Expression Of LIPA In Skin Melanoma And Its Biological Effect

BackgroundMelanoma is a relatively common malignant tumor that mainly involves the skin and internal organs.Among them,the skin system as the primary malignant melanoma accounts for 2.3%of all skin cancers,and it is the most malignant and life-threatening skin cancer[1,2]In recent years,the incidence of skin cancer has increased year by year,and the incidence of melanoma has increased faster than that of the other two types of skin cancer[3]According to data from the World Health Organization(WHO),132,000 new patients are diagnosed each year worldwide[4]In the past 30 years,especially men over the age of 60 and[5]and relatively young patients accounted for a relatively high proportion[6].Studies have shown that the incidence of some melanomas is related to sun exposure and sunbathing[7]fair skin,and overexposed whites have a higher risk of illness.In addition,about 2%of melanoma patients have a clear family history,and the relatives of melanoma patients who have a blood relationship are at increased risk of melanoma than others.The occurrence,development and evolution of melanoma are the result of a combination of factors,including genetic mutation,inheritance,immunity and external environmeny[9].At present,we have not yet known these complex mechanisms.The cyclin-dependent kinase inhibitor 2A(CDKN2A)gene is the most common familial melanoma pathogenic gene,with a positive rate of about 20%to 40%in patients with a family history of melanoma[8].Although we have affirmed the role of cyclin-dependent kinase inhibitor 2A(CDKN2A),CDK4,TERT,MITF,and BAP1 in melanoma,there are still many genes in the development of melanoma Plays an important role.Therefore,we need to clarify the correlation between multiple disease-causing genes,determine the underlying molecular mechanism,and find important biomarkers,which are helpful for the early diagnosis,early treatment,and targeted therapy of melanoma.Provide clinical help.A few years ago,the treatment of malignant melanoma was mainly surgical resection.However,surgical treatment is only effective in the early stages of the onset of melanoma,and metastatic melanoma still lacks effective treatment methods.It is gratifying that in recent years,significant progress has been made in the development of new targeted therapies and immunotherapy[10].More and more drugs are being studied for the treatment of clinical melanoma:oncolytic immunotherapy,indoleamine Selective inhibitors of 2,3-dioxygenase,anti-PD-(L)1,etc.,undoubtedly bring hope to patients with advanced malignant melanoma and significantly change the future development direction of contemporary oncology.Although new treatments have been introduced and many studies have confirmed their effectiveness,there is still a need for more effective and precise treatments for melanoma patients.The protein encoded by the LIPA gene is Lysosomal acid lipase(LAL).LAL is the only known essential intracellular lipase that is active in an acidic environment.LAL can hydrolyze cholesterol esters and triglycerides in lysosomes to produce fatty acids and store them in the form of lipid droplets.LAL mediates intracellular and extracellular lipid catabolism in a tissue-and cell-specific manner.Studies have shown that cancer cells synthesize fatty acids(FAs)when they are in a harsh environment,and rely on fatty acids in the intracellular and extracellular microenvironments to increase their viability[30,31].It can be seen from this that cancer cells have a strong ability to obtain extracellular lipids,and can improve tumor viability through intracellular lipid mobilization and recycling.Therefore,the regulation of lipid metabolism is an attractive target for the treatment of cellular stress in cancer,and LIPA may play an important role in the direction of tumor lipid metabolism.ObjectiveUsing bioinformatics methods to determine the important pathogenic gene LIPA in melanoma,through immunohistochemistry,Western blot and PCR experiments to study the expression of LIPA gene in melanoma,so as to evaluate and clarify the LIPA protein in melanoma Significance and value;through cell experiments to further clarify the expression of LIPA in cutaneous melanoma and its effect on the activity,proliferation and migration of cutaneous melanoma cells and the mechanism of biological action,thus providing an important target for melanoma To improve the patient’s quality of life and bring the gospel to patients.Methods1)Determine the differentially expressed genes(DEGs)potentially co-expressed in primary melanoma of the skin through bioinformatics analysis,and determine the target genes to be studied in the next step;2)Immunohistochemical method was used to detect the expression of LIPA protein in the skin lesions of the above 25 cases of malignant melanoma and 25 cases of skin pigment nevus.Relevance of type and clinical stage;4)Culture melanoma cells and pigmented nevus cells in vitro,and use Western blot and PCR methods to detect the protein and gene expression levels of LIPA in in vitro model cells;construct the LIPA using techniques such as plasmid synthesis,plasmid extraction,and competent cell preparation Silent cell model,then use the cell scratch test,MTT test,flow cytometry to detect tumor cell activity,proliferation and migration ability.Results1.Through bioinformatics analysis,4 gene chips GSE100050,GSE114445,GSE3189,GSE4587 co-expressed DEGs with 256 genes.Compared with normal skin tissue,there are 109 co-overexpressed genes,which are commonly expressed with low There are 142 genes,and LIPA is an overexpressed gene;2.The expression of LIPA protein in cutaneous malignant melanoma tissue is significantly higher than that of pigmented nevus tissue(P=0.001),the difference is statistically significant;3.25 cases of skin malignant melanoma tissue.The expression of LIPA protein in melanoma tissues is related to the age,clinical stage,and location of the melanoma patients.Patients younger than 60 years old had higher expression of LIPA protein than patients over 60 years old.Clinically diagnosed patients with stage Ⅲ,stage Ⅳhad higher expression of LIPA protein than patients with stage Ⅰ and stage Ⅱ.Patients with diseased parts located on the extremities had higher expression of LIPA protein than patients with disease on the trunk High(P=0.023,0.006,0.010),the difference is statistically significant.There was no significant correlation between the expression of LIPA protein in melanoma tissue and the depth of melanoma tumor infiltration,tumor size,clinical phenotype(ulcer type,non-ulcer type),the presence of recurrence and metastasis(P=0.470,1.000,0.250,0.428,0.740,0.350),the difference is not statistically significant;4.The expression of LIPA protein and mRNA in melanoma cell A375 is higher than that of pigmented nevus cell HEM(P=0.000,0.000);the expression of LIPA protein and mRNA in melanoma cell MV3 is higher than that of pigment Mole cell HEM(P=0.000,0.001),the difference is statistically significant;5.After transfection of melanoma cells A3 75 and MV3 with LIPA-ncRNA and LIPA-shRNA plasmids,the proliferation ability of melanoma A3 75 cells and MV3 cells decreased compared with the control group(P=0.000,0.000),the difference is statistically significant;6.Compared with pigmented nevus cells,the activity and migration ability of melanoma cells are higher than those of control group(P=0.000,0.000),the difference is statistically significant.Conclusion1.LIPA,BBOX1,CDK1,CPEB3 and other DEGs play an important role in the pathogenesis of melanoma.Among them,LIPA,CDK1,etc.are overexpressed genes;2.The increased expression of LIPA in cutaneous melanoma may be related to the age,location and clinical stage of malignant melanoma;3.Inhibiting the expression of LIPA in melanoma cells can reduce the activity,proliferation and migration ability of melanoma cells in vitro.