Study On The Role And Mechanism Of S100A12 And HAX-1 In Aggressiveness Of Glioma

Background: Gliomas are the most common tumor in central nervous systems in adults,and that were divided to high grade glioma(Grades III and IV)and low grade glioma(Grades I and II)by the World Health Organization(WHO).The glioblastoma is belonged to the high grade glioma.The current standard of care for patient of glioblastoma followed by operation,radiotherapy,chemotherapy,and the prognosis was poor.In the 2016 world health organization of tumors of the central nervous system,the authors used molecular parameters in addition to histology to define the glioma for the first time.The previous abundant dedicated research into the molecular biology of gliomas has caused a rapid acceleration of the discovery of some of the key molecular mechanisms as well as the genetic and epigenetic underpinnings of these tumors.Despite many advances in medical treatments such as surgery,radiotherapy and chemotherapy,the prognosis of glioma is still poor especially in glioblastoma with a median survival of14.6?months.Thus,identification of novel mechanisms to suggest new possibilities for treatment of glioma is urgently needed.The S100 protein family belongs to inflammatory molecules subgroup which predominantly comprises calcium-binding proteins.S100 protein has been reported to be participated in a series of pathological progresses,such as chronic inflammation,autoimmune diseases and malignancies.S100A12 is a member of this family and is widely expressed in neutrophil and low expressed in lymphocytes and monocyte.S100A12 has also been found to be involved in multiple cancers.S100A12 is close linked to inflammation and vascular invasion and contribute to cancer metastasis.It has been reported that high levels of S100A12 has been correlated with good prognosis for patients with oropharyngeal squamous cell carcinoma.In addition,S100A12 was significantly increased in colorectal cancer samples when compared adjacent normal colon tissues.It has been reported that the expression of S100A4 is highly correlated with the progression of glioma which indicated that S100A4 plays a vital role in the pathogenesis of glioma.However,the role of S100A12 in glioma hasn't been fully illuminated.Therefore,the aim of this study was to explore the role of S100A12 in glioma.We assumed that S100A12 was upregulated in glioma tissues,and that knockdown of S100A12 resulted in a repression of apoptosis and a elevation of proliferation of glioma cells.The HAX-1 protein has a variety of biological functions,such as anti-apoptosis,regulation of cell migration and endocytosis,and is involved in invasion,metastasis and tumorigenesis in different types of tumors.However,there are few studies on the regulatory mechanisms of S100A12 and HAX-1 proteins in glioma cells,and their mechanisms of action have not been fully elucidated.Objective:1.S100 calbindin A12(S100A12)is a member of the S100 protein family,widely expressed in neutrophils,and lowly expressed in lymphocytes and monocytes.However,the role of S100A12 in glioma has not been clarified.We performed an immunohistochemical study of S100A12 in 81 glioma tissues to determine the expression of S100A12 in glioma cells and to evaluate the clinical significance of S100A12 in glioma patients.2.Pretend a novel prospect that HAX-1 plays a vital role in glioma progression.HAX-1 is up-regulated in glioma samples and might contributed to the prognosis of the glioma patients.Moreover,silencing of HAX-1 inhibits the proliferation and the EMT process.It also promotes the apoptosis of the glioma cell lines.Method: In the present study,we carried out immunohistochemical investigation of S100A12 and HAX-1 in 81 glioma tissues to determine the expression of S100A12 and HAX-1 in glioma cells,and evaluate the clinical significance of S100A12 and HAX-1in glioma patients.Futher we knockdown the S100A12 by sh RNA,and evaluated cell proliferation,cell migration and cell apoptosis by MTT,colony formation assay,transwell assay,flow cytometry assa and western blot.1.Glioma cells used in this study were cultured by cell culture;2.The transcription levels of S100A12 and HAX-1 genes in glioma cells were detected by real-time polymerase chain reaction;3.The expression of S100A12 and HAX-1 protein was investigated by using immunohistochemical staining;4.By detecting the expression of S100A12 and HAX-1 protein in glioma cells and bystander tissues using western blot western blot technique,the changes of S100A12 and HAX-1 protein expression after the construction of S100A12 and HAX-1 gene mutation strains have been detected;5.To explore the changes in cell growth ability before and after S100A12 gene knockout by using colony formation assay.6.Changes in cell growth cycle and apoptosis were measured using the Cell Loss Detection technique.Results:S100A12:It was found that the expression of S100A12 in tumor cells of glioma patients was directly correlated with the survival rate of patients,the survival rate of patients with low S100A12 expression was more than 50%,while the survival rate of patients with high S100A12 expression was less than 10%,the tumor size and KPS value in tumor cells of81 patients were correlated with the S100A12 gene expression of patients,and the number of patients with grade III ~ IV was significantly increased in patients with high S100A12 expression.In addition,by detecting the expression of S100A12 in glioma cells,MTT results showed that S100A12 could significantly enhance the growth of glioma cells,western blot analysis of protein expression levels we found that S100A12 could regulate the process of apoptosis,and the number of apoptotic cells