Effect Of WIPI1 Gene On Proliferation And Apoptosis Of Human Melanoma A375 Cells

Objective: Malignant melanoma(MM)is a highly malignant tumor,most frequently occurring in the skin,and it is the third most common skin malignant tumor(6.8%~20%).In recent years,the morbidity and mortality of malignant melanoma have shown a trend of continuous growth all over the world.Malignant melanoma has high malignant degree,early metastasis,and is not sensitive to radiotherapy and chemotherapy,which seriously threatens people’s life and health.At present,The occurrence of malignant melanoma involves many factors such as race and heredity,trauma and stimulation,viral infection,sunlight,immunity.In China,people generally lack understanding and attention to malignant melanoma,excessive ultraviolet irradiation,sunburn,long-term local stimulation,trauma,etc.,can cause malignant changes in benign nevus to and induce malignant melanoma.Researches have shown that early diagnosis and appropriate surgical resection can make the cure rate of low-risk melanoma over 90%,while the 5-year survival rate for high-risk and extremely high-risk patients ranges from 10% to 50%.Therefore,the improvement of early diagnosis techniques and methods,and the research of effective molecular biological targets and genes are of great significance to the prognosis of malignant melanoma patients.It has been found that autophagy related gene WIPI1 can regulate the biological behavior of colon cancer and other tumors,In this study,we silenced WIPI1 gene by RNA interference technology to detect its effect on the proliferation and appotosis of human melanoma A375 cells,in order to find out the relationship and mechanism of action between WIPI1 gene and malignant melanoma,and provide theoretical basis for gene therapy of malignant melanoma。Methods:1.Detect the expression of WIPI1 gene in three strains of human melanoma cells A375,MUM-2B and MUM-2C by q PCR;with WIPI1 gene as the template,the corresponding interference targets were designed,and the RNA interfering lentivirus vector of WIPI1 gene was constructed;in tool cell 293 T,Western Blot was used to verify the effectiveness of the interfering target。2.This experiment was divided into two groups: sh WIPI1 group(normal A375 cells,with additional cells infected by WIPI1 gene sh RNA virus)and sh Ctrl group(normal A375 cells,with additional cells infected by negative control virus),q PCR was used to detect the m RNA expression of WIPI1 gene in A375 cells of the two groups;Celigo method was used to detect the effect of WIPI1 gene knock-down on cell proliferation;the effect of WIPI1 gene knock-down on apoptosis was detected by Caspase3/7;the effect of WIPI1 gene knock-down on apoptosis was detected by Annexinv-apc method;PI-FACS was used to detect the effect of WIPI1 gene knock-down on cell cycle。Results:1.QPCR results confirmed that WIPI1 gene was highly expressed in three human melanoma cell strains;2.After silencing WIPI1 gene,the m RNA expression of WIPI1 gene was significantly inhibited(P<0.05),with the knock-down efficiency of 88.5%;3.After silencing WIPI1 gene,the proliferation of A375 cells was inhibited obviously;4.Compared with sh Ctrl group,the number of apoptosis in sh WIPI1 group increased(P<0.05);5.After silencing WIPI1 gene,the apoptosis rate of sh WIPI1 group was increased(P<0.05);6.Cell cycle detection by PI-FACS: in sh WIPI1 group,the number of cells increased in G1 phase(P<0.05),decreased in S phase(P<0.05),and decreased in G2/M phase(P<0.05)。Conclusions:1.Silencing WIPI1 gene can effectively reduce the activity of human melanoma A375 cells,inhibit cell proliferation,and induce cell apoptosis。2.WIPI1 gene may be a potential target for malignant melanoma,which can provide help for early diagnosis of malignant melanoma and molecular targeted therapy for patients with advanced malignant melanoma。...