AGPAT4对肝细胞癌生长和仑伐替尼耐药的调控作用

The regulation of APGAT4 on the growth and lenvatinib resistance of hepatocellular carcinoma

目的探讨溶血磷脂酸酰基转移酶D(APGAT4)对肝细胞癌生长和仑伐替尼耐药的影响,为肝细胞癌临床治疗提供新的靶点。方法TCGA数据库中分析APGAT4的表达,及其与肝细胞癌患者预后的关系;免疫组化检测APGAT4在肝癌组织的表达,并分析其表达强弱与患者预后的关系;GEO分析仑伐替尼耐药后APGAT4的表达;使用CCK8、EdU、细胞周期,细胞凋亡实验检测APGAT对肝癌细胞生长和仑伐替尼耐药的影响;使用慢病毒构建APGAT4稳定敲低细胞株并建立裸鼠皮下瘤模型,予仑伐替尼治疗,评估APGAT4对肝癌仑伐替尼疗效的影响。多组间数据比较使用方差分析。结果APGAT4的mRNA水平和蛋白在肿瘤组织显著高于癌旁组织,提示肝癌患者预后不良(P<0.05)。使用小干扰RNA可以显著敲低肝癌细胞Hep3B和HCCLM3中APGAT4的mRNA和蛋白表达水平。APGAT4敲低组肝癌细胞(Hep3B、HCCLM3)与对照组相比,增殖能力明显受抑(P<0.05),细胞周期阻滞于G2/M期。GEO数据分析提示APGAT4在仑伐替尼耐药细胞中表达显著上调(P<0.05)。与对照组相比,APGAT4敲低组肝癌细胞(Hep3B,HCCLM3)更容易被仑伐替尼诱导细胞凋亡(P<0.05)。此外,在肝癌裸鼠皮下移植瘤模型中,与对照组相比,仑伐替尼显著抑制APGAT4稳定敲低组肿瘤生长和诱导细胞凋亡(P<0.05)。结论APGAT4促进肝癌细胞的生长和仑伐替尼耐药,是肝癌治疗的潜在治疗靶点。靶向APGAT4治疗有助于抑制肝癌生长和仑伐替尼耐药。

Objective To investigate the effect of 1-acyl-sn-glycerol-3-phosphate acyltransferaseδ(APGAT4)on the growth and lenvatinib resistance of hepatocellular carcinoma(HCC),and provide novel targets for HCC treatment.Methods Using the bioinformatics methods to screen out upregulated genes in lenvatinib resistant cell lines from GEO dataset and survival related genes from TCGA dataset.Immumohistochemical staining was used to detect the expression AGPAT4 in HCC tissues,and its correlation with patients’survival.CCK8,EdU,cell cycle,and cell apoptosis assays were used to investigate the impact of role AGPAT4 on the proliferation and lenvatinib reistance of HCC cells.AGPAT4 stable knockdown cell line and subcutaneous nude mouse model were established to test the therapeutic effects of Lenvatinib.Analysis of variance was used to compare the differences between data sets.Results APGAT4 was the common factor that predicted poor survival and Lenvatinib resistance.The mRNA and protein levels of APGAT4 were significantly upregulated in HCC tissues compared to the para-tumor tissues(P<0.05).Using siRNA could significantly knocked down the mRNA and protein expression of APGAT4 in HCC cell lines Hep3B and HCCLM3.Compared with the control group,the proliferation ability of HCC cell lines(Hep3B and HCCLM3)in APGAT4 knockdown group was significantly inhibited,and the cell cycle was arrested in G2/M phase(P<0.05).In addition,compared to the control group,HCC cell lines(Hep3B and HCCLM3)in APGAT4 knockdown group showed significant decrease in the Lenvatinib half maximal inhibitory concentration,and were more sensitive to lenvatinib-induced apoptosis(P<0.05).In HCC subcutaneous nude mouse model,compared to the control group,the growth of tumor in APGAT4 knockdown group was significantly suppressed,and more apoptosis cells were induced(P<0.05).Conclusion APGAT4 promotes the growth and Lenvatinib resistance of HCC,which is a potential target for HCC treatment.Targeting APGAT4 treatment is conducive to inhibit the growth and Lenvatinib resistance of HCC.