Mouse MiR-763 Suppresses The Proliferation And Metastasis Of Human Melanoma Cells By Targeting GRB2

BackgroundMalignant melanoma belongs to skin cancer,which is highly metastatic and difficult to cure.Surgery is the primary treatment for patients with early melanoma.Most patients with advanced melanoma are usually treated with chemotherapy and biological therapy.However,the prognosis is very poor.Micro RNAs（miRNAs）play an important role in controlling the expression of key genes and all kinds of cellular activities in the secondary process of human cancer.It can strictly control the expression of target genes and regulate the basic functions of cells.Previous studies have shown that the expression level of miR-763 in B16F10,a mouse melanoma cell with rapid proliferation,was significantly reduced,and it was predicted by bioinformatics that GRB2 might be its target gene,which suggested that miR-763 might have anti-tumor activity.However,the specific mechanism by which miR-763 functions remains unknown.Growth factor receptor bound protein 2（GRB2）is a 25 KD adaptor protein,which was originally found to complete basic cell activities such as cell growth,cell proliferation and metabolism.Recent studies have proved that GRB2 plays an important role in a variety of malignant tumors.However,its biological role in melanoma is not clear.Therefore,we deeply study whether mouse miR-763 has tumor inhibition in human melanoma cells and the exact molecular mechanism of its action.This will help to design new diagnostic methods for melanoma patients.Objectives1.To explore the effect of miR-763 on the proliferation and migration of human melanoma cells.2.To explore whether miR-763 targets human GRB2 gene and the specific molecular mechanism of antitumor effect.Methods1.Screening of stable cell lines with overexpression of miR-763:miR-763 overexpression lentivirus and its control group infected human melanoma cells SK-MEL-5 and SK-MEL-28,and the stable cell lines were screened by puromycin to see if there was green fluorescence.2.The effect of miR-763 overexpression on the proliferation and migration of melanoma cells: CCK8 experiment and Transwell chamber experiment were carried out with stable cell lines to detect the change of cell proliferation and migration ability.3.To detect whether GRB2 is the objective gene of miR-763: The impact of miR-763 on GRB2 expression was observed by double luciferase experiment and Western blot.The activity of luciferase was compared to express the expression level of GRB2.Western blot was used to detect the effect of miR-763 overexpression on GRB2 expression level.4.Whether miR-763 targets GRB2 to inhibit melanoma: miR-763 was overexpressed in melanoma cells SK-MEL-5 and SK-MEL-28.CCK8 experiment and Transwell chamber experiment were carried out to detect whether miR-763 inhibited melanoma through GRB2.5.The specific mechanism of miR-763 in human melanoma cells: The protein expression level of cyclin D3 downstream of GRB2 and the phosphorylation level of p38 were detected by Western blot.6.In vivo experiment in nude mice,the effect of overexpression of miR-763 on the proliferation of melanoma was detected: The subcutaneous transplanted tumor model in nude mice was established,and the tumor growth was observed and recorded.The protein of tumor tissue was extracted for Western blot experiment.Results1.In the stably transfected human melanoma cells,the proliferation and migration abilities of the miR-763 overexpression group were weakened compared with those of the control group,indicating that the overexpression of miR-763 might inhibit the proliferation and migration abilities of human melanoma cells SK-MEL-5 and SK-MEL-28.2.the double luciferase experiment showed that there was a targeting relationship between miR-763 and GRB2,and the overexpression of miR-763 down regulated the protein expression level of GRB2.GRB2 overexpression can reverse the inhibition of proliferation and migration induced by miR-763 mimic,which further proves that GRB2 is the target gene of miR-763.3.Western blot analysis showed that the overexpression of miR-763 down-regulated the protein expression level of cyclin D3,a downstream signaling molecule of GRB2,and the phosphorylation level of p38,proving that miR-763 exerted the inhibitory effect on melanoma by mediating p38 MAPK signaling pathway.4.The in vivo experimental results in nude mice showed that the tumor volume and weight in the miR-763 overexpression group were smaller than those in the control group.In addition,the Western blot experimental results of tumor tissues showed that miR-763 overexpression down-regulated the protein expression level of cyclin D3,a downstream signaling molecule of GRB2,and the phosphorylation level of p38,proving that miR-763 overexpression inhibited the growth of melanoma in nude mice.ConclusionThis study found that overexpression of mouse miR-763 inhibited the proliferation and migration of human melanoma cells,and further proved that GRB2 was the target gene of miR-763.Mi R-763 may play an anti-tumor role by targeting GRB2 to regulate p38 MAPK signaling pathway,thereby inhibiting the proliferation and migration of human malignant melanoma cells.Therefore,these results may contribute to the development of drugs in the treatment of melanoma.