miR-34a-3p通过靶基因YAP1调控黑色素瘤A375细胞的增殖和凋亡
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摘要
目的探讨miR-34a-3p通过靶基因YAP1调控黑色素瘤A375细胞的增殖和凋亡。方法通过荧光定量PCR检测miR-34a-3p在黑色素瘤和正常皮肤组织中的表达,并通过双荧光素酶报告基因检测miR-34a-3p的靶基因,黑色素瘤A375细胞转入miR-34a-3p模拟剂(miR-34a-3p mimics组),转入对照物miR-NC(对照组miR-NC),miR-34a-3p mimics组转染YAP1表达载体(miR-34a-3p mimics+YAP1组)。通过MTT和流式细胞术分别检测miR-34a-3p和YAP1对黑色素瘤A375细胞增殖和凋亡的影响,并通过Western blot检测miR-34a-3p对YAP1蛋白表达的影响。结果黑色素瘤组中的miR-34a-3p的表达量(0. 67±0. 21)低于正常皮肤组织(1. 35±0. 47),差异具有统计学意义(P <0. 05)。双荧光素酶报告基因显示,miR-34a-3p mimics与YAP1 3'UTR WT载体共转染组荧光强度(0. 39±0. 11)明显低于对照miR-NC与WT载体共转染组(0. 98±0. 35),差异具有统计学意义(P <0. 05)。而miR-34a-3p mimics与YAP1 3'UTR MUT载体共转染组(1. 31±0. 23)与其对照miR-NC与MUT载体共转染组(1. 22±0. 21)之间的荧光强度无显著差异(P> 0. 05)。在培养24 h、48 h和72 h后,miR-34a-3p mimics组细胞细胞活力低于对照组miR-NC,差异具有统计学意义(P <0. 05); miR-34a-3p mimics+YAP1组细胞活力高于miR-34a-3p mimics组,差异具有统计学意义(P <0. 05)。miR-34a-3p mimics组细胞的凋亡率高于对照组miR-NC,差异具有统计学意义(P <0. 05)。miR-34a-3p mimics+YAP1组细胞凋亡率低于miR-34a-3p mimics组,差异具有统计学意义(P <0. 05)。与对照组miR-NC比,miR-34a-3p mimics组细胞的YAP1蛋白表达量显著降低,cleaved caspase 9表达水平显著升高,差异具有统计学意义(P <0. 05)。而与miR-34a-3p mimics组比较,miR-34a-3p mimics+YAP1组细胞凋YAP1蛋白表达量显著增加,cleaved caspase 9表达水平显著降低,差异具有统计学意义(P <0. 05)。结论miR-34a-3p能够通过调控靶基因YAP1的表达,抑制黑色素瘤A375细胞的增殖,促进细胞凋亡。
Objective To investigate the effect of miR-34a-3p on proliferation and apoptosis of melanoma A375 cells by targeting YAP1. Methods The expression of miR-34a-3p in melanoma A375 cells and paracancerous tissues was detected by Q-PCR. The downstream target gene of miR-34a-3p was detected by dual luciferase reporter gene assay. And melanoma A375 cells were transferred to miR-34a-3p mimetic( miR-34a-3p mimics group),transferred to control miR-NC( control group miR-NC),miR-34a-3p mimics group transfected YAP1 expression vector( miR-34a-3p mimics + YAP1 group). The effects of miR-34a-3p and YAP1 on proliferation and apoptosis of melanoma A375 cells were detected by MTT and flow cytometry,respectively. The effect of miR-34a-3p on YAP1 protein expression was detected by Western blot. Results The expression of miR-34a-3p in the melanoma group( 0. 67 ± 0. 21) was lower than that in normal skin tissues( 1. 35 ± 0. 47),and the difference was statistically significant( P < 0. 05). The dual luciferase reporter gene showed that the fluorescence intensity of the co-transfection group of miR-34a-3p mimics and YAP1 3'UTR WT vector( 0. 39 ± 0. 11) was significantly lower than that of the control miR-NC and WT vector co-transfection group( 0. 98 ± 0. 35),and the difference was statistically significant( P < 0. 05). There was no significant difference in fluorescence intensity between the co-transfected group of miR-34a-3p mimics and YAP1 3'UTR MUT( 1. 31 ±0. 23) and the co-transfected group of miR-NC and MUT vector( 1. 22 ± 0. 21). After 24 h,48 h and 72 h,the cell viability of miR-34a-3p mimics group was lower than that of control group miR-NC,and the difference was statistically significant( P < 0. 05). The cell viability of miR-34a-3p mimics + YAP1 group was higher than that of miR-34a-3p mimics group,and the difference was statistically significant( P <0. 05). The apoptosis rate of miR-34a-3p mimics group was higher than that of control group miR-NC,and the difference was statistically significant( P < 0. 05). The apoptotic rate of miR-34a-3p mimics + YAP1 group was lower than that of miR-34a-3p mimics group,and the difference was statistically significant( P < 0. 