TRIM14 Inhibits Non-small Cell Lung Cancer Cell Growth Through Regulating Hexosamine Biosynthetic Pathway

Background and Objective: Tripartite motif(TRIMs)is a kind of containing the RING structure domain of E3 ubiquitin ligase, usually contains a RING domain,which has one or two B- box and a coiled-coil structure.As we all known,TRIMs family protein is play the function of E3 ubiquitin ligase activity, regulating multiple signaling pathways in cells, involved in cell differentiation, proliferation, apoptosis, etc. However, the biological functions of many members of the TRIMs family are still not clear. Interestingly, TRIM14 is the member that does not contain a RING domain. In the present study, the expressions of TRIM14 in non-small cell lung cancer(NSCLC) and its tissue adjacent to carcinoma and furthermore, we explored the roles and mechanisms of TRIM14 in NSCLC. Certainly, our results will provide important evidences for diagnosis and treatment of NSCLC clinically.Methods and Results:1. Immunohistochemistry and western blot were used to detect the expressions of TRIM14 in the non-small cell lung cancer tissues and adjacent lung tissue. The results showed that the expressions of TRIM14 were downregulated in non-small cell lung cancer tissues compared with adjacent tissues.2. Western blot and real-time fluorescence quantitative PCR were used to detect the expressions of TRIM14 in NSCLC cells. We observed that the expressions of TRIM14 in A549 and H1299 were the highest and the lowest, respectively.3. TRIM14 overexpressions in H1299 and knockdown in A549 were reached by transfacting corresponding plasmids respectively and then the cell growth, migration and clone formation were detected in the cell lines. Our results showed that overexpressions of TRIM14 can significantly inhibit the growth, migration and clone formation of H1299 cells. Knockdown of TRIM14 can significantly promote the growth, migration and clone formation of A549 cells.4. The expressions of O-GlcNAc and GFAT1 were detected by western blot in H1299 cells overexpressing TRIM14 and in A549 cells of knockdown of TRIM14. The results showed that the levels of O-GlcNAc and GFAT1 were reduced in H1299 cells of overexpressing TRIM14.5. With H1299 cells of overexpressing TRIM14, CHX and MG132 were added and incubated at the various time, then the expression of GFAT1 and O-GlcNAc were detected. The results showed that MG132 can block the degradation of GFAT1 by TRIM14.6. The expressions of O-GlcNAc were detected in H1299 cells of overexpressing TRIM14 with various concentrations of N-Acetyl-D-glucosamin. The results showed that N-acetyl glucosamine could restore the decreased level of O-GlcNAc induced by TRIM14 in NSCLC cells.Conclusion:Our results showed that the expression of TRIM14 was downregulated in non-small cell lung cancer tissues compared with adjacent tissues and that TRIM14 can significantly inhibit the cell growth, migration and colony formation of NSCLC cell lines. We demonstrated that TRIM14 as an E3 ligase reduced the O-linked ?-N-acetylglucosamine(O-GlcNAc) level of NSCLC cells through degrading the limited enzyme GFAT1 of the hexosamine biosynthetic pathway(HBP), indicating that TRIM14 maybe a new suppressor gene in non-small lung cancer, which inhibits the growth and migration of NSCLC cell through degrading GFAT1 to control the level of O-GlcNAc.