shRNA-FAM38A基因对肺癌A549细胞系增殖、迁移和凋亡的影响
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摘要
目的构建shRNA-FAM38A慢病毒载体,对肺腺癌A549细胞中FAM38A基因表达进行沉默干扰,研究其对肿瘤细胞增殖、迁移和凋亡的影响。方法采用慢病毒载体构建的方法,构建shRNA-FAM38A慢病毒载体,免疫组织化学染色检测FAM-38A的表达; RT-qPCR用来检测凋亡因子Bcl-2,Caspase-3和Caspase-9的表达量; CCK-8试剂盒检测肺癌细胞的增殖; Transwell小室检测肺癌细胞的迁移; AV-PI凋亡试剂盒检测肺癌细胞的凋亡。结果 (1)慢病毒转染效率较高,以肺癌细胞A549为靶点种子细胞,慢病毒转染率为90%。这说明慢病毒转染细胞具有较高的转染效率。(2)免疫组织化学方法检测到FAM-38A可以在肺癌组织中过度表达;(3)阴性对照组(shRNAFAM38A-Con),空质粒组,shRNAFAM38A1组和shRNAFAM38A 2组的FAM38A,凋亡因子Bcl-2,Caspase-3和Caspase-9的mRNA的相对表达量具有统计学差异(F=17. 967; P <0. 05)。shRNA FAM-38A1组中FAM38A,凋亡因子Bcl-2,Caspase-3和Caspase-9的mRNA的相对表达量与shRNAFAM38A 2组相比,表达量降低,表明shRNA FAM-38A1可以有效抑制FAM38A的表达,抑制Bcl-2的表达,促进Caspase-3和Caspase-9的表达,进而促进肿瘤细胞凋亡。然而shRNA FAM-38A2是个无效干扰序列,没有起到沉默FAM38A基因表达的作用。(4) Transwell实验,CCK-8实验和AV-PI实验的结果显示,shR-NA FAM-38A1可以抑制肺癌肿瘤细胞的增殖,抑制肿瘤细胞的迁移,并且可以促进肿瘤细胞的过度凋亡。结论 shRNA FAM38A1可以有效的抑制肺癌细胞的迁移和增殖,并且提高其凋亡率。
Objective To study its effect on proliferation,migration and apoptosis of tumor cells by constructing shRNA-FAM38 A lentivirus vector to interfere with the expression of FAM38 A gene in lung adenocarcinoma A549 cells. Methods It used constructing lentivirus vector method to construct shRNA-FAM38 A lentivirus vector,and the expression of FAM-38 A was detected by immunohistochemistry. RT-qPCR was used to detect the expression of apoptosis factor Bcl-2 Caspase-3 and Caspase-9. CCK-8 kit was used to detect the proliferation of lung cancer cells. Transwell chamber was used to detect the migration of lung cancer cells and AV-PI apoptosis kit was used to detect the apoptosis of lung cancer cells. Results( 1) The efficiency of lentivirus transfection was high,and the lentivirus transfection efficiency was 90 when the lung cancer cell A549 was used as the target seed cell. It indicated that lentivirus transfection cells had higher transfection efficiency.( 2) The expression of FAM-38 A in lung cancer tissues was detected by immunohistochemistry.( 3) In the negative control group,the empty plasmid group,the shRNAFAM38 A1 group and the shRNAFAM38 A2 group,the relative expression of mRNA of Bcl-2 Caspase-3 and Caspase-9 were significantly different( P < 0. 05). The relative expression of FAM38 A,Bcl-2 Caspase-3 and Caspase-9 mRNA had the same trend. The shRNA FAM-38 A1 group was lower than that the shRNAFAM38 A2 group,and the relative expression of FAM38 A,Bcl-2,Caspase-3 and Caspase-9 in the FAM-38 A1 group was lower than that in the shRNAFAM38 A2 group,and the relative expression of FAM38 A,Bcl-2,Caspase-3 and Caspase-9 in the FAM-38 A1 group was significantly lower than that in the shRNAFAM38 A2 group. These Results suggested that shRNA FAM-38 A1 could effectively inhibit the expression of FAM38 A and Bcl-2,promote the expression of Caspase-3 and Caspase-9,and then promote the apoptosis of tumor cells. However,shRNA FAM-38 A2 was an invalid interference sequence and did not silence the expression of FAM38 A gene.( 4) The Results of Transwell assay,CCK-8 test and AV-PI assay showed that shRNA FAM38 A1 could inhibit the proliferation of lung cancer cells,inhibit the migration of tumor cells,and promote the excessive apoptosis of tumor cells. Conclusion shRNA-FAM38 A can effectively inhibit the migration and proliferation of lung cancer cells and increase its apoptosis rate.
