摘要
目的:检测肺腺癌中SETDB1 和SPG20 甲基化水平,探究下调SETDB1 对SPG20 甲基化及A549 细胞迁移和侵袭的影响.方法:选取60 例肺腺癌组织和正常肺组织,qRT-PCR检测 mRNA相对表达量;免疫组织化学法和Western blot检测蛋白表达量;基因甲基化水平应用焦磷酸测序法检测;构建并合成SETDB1 的小干扰RNA,脂质体介导法转染细胞,qRT-PCR 检测 mRNA 相对表达量、Wesrern blot检测蛋白表达、焦磷酸测序法检测基因甲基化、MTT 检测细胞增殖活性、划痕愈合检测细胞迁移、Transwell小室实验检测细胞侵袭.结果:qRT-PCR 结果显示肺腺癌组织中 SPG20 表达下调(P<0.05),而SETDB1 表达上调(P<0.05);Western blot结果显示肺腺癌组织中SETDB1 蛋白表达量显著高于正常肺组织,而SPG20 蛋白在癌组织中呈低表达,低于正常肺组织.在癌组织中 SPG20 基因甲基化率显著高于正常肺组织,差异有统计学意义.SPG20 甲基化水平与SETDB1 表达呈正相关性;肺腺癌细胞中SETDB1 呈高表达,而SPG20 呈低表达;正常支气管上皮细胞中 SETDB1 呈低表达,SPG20 则呈高表达.RNA干扰SETDB1 可使A549 细胞内SETDB1 mRNA和蛋白表达量显著降低,SPG20 基因甲基化率明显下降,抑制了细胞的增殖活性、迁移和侵袭能力.结论:在肺腺癌中甲基转移酶 SETDB1 和 SPG20甲基化存在相关性,SETDB1 可能是SPG20 发生甲基化的催化酶.
Abstract
Objective:To investigate the methylation levels of SETDB1 and SPG20 in lung adenocarci-noma and explore the impact of downregulating SETDB1 on SPG20 methylation,A549 cell migration,and in-vasion.Methods:Sixty cases of lung adenocarcinoma tissues and normal lung tissues were selected.qRT-PCR was used to detect mRNA relative expression levels,immunohistochemistry and western blot were used to detect protein expression levels,and gene methylation levels were assessed by bisulfite sequencing.Small in-terfering RNA(siRNA)targeting SETDB1 was constructed and transfected into cells using liposomes.qRT-PCR,Western blot,bisulfite sequencing,MTT assay,scratch healing assay,and Transwell chamber assay were performed to evaluate mRNA and protein expression,gene methylation,cell proliferation,migration,and invasion,respectively.Results:qRT-PCR results showed that SPG20 expression was downregulated(P<0.05),while SETDB1 expression was upregulated(P<0.05)in lung adenocarcinoma tissues.Western blot re-sults indicated that SETDB1 protein expression in cancer tissues was significantly higher than in normal lung tissues,whereas SPG20 protein exhibited lower expression in cancer tissues,lower than in normal lung tis-sues.In cancer tissues,the SPG20 gene methylation rate was significantly higher than in normal lung tissues,with a statistically significant difference.SPG20 methylation level was positively correlated with SETDB1 ex-pression.SETDB1 was highly expressed,while SPG20 was lowly expressed in lung adenocarcinoma cells.Conversely,in normal bronchial epithelial cells,SETDB1 was lowly expressed,and SPG20 was highly ex-pressed.RNA interference with SETDB1 significantly reduced SETDB1 mRNA and protein expression in A549 cells,decreased SPG20 gene methylation,and inhibited cell proliferation,migration,and invasion.Conclu-sion:There is a correlation between methyltransferase SETDB1 and SPG20 methylation in lung adenocarcino-ma.SETDB1 may serve as a catalytic enzyme for SPG20 methylation.
基金项目
河北省自然科学基金(H2021406045)
承德市科学技术研究与发展计划(201904A029)
国家科技期刊平台
NSTL
维普
万方数据