肉灵芝提取物调控长链非编码RNA BRCA1相邻基因2/双特异性磷酸酶4/细胞外信号调节激酶通路对肝癌细胞Hep3B增殖及凋亡的影响

Effects of Ganoderma lucidum extract on the proliferation and apoptosis of Hep3B hepatocellular carcinoma cells by regulating the lncRNA NBR2/DUSP4/ERK pathway

目的探讨肉灵芝提取物是否可通过长链非编码RNA BRCA1相邻基因2(LncRNA NBR2)/双特异性磷酸酶4/细胞外信号调节激酶(DUSP4/ERK)通路而影响肝癌HepB细胞增殖及凋亡。方法体外培养肝癌HepB细胞,分为1 mg/L肉灵芝提取物组、2 mg/L肉灵芝提取物组、4 mg/L肉灵芝提取物组、si-NC组、si-LncRNA NBR2组、pcDNA-NC组、pcDNA-LncRNA NBR2组、肉灵芝提取物+si-NC组、肉灵芝提取物+si-LncRNA NBR2组;采用MTT检测肝癌HepB细胞增殖;流式细胞术检测肝癌HepB细胞凋亡;RT-qPCR检测LncRNA NBR2的表达量;蛋白质印迹法(Western blotting)检测增殖细胞核抗原(PCNA)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-caspase-3)、DUSP4、p-ERK蛋白表达量。结果肉灵芝提取物可明显降低肝癌HepB细胞增殖率与PCNA、DUSP4、p-ERK蛋白水平(P<0.05),提高肝癌HepB细胞凋亡率、Cleaved-caspase-3蛋白水平及LncRNA NBR2的表达水平(P<0.05);转染si-LncRNA NBR2后可明显提高肝癌HepB细胞增殖率及PCNA、DUSP4、p-ERK蛋白水平(P<0.05),降低凋亡率及Cleaved-caspase-3蛋白水平(P<0.05),而转染pcDNA-NBR2可明显减弱转染si-LncRNA NBR2对肝癌HepB细胞增殖及凋亡的作用;与肉灵芝提取物+si-NC组比较,肉灵芝提取物+si-LncRNA NBR2组肝癌HepB细胞增殖率及PCNA、DUSP4、p-ERK蛋白水平升高(P<0.05),凋亡率及Cleaved-caspase-3蛋白水平降低(P<0.05)。结论肉灵芝提取物可通过上调LncRNA NBR2的表达而抑制DUSP4/ERK信号通路的活化从而抑制肝癌HepB细胞增殖及诱导细胞凋亡。

Objective To investigate whether Ganoderma lucidum extract can affect the proliferation and apoptosis of Hep3B liver cancer cells through the long noncoding RNA BRCA1 adjacent gene 2(lnc RNA NBR2)/dual specificity phosphatase 4/extracellular signal-regulated kinase(DUSP4/ERK)pathway.Methods Hep3B cells were cultured in vitro and divided into the 1 mg/L Ganoderma lucidum extract group,2 mg/L Ganoderma lucidum extract group,4 mg/L Ganoderma lucidum extract group,si-NC group,si-lnc RNA NBR2 group,pc DNA-NC group,pc DNA-lnc RNA NBR2 group,Ganoderma extract+si-NC group,and Ganoderma extract+si-lnc RNA NBR2 group.MTT was used to detect the proliferation of Hep3B cells,and flow cytometry was used to detect the apoptosis of Hep3B cells.RT-qPCR was used to detect the expression of lnc RNA NBR2;Western blot was used to detect proliferating cell nuclear antigen(PCNA),activated cysteine-containing caspase-3(Cleaved-caspase-3),DUSP4,and p-ERK protein expression.Results Ganoderma lucidum extract can significantly reduce the proliferation rate of Hep3B cells and the protein levels of PCNA,DUSP4,and p-ERK(P<0.05)and increased the apoptosis rate and the expression levels of cleaved caspase-3 and lnc RNA NBR2 in Hep3B cells(P<0.05).Transfection of si-lncRNA NBR2significantly increased the proliferation rate and the protein levels of PCNA,DUSP4,and p-ERK(P<0.05)and decreased the apoptosis rate and the protein level of cleaved-caspase-3(P<0.05).Transfection of pc DNA-lnc RNA NBR2 significantly attenuated the effect of si-lnc RNA NBR2 transfection on the proliferation and apoptosis of Hep3B cells.Compared with the Ganoderma lucidum extract+si-NC group,the cell proliferation rate and the protein levels of PCNA,DUSP4,and p-ERK in the Ganoderma lucidum extract+si-Lnc RNA NBR2 group increased(P<0.05),while apoptosis and the protein level of cleaved-caspase-3 were reduced(P<0.05).Conclusion Ganoderma lucidum extract significantly reduced the activation of the DUSP4/ERK signaling pathway by upregulating the expression of LncRNA NBR2,thereby inhibiting the proliferation and increased the apoptosis of Hep3B cells.