Researches on the Mechanism of Overexpression of Tripartite Motif Protein 48 Regulating p-ERK1/2 to Inhibit Glioma Growth
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摘要:
目的 探究过表达三结构域蛋白48（tripartite motif protein，TRIM）对胶质瘤生长的影响及其相关机制。 方法 将12只裸鼠随机均分为2组，分别接种过表达TRIM48的U87胶质瘤稳转株（oeTRIM48组）及其对照细胞株（Vector组）。接种后每3 d测定肿瘤体积，4周后取出肿瘤组织并记录瘤重。肿瘤组织做HE染色，并通过免疫荧光法检测Ki-67的表达，使用Western blot和免疫组化分别检测裸鼠肿瘤和人胶质瘤组织芯片中的TRIM48、ERK1/2和p-ERK1/2水平。 结果 oeTRIM48组裸鼠肿瘤体积、重量比Vector组裸鼠明显降低（P < 0.0001）；HE染色结果显示oeTRIM48组细胞核减小、核分裂象减少；Ki-67阳性区域显著降低（P < 0.0001），而且oeTRIM48组p-ERK1/2蛋白水平比Vector组显著降低（P < 0.01）。组织芯片免疫组化显示，TRIM48和p-ERK1/2在癌旁组织分别呈高表达和低表达，在肿瘤组织则相反。 结论 过表达TRIM48能够抑制胶质瘤生长、增殖，其作用机制可能与ERK1/2信号通路有关。 Abstract:Objective To investigate the impact of TRIM48 overexpression on glioma and explore the underlying mechanism. Methods Twelve nude mice were randomly assigned into two groups: one inoculated with U87 glioma cells stably overexpressing TRIM48 （oeTRIM48 group） and the other with the control cell line （Vector group）. Tumor volume was measured every 3 days post-inoculation, and after 4 weeks, the tumors were excised and weighed. Tumor tissues underwent hematoxylin and eosin （H&E） staining, and Ki-67 expression was assessed using immunofluorescence. Additionally, the expressions of TRIM48, ERK1/2, and phosphorylated ERK1/2 （p-ERK1/2） in the tumors of nude mice and human glioma tissue arrays were analyzed through Western blot and immunohistochemistry respectively. Results Compared to the Vector group, the oeTRIM48 group exhibited significantly reduced tumor volume and weight （P < 0.0001）. H&E staining indicated a decrease in nuclei and mitotic count in the oeTRIM48 group. Furthermore, Ki-67 expression was significantly lower in the oeTRIM48 group than that in the Vector group （P < 0.0001）. The oeTRIM48 group also showed a significantly lower expression of p-ERK1/2 protein compared to the Vector group （P < 0.01）. Immunohistochemistry on tissue microarrays revealed the high expression of TRIM48 and low expression of p-ERK1/2 in adjacent non-tumoral tissues, with the opposite pattern observed in tumor tissues. Conclusion Overexpression of TRIM48 inhibits the growth and proliferation of glioma, potentially through modulation of the ERK1/2 signaling pathway. -
图 1 过表达TRIM48对胶质瘤生长的影响
A：肿瘤组织重量；B：肿瘤组织体积；C：肿瘤组织HE染色（×200）；n=6； ****P < 0.0001。
Figure 1. Effect of TRIM48 overexpression on glioma growth
图 2 过表达TRIM48影响胶质瘤组织增殖活性
n=5，****P < 0.0001。
Figure 2. Overexpression of TRIM48 affects the proliferation activity of glioma tissues
图 3 过表达TRIM48对ERK1/2通路的调控
A：qRT-PCR和Western blot检测转染结果；B：Western blot检测过表达TRIM48对ERK1/2信号通路激活情况。n=6； ns：差异无统计学意义；*P < 0.05；**P < 0.01；****P < 0.0001。
Figure 3. Regulation of ERK1/2 pathway by overexpression of TRIM48
图 4 胶质瘤患者组织芯片中TRIM48与ERK1/2信号通路之间的关系（×200）
A：通过免疫组化检测组织芯片中胶质瘤患者肿瘤组织及匹配癌旁组织TRIM48与p-ERK1/2表达情况；B：p-ERK1/2在肿瘤组织与癌旁组织中的阳性区域；C：TRIM48在肿瘤组织和癌旁组织中的阳性区域；n=6；****P < 0.0001。
Figure 4. Direct relationship between TRIM48 and ERK1/2 signaling pathway in glioma tissue microarray （×200）
表 1 PCR引物
Table 1. PCR primers
引物名称 目的基因 序列（5'→3'） 产物大小（bp） Forward primer 1 TRIM48 AGCACCGGTATCACAGACAC 162 Reverse primer 1 TRIM48 TGTCTCCAAAAGCCTTCCAGTG 162 Forward primer 2 β-actin AGGATTCCTATGTGGGCGAC 273 Reverse primer 2 β-actin ATAGCACAGCCTGGATAGCAA 273 
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