Study On Clinical Significance And Effect Of MiR-612 In Melanoma

Melanoma is a kind of malignant tumor formed by mutation of melanocytes.It is the most aggressive skin cancer with the highest mortality.Despite the development of prevention and treatment,the incidence rate of melanoma continues to increase by about 3%annually.It is still a serious public health problem,which brings heavy financial burden to patients and society.Melanoma is highly malignant and easy to metastasize,and clinical treatment is quite tricky.On the basis of studying the pathogenesis of melanoma,it is of great significance to explore stable and effective biomarkers to provide new ideas for its diagnosis and treatment.microRNAs(miRNAs)are small RNA molecules which play roles in gene silencing and translation by binding to target mRNAs.Since miRNAs discovery in 1993,miRNAs have been found in all conserved eukaryotic cells throughout the species.In recent years,extensive researches have been conducted on the regulatory roles of miRNAs in biological processes and the occurrence and development of various human diseases.Studies have reported that the expression of miR-612 in a variety of human malignant tumors shows a downward trend,and it mediates the biological development of malignant tumors.However,no researchers have reported the expression and effect of miR-612 in melanoma and its correlation with clinicopathological characteristics of melanoma.Therefore,this study conducted research from the above aspects and tried to investigate the target genes of miR-612 in melanoma,aiming to provide an experimental basis for miR-612 in the diagnosis and treatment of melanoma.Part Ⅰ Expression and Clinical Significance of miR-612 in MelanomaObjective1.To investigate the expression of miR-612 in melanoma and non-cancerous normal tissues.2.To investigate the relationship between the expression of miR-612 and clinicopathological parameters of melanoma.3.To investigate the relationship between the expression of miR-612 and the prognosis of patients with melanoma.MethodsMelanoma tissue samples and matched normal tissue samples were collected from 89 patients with melanoma confirmed by histopathology or cytology.Total RNA was extracted from tissue samples,and the expression of miR-612 was detected by RT-PCR.The patients with melanoma were followed up by outpatient or telephone to collect the information of overall survival(OS)and disease-free survival(DFS).The clinical and pathological characteristics of patients in miR-612 high expression group and miR-612 low expression group were compared by chi square analysis.Kaplan-Meier method was used to draw the survival curve,and the log rank test was used to compare survival rate.Results1.The expression level of miR-612 in melanoma tissue was significantly lower than that in matched non-cancerous normal tissues(P=0.0154).2.There were no significant correlation between the expression of miR-612 and the age,gender,lesion location,ulceration,tumor volume,and TNM stage of melanoma patients(P>0.05),However,the low expression of miR-612 were significantly related to the thickness of melanoma(P=0.0013)and lymph node metastasis(P=0.0169).3.The overall survival rate of patients in the miR-612 low expression group was significantly lower than that in the miR-612 high expression group(P=0.0138),and the disease-free survival rate was also significantly lower than that in the miR-612 high expression group(P=0.0022).ConclusionThe expression of miR-612 is low in melanoma,which is closely related to the thickness of melanoma and lymph node metastasis.Down-regulation of miR-612 expression indicates poor prognosis.Part Ⅱ Study on the Mechanism of miR-612 in MelanomaObjective1.To compare the expression of miR-612 in melanoma cells and human epidermal melanocytes.2.To investigate the target genes of miR-612 in melanoma cells.3.To investigate the effect of miR-612 on the growth,migration,invasion,and drug resistance of melanoma cells or transplanted tumors and its possible mechanisms.Methods1.Under the conditions of 5%CO2 and 37℃,human melanoma cells(SK-MEL-28,SK-MEL-3,A375,HT-144 and Hs294T)were cultured in DMEM medium containing 10%fetal bovine serum,and the human epidermal melanocyte cells were all cultured in melanocyte culture medium containing 10%of the fetal bovine serum cultivates.2.The melanoma cells were treated with different concentrations of doxorubicin(0,1,2,5 and 10 μM)for 24 h,and the half maximum inhibitory concentration(IC50)of doxorubicin was calculated.3.The qRT-PCR method was used to detect the expression level of miR-612 in melanoma cells and tissues.4.Vector plasmid,miR-612 expression plasmid and Espin+miR-612 expression plasmid were prepared and transfected into melanoma cells by Lipofectamine 2000.5.The BrdU-ELISA method was used to detect cell proliferation;the plate colony formation