05). Compared with the control group miR-NC,the expression of YAP1 protein in the miR-34a-3p mimics group was significantly decreased,and the expression level of cleaved caspase 9 was significantly increased( P < 0. 05). Compared with the miR-34a-3p mimics group,the expression of YAP1 protein in the miR-34a-3p mimics + YAP1 group was significantly increased,and the expression level of cleaved caspase 9 was significantly decreased( P < 0. 05). Conclusion miR-34a-3p can inhibit the proliferation of melanoma A375 cells and promote apoptosis by regulating the expression of target gene YAP1.
Objective To investigate the effect of miR-34a-3p on proliferation and apoptosis of melanoma A375 cells by targeting YAP1. Methods The expression of miR-34a-3p in melanoma A375 cells and paracancerous tissues was detected by Q-PCR. The downstream target gene of miR-34a-3p was detected by dual luciferase reporter gene assay. And melanoma A375 cells were transferred to miR-34a-3p mimetic( miR-34a-3p mimics group),transferred to control miR-NC( control group miR-NC),miR-34a-3p mimics group transfected YAP1 expression vector( miR-34a-3p mimics + YAP1 group). The effects of miR-34a-3p and YAP1 on proliferation and apoptosis of melanoma A375 cells were detected by MTT and flow cytometry,respectively. The effect of miR-34a-3p on YAP1 protein expression was detected by Western blot. Results The expression of miR-34a-3p in the melanoma group( 0. 67 ± 0. 21) was lower than that in normal skin tissues( 1. 35 ± 0. 47),and the difference was statistically significant( P < 0. 05). The dual luciferase reporter gene showed that the fluorescence intensity of the co-transfection group of miR-34a-3p mimics and YAP1 3'UTR WT vector( 0. 39 ± 0. 11) was significantly lower than that of the control miR-NC and WT vector co-transfection group( 0. 98 ± 0. 35),and the difference was statistically significant( P < 0. 05). There was no significant difference in fluorescence intensity between the co-transfected group of miR-34a-3p mimics and YAP1 3'UTR MUT( 1. 31 ±0. 23) and the co-transfected group of miR-NC and MUT vector( 1. 22 ± 0. 21). After 24 h,48 h and 72 h,the cell viability of miR-34a-3p mimics group was lower than that of control group miR-NC,and the difference was statistically significant( P < 0. 05). The cell viability of miR-34a-3p mimics + YAP1 group was higher than that of miR-34a-3p mimics group,and the difference was statistically significant( P <0. 05). The apoptosis rate of miR-34a-3p mimics group was higher than that of control group miR-NC,and the difference was statistically significant( P < 0. 05). The apoptotic rate of miR-34a-3p mimics + YAP1 group was lower than that of miR-34a-3p mimics group,and the difference was statistically significant( P < 0. 05). Compared with the control group miR-NC,the expression of YAP1 protein in the miR-34a-3p mimics group was significantly decreased,and the expression level of cleaved caspase 9 was significantly increased( P < 0. 05). Compared with the miR-34a-3p mimics group,the expression of YAP1 protein in the miR-34a-3p mimics + YAP1 group was significantly increased,and the expression level of cleaved caspase 9 was significantly decreased( P < 0. 05). Conclusion miR-34a-3p can inhibit the proliferation of melanoma A375 cells and promote apoptosis by regulating the expression of target gene YAP1.
