LINC00324对黑色素瘤细胞增殖、迁移和侵袭能力的影响及分子机制

Effect of LINC00324 on proliferation,migration and invasion of melanoma cell and its molecular mechanism

目的探讨LINC00324对黑色素瘤增殖、迁移和侵袭能力的影响及分子机制。方法基于基因表达谱交互式分析平台(GEPIA)数据库分析LINC00324在黑色素瘤组织和癌旁组织中的表达水平以及对患者预后的影响。在黑色素瘤细胞株SK-MEL-2中转染LINC00324过表达载体和siRNA,并标记为过表达阴性对照组(NC组)、过表达组(OE组)、沉默阴性对照组(si-NC组)和沉默组(si-LINC00324组);实时荧光定量PCR(qRT-PCR)检测LINC00324转染效率以及Beclin1、LC3A/B、Bcl-2的mRNA表达水平;CCK-8实验检测细胞增殖能力;划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力;Western blot实验检测关键蛋白表达水平。结果与癌旁组织相比,LINC00324在黑色素瘤组织中低表达(P<0.05),且低表达患者的预后较差。qRT-PCR结果显示,LINC00324的过表达载体和siRNA在黑色素瘤细胞中转染成功(P<0.01)。与NC组相比,OE组SK-MEL-2细胞增殖、迁移以及侵袭能力明显减弱(P<0.01);与si-NC组相比,si-LINC00324组SK-MEL-2细胞增殖、迁移能力提高(P<0.01)。与NC组相比,OE组细胞中Beclin1、LC3A/B、Bcl-2的mRNA水平下调(P<0.01);与si-NC组相比,si-LINC00324组细胞中Beclin1、LC3A/B、Bcl-2的mRNA水平上调(P<0.01)。Western blot结果显示,与NC组相比,OE组细胞中p62和Bcl-2的蛋白水平上调(P<0.05),LC3BⅡ蛋白水平下调(P<0.01);与si-NC组相比,si-LINC00324组细胞中p62和Bcl-2的蛋白水平下调(P<0.05),LC3BⅡ蛋白水平上调(P<0.05)。结论LINC00324在黑色素瘤组织和细胞系中低表达,可以通过上调Bcl-2抑制自噬影响黑色素瘤细胞的增殖、迁移和侵袭能力。

Objective To investigate the influence and its molecular mechanism of LINC00324 on the proliferation,migration and invasion abilities of melanoma cells.Methods The expression level of LINC00324 in melanoma tissues and paracancerous tissues,and its effect on patient prognosis were analyzed using gene expression profiling interactive analysis(GEPIA).The melenoma SK-MEL-2 cells were divided into four groups:negative control(NC)group,overexpression(OE)group(transfected with overexpression vector),silent negative control(si-NC)group and si-LINC00324 group(transfected with LINC00324 siRNA).The transfection efficiency of LINC00324 and the mRNA expression levels of Beclin1,LC3A/B,and Bcl-2 were determined using real-time fluorescence quantitative PCR(qRT-PCR).CCK-8 was performed to detect the cell proliferation ability.Scratch assay was used to detect the cell migration ability.Transwell assay was used to detect the cell invasion ability.The expression levels of key proteins were detected using Western blot.Results Compared with paracancerous tissues,the expression of LINC00324 was decreased in melanoma tissues(P<0.05),and the patients with low expression had a poor prognosis.The qRT-PCR results revealed that the LINC00324 overexpression vetor and siRNA were successfully transfected into melanoma cells(P<0.01).Compared with NC group,the proliferation,migration,and invasion abilities of SK-MEL-2 cell were significantly decreased in OE group(P<0.01).Compared with si-NC group,the proliferation and migration abilities of SK-MEL-2 cell were increased in si-LINC00324 group(P<0.01).Compared with NC group,the mRNA levels of Beclin1,LC3A/B,and Bcl-2 were reduced in OE group(P<0.01).Compared with si-NC group,the mRNA levels of Beclin1,LC3A/B,and Bcl-2 were increased in si-LINC00324 group(P<0.01).Compared with NC group,the protein levels of p62 and Bcl-2 were upregulated(P<0.05),while the protein level of LC3BⅡ was downregulated in OE group(P<0.01).Compared with si-NC group,the protein levels of p62 and Bcl-2 were downregulated(P<0.05),while the protein level of LC3BⅡ was upregulated in si-LINC00324 group(P<0.05).Conclusion The expression of LINC00324 is low in melanoma tissues and cells,which can affect melanoma cell proliferation,migration,and invasion by suppressing autophagy via Bcl-2 upregulation.