experiment was used to observe cell growth,the scratch tests and the Transwell experiments were used to detect cell migrations and invasions;the Western Blot methods was used to detect E-cadherin,vimentin and Espin protein expression;immunohistochemical method was used to detect Ki-67 expression in transplanted tumor tissue.6.The Pearson method was used to analyze the correlation between miR-612 and Espin expression.Results1.The expression of miR-612 in melanoma cells SK-MEL-28,A375,HT-144,Hs294T were significantly lower than those in human epidermal melanocytes,two sets of data between a statistically significant diffience(P<0.05).2.In SK-MEL-28 and A375 cells,the expression level of miR-612 transfected with miR-612 expression plasmid was significantly higher than that of cells in the single-transfected Vector plasmid group,and two sets of data between a statistically significant diffience(P<0.05).3.The proliferation of SK-MEL-28 and A375 cells transfected with miR-612 expression plasmid was significantly less than that of cells transfected with Vector plasmid alone,and two sets of data between a statistically significant diffience(P<0.05).4.The number of colonies of SK-MEL-28 and A375 cells transfected with miR-612 expression plasmid was significantly less than that of cells transfected with Vector plasmid alone,and two sets of data between a statistically significant diffience(P<0.05).5.The tumor volume and weight of A375 cells inoculated with miR-612 expression plasmid were significantly smaller than those of the single-transfected Vector group,and two sets of data between a statistically significant diffience(P<0.05).The Ki-67 positive expression rate in transplanted tumors inoculated with A375 cells transfected with miR-612 expression plasmid was significantly lower than that of transplanted tumors in the single-transfected Vector plasmid group,and sets of data between a statistically significant diffience(P<0.05).6.The wound healing rate and invasion rate of SK-MEL-28 and A375 cells transfected with miR-612 expression plasmid were significantly lower than those of cells transfected with Vector plasmid alone,and two sets of data between a statistically significant diffience(P<0.05).However,the protein expression level of E-cadherin vimentin in the cells is not significantly different from that of cells transfected with Vector plasmid alone(P>0.05).7.Espin 3’-UTR has a miR-612 binding site.Overexpression of miR-612 significantly inhibited wild-type Espin 3’-UTR luciferase activity in HEK293T cells(P<0.05),but it has no effect on Espin 3’-UTR mutant luciferase activity(P>0.05).8.The expression level of Espin protein in SK-MEL-28 and A375 cells transfected with miR-612 expression plasmid was significantly lower than that in cells transfected with Vector plasmid alone,and two sets of data between a statistically significant diffience(P<0.05).9.There was a significant negative correlation between miR-612 and Espin protein expression in melanoma tissue(r=-0.376,P=0.018).10.The expression level of Espin protein in A375 cells co-transfected with Espin and miR-612 expression plasmids was significantly higher than that in cells co-transfected with Vector plasmid,and sets of data between a statistically significant diffience(P<0.05).11.The proliferation of A375 cells co-transfected with Espin and miR-612 expression plasmids was significantly higher than that of the cells co-transfected with Vector plasmid,and sets of data between a statistically significant diffience(P<0.05).12.The size of transplanted tumors of A375 cells co-transfected with Espin and miR-612 expression plasmids was significantly larger than that of the co-transfected Vector plasmid group,and sets of data between a statistically significant diffience(P<0.05).13.The invasion rate of A375 cells co-transfected with Espin and miR-612 expression plasmids was significantly higher than that of cells co-transfected with Vector plasmids,and sets of data between a statistically significant diffience(P<0.05).14.The doxorubicin IC50 value of SK-MEL-28 and A375 cells transfected with miR-612 plasmid was significantly lower than that of cells transfected with Vector plasmid alone,and the difference was statistically significant(P<0.05).The doxorubicin IC50 values of SK-MEL-28 and A3 75 cells transfected with Espin siRNA overexpressing miR-612 were significantly lower than those in the control siRNA group,and sets of data between a statistically significant diffience(P<0.05).Conclusion1.The expression of miR-612 is down-regulated in melanoma cells.2.miR-612 inhibits the growth of melanoma cells/transplanted tumors by regulating its target gene Espin,prevents the migration and invasion of melanoma cells,and enhances the sensitivity of melanoma cells to the chemotherapeutic drug doxorubicin,so as to play the role of tumor suppressor gene in melanoma.