Effect of Trim31 on sorafenib resistance in hepatocellular carcinoma cells and its related mechanism
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摘要: 目的 探讨三基序蛋白31(Trim31)对肝癌细胞索拉非尼耐药的影响及相关机制。方法 采用GEPIA数据库分析肝癌中Trim31 mRNA表达水平,采用Western blot检测肝癌组织中Trim31蛋白的表达水平。构建过表达Trim31的肝癌细胞系,采用CCK-8实验、集落形成实验和EdU实验评估过表达Trim31对肝癌细胞增殖的影响,采用蛋白富集分析及Western blot筛选验证Trim31下游信号通路。筛选Trim31下调质粒,并构建稳定沉默Trim31的肝癌细胞系,采用Western blot、CCK-8实验、集落形成实验和EdU实验评估沉默Trim31对肝癌细胞增殖的影响。在过表达Trim31的基础上进一步过表达P38,观察其对肝癌细胞增殖的影响,明确Trim31与P38通路之间的调控关系。用索拉非尼处理过表达Trim31的肝癌细胞系,采用CCK-8实验、集落形成实验、EdU实验和Western blot评估过表达Trim31对索拉非尼处理后肝癌细胞增殖的影响及机制。构建索拉非尼耐药肝癌细胞株,并在此基础上构建沉默Trim31的肝癌细胞系,采用CCK-8实验、集落形成实验、EdU实验和Western blot评估沉默Trim31对索拉非尼耐药细胞株增殖的影响及机制。结果 Trim31 mRNA在肝癌组织中呈高表达。过表达Trim31能促进肝癌细胞的增殖,而沉默Trim31可抑制肝癌细胞的增殖;过表达P38可抑制过表达Trim31对肝癌细胞的促增殖作用;Trim31与P38通路之间可能存在负调控作用。过表达Trim31可减弱索拉非尼对肝癌细胞增殖的抑制作用。索拉非尼并不影响肝癌耐药细胞株的增殖,但沉默Trim31后索拉非尼肝癌耐药细胞株的增殖受到显著抑制。结论 Trim31 mRNA在肝癌中呈高表达,沉默Trim31可减弱肝癌细胞对索拉非尼的耐药性,其机制可能与激活P38通路有关。Abstract: Objective To investigate the effect of tripartite motif-containing protein 31 (Trim31) on sorafenib resistance in hepatocellular carcinoma (HCC) cells and the underlying mechanisms. Methods The GEPIA database was used to analyze the levels of Trim31 mRNA in HCC. The protein levels of Trim31 in HCC tissues were detected by Western blot. A Trim31-overexpressing HCC cell line was constructed, and the effect of Trim31 overexpression on HCC cell proliferation were assessed by CCK-8, colony formation, and EdU assays. Protein enrichment analysis and Western blot were used to screen and validate the downstream signaling pathways of Trim31. Furthermore, Trim31-targeting shRNA was screened, and a stable Trim31-silenced HCC cell line was established. The effect of Trim31 silencing on HCC cell proliferation was evaluated by Western blot, and CCK-8, colony formation, and EdU assays. Moreover, the effect of P38 overexpression on HCC cell proliferation in the context of Trim31 overexpression was observed to elucidate the regulatory relationship between Trim31 and the P38 pathway. Sorafenib-treated Trim31-overexpressing HCC cell lines were used to assess the impact of Trim31 overexpression on HCC cell proliferation post-sorafenib treatment through CCK-8, colony formation, EdU assays, and Western blot. A sorafenib-resistant HCC cell line was established, where Trim31 was silenced in this cell line. The effect of Trim31 silencing on the proliferation of the sorafenib-resistant cell line was evaluated by CCK-8, colony formation, EdU assays, and Western blot. Results Trim31 mRNA was highly expressed in HCC tissues. Trim31 overexpression promoted HCC cell proliferation, while Trim31 silencing inhibited it. P38 overexpression suppressed the proliferative effect of Trim31 overexpression in HCC cells, suggesting a possible negative regulatory relationship between Trim31 and the P38 pathway. Trim31 overexpression attenuated the inhibitory effect of sorafenib on HCC cell proliferation. Although sorafenib did not affect the proliferation of the resistant HCC cell line, Trim31 silencing significantly inhibited the proliferation of the sorafenib-resistant HCC cells. Conclusions Trim31 mRNA is highly expressed in HCC, and silencing Trim31 can reduce the resistance of HCC cells to sorafenib, potentially through the activation of the P38 pathway.
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Keywords:
- liver cancer /
- tripartite motif-containing protein 31 /
- P38 /
- sorafenib /
- drug resistance /
- proliferation
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