摘要
Aim To study the mechanism and target of apoptosis induced by berberine ( BBR) in cervical cancer HeLa cells. Methods Drug affinity responsive target stability (DARTS) and mass spectrometry (MS) were used to identify the potential binding proteins of berberine. The binding affinity between berberine and candidate target protein was detected by microscale thermophoresis technique (MST) , and cellular thermal shift assay (CETSA) was used to detect the binding of berberine to candidate target proteins in living cells. CRISPR/Cas9 gene editing technique was used to establish candidate target protein TRIM25-deficient tumor cell lines. CCK-8 assay and Annexin V/propidium iodide combined with flow cytometry were used to detect the inhibitory and apoptotic effects of berberine on wild-type and TRIM25-KO cells. Western blot was used to detect the effect of berberine on TRIM25 and its substrate protein levels.Results DARTS found that after berberine treatment, the sensitivity of TRIM25 to pronase proteolysis showed the most significant change. MST and CETSA assays showed that berberine directly bound to TRIM25 at molecular and cellular levels, and its dissociation constant was 4.02 μmol • L
摘要
Objective:To investigate the expressions of TRIM25 and RIG-Ⅰ in liver cancer tissues and their relationship with the prognosis of patients.Methods:The data of 82 patients with liver cancer who were admitted to the Affiliated Huai'an No. 1 People's Hospital of Nanjing Medical University from January 2017 to January 2018 were retrospectively analyzed, and their cancer tissue and paracancerous tissue (>5 cm from the edge of the tumor) specimens were collected. The protein expressions of TRIM25 and RIG-Ⅰ in cancer tissues and paracancerous tissues were detected by immunohistochemistry. The relationship between TRIM25 and RIG-Ⅰ expressions in cancer tissues of patients and clinicopathological features was analyzed. Kaplan-Meier method was used to analyze the overall survival (OS) of patients with different TRIM25 and RIG-Ⅰ expression status.Results:The positive rate of TRIM25 in cancer tissues was higher than that in paracancerous tissues [68.29% (56/82) vs. 21.95% (18/82), P < 0.001], and the positive rate of RIG-Ⅰ in cancer tissues was lower than that in paracancerous tissues [31.71% (26/82) vs. 74.39% (61/82), P < 0.001]. The positive rate of TRIM25 in cancer tissues of poorly differentiated patients was higher than that of highly and moderately differentiated patients ( P < 0.05), and the positive rate of RIG-Ⅰ was lower than that of highly and moderately differentiated patients ( P < 0.05). The positive rate of TRIM25 in cancer tissues of patients with extrahepatic metastasis was higher than that of patients without extrahepatic metastasis ( P < 0.05), but the positive rate of RIG-Ⅰ was lower than that of patients without extrahepatic metastasis ( P < 0.05). The positive rate of TRIM25 in patients with clinical Ⅲ-Ⅳ was higher than that in patients with stage Ⅰ-Ⅱ ( P < 0.05), but the positive rate of RIG-Ⅰ was lower than that in patients with stage Ⅰ-Ⅱ ( P < 0.05). The median follow-up time was 27 months (4-48 months), 2 patients were lost to follow-up. At the end of follow-up in January 2022, the overall survival rate was 43.75% (35/80). The survival rates of patients with TRIM25-positive and TRIM25-negative cancer tissues were 33.33% (18/54) and 65.38% (17/26), respectively. The survival rates of patients with RIG-Ⅰ-positive and RIG-Ⅰ-negative cancer tissues were 64.00% (16/25) and 34.55% (19/55), respectively. Kaplan-Meier analysis showed that the OS of patients with TRIM25-negative cancer tissues was better than that of patients with TRIM25-positive cancer tissues, and the OS of patients with RIG-Ⅰ-positive cancer tissues was better than that of patients with TRIM25-negative cancer tissues, and the differences were statistically significant (both P < 0.05). Conclusions:The expression of TRIM25 is increased and the expression of RIG-Ⅰ is decreased in liver cancer tissues. The expressions of TRIM25 and RIG-Ⅰ in liver cancer tissues are associated with prognosis.