The Role Of Histone Methyltransferase SMYD3 In Regulation Of Proliferation In Neuroblastoma Cells

Neuroblastoma, developed from the sympathetic nervous system, is a common pediatric extracranial solid outer peripheral nervous system malignant tumor, accounting for childhood cancer rate of 8% ~ 10%, and the children’s cancer death 15%. Neuroblastoma is heterogeneous in the process of tumor formation, and its strong ability of proliferation and metastasis are the main factor leading to the death of the patients. Among them, 60% children were more than 1 years old at the time of initial diagnosis, and 45% had distant metastasis. The clinical features of neuroblastoma differ from each other. In the later stage of the patients, it commonly has strong resistance to traditional treatments, including surgery, chemotherapy and radiotherapy. Therefore, in recent years, a lot of experiments are devoted to explore new therapeutic methods for neuroblastoma.SMYD3(SET and MYND domain-containing protein 3) is a newly discovered histone methyl-transferase in recent years. Studies have shown that SMYD3 is highly expressed in colon cancer, liver cancer, breast cancer, gastric cancer, colon cancer and other cancer tissues or cells, but in the corresponding normal tissue with low expression or even undetectable, suggesting that SMYD3 and tumor occurrence as well as development are closely related. In this study, we explored the effect of SMYD3 on the proliferation and tumorigenesis of neuroblastoma cells in order to provide a new way for the treatment of neuroblastoma. This paper reveals the SMYD3 plays an important role in the process cells proliferation and apoptosis on neuroblastoma. Based on the analysis of the database of tumor prognosis, we found that SMYD3 can significantly affect the prognosis of neuroblastoma patients. We also detected the expression level of SMYD3 in different neuroblastoma cell lines. By means of SMYD3 knockdown andrestore expression experiments in neuroblastoma cell line BE(2)C and SHEP1, we detected the function of SMYD3 on cell growth, proliferation and apoptosis in vitro and in vivo. The main results are as follows:1. SMYD3 affects the prognosis of neuroblastoma and is commonly expressed in neuroblastoma cell linesThrough analysis of the neuroblastoma prognosis database Seeger and Versteeg, we found that high expression level of SMYD3 in neuroblastoma patients is closely related to poor prognosis, suggesting that SMYD3 may be associated with neuroblastoma malignant degree. RT-PCR, Western blot and immunofluorescence assay were used to detect the expression level of SMYD3, and the results showed that SMYD3 was widely expressed in neuroblastoma cell lines.2. SMYD3 promotes the growth of neuroblastoma cellsTo sdudy the mechanism of SMYD3 promoting neuroblastoma cells proliferation, we constructed the SMYD3 konck-down and corresponding overexpression vector, and packaged lentivirus. Thereafter, human NB cell lines were infected with these viruses to address whether the changes of SMYD3 expression levels had an impact on neuroblastoma cells growth or not. Cell proliferation curves and Brd U labeling experiments showed that interferencing SMYD3 significantly inhibited the proliferation of cells in vitro. The cell cycle distribution was detected by flow cytometry also. When knockdown SMYD3, the cell cycle was inhibited in G1 phase in BE(2)C and SHEP1 cells. These results indicated that SMYD3 can promote the growth of neuroblastoma, so the targeted therapy for SMYD3 is expected to play a role in further development of neuroblastoma.3. SMYD3 can promote the growth of neuroblastoma by inhibiting the p53/p21 pathwayThrough the review of the literature we found that SMYD3 may promote cell proliferation by inhibiting the p53 protein level in gastric cancer cells. So we detected the effection of SMYD3 interference and recovery on p53 and p21 protein levels by Western blot analysis. Knockdown of SMYD3 resulted in an increased p53 and p21 level, and the recovery of SMYD3 led to decreaseed p53 and p21 level in BE(2)C and SHEP1 cell lines. Real time RT-PCR and Western blot analysis found that MDM2, the p53 canonical ubiquitin enzyme, did not change when interferencing SMYD3 expression, whereas ubiquitination protease TRIM24 expression level was down regulated. When restoring the expression of SMYD3, TRIM24 expression increased,and MDM2 did not change. These results suggest that the regulation of p53 on SMYD3 is mediated by TRIM24 rather than MDM2. SMYD3 promoted neuroblastoma cells growth maybe by TRIM24 mediated p53/p21 signaling pathway.In summary, SMYD3 plays an important role in cell proliferation, apoptosis and tumorigenesis in neuroblastoma. These results suggested that SMYD3 may be used as a clinical marker for neuroblastoma diagnosis and can also provide a theoretical basis for neuroblastoma molecular therapy.