Study On Expression Of Hypoxia Inducible Factor-1? And Relationship With Angiogenesis-Related Protein In Skin Neoplasm

Part ?Expression of Hypoxia Inducible Factor-la and Relationship with Angiogenesis-Related Protein in Skin neoplasmObjectives:Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) are the most common malignant tumors of human skin. Their growth is divided into no vascular and vascular phase, which refers to the transformation from the vessels of the slow growth stage to rapid growth stage, the same as other malignant tumors. When the blood supply is insufficient to meet the needs of tumor growth, this will lead to hypoxia. Secondary hypoxia can increase the expression of a series of angiogenic factors to induce tumor angiogenesis. The tumor can get adequate nutrition from the formation of new blood vessels. Therefore, the growth of tumor can not be separated from the establishment of its related blood vessels. So the tumor can grow rapidly and can be transferred to the surrounding tissues. With the deepening of research, it was found that the vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-la) play very important roles in tumor angiogenesis. So this study is to investigate the expression of Hypoxia Inducible Factor-1? (HIF-1 a)and vascular endothelial growth factor (VEGF) protein in lesions of basal cell carcinoma(BCC) and cutaneous squamous cell carcinoma(cSCC) and its correlations with angiogenesis.Methods:One hundred and two paraffin-enbeded lesions collected for diagnostic histopathology from the files of our pathology department and skin pathology laboratory in January 2010-December 2014, including 56 cases of BCC,46 cases of cSCC and 40 specimens of normal skin. In BCC and cSCC cases, there were 61 males and 41 females, whose median age was 58 (26-90) years old. In the normal control group, there were 22 males and 18 females, whose median age 44 (20-68) years old. In the case group and control group, there were no associated with major organ disease history, no radiotherapy and chemotherapy treatments histories. All specimens were identified by two pathological experts. There was no significant difference in age and sex between the three groups. Samples were drawn from the regions of the head, face, trunk, limbs and so on. The specimens were examined for HIF-1? and VEGF by the immunihistochemical MaxVisionTM method. All the statistical analyses in this study were performed by SPSS 17.0 software packages. Comparison of two sample mean of measurement data was used t test, The comparison of enumeration data was analyzed bychi-square test and Scheffe test. Spearman was used for the correlation coefficient, which is not consistent with the normal distribution data of the two variables. P value< 0.05 was defined as statistically significant.Results:1 VEGF positive staining cells were mainly located in cytoplasm, the visible part of the vascular endothelial cells, trichilemmal weak expression of new blood vessels near the high expression of tumor cells. The positive espression rate of VEGF in BCC, cSCC and normal skin tissue was 33.93%(19/56),67.39% (31/46) and 12.50%(5/40). There were significant differences of expressions of VEGF among them(P< 0.05).2 HIF-1? positive staining were mainly located in the tumor cell cytoplasm, accidentally in staining nucleus and the surrounding of tumor necrosis area. Part of the vascular endothelial cells positive staining was also seen. Normal skin was negatively expressed. The positive espression rate of HIF-1? in BCC, cSCC and normal skin tissue was 48.32%(27/56), 82.60% (38/4)and 0. There were significant differences of expressions of VEGF among them(P<0.05). And the expression of HIF-l a had a positive relationship with the expression of VEGF in cSCC and BCC(rs=0.536, P< 0.05).Conclusions:Overexpression of HIF-1? and VEGF may play key roles in the development and the invasion of skin carcinoma.Part IIEffect of RNA Interference for Hypoxia Inducible Factor-1? on Expression of Angiogenesis-Related Protein in Skin neoplasmObjectives:Due to hypoxia inducible expression of a variety of genes are controlled by HIF-1, so it is possible to provide a more accurate means of detection of the tumor and other pathological tissues as a result of the inherent signs of hypoxia. Many studies have indicated that HIF-1 may regulate angiogenesis, placental growth factor, platelet derived growth factor and vascular endothelial growth factor (VEGF). Preliminary research found that, in BCC and cSCC tissues, the expressions of VEGF