缺氧环境下miR-210调控HDAC2对肝癌VEC细胞血管通透性、血管形成及放疗耐药性的影响

Effects of miR-210 on vascular permeability,angiogenesis and radiation resistance of liver cancer VEC cells by regulating HDAC2 in hypoxic environment

目的探究缺氧环境下miR-210调控组蛋白去乙酰化酶2(histone deacetylase 2,HDAC2)对肝癌VEC细胞血管通透性、血管形成及放疗耐药性的影响。方法分别在缺氧环境和正常环境下培养VEC细胞,以RT-qPCR和Western blotting检测两种培养环境下VEC细胞miR-210和HDAC2表达。其中在缺氧环境下培养VEC细胞并随机分为对照组、阴性对照组、miR-210 inhibitor组、HDAC2敲低组、miR-210 inhibitor+HDAC2过表达组,采用MTT法和Edu染色检测缺氧环境下各组VEC细胞增殖;Transwell小室法、小管形成实验分别检测各组VEC细胞血管通透性和血管形成;Western blotting和ELISA检测各组VEC细胞血管VEGF表达释放;MTT法测定各组细胞活力并检测其放疗耐药指数。结果与正常环境下培养的VEC细胞相比,缺氧环境下VEC细胞miR-210表达、HDAC2 mRNA及蛋白表达升高(P<0.05)。与对照组相比,miR-210 inhibitor组、HDAC2敲低组细胞HDAC2 mRNA及蛋白表达、细胞活力、增殖率、通透性强度、成管长度、VEGF蛋白表达、细胞培养基中VEGF水平、放疗耐药指数降低(P<0.05),阴性对照组细胞各指标差异无统计学意义(P>0.05);与miR-210 inhibitor组相比,miR-210 inhibitor+HDAC2过表达组细胞HDAC2 mRNA及蛋白表达、细胞活力、增殖率、通透性强度、成管长度、VEGF蛋白表达、细胞培养基中VEGF水平、放疗耐药指数升高(P<0.05)。结论缺氧环境下下调miR-210可通过降低HDAC2表达而抑制肝癌VEC细胞增殖、血管通透性、血管形成及放疗耐药性。

Objective To investigate the effects of miR-210 on vascular permeability,angiogenesis and radiation re-sistance of liver cancer VEC cells by regulating histone deacetylase 2(HDAC2)in hypoxic environments.Methods VEC cells were cultured in hypoxic and normal environments,and the expression of miR-210 and HDAC2 in VEC cells was detected using RT-qPCR and Western blotting.VEC cells were cultured in hypoxic environment and randomly separated into control group,negative control group,miR-210 inhibitor group,HDAC2 knockdown group and miR-210 inhibitor+HDAC2 overexpression group.MTT method and Edu staining were applied to detect the proliferation of VEC cells in va-rious groups under hypoxic environment;Transwell chamber method and tubular formation experiment were applied to detect the vascular permeability and angiogenesis of VEC cells in each group;Western blotting and ELISA were applied to detect the expression and release of VEGF in VEC cells in each group.The viability of cells in each group was meas-ured using MTT method and their radiation resistance index was measured.Results Compared with normal cultured VEC cells,the expression of miR-210,HDAC2 mRNA and protein in VEC cells increased under hypoxic conditions(P<0.05).Compared with the control group,the miR-210 inhibitor group and HDAC2 knockdown group showed a de-crease in HDAC2 mRNA and protein expression,cell viability,proliferation rate,permeability intensity,tubular length,VEGF protein expression,VEGF level in cell culture medium and radiation resistance index(P<0.05),there was no obvious difference in all indicators of cells in the negative control group(P>0.05).Compared with the miR-210 inhibi-tor group,the miR-210 inhibitor+HDAC2 overexpression group showed an increase in HDAC2 mRNA and protein ex-pression,cell viability,proliferation rate,permeability intensity,tubular length,VEGF protein expression,VEGF level in cell culture medium and radiation resistance index(P<0.05).Conclusion Downregulation of miR-210 in hypoxic environments can inhibit the proliferation,vascular permeability,angiogenesis and radiation resistance of liver cancer VEC cells by reducing the expression of HDAC2.