STAT3诱导巨噬细胞M2型极化促进宫颈癌细胞的放疗抵抗及机制研究

The function and mechanism of STAT3 inducing M2 polarization of macrophages to promote radiotherapy resistance of cervical cancer cells

目的:探究STAT3诱导的巨噬细胞极化对宫颈癌细胞放疗抵抗的影响及机制。方法:THP1与100 ng/mL PMA孵育48 h诱导其分化为M0型巨噬细胞,LPS诱导M0型巨噬细胞分化为M1型,IL-4诱导M0型巨噬细胞分化为M2型,qRT-PCR和Western blot检测M0、M1、M2型巨噬细胞中STAT3表达,M0型巨噬细胞中转染空白质粒(对照组)和STAT3过表达质粒(STAT3组),qRT-PCR检测各组细胞Arg-1、CD206、IL-1β和TGF-βmRNA表达,Western blot检测STAT3、SOCS3表达以及STAT3磷酸化水平,克隆形成实验检测0 Gy、2 Gy、4 Gy、6 Gy、8 Gy 6 MV-X射线照射剂量下宫颈癌细胞Hela和SiHa的克隆形成率(PE)和细胞存活分数(SF),将M0、M1、M2型巨噬细胞以及对照组和STAT3组巨噬细胞与Hela和SiHa细胞共孵育后,用6 Gy剂量照射Hela和SiHa细胞,CCK8法检测照射后Hela和SiHa细胞的增殖能力。结果:M0、M1、M2型巨噬细胞诱导成功,M2型巨噬细胞中STAT3高表达(P<0.001),M1型巨噬细胞中STAT3低表达(P<0.001),相比于对照组,STAT3组巨噬细胞中Arg-1、CD206、IL-1β、TGF-β、SOCS3表达显著上调(P<0.001),STAT3磷酸化水平显著增加(P<0.001)。不同剂量(0 Gy、2 Gy、4 Gy、6 Gy、8 Gy)照射后,Hela和SiHa细胞的PE、SF水平随照射剂量升高而逐渐降低(P<0.05),6 Gy和8 Gy条件下PE和SF无显著差异。在6 Gy照射剂量下,相比于M0型和对照组巨噬细胞共孵育的Hela和SiHa细胞,M2型巨噬细胞和STAT3组巨噬细胞共孵育后的Hela和SiHa细胞增殖能力显著增加(P<0.05)。结论:STAT3可诱导M2型巨噬细胞极化而增加宫颈癌细胞的放疗抵抗。

Objective:To explore the effect and mechanism of STAT3-induced macrophage polarization on the radiotherapy resistance of cervical cancer cells.Methods:THP1s were incubated with 100 ng/mL PMA for 48 hours to induce differentiation into M0 type macrophages,LPS induced M0 type macrophages to differentiate into M1 type,IL-4 induced M0 type macrophages to differentiate into M2 type,qRT-PCR and Western blot was used to detect the expression of STAT3 in M0,M1,and M2 macrophages,blank plasmids(control group)and STAT3 overexpression plasmids(STAT3 group)were transfected in M0 macrophages,and Arg-1,CD206,IL-1βand TGF-βmRNA expression in each group was detected by qRT-PCR.Western blot was used to detect STAT3,SOCS3 expression and STAT3 phosphorylation level.Clone formation experiment was used to detect the colony formation rate(PE)and cell survival fraction(SF)of cervical cancer cells Hela and SiHa cells at 0 Gy,2 Gy,4 Gy,6 Gy,8 Gy 6 MV-X ray irradiation.M0,M1,M2 type macrophages,control group and STAT3 group macrophages were incubated with Hela and SiHa cells,then Hela and SiHa cells were irradiated with a dose of 6 Gy,and the proliferation ability of Hela and SiHa cells after irradiation was detected by CCK8 method.Results:M0,M1,and M2 macrophages were successfully induced.STAT3 was highly expressed in M2 macrophages(P<0.001),and STAT3 was lower expression in M1 macrophages(P<0.001).Compared with the control group,the expressions of Arg-1,CD206,IL-1β,TGF-βand SOCS3 in macrophages of the group were significantly up-regulated(P<0.001),and the phosphorylation level of STAT3 was significantly increased(P<0.001)in STAT3 group.After irradiation with different doses(0 Gy,2 Gy,4 Gy,6 Gy,8 Gy),the PE and SF levels of Hela and SiHa cells gradually decreased with the increase of the irradiation dose(P<0.05),6 Gy and 8 Gy conditions were no significant difference between PE and SF.At a dose of 6 Gy,compared with Hela and SiHa cells incubated with M0 and control macrophages,Hela and SiHa cells incubated with M2 macrophages and STAT3 macrophages had significantly greater proliferation ability(P<0.05).Conclusion:STAT3 can induce polarization of M2 type macrophages and increase the radiotherapy resistance of cervical cancer cells.