC-1-B and JEC cells(P<0.01). Suspension of stable transfection siRN A IRAK1 or siNC HEC-1-B and JEC was injected in the 24 nude mice, the positive of tumor formation was 100%. Compared with the siNC treatment group, the nude mice in the siRNA-IRAK1 group showed good mental state and activity ability with no obvious abnormal activity and no progressive weight loss. The average weight on the50 th day was respectively( 16.3 ±3.5) g and( 16.6±4.1) g(P<0.05). Tumor growth rate was slow,and tumor volume was significantly smaller than that of the control group(P<0.01). Western blot results showed that compared with the siNC group, the expression levels of IRAK1 protein in the tumor tissues of siRNA IRAK1 transfected HEC-1-B and siRNA IRAK1 transfected JEC cells were decreased by 59.3% and 64.5%, respectively, and the difference was statistically significant(P<0.05). Conclusion Inhibition of IRAK1 gene could decrease the proliferation rate of endometrial adenocarcinoma cells and the growth of the xenografts in nude mice.
Objective To investigate the effect of siRNA IRAKI gene transfected human endometrioid adenocarcinoma HEC-1-B and JEC cells on tumor formation in nude mice. Methods Endometrial adenocarcinoma cell lines HEC-1-B and JEC cultured in vitro were randomly divided into the blank control group, negative control group, and siRNA-IRAK1 group.Recombinant lentivirus virus vectors(NC-siRNA and IRAK1-siRNA) were constructed and were respectively transfected into negative control group and siRNA-IRAK1 group.The expression levels of IRAK1 protein were measured by Western blotting. Human endometrial adenocarcinoma cell lines HEC-1-B and JEC transfected with lentiviral vectors expressing siRNA IRAK1, or empty lentiviral vectors were transplanted subcutaneously into nude mice to generate tumor xenografts,respectively. The status of mice and the growth of the xenografts in each group of mice was observed. At 50 d after transplantation, the mice were sacrificed and their xenografts were isolated. The size of the xenografts was measured and the expressions of IRAK1 were determined by Western blot. Results Compared with the control group, the expression of IRAK1 in siRNA-IRAK1 group was effectively down-regulated in both HEC-1-B and JEC cells(P<0.01). Suspension of stable transfection siRN A IRAK1 or siNC HEC-1-B and JEC was injected in the 24 nude mice, the positive of tumor formation was 100%. Compared with the siNC treatment group, the nude mice in the siRNA-IRAK1 group showed good mental state and activity ability with no obvious abnormal activity and no progressive weight loss. The average weight on the50 th day was respectively( 16.3 ±3.5) g and( 16.6±4.1) g(P<0.05). Tumor growth rate was slow,and tumor volume was significantly smaller than that of the control group(P<0.01). Western blot results showed that compared with the siNC group, the expression levels of IRAK1 protein in the tumor tissues of siRNA IRAK1 transfected HEC-1-B and siRNA IRAK1 transfected JEC cells were decreased by 59.3% and 64.5%, respectively, and the difference was statistically significant(P<0.05). Conclusion Inhibition of IRAK1 gene could decrease the proliferation rate of endometrial adenocarcinoma cells and the growth of the xenografts in nude mice.
引文
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[2]王建六.客观评价子宫内膜癌筛查方法[J].中国实用妇科与产科杂志,2016,32(5):402-405.
[3]Siegel RL,Fedewa SA,Miller KD,et al.Cancer statistics for Hispanics/Latinos,2015[J].Ca Cancer J Clin,2016,65(6):457-480.
[4]Li Q,Zhang C,Chen R,et al.Disrupting MALAT1/miR-200c sponge decreases invasion and migration in endometrioid endometrial carcinoma[J].Cancer Letters,2016,383(1):28-40.
[5]黄佳明,耿慧珍,柯珮琪,等.MACC1与CUL4B蛋白在子宫内膜样腺癌中的表达及临床意义[J].热带医学杂志,2015,15(1):50-52.
[6]李莉莉,范江涛.炎症与子宫内膜癌的相互影响[J].国际妇产科学杂志,2016,43(3):323-326.
[7]鲍雷,史明,姚媛菲,等.Toll样受体与肿瘤免疫[J].现代生物医学进展,2015,15(4):771-773.
[8]Gottipati S,Rao NL,Fungleung WP.IRAK1:a critical signaling mediator of innate immunity[J].Cellular Signalling,2008,20(2):269-276.
[9]Kawai T,Akira S.Signaling to NF-kappaB by Toll-like receptors[J].Trends in Molecular Medicine,2007,13(11):460-469.
[10]Dussiau C,Trinquand A,Lhermitte L,et al.Targeting IRAK1 in T-Cell acute lymphoblastic leukemia[J].Oncotarget,2015,6(22):18956-18965.
[11]王齐敏,于香莉,吕丽,等.靶向治疗药物的分子靶点蛋白在三阴性乳腺癌中的表达及临床意义[J].肿瘤学杂志,2014,20(9):710-714.
[12]王娜,刘刚,刘欢,等.人参皂苷下调IL-1受体相关激酶1基因表达抑制肝癌细胞增殖及促进其凋亡作用及机制[J].现代肿瘤医学,2017,25(8):1183-1187.
[13]王娜.IRAK1激酶在结肠癌中作为西妥昔单抗协同作用靶标机制的研究[D].西安:西安第四军医大学,2017.