ZMYND10 Inhibits The Proliferation Of Melanoma Cells By Regulating IGF-1R/ERK Signaling Pathway

BackgroundMelanoma is a malignant tumor which is usually caused by abnormal proliferation of melanocytes.It is a common malignant tumor in skin and visceral mucosa.Incidence rate is high.Although melanoma can be cured by local excision at the early stage of the disease,the treatment is usually not satisfactory when it develops to the late stage of metastasis.Melanoma bears a heavy health and economic burden.Therefore,it is of great significance to study the pathogenesis of melanoma and find new treatment methods and drugs to improve the therapeutic effect of melanoma.ZMYND10,also known as BLU,can inhibit transcription.ZMYND10 is located in 3p21.3 region and is inactivated or down regulated in many solid tumors,such as lung cancer,glioma,ovarian cancer,liver cancer,esophageal squamous cell carcinoma,neuroblastoma,myelodysplastic syndrome,gastric cancer and nasopharyngeal carcinoma.Previous studies have confirmed that ZMYND10 can induce apoptosis,block cell cycle,inhibit cell proliferation and angiogenesis in different tumors,but its biological function and molecular mechanism in melanoma are still unclear.At present,80% of melanoma is caused by abnormal activation of MAPK/ERK signaling pathway,and our previous experimental results also showed that zmynd10 overexpression can inhibit the tumorigenesis and metastasis of melanoma cells,and is related to the down-regulation of MAPK/ERK signaling pathway;however,the specific molecular mechanism of how ZMYND10 regulates MAPK/ERK signaling pathway is still unclear.Therefore,it is necessary to further study the relationship between ZMYND10 and melanoma cells The relationship between various melanoma related abnormal molecules will help to reveal the pathogenesis of melanoma and help us find out how ZMYND10 regulates MAPK/ERK signaling pathway to inhibit the proliferation of melanoma.Objective1.To explore the effect of ZMYND10 on the proliferation of B16F10 melanoma cells;to explore the molecular mechanism underlying ZMYND10 inhibits the proliferation of B16F10 cells.Method1.Screening of stable cell line B16F10 with ZMYND10 overexpression: Mouse melanoma B16F10 cells were infected with lentivirus containing ZMYND10,The stable cell lines were screened by puromycin,ZMYND10 antibody and flag tag antibody were used to confirm ZMYND10 overexpression.2.The effect of ZMYND10 overexpression on the proliferation of melanoma cells:After digestion and cell counting,ZMYND10 overexpression stably transformed cell lines were inoculated into 96 well plates,MTT assay was used to analyze the effect of zmynd10 overexpression on the proliferation of melanoma cells;Clonogenic assay was used to analyze the effect of ZMYND10 overexpression on the clonogenic ability of melanoma cells,Two weeks after subcutaneous injection of ZMYND10 overexpressed cells,the tumor was dissected and the tumor size was measured to explore the effect of ZMYND10 overexpression on the proliferation of melanoma cells in mice.3.Signal pathway of ZMYND10 overexpression inhibiting melanoma proliferation:B16F10 cell line with ZMYND10 overexpression was collected,and the effects of ZMYND10 overexpression on MAPK/ERK phosphorylation levels were analyzed by Western Blot;To further elucidate the molecular mechanism of ZMYND10 inhibiting the proliferation of melanoma cells,we added inhibitors of corresponding signaling pathways to B16F10 and B16F10 cell lines with ZMYND10 overexpression and zmynd10 silencing,respectively.4.the whole transcriptional group was sequenced and the expression of transcription factors in stable cell lines was detected;The RNA was extracted from B16F10 cell line of ZMYND10 overexpression,sequenced by the company,the expression level of relevant transcription factors was detected,and the most obvious change was screened for test verification;5.ZMYND10 regulates MAPK /ERK signaling pathway by regulating IGF-1R level to inhibit proliferation of B16F10 cells;The expression of ZMYND10 was collected and the effect of overexpression and silencing on IGF-1R protein and m RNA level was analyzed by q PCR;IGF-1R inhibitors were added to the stable cell lines B16F10 and ZMYND10 silenced stable cell lines B16F10,which were overexpressed by ZMYND10,respectively,to further elucidate the mechanism of ZMYND10 in inhibiting the proliferation of melanoma cells.Result1.B16F10 cells were screened by lentivirus infection and puromycin,and ZMYND10 overexpression cell line was obtained;2.By MTT assay,colony formation assay and subcutaneous tumorigenesis assay,it was found that ZMYND10 overexpression could inhibit the proliferation of B16F10 cells;3.Western blot showed that ZMYND10 overexpression could reduce the phosphorylation levels of ERK1/2,MEK1/2 and c-Raf;4.Transcriptome sequencing showed that ZMYND10 overexpression could inhibit the expression of IGF-1R at m RNA level;further verification by real-time PCR and Western blot showed that ZMYND10 overexpression could inhibit the expression of IGF-1R,while ZMYND10 gene silencing could increase the expression of IGF-1R;5.IGF-1R inhibitor picropodophyllin can reverse the promotion of ZMYND10 gene silencing on the phosphorylation of ERK1/2,MEK1/2 and c-Raf in MAPK/ERK signaling pathway and cell proliferation.ConclusionZMYND10 overexpression can inhibit the proliferation of mouse melanoma B16F10 cells by inhibiting the expression of IGF-1R and regulating MAPK/ERK signaling pathway.The research results of this paper will help to further clarify the molecular mechanism of melanoma proliferation,and provide a theoretical basis for the research and development of anti melanoma drugs and the targeted therapy of anti-tumor drugs.