LINC00467调控CDK1和CDC45促进肝细胞癌对索拉非尼耐药的研究

LINC00467 promotes hepatocellular carcinoma resistance to sorafenib by regulating CDK1 and CDC45

目的探讨LINC00467调控肝细胞癌(肝癌)对索拉非尼耐药的作用机制。方法通过加权基因共表达网络分析GEO数据集GSE73571,筛选与索拉非尼耐药相关基因;并通过GO和KEGG富集分析确认与LINC00467相关的功能基因及其调控的下游基因。构建索拉非尼耐药细胞株HepG2-R,正常HepG2细胞设为HepG2-。CCK-8检测索拉非尼细胞毒性和计算IC50值;荧光定量RTPCR检测LINC00467、周期蛋白依赖性激酶1(CDK1)和细胞分裂周期蛋白45(CDC45)的mRNA表达水平。两组LINC00467、CDK1、CDC45 mRNA相对表达量比较采用t检验。结果加权基因共表达网络分析筛选与索拉非尼耐药相关基因LINC00467,GO和KEGG分析确认与LINC00467相关功能及其调控下游基因CDK1和CDC45。荧光定量RT-PCR检测发现HepG2-R细胞LINC00467 mRNA相对表达量为2.49±0.30,明显高于HepG2-P细胞的1(t=4.91,P<0.05);siRNA干扰后LINC00467 mRNA相对表达量为0.36±0.04,干扰前为1,LINC00467 mRNA表达明显降低(t=-14.60,P<0.05)。转染siRNA-LINC00467可将耐药细胞对索拉非尼的IC50值从14.03μmol/L降低至7.33μmol/L。CDK1和CDC45在HepG2-R耐药细胞中表达升高,而siRNA干扰LINC00467的表达导致CDK1和CDC45在HepG2耐药细胞中的表达明显降低。结论基于公共数据库挖掘发现LINC00467在肝癌中高表达,LINC00467可能通过上调CDK1和CDC45参与调控细胞周期以及DNA损伤修复过程,从而促进肝癌对索拉非尼耐药。

Objective To explore the mechanism of LINC00467 in regulating the resistance of hepatocellular carcinoma(HCC)cells to sorafenib.Methods GEO dataset GSE73571 was analyzed by weighted gene co-expression network analysis(WGCNA)to screen the genes related to sorafenib resistance.The functional genes related to LINC00467 and the downstream genes were confirmed by GO/KEGG enrichment analysis.The sorafenib-resistant cell line HepG2-R was constructed and normal HepG2 cells were assigned as HepG2-P.The cytotoxicity of sorafenib was determined by CCK-8 assay and the IC50 value was calculated.The mRNA expression of LINC00467,cyclin-dependent kinase 1(CDK1)and cell division cyclin 45(CDC45)were detected by fluorescent quantitative RT-PCR.The mRNA relative expression of LINC00467,CDK1 and CDC45 between two groups were statistically compared by t test.Results WGCNA identified LINC00467 as the sorafenib resistance-related gene.GO/KEGG enrichment analysis confirmed CDK1 and CDC45 as the functional genes related to LINC00467 and the downstream genes.Fluorescent quantitative RT-PCR found that the relative expression level of LINC00467 mRNA in HepG2-R cells was 2.49±0.30,significantly higher than 1 in HepG2-P cells(t=4.91,P<0.05).The relative expression level of LINC00467 mRNA was 0.36±0.04 after siRNA interference,significantly lower than 1 before siRNA interference(t=-14.60,P<0.05).siRNA-LINC00467 transfection could reduced the IC50 value of sorafenibresistant cells from 14.03μmol/L to 7.33μmol/L.The expression levels of CDK1 and CDC45 were upregulated in sorafenib-resistant HepG2-R cells,whereas it was significantly down-regulated when LINC00467 interfered by siRNA.Conclusions Based on the mining of public database,it is found that LINC00467 is highly expressed in HCC cells.LINC00467 participates in the regulation of cell cycle and DNA damage repair process probably by up-regulating the expression of CDK1 and CDC45,thereby promoting the resistance of HCC cells to sorafenib.