B7-H4活化NF-κB/IL-6/STAT3途径促进MFC细胞增殖和肿瘤形成

B7-H4 activates the NF-κB/IL-6/STAT3 pathway to promote MFC cell proliferation and tumor formation

摘要

目的 探究协同共刺激分子B7-H4对MFC细胞增殖、迁移以及肿瘤形成的影响并探究其可能的作用机制。方法 Western blot检测MFC细胞中B7-H4的表达水平,通过慢病毒转染敲低MFC细胞中B7-H4的表达并筛选稳转细胞株,转染B7-H4敲低慢病毒为实验组(sh-B7-H4),转染空载慢病毒为对照组(sh-NC)。通过CCK-8、细胞划痕实验检测敲低B7-H4对MFC细胞增殖和迁移能力的影响。通过小鼠皮下成瘤实验探究敲低B7-H4对肿瘤形成的影响;RT-PCR和Western blot检测敲低B7-H4后,B7-H4、NF-κB、IL-6、STAT3等基因在细胞和肿瘤组织中表达水平的变化。结果 RT-PCR和Western blot检测结果显示,相较于sh-NC组,sh-B7-H4组的B7-H4表达水平降低(P<0.001),成功构建B7-H4敲低的细胞株。MFC细胞敲低B7-H4后,CCK-8实验结果显示,sh-B7-H4组较sh-NC组细胞增殖能力明显被抑制(P<0.001);细胞划痕实验结果显示,sh-B7-H4组较sh-NC组细胞迁移能力明显降低(P<0.001)。小鼠皮下成瘤实验中,实验组中小鼠的肿瘤体积及重量明显低于对照组(P0.05)。结论 低表达B7-H4抑制MFC细胞的增殖和迁移能力,同时抑制MFC细胞肿瘤的形成,这种抑癌的作用机制可能是通过阻断NF-κB/IL-6/STAT3途径的激活而实现的。

Abstract

Objective To investigate the effects of co-stimulatory molecule B7-H4 on proliferation, migration, and tumorigenesis of MFC cells and explore its potential mechanisms. Methods Western blot was used to detect the expression level of B7-H4 in MFC cells. B7-H4 expression was knocked down in MFC cells by lentiviral transfection, and stable transfection cell lines were screened. The lentivirus transfecting B7-H4 knockdown was designated as the experimental group (sh-B7-H4), and the lentivirus transfecting empty vector was designated as the control group (sh-NC). The effects of B7-H4 knockdown on MFC cell proliferation and migration were assessed using CCK-8 and cell scratch assays. The impact of B7-H4 knockdown on tumor formation was investigated through mouse subcutaneous tumorigenesis experiments. RT-PCR and Western blot were employed to detect changes in the expression levels of B7-H4, NF-$\kappa$ B, IL-6, STAT3 and other genes in cells and tumor tissues after B7-H4 knockdown. Results RT-PCR and Western blot results demonstrated that the expression level of B7-H4 was significantly reduced in the sh-B7-H4 group compared to the sh-NC group (P<0.001), indicating the successful establishment of a B7-H4 knockdown cell line. After B7-H4 knockdown in MFC cells, CCK-8 assay results showed that the proliferative capacity of cells in the sh-B7-H4 group was significantly inhibited compared to the sh-NC group (P<0.001). Cell scratch assay results indicated that the migratory capacity of cells in the sh-B7-H4 group was significantly reduced compared to the sh-NC group (P<0.001). In mouse subcutaneous tumorigenesis experiments, the tumor volume and weight in the experimental group were significantly lower than those in the control group (P<0.001). RT-PCR and Western blot results revealed that in MFC cells and mouse tumor tissues, the expression levels of B7-H4, p-N F-$\kappa$ B, IL-6, and p-STAT3 were significantly reduced in the sh-B7H4 group compared to the sh-NC group (P<0.001), while the expression levels of total N F-$\kappa$ B and total STAT3 showed no significant changes (P>0.05). Conclusion Low expression of B7-H4 inhibits the proliferation and migration of MFC cells, and also impairs tumor formation in MFC cells. This tumor-suppressive mechanism may be achieved by blocking the activation of the NF-кB/IL-6/STAT3 pathway.

关键词

B7-H4 / 细胞增殖 / 细胞迁移 / 肿瘤形成 / NF-κB/IL-6/STAT3

Key words

B7-H4 / cell proliferation / cell migration / tumor formation / NF-кB/IL-6/STAT3

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基金资助

河北省自然科学基金资助项目(H2021206119)