| Purpose: Thyroid cancer is the most common malignant tumor of endocrine system,accounting for 1%-2% of all cancer cases.Papillary thyroid cancer(PTC)is the most common pathological type,accounting for more than 90% of all thyroid cancer.Although the majority of PTC patients can survive for a long time through surgical resection and postoperative thyroid stimulating hormone suppression combined with radioiodine therapy,there are still some PTC patients who are ineffective in this treatment and lead to death.Therefore,it is very important to elucidate the pathogenesis of PTC at the molecular level.Methods: 1.100 pairs of PTC tissues and paired para cancerous tissues were collected from the thyroid surgery department of the First Affiliated Hospital of China Medical University.2.The expressions of RBM47,SNHG5,FOXO3,ATG3 and ATG5 in 100 pairs of PTC tissues and adjacent tissues were detected by q RT-PCR.The relationship between the expressions of RBM47 or SNHG5 in PTC tissues and the clinicopathological data of PTC patients was analyzed by chi square test.3.Lentivirus was used to construct PTC cell lines with low and over expression of RBM47 or SNHG5. Small interfering RNA(si RNA)knockdown and plasmid over expression of FOXO3 and USP21 were used to detect the transfection efficiency.4.CCK-8 assay was used to detect the effects of RBM47 or SNHG5 on the proliferation of PTC cells;Western blot,immunofluorescence and transmission electron microscopy were used to detect the effects of RBM47 or SNHG5 on the autophagy of PTC cells.5.RIP confirmed the binding ability of SNHG5 to different proteins.6.q RT-PCR and Western blot were used to verify the regulation of genes in the cell line.7.Western blot and immunoprecipitation were used to detect the effect of USP21 on ubiquitination of FOXO3.8.The luciferase reporter gene showed that FOXO3 could bind to the promoters of RBM47,ATG3 and ATG5 and play a role in transcriptional regulation.9.The effects of RBM47 or SNHG5 on the proliferation of subcutaneous tumor in nude mice were detected.Results: 1.The expression of RBM47 in PTC tissue was significantly lower than that in matched paracancerous tissue,and the expression of RBM47 in PTC tissue was correlated with tumor diameter and TNM stage.2.RBM47 can inhibit the growth of PTC cells and activate autophagy.3.The expression of FOXO3 in PTC tissues was significantly lower than that in matched paracancerous tissues,and RBM47 was positively correlated with FOXO3 expression in PTC tissues.4.RBM47 can indirectly regulate the expression of FOXO3.5.RBM47 binds and promotes the stability of SNHG5.6.The expression of SNHG5 in PTC tissues was significantly lower than that in matched para cancerous tissues.The expression of SNHG5 in PTC tissues was correlated with tumor diameter and lymph node metastasis.7.SNHG5 can inhibit the growth of PTC cells and activate autophagy.8.SNHG5 regulates FOXO3 at the post transcriptional level.9.Both in vivo and in vitro experiments confirmed that RBM47 affected the proliferation,autophagy and FOXO3 expression of PTC cells through SNHG5.10. SNHG5 can inhibit the ubiquitination of FOXO3.11.SNHG5 can recruit USP21.12. USP21 promotes the intranuclear transfer of FOXO3.13.USP21 can directly bind and inhibit the ubiquitination of FOXO3.14.FOXO3 can bind to ATG3 and ATG5 promoters and activate transcription.15.FOXO3 can bind to the promoter of RBM47 and activate RBM47 to form a positive feedback loop.Conclusion: RBM47 binds and regulates the stability of SNHG5.SNHG5 inhibits the ubiquitination of FOXO3 by recruiting USP21,thereby promoting its entry into the nucleus.On the one hand,FOXO3 promotes the expression of ATG3 and ATG5,and activates autophagy;on the other hand,FOXO3 transcription activates RBM47,forming a positive feedback loop.This experiment reveals a new pathway of RBM47/SNHG5/USP21/ FOXO3 axis promoting PTC autophagy,which provides a new theoretical and experimental basis for the treatment of thyroid cancer. |
PDF Full Text Request