灵芝乙醇提取物对宫颈癌细胞生物学行为和JAK1/STAT3信号通路的影响

Effects of ganoderma lucidum ethanol extract on biological behavior and JAK1/STAT3 signaling pathway of cervical cancer cells

目的:探讨灵芝乙醇提取物(GLEE)对宫颈癌细胞生物学行为和Janus激酶1(JAK1)/信号传导和转录激活因子3(STAT3)信号通路的影响,并阐明其可能的作用机制。方法:将人宫颈癌HeLa细胞随机分为对照组(给予完全培养液)和低、中及高剂量GLEE组(给予25、50、100 mg·L^(-1) GLEE)。MTT法检测各组细胞增殖抑制率,平板克隆实验检测各组克隆形成率,Transwell小室实验检测各组迁移细胞数和侵袭细胞数,实时荧光定量PCR(RT-qPCR)法检测各组细胞中JAK1和STAT3 mRNA表达水平,Western blotting法检测各组细胞中JAK1、磷酸化JAK1(p-JAK1)、STAT3、磷酸化STAT3(p-STAT3)、细胞因子信号传导抑制蛋白3(SOCS3)和活化STAT蛋白抑制因子1(PIAS1)蛋白表达水平。结果:与对照组比较,低、中和高剂量GLEE组宫颈癌HeLa细胞增殖抑制率升高(P<0.05),细胞克隆形成率、迁移细胞数和侵袭细胞数降低(P<0.05),细胞中JAK1和STAT3 mRNA表达水平降低(P<0.05),细胞中p-JAK1和p-STAT3蛋白表达水平降低(P<0.05),细胞中SOCS3和PIAS1蛋白表达水平升高(P<0.05)。与低剂量GLEE组比较,中和高剂量GLEE组宫颈癌HeLa细胞增殖抑制率升高(P<0.05),细胞克隆形成率、迁移细胞数和侵袭细胞数降低(P<0.05),细胞中JAK1和STAT3 mRNA表达水平降低(P<0.05),细胞中p-JAK1和p-STAT3蛋白表达水平降低(P<0.05),细胞中SOCS3和PIAS1蛋白表达水平升高(P<0.05)。与中剂量GLEE组比较,高剂量GLEE组宫颈癌HeLa细胞增殖抑制率升高(P<0.05),细胞克隆形成率、迁移细胞数和侵袭细胞数降低(P<0.05),细胞中JAK1和STAT3 mRNA表达水平降低(P<0.05),细胞中p-JAK1和p-STAT3蛋白表达水平降低(P<0.05),细胞中SOCS3和PIAS1蛋白表达水平升高(P<0.05)。结论:GLEE对宫颈癌HeLa细胞增殖、克隆形成、迁移和侵袭均有一定的抑制作用,其可能是通过上调细胞中SOCS3及PIAS1表达水平,进而影响JAK1/STAT3信号通路发挥作用。

Objective:To investigate the effects of ganoderma lucidum ethanol extract(GLEE)on the biological behavior and Janus kinase 1(JAK1)/signal transduction and transcription activator 3(STAT3)signaling pathway of the cervical cancer cells,and to clarify its possible mechanisms.Methods:The human cervical cancer HeLa cells were randomly divided into control group(given complete culture medium)and low,medium and high doses of GLEE groups(given 25,50,and 100 mg·L^(-1) GLEE).The inhibitory rates of proliferation of the human cervical cancer HeLa cells in various groups were detected by MTT assay;the clone formation rates of the human cervical cancer HeLa cells in various groups were detected by plate cloning experiment;Transwell chamber assay was used to detect the numbers of migration cells and invasion cells in various groups;the expression levels of JAK1 and STAT3 mRNA in the human cervical cancer HeLa cells in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method;the expression levels of JAK1,phophorylated JAK1(p-JAK1),STAT3,phophorylated STAT3(p-STAT3),suppressor of cytokine signaling 3(SOCS3),and protein inhibitor of activated STAT1(PIAS1)proteins in the human cervical cancer HeLa cells in various groups were detected by Western blotting method.Results:Compared with control group,the inhibitory rates of proliferation of the cervical cancer HeLa cells in low,medium and high doses of GLEE groups were increased(P<0.05),the clone formation rates the cervical cancer HeLa cells were decreased(P<0.05),the numbers of migration cells were decreased(P<0.05),the numbers of invasion cells were decreased(P<0.05),the expression levels of JAK1 and STAT3 mRNA in the cervical cancer HeLa cells were decreased(P<0.05),and the expression levels of SOCS3 and PIAS1 proteins in the cervical cancer HeLa cells were increased(P<0.05).Compared with low dose of GLEE group,the inhibitory rates of proliferation of the cervical cancer HeLa cells in medium and high doses of GLEE groups were increased(P<0.05),the clone formation rates of the cervical cancer HeLa cells were decreased(P<0.05),the numbers of migration cells were decreased(P<0.05),the numbers of invasion cells were decreased(P<0.05),the expression levels of JAK1 and STAT3 mRNA in the cervical cancer HeLa cells were decreased(P<0.05),and the expression levels of SOCS3 and PIAS1 proteins in the cervical cancer HeLa cells were increased(P<0.05).Compared with midium dose of GLEE group,the inhibitory rate of proliferation of the cervical cancer HeLa cells in high dose of GLEE group was increased(P<0.05),the clone formation rate of the cervical cancer HeLa cells was decreased(P<0.05),the number of migration cells was decreased(P<0.05),the number of invasive cells was decreased(P<0.05),the expression levels of JAK1 and STAT3 mRNA in the cervical cancer HeLa cells were decreased(P<0.05),and the expression levels of SOCS3 and PIAS1 proteins in the cervical cancer HeLa cells were increased(P<0.05).Conclusion:GLEE can inhibit the proliferation,cloning,migration and invasion of the cervical cancer HeLa cells to some extent,which may play a role by up-regulating the expression levels of SOCS3 and PIAS1 in the cells,thereby affecting the JAK1/STAT3 signaling pathway.