LINC00467调控CDK1和CDC45促进肝细胞癌对索拉非尼耐药的研究

doi:10.3877/cma.j.issn.2095-3232.2020.03.019

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RT-PCR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14.03 μmol/L降低至7.33 μmol/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。

关键词: 癌，肝细胞, 索拉非尼, 耐药, LINC00467, CDK1, CDC45

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 sorafenib-resistant cells from 14.03 μmol/L to 7.33 μmol/L. The expression levels of CDK1 and CDC45 were up-regulated 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.

Key words: Carcinoma, hepatocellular, Sorafenib, Drug resistance, LINC00467, CDK1, CDC45

图1 String数据库分析与LINC00467相关mRNA的核心网络图

图2 肝癌组织LINC00467 mRNA表达

图3 CCK-8检测肝癌索拉非尼耐药细胞及其干扰后细胞毒性

图4 LINC00467可调控肝癌细胞CDK1和CDC45 mRNA的表达

[1]	Rimassa L, Santoro A. Sorafenib therapy in advanced hepatocellular carcinoma: the SHARP trial[J]. Expert Rev Anticancer Ther, 2009, 9(6):739-745.
[2]	Marisi G, Cucchetti A, Ulivi P, et al. Ten years of sorafenib in hepatocellular carcinoma: are there any predictive and/or prognostic markers? [J]. World J Gastroenterol, 2018, 24(36):4152-4163.
[3]	朱思聪，谭文亮，商昌珍，等．分子靶向药物治疗肝细胞癌的临床应用进展[J]．中华实验外科杂志，2018, 35(9):1775-1777.
[4]	张丽红，董学君，王宏斌，等．长链非编码RNA在肝细胞癌诊断及预后中的研究进展 [J/CD]．中华临床实验室管理电子杂志，2019, 7(3):129-132.
[5]	张争运，张国梁，常越．长链非编码RNA在肝细胞癌中的研究进展[J]．肝脏，2019, 24(7):825-827.
[6]	Faranda T, Grossi I, Manganelli M, et al. Differential expression profiling of long non-coding RNA GAS5 and miR-126-3p in human cancer cells in response to sorafenib[J]. Sci Rep, 2019, 9(1):9118.
[7]	Li W, Dong X, He C, et al. LncRNA SNHG1 contributes to sorafenib resistance by activating the Akt pathway and is positively regulated by miR-21 in hepatocellular carcinoma cells[J]. J Exp Clin Cancer Res, 2019, 38(1):183.
[8]	Zhi Y, Abudoureyimu M, Zhou H, et al. FOXM1-mediated LINC-ROR regulates the proliferation and sensitivity to sorafenib in hepatocellular carcinoma[J]. Mol Ther Nucleic Acids, 2019(16):576-588.
[9]	Yin L, Cai Z, Zhu B, et al. Identification of key pathways and Genes in the Dynamic Progression of HCC Based on WGCNA[J]. Genes, 2018, 9(2):pii: E92.
[10]	阮班来，王桂玲．利用WGCNA构建与胃癌发展进程相关的关键lncRNA网络[J]．解剖科学进展，2019, 25(6): 680-684.
[11]	Perl AL, O'Connor CM, Fa P, et al. Protein phosphatase 2A controls ongoing DNA replication by binding to and regulating cell division cycle 45 (CDC45) [J]. J Biol Chem, 2019, 294(45):17043-17059.
[12]	周琎．二甲双胍诱导miR-378下调CDK1水平抑制肝细胞肝癌的细胞增殖[D]．南京：南京医科大学，2018.
[13]	Liu L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5[J]. Cancer Res, 2006, 66(24):11851-11858.
[14]	Brunetti O, Gnoni A, Licchetta A, et al. Predictive and prognostic factors in HCC patients treated with sorafenib[J]. Medicina, 2019, 55(10):pii: E707.
[15]	Tan W, Luo X, Li W, et al. TNF-α is a potential therapeutic target to overcome sorafenib resistance in hepatocellular carcinoma[J]. EBioMedicine, 2019(40):446-456.
[16]	Atmadibrata B, Liu PY, Sokolowski N, et al. The novel long noncoding RNA linc00467 promotes cell survival but is down-regulated by N-Myc[J]. PloS one, 2014, 9(2):e88112.
[17]	Chang Y, Yang L. LINC00467 promotes cell proliferation and stemness in lung adenocarcinoma by sponging miR-4779 and miR-7978[J]. J Cell Biochem, 2019, DOI: 10.1002/jcb.29510[Epub ahead of print].
[18]	Ding H, Luo Y, Hu K, et al. Linc00467 promotes lung adenocarcinoma proliferation via sponging miR-20b-5p to activate CCND1 expression[J]. Onco Targets Ther, 2019(12):6733-6743.
[19]	Cai K, Li T, Guo L, et al. Long non-coding RNA LINC00467 regulates hepatocellular carcinoma progression by modulating miR-9-5p/PPARA expression [J]. Open Biol, 2019, 9(9):190074.
[20]	Jiang W, Cheng X, Wang T, et al. LINC00467 promotes cell proliferation and metastasis by binding with IGF2BP3 to enhance the mRNA stability of TRAF5 in hepatocellular carcinoma[J]. J Gene Med, 2019:e3134.

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

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