and HIF-1 alpha are increased. The research also found that, HIF-1 alpha can activate the expression of VEGF transcription on the promoter binding sites, making tumor cells adapt to hypoxia environment. The expression of VEGF and HIF-1 alpha in BCC and cSCC was positively correlated, and we speculated that they together to promote tumor angiogenesis and cell proliferation. At present, the treatment of BCC and cSCC is still mainly based on surgery, which can not be operated or have a distant metastasis, treated with radiotherapy and chemotherapy. Radiotherapy and chemotherapy can effectively eliminate tumor cells, but it is difficult to achieve accurate target therapy. In the treatment process, there will be the deaths of a lot of normal cells, which damages the human body. Therefore, it is an important development direction to achieve the targeted therapy of tumor. Previous studies suggested that hypoxia may be involved in the occurrence and development of BCC and cSCC. In view of the important role of HIF-1 in the occurrence and development of tumor, the biological treatment of tumor with HIF-1 as the target is more and more concerned by people. Inhibiting the expression of HIF-1 alpha and blocking the transmission of hypoxia signal have become a new research hotspot. Therefore, RNA interference(RNAi) method targeting the HIF-1 alpha can inhibit the expression of HIF-1 alpha or make its domain inactivation, thereby blocking the biological role of HIF-1. This may be an effective way to control the malignant tumor and improve the survival of the patients. So this study is to investigate the influence of RNA Interference(RNAi) method targeting HIF-1? and VEGF mRNA and proteins in skin carcinoma.Methods:According to the design principle of siRNA, construct gene siRNA and negative control NC (Neegative Control) by the application of Guangzhou ribobio Co. Ltd. designed software. Sequences are designed and synthesized by Guangzhou ribobio Co. Ltd. Small interfering RNA(siRNA) targeting HIF-1? was designed and synthesized, which was then transfected into cutaneous squamous cell carcinoma A431 cells by using riboFectTM CP reagent, at the same time, the control group was set up. Total celluar RNA and whole cell protein was extracted from the treated cutaneous squamous cell carcinoma A431 cells 72hrs after which were cultured under Hypoxia conditions. Rt-qPCR and Western Blot were used to detect the expression levels of HIF-1? and VEGF mRNA and proteins. RT-qPCR experimental results were analyzed by the strip light density value with using gel analysis software. Western Blot experimental results were analyzed by the reference protein and beta-actin band gray value with using gel image processing system. All the statistical analyses in this study were performed by SPSS 17.0 software packages. Comparison of two sample mean of measurement data was used t test, The comparison of enumeration data was analyzed bychi-square test and Scheffe test. Spearman was used for the correlation coefficient, which is not consistent with the normal distribution data of the two variables. P value< 0.05 was defined as statistically significant.Results:1)RT-qPCR and Western Blot results showed that the transfection of HIF-1 alpha-siRNA skin tumor cells cultured under hypoxia, compared with empty transfection control, the expression of mRNA and protein HIF-1 alpha of siRNA-1, siRNA-2 and siRNA-3 were inhibited (P<0.05).2) RT-qPCR and Western Blot results showed that compared with the transfection with empty control group and transfected with siRNA negative control group, the expression levels of VEGF mRNA and protein in human skin squamous cell carcinoma cells had significant difference in the transfected HIF-1 alpha-siRNA group (P<0.05).Conclusion:The expression of RNA silencing HIF-1 gene, upregulation of mechanism can make the cutaneous squamous cell carcinoma A431 cells under hypoxia and angiogenesis related gene expression of VEGF was inhibited, VEGF expression of both mRNA and protein levels were significantly reduced. It has also been proved that HIF-1 a is involved in the formation of VEGF in the cutaneous squamous cell carcinoma A431 cells induced by the activation of transcription, which stimulates the formation of tumor angiogenesis. Downregulation of HIF-1? expression can inhibit expression of VEGF mRNA and proteins in cutaneous squamous cell carcinoma A431 cells under hypoxia. We speculated expression of VEGF was regulated by HIF-1 a in cutaneous squamous cell carcinoma A431 cells under hypoxia.