β-D-葡聚糖通过下调HPV16 E6和重新激活p53调节HPV16阳性宫颈癌细胞的生物学行为

β-D-glucan regulates the biological behavior of HPV16-positive cervical cancer cells by regulating HPV16 E6 and reactivating p53

目的:研究β-D-葡聚糖在宫颈癌细胞中的作用及可能机制。方法:CCK-8法检测β-D-葡聚糖对宫颈癌细胞CaSki、HeLa和SiHa细胞增殖的影响,伤口愈合试验检测细胞迁移情况,流式细胞术检测细胞周期和细胞凋亡情况。采用实时荧光定量聚合酶链式反应(qRT-PCR)检测细胞中HPV16 E6/E7、HPV18 E6/E7、p53和p21 mRNA表达水平,Western blot检测N-钙黏蛋白、E-钙黏蛋白、p53、波形蛋白、p21蛋白表达水平。以宫颈癌CaSki细胞为研究对象,在雌性BALB/c小鼠皮下注射成瘤,验证β-D-葡聚糖在体内对肿瘤生长的抑制作用。结果:β-D-葡聚糖对不同宫颈癌细胞增殖的影响不同,对CaSki细胞增殖有显著的时间依赖性抑制作用,对HeLa细胞增殖无显著影响,对SiHa细胞作用72h后表现为促进增殖作用(P<0.05)。β-D-葡聚糖对CaSki、HeLa和SiHa细胞的迁移都有抑制作用(P<0.05)。β-D-葡聚糖对CaSki、SiHa的细胞周期有调节作用并能促进其凋亡(P<0.05),但对HeLa细胞无显著影响。与对照组相比,β-D-葡聚糖处理组CaSki和HeLa细胞中HPV16 E6 mRNA表达水平降低,p53和p21 mRNA表达水平在3种细胞中均升高(P<0.05)。与对照组相比,β-D-葡聚糖处理组3种细胞波形蛋白表达量降低,E-钙黏蛋白、p53和p21表达量增加(P<0.05)。结论:β-D-葡聚糖抑制宫颈癌细胞中HPV16 E6表达并上调p53和p21表达,抑制HPV16阳性宫颈癌细胞的增殖、迁移,调节细胞周期,促进细胞凋亡,并在体内发挥抗肿瘤作用。

Objective:To investigate the possible mechanism of action ofβ-D-glucan in cervical cancer cells.Methods:CCK-8 was carried out to evaluate the influence ofβ-D-glucan on proliferation.Wound healing assay was performed to evaluate migration.Flow cytometry was used to test cell cycle and apoptosis.Quantitative reverse transcriptase polymerase chain reaction and Western blot were conducted to determine mRNA expression and protein levels,respectively.Tumor cell grafting was conducted to test the function ofβ-D-glucan in vivo.Results:β-D-glucan exerted a significant effect on inhibition of proliferation,cell cycle and migration in HPV16 positive cervical cancer cells,but promoted proliferation after 72 hours in SiHa(P<0.05).β-D-glucan inhibited the migration of the CaSki,HeLa,and SiHa(P<0.05).β-D-glucan regulated the cell cycle of CaSki,SiHa and promoted its apoptosis(P<0.05).The expression of HPV16 E6 mRNA were reduced in CaSki and HeLa inβ-D-glucan group,and the p53 and p21 mRNA were increased in all cervical cancer cells(P<0.05).Compared with the control group,the expression of vimentin was decreased inβ-D-gluan group,while E-cadherin,p53,and p21 increased(P<0.05).Conclusion:β-D-gluccan suppresses HPV16 E6 and upregulates p53 and p21 in cervical cancer cells,suppresses proliferation and migration of HPV16-positive cervical cancer cells,regulates cell cycle,promotes apoptosis,and exerts antitumor effects in vivo.