OBJECTIVE: To verify whether ZQLHR regulates granulosa cells (GCs) proliferation and apoptosis through the Krüppel-like factor 4 (KLF4) - CCATT enhancer-binding proteinβ (C/EBPβ) pathway, and to provide in vitro molecular mechanism supporting for the effects of ZQLHR to enhance follicular development and treat patients with PCOS.
METHODS: Based on previous experiments, we performed the following experiments. Firstly, we treated KGN cells (a steroidogenic human granulosa-like tumor cell line) for 48 h using different concentrations of ZQLHR in order to observe apoptosis in each group. Secondly, the mRNA and protein expression levels of KLF4 and C/EBPβ in KGN cells after administrated with ZQLHR were examined by quantitative real-time PCR(q-PCR) and Western blot assay. Thirdly, after knocking down KLF4 and C/EBPβ using siRNAs, the relationship between KLF4 and C/EBPβ in KGN cells was detected. Further, cell counting kit-8 assay, colony formation assay and flow cytometry were used to verify whether ZQLHR promotes proliferation and facilitates apoptosis in KGN cells through the KLF4-C/EBPβ pathway. Finally, q-PCR and Western blot were used to test whether ZQLHR mediated proliferation and apoptosis-related factors such as B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (BAX), proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 to affect the proliferation and apoptosis of KGN cells through the KLF4-C/EBPβ pathway.
CONCLUSIONS: ZQLHR, containing 0.2% by volume, administered to KGN cells resulted in the lowest rate of apoptosis. The expression levels of KLF4 and C/EBPβ were increased in KGN cells following ZQLHR treatment. Additionally, ZQLHR promoted proliferation and inhibited apoptosis of KGN cells by modulating proliferation and apoptosis-related factors via the KLF4-C/EBPβ pathway. Furthermore, we confirmed that KLF4 and C/EBPβ regulate each other in KGN cells. These findings indicate that ZQLHR enhances the proliferation of GCs and suppresses their apoptosis, which constitutes a therapeutic mechanism for treating patients with PCOS.
目的:验证ZQLHR是否通过Krüppel样因子4(KLF4)-CCATT增强子结合蛋白β(C/EBPβ)通路调节颗粒细胞(GCs)的增殖和凋亡,并为ZQLHR促进卵泡发育和治疗PCOS患者提供体外分子机制支持。
方法:基于先前的实验,我们进行了以下实验。首先,我们使用不同浓度的ZQLHR处理KGN细胞(类固醇生成的人颗粒样肿瘤细胞系)48小时,以观察各组的细胞凋亡。其次,通过定量实时PCR(q-PCR)和Westernblot检测ZQLHR给药后KGN细胞中KLF4和C/EBPβ的mRNA和蛋白表达水平。第三,在使用siRNA敲除KLF4和C/EBPβ后,检测KGN细胞中KLF4与C/EBPβ的关系。Further,细胞计数试剂盒-8测定,使用集落形成测定和流式细胞术验证ZQLHR是否通过KLF4-C/EBPβ途径促进KGN细胞的增殖和促进凋亡。最后,q-PCR和Westernblot检测ZQLHR是否介导B细胞淋巴瘤-2(Bcl-2)等增殖和凋亡相关因子,Bcl-2相关X(BAX),增殖细胞核抗原(PCNA)和caspase-3通过KLF4-C/EBPβ途径影响KGN细胞的增殖和凋亡。
结论:ZQLHR,按体积计含0.2%,给予KGN细胞导致最低的凋亡率。在ZQLHR处理后,KGN细胞中KLF4和C/EBPβ的表达水平增加。此外,ZQLHR通过KLF4-C/EBPβ途径调节增殖和凋亡相关因子,促进KGN细胞增殖,抑制凋亡。此外,我们证实KLF4和C/EBPβ在KGN细胞中相互调节。这些发现表明ZQLHR增强了GCs的增殖并抑制了它们的凋亡,这构成了治疗PCOS患者的治疗机制。