Objective To study its effect on proliferation,migration and apoptosis of tumor cells by constructing shRNA-FAM38 A lentivirus vector to interfere with the expression of FAM38 A gene in lung adenocarcinoma A549 cells. Methods It used constructing lentivirus vector method to construct shRNA-FAM38 A lentivirus vector,and the expression of FAM-38 A was detected by immunohistochemistry. RT-qPCR was used to detect the expression of apoptosis factor Bcl-2 Caspase-3 and Caspase-9. CCK-8 kit was used to detect the proliferation of lung cancer cells. Transwell chamber was used to detect the migration of lung cancer cells and AV-PI apoptosis kit was used to detect the apoptosis of lung cancer cells. Results( 1) The efficiency of lentivirus transfection was high,and the lentivirus transfection efficiency was 90 when the lung cancer cell A549 was used as the target seed cell. It indicated that lentivirus transfection cells had higher transfection efficiency.( 2) The expression of FAM-38 A in lung cancer tissues was detected by immunohistochemistry.( 3) In the negative control group,the empty plasmid group,the shRNAFAM38 A1 group and the shRNAFAM38 A2 group,the relative expression of mRNA of Bcl-2 Caspase-3 and Caspase-9 were significantly different( P < 0. 05). The relative expression of FAM38 A,Bcl-2 Caspase-3 and Caspase-9 mRNA had the same trend. The shRNA FAM-38 A1 group was lower than that the shRNAFAM38 A2 group,and the relative expression of FAM38 A,Bcl-2,Caspase-3 and Caspase-9 in the FAM-38 A1 group was lower than that in the shRNAFAM38 A2 group,and the relative expression of FAM38 A,Bcl-2,Caspase-3 and Caspase-9 in the FAM-38 A1 group was significantly lower than that in the shRNAFAM38 A2 group. These Results suggested that shRNA FAM-38 A1 could effectively inhibit the expression of FAM38 A and Bcl-2,promote the expression of Caspase-3 and Caspase-9,and then promote the apoptosis of tumor cells. However,shRNA FAM-38 A2 was an invalid interference sequence and did not silence the expression of FAM38 A gene.( 4) The Results of Transwell assay,CCK-8 test and AV-PI assay showed that shRNA FAM38 A1 could inhibit the proliferation of lung cancer cells,inhibit the migration of tumor cells,and promote the excessive apoptosis of tumor cells. Conclusion shRNA-FAM38 A can effectively inhibit the migration and proliferation of lung cancer cells and increase its apoptosis rate.
引文
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[2]MERCER B A,LEMATRE V,POWELL C A,et al.The epithelial cell in lung health and emphysema pathogenesis[J].Curr Respir Med Rev,2006,2(2):101-142.
[3]郭彩霞,石红梅,李飞,等.吸烟对肺癌患者血清转化生长因子-β、白细胞介素-6水平及肺功能的影响[J].中国老年学杂志,2017,37(4):924-925.
[4]李放,谢双华,王刚,等.BMI与吸烟男性肺癌发病关系的前瞻性队列研究[J].中华预防医学杂志,2016,50(5):385-390.
[5]RAWLINS E L,HOGAN B L.Epithelial stem cells of the lung:privileged few or opportunities for many[J].Development,2006,133(13):2455-2465.
[6]张亚娟,常德,张健鹏.肺癌化疗中铂类耐药的研究进展[J].中国医学科学院学报,2017,39(1):150-155.
[7]NOVELLO S,MAZIRES J,OH I J,et al.Alectinib versus chemotherapy in crizotinib-pretreated anaplastic lymphoma kinase(ALK)-positive non-small-cell lung cancer:Results from the phaseⅢALUR study[J].Ann Oncol,2018,29(6):1409-1416.
[8]PETRUSEVSKA M,STAVRIDIS I P,MLADENOVSKA K,et al.Secondary Hodgkin Lymphoma and Myelodysplastic Syndrome(MDS)After paclitaxel-carboplatin treatment in a patient with small cell lung cancer[J].Pril(Makedon Akad Nauk Umet Odd Med Nauki),2017,38(3):97-103.
[9]CHEN Q,ZHANG H.Smac combined with DDP can inhibit drug resistance of ovarian cancer through regulation of survivin expression[J].Cancer Biomark,2018,22(1):1-6.
[10]COSTE B,MATHUR J,SCHMIDT M,et al.Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels[J].Science,2010,330(6000):55-60.
[11]JIANG L,ZHAO Y D,CHEN W X.The function of the novel mechanical activated ion channel Piezo1 in the human osteosarcoma cells[J].Med Sci Monit,2017,23:5070-5082.
[12]WANG Q,YANG C S,MA Z X,et al.Inhibition of prostate cancer DU145 cell growth with small interfering RNA targeting the SATB1gene[J].Exp Ther Med,2018,15(3):3028-3033.
[13]唐益庭,申文豪,何燕,等.用shRNA下调泛素表达抑制人肺癌H1299细胞生长并促进其凋亡[J].细胞与分子免疫学杂志,2014,30(12):1287-1290.
[14]COSTE B,XIAO B,SANTOS J S,et al.Piezo proteins are poreforming subunits of mechanically activated channels[J].Nature,2012,483(7388):176-181.
[15]MIYAMOTO T M,NAKAGOMI H,KIRA S,et al.71 Piezo1,a novel mechanosensor in the bladder urothelium,transmits signals of bladder sensation[J].Eur Urol Suppl,2013,12(1):e71.
[16]王莉,李素云,彭田红,等.7-二氟亚甲基-5,4'-二甲氧基染料木黄酮通过下调Caspase-3的表达拮抗H2O2诱导的血管内皮细胞凋亡[J].中国动脉硬化杂志,2017,25(12):1219-1224.
[17]郭晓东,杨美,余灵祥,等.Caspase-9和Survivin在肝细胞癌患者癌组织中的表达[J].肝脏,2014,19(9):661-663.
[18]王俊钢,李聪聪,毛广显,等.长链非编码MALAT-1调控miR-205的表达影响非小细胞肺癌细胞的生长和迁移[J].中国免疫学杂志,2018,34(2):227-232.