引文
[1] Delishaj D,Rembielak A,Manfredi B,et al. Non-melanoma skin cancer treated with high-dose-rate brachytherapy:a review of literature[J]. J Contemp Brachytherapy,2016,8(6):533-540.
[2] Deken MA,Gadiot J,Jordanova ES,et al. Targeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma[J].Oncoimmunology,2016,5(12):e1238557.
[3] Nonomura Y,Otsuka A,Nakashima C,et al. Peripheral blood Th9cells are a possible pharmacodynamic biomarker of nivolumab treatment efficacy in metastatic melanoma patients[J]. Oncoimmunology,2016,5(12):e1248327.
[4] Baldwin S,Deighan C,Bandeira E,et al. Analyzing the miRNA content of extracellular vesicles by fluorescence nanoparticle tracking[J].Nanomedicine,2017,13(2):765-70.
[5] Roncati L,Pusiol T,Piscioli F. Thin Melanoma:A generic term including four histological subtypes of cutaneous melanoma[J]. Acta Dermatovenerol Croat,2016,24(4):169-174.
[6] Huang Q,Zheng Y,Ou Y,et al. miR-34a/Bcl-2 signaling pathway contributes to age-related hearing loss by modulating hair cell apoptosis[J]. Neurosci Lett,2017,661:51-66.
[7] Nasr-Esfahani E,Samavi S,Karimi N,et al. Melanoma detection by analysis of clinical images using convolutional neural network[J]. Conf Proc IEEE Eng Med Biol Soc,2016,2016:1373-1376.
[8] Hoel DG. Commercial tanning salons and melanoma risk[J]. Dermatoendocrinol,2017,9(1):e1270485.
[9] Prabhakaran S,Fulp WJ,Gonzalez RJ,et al. Resection of gastrointestinal metastases in stage iv melanoma:correlation with outcomes[J]. Am Surg,2016,82(11):1109-1116.
[10] Tallerico R,Cristiani CM,Staaf E,et al. IL-15,TIM-3 and NK cells subsets predict responsiveness to anti-CTLA-4 treatment in melanoma patients[J]. Oncoimmunology,2017,6(2):e1261242.
[11]赵立涵,刘立,蒋波,等. MicroRNA-144-3p对肝癌细胞增殖、侵袭转移的影响及其机制[J].临床和实验医学杂志,2018,17(14):1493-1496.
[12] Li D,Li J. Association of miR-34a-3p/5p,miR-141-3p/5p,and miR-24 in decidual natural killer cells with unexplained recurrent spontaneous abortion[J]. Med Sci Monit,2016,22:922-929.
[13] Zhu Y,Han Y,Tian T,et al. MiR-21-5p,miR-34a,and human telomerase RNA component as surrogate markers for cervical cancer progression[J]. Pathol Res Pract,2018,214:374-9.
[14] Lee TF,Tseng YC,Chang WC,et al. YAP1 is essential for tumor growth and is a potential therapeutic target for EGFR-dependent lung adenocarcinomas[J]. Oncotarget,2017,8(52):89539-89551.
[15] Ooki A,Del Carmen Rodriguez Pena M,Marchionni L,et al. YAP1and COX2 coordinately regulate urothelial cancer stem-like cells[J].Cancer Res,2018,78(1):168-181.
[16] Liu T,Xu J,Guo JL,et al. YAP1 up-regulation inhibits apoptosis of aortic dissection vascular smooth muscle cells[J]. Eur Rev Med Pharmacol Sci,2017,21:4632-4639.
[17] Tang HL,Tang HM,Fung MC,et al. In vivo biosensor tracks nonapoptotic caspase activity in drosophila[J]. J Vis Exp,2016,(117).
