The Functional Analysis Of E3 Ubiquitin Ligase TRIM25 In The Development Of Non-small Cell Lung Cancer

Background ubiquitin-proteasome pathway (UPP) is the main pathway for protein degradation in eukaryotic cells. In eukaryotic cells,80%-90% protein is degraded through the ubiquitin proteasome pathway, and the selective degradation of protein participates in many physiological and biochemical processes. With the intensive research,it is found that the disorder of UPP function in eukaryotic cells is related to the occurrence and development of many diseases. Among them, UPP acts a very important role in the development of cancers. The ubiquitin proteasome system (UPS) includes ubiquitin (ub), ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme(E2), ubiquitin-ligase enzyme (E3), 26S proteasome and deubiquitinating enzymes(DUBs).TRIM25 (Tripartite motif 25), also known as EFP (Estrogen-responsive Finger Protein),encoded by gene TRIM25, belonging to the TRIM family. Many TRIM family proteins have a function of E3 ligase. It is reported that the E3 ligase TRIM25 plays an important role in the development of breast cancer. So far, the relationship between TRIM25 and non-small cell lung cancer (NSCLC) is far from known. According to this, this object investigates the function and mechanism of ubiquitin ligase TRIM25 in the development of NSCLC.Part 1 The expression level of TRIM25 in clinical NSCLCObjective The relationship between TRIM25 and NSCLC is still not clear. In this part, we compared the protein level of TRIM25 in lung cancer tissues and adjacent tissues.Method The protein levels of TRIM25 in carcinoma tissue and para-carcinoma tissue were detected by western blot.Result According to the results of western blotting, the protein level of TRIM25 in carcinoma tissue was higher than para-carcinoma tissue in 19/25(76.00%) of lung adenocarcinoma and 12/17 (70.59%) of lung squamous carcinoma.Conclusion TRIM25 was highly expressed in clinical NSCLC carcinoma tissue.Part 2 Effect of TRIM25 on migration ability of lung adenocarcinomaObjective To explore the effect of TRIM25 on the migration ability in lung cancer cells.Methods Selected human lung adenocarcinoma H1299 cell line to construct the stable high lung adenocarcinoma cell line expressing enhanced TRIM25 and its control,which named H1299(TRIM25) and H1299 (control).Compared the migration ability between the two cell lines by wound healing assay.Results The migration ability of H1299 (TR1M25) cells was significantly stronger than that of H1299 (control). Conclusion Overexpression of TRIM25 could promote the migration ability of lung cancer cells.Part 3 The role and mechanism of TRIM25 in the development of lung cancerObjective To explore the mechanism of TRIM25 in the development of NSCLC and to find its downstream signaling pathway.Methods (1)Over-expressed TR1M25 in lung adenocarcinoma cell lines and detected the protein level of P53、p-AKT、p-mTOR by western blot;(2) Treated the H1299 (TRIM25) cell line and the control with Rapamycin to induce autophagy and detecte the effects by western blot;(3) The effects of TRIM25 on tumor suppressor PTEN ubiquitination and stability were detected through the western blot and co-immunoprecipitation methods; (4) The effects of over-expressed TRIM25 and TRIM25 silence on sensitivity of cisplatin in lung cancer cell were assessed by MTT assays.Results (1) Overexpression of TRIM25 obviously down-regulated the protein level of P53 in A549 cell and over expressed TRIM25 could up-regulate the protein level of p-AKT and p-mTOR in H1299 cell;(2) Treated the H1299 (TRIM25) cell line and its control with Rapamycin,the result shown that under the same concentration of Rapamycin,the level of autophagy in H1299 (TRIM25) cell line was significantly lower than that in the control cell line and theconcentration of Rapamycin was needed to induce autophagy was also higher than that in the control cell line; (3)Co-transfected myc-TRIM25、Flag-PTEN、HA-ub in HEK293T cell,the result shown that TRIM25 was an E3 ligase of PTEN,which can mediated ubiquitination of PTEN but did not affect the stability of PTEN; (4) Overexpression of TRIM25 could decrease the sensitivity to cisplatin in H1299 cell and the silence of TRIM25 could enhance the cytotoxic effects of cisplatin in H460 cell.Conclusion Overexpression of TRIM25 in lung cancer cells down-regulated the protein stability of p53 and activate AKT-mTOR signaling pathway to inhibit autophagy. TRIM25 mediated the ubiquitination of PTEN and did not affect the protein stability of PTEN. TRIM25 negative regulated the sensitivity to cisplatin of lung cancer cells.