The Expression Of Ees-3 Gene In Cutaneous Squamous Cell Carcinoma And Its Effect On The Proliferation Of Human Skin Keratinocytes

BackgroundSkin squamous cell carcinoma,also known as skin squamous carcinoma,is one of the common skin malignant tumor mainly occurring on light exposure area in the elderly,such as hair on the scalp,face,hand.Due to various reasons such as environmental impact in recent years,there is an upward trend in the incidence of this disease.Moreover,the diversity of clinical manifestation may lead to misdiagnosis and misses the opportunity of treatment.Ese-3 is a member of the ETS family of transcription factors,which expresses in prostate,pancreatic,salivary glands,tracheal epithelial cells specifically.Ese-3 can enhance or inhibit the purpose gene expression,affecting the cell cycle,proliferation,differentiation,apoptosis,invasion,migration and angiogenesis and other physiological processes in combination with specific sequences of ETS binding sites of purpose gene promoter regions.Giovanna Chiorino etc study found that in prostate cancer,Ese-3 molecules by inhibiting the expression of genes such as POU5F1,TWIST1,ZEB2 and BMI1 in the process of mesenchymal-epithelial transformation and tumor stem cells form to suppress tumor development.And the prostate cancer patients who loss of Ese-3 expression more prone to relapse after treatment.But researchers found that in ovarian cancer patients,the excessive expression of Ese-3 promotes cancer development and the higher expression level associated with poor prognosis.Knocking-down the expression of Ese-3 molecules in ovarian cancer cell can reduce the proliferation and invasion ability significantly.Therefore,the role of Ese – 3 molecule in tumor differs in different organs and organizations.In the patient of prostate cancer,lung cancer,esophageal cancer,Ese-3 gene plays a role of tumor suppressor by regulating kinds of downstream molecular.While the excessive expression of Ese-3 gene in ovarian cancer patients and breast cancer patient promote the development of these disease.But there is no report on the Ese-3 gene expression and its effect on downstream gene in development of skin and skin tumor.PurposeBy immunohistochemistry and Western blot methods,detect Ese-3 expression in squamous cell carcinoma of the skin tissue and SSCC cell A431 l.Establish Ese-3 knock-down cell model in immortalized human keratinocyte HaCaT cell.Detect the influence of Ese-3 genes on the biological characteristics of HaCaT cell such as clone formation,proliferation,apoptosis,etc.Explore the role of Ese-3 in skin squamous cell carcinoma preliminary.MethodsUsing immunohistochemical method to detect the expression of Ese-3 in skin squamous cell carcinoma tissue and tissue adjacent to carcinoma.Western blot detect expression of Ese-3 molecule in the squamous cancer cells A431 and immortalized human keratinocyte HaCaT cells.Establish Ese-3 gene knockdown HaCaT cells lineages by infecting Ese-3 sh RNA lentivirus.Using MTT colorimetric method,clone formation and flow cytometry detect the change of biological characteristics of HaCaT cell such as cell cycle,proliferation,apoptosis,when the expression of Ese-3 molecule reduced.Results(1)Immunohistochemical staining showed that in 21 cases of normal skin tissue,Ese-3 present positive or strong positive expression,and tan dyeing is located in cell nucleus.While in 51 cases of skin squamous cell carcinoma tissue,Ese-3 weakly express or even missing.Evaluate the staining of SSCC tissue and normal skin tissue according to the degree of staining and the proportion of coloring cell number,statistically tested by the chi-square test,and the difference was significant,p < 0.05(2)Western blot results revealed that the expression of Ees-3 was reduced in the skin squamous carcinoma cells A431 cells,while the expression was normal in the human keratinocytes HaCaT cells.(3)Real-time PCR and Western blot test were used to detect the changes in mRNA levels and protein levels of the HaCaT cells after infected with Ees-3 shRNA lentivirus.The results showed that NO.1 of the 4 target loci was able to reduce the efficiency of the mRNA and protein level of Ees-3 in HaCaT cells.(4)MTT colorimetry was used to determine the proliferation of HaCaT cells and control cells after the expression decreased of Ees-3 gene,and the results showed that HaCaT Ese-3 Si cells were growing faster than HaCaT CON cells.(5)Clone formation assay showed that the ability of the HaCaT cell clone formation increased after the expression of the es-3 gene was decreased compared to the control group HaCaT CON cells.(6)Flow cytometry was used to detect the cell cycle finding that HaCaT cell growth is promoting after reduction of Ese-3 expression.The percentage of HaCaT Ese-3 Si cells in G0 / G1 phase was 51.80%,in S phase was 31.12%,while the control group HaCaT CON cells in G0 / G1 phase was 62.11%,21.77% S phase,the difference was significant.The cell apoptosis was detected by flow cytometry aslo,and there was no significant difference between the HaCaT Ese-3 Si cells and the control group cells.ConclusionThe expression of Ese-3 in squamous cell carcinoma of the skin tissue and CSCC A431 cells decreased.Reduce Ese-3 gene expression formed in immortalized human keratinocyte cell HaCaT cell can promote HaCaT cell proliferation,increase the clone forming ability and increase the proportion of S phase cells,indicating that the loss of Ese – 3 gene expression can promote the development of skin squamous cell carcinoma.