[18] Chao ML,Guo J,Cheng WL,et al. Loss of caspase-activated dnase protects against atherosclerosis in apolipoprotein e-deficient Mice[J].J Am Heart Assoc,2016,5(12):e004362.
[2] Deken MA,Gadiot J,Jordanova ES,et al. Targeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma[J].Oncoimmunology,2016,5(12):e1238557.
[3] Nonomura Y,Otsuka A,Nakashima C,et al. Peripheral blood Th9cells are a possible pharmacodynamic biomarker of nivolumab treatment efficacy in metastatic melanoma patients[J]. Oncoimmunology,2016,5(12):e1248327.
[4] Baldwin S,Deighan C,Bandeira E,et al. Analyzing the miRNA content of extracellular vesicles by fluorescence nanoparticle tracking[J].Nanomedicine,2017,13(2):765-70.
[5] Roncati L,Pusiol T,Piscioli F. Thin Melanoma:A generic term including four histological subtypes of cutaneous melanoma[J]. Acta Dermatovenerol Croat,2016,24(4):169-174.
[6] Huang Q,Zheng Y,Ou Y,et al. miR-34a/Bcl-2 signaling pathway contributes to age-related hearing loss by modulating hair cell apoptosis[J]. Neurosci Lett,2017,661:51-66.
[7] Nasr-Esfahani E,Samavi S,Karimi N,et al. Melanoma detection by analysis of clinical images using convolutional neural network[J]. Conf Proc IEEE Eng Med Biol Soc,2016,2016:1373-1376.
[8] Hoel DG. Commercial tanning salons and melanoma risk[J]. Dermatoendocrinol,2017,9(1):e1270485.
[9] Prabhakaran S,Fulp WJ,Gonzalez RJ,et al. Resection of gastrointestinal metastases in stage iv melanoma:correlation with outcomes[J]. Am Surg,2016,82(11):1109-1116.
[10] Tallerico R,Cristiani CM,Staaf E,et al. IL-15,TIM-3 and NK cells subsets predict responsiveness to anti-CTLA-4 treatment in melanoma patients[J]. Oncoimmunology,2017,6(2):e1261242.
[11]赵立涵,刘立,蒋波,等. MicroRNA-144-3p对肝癌细胞增殖、侵袭转移的影响及其机制[J].临床和实验医学杂志,2018,17(14):1493-1496.
[12] Li D,Li J. Association of miR-34a-3p/5p,miR-141-3p/5p,and miR-24 in decidual natural killer cells with unexplained recurrent spontaneous abortion[J]. Med Sci Monit,2016,22:922-929.
[13] Zhu Y,Han Y,Tian T,et al. MiR-21-5p,miR-34a,and human telomerase RNA component as surrogate markers for cervical cancer progression[J]. Pathol Res Pract,2018,214:374-9.
[14] Lee TF,Tseng YC,Chang WC,et al. YAP1 is essential for tumor growth and is a potential therapeutic target for EGFR-dependent lung adenocarcinomas[J]. Oncotarget,2017,8(52):89539-89551.
[15] Ooki A,Del Carmen Rodriguez Pena M,Marchionni L,et al. YAP1and COX2 coordinately regulate urothelial cancer stem-like cells[J].Cancer Res,2018,78(1):168-181.
[16] Liu T,Xu J,Guo JL,et al. YAP1 up-regulation inhibits apoptosis of aortic dissection vascular smooth muscle cells[J]. Eur Rev Med Pharmacol Sci,2017,21:4632-4639.
[17] Tang HL,Tang HM,Fung MC,et al. In vivo biosensor tracks nonapoptotic caspase activity in drosophila[J]. J Vis Exp,2016,(117).
[18] Chao ML,Guo J,Cheng WL,et al. Loss of caspase-activated dnase protects against atherosclerosis in apolipoprotein e-deficient Mice[J].J Am Heart Assoc,2016,5(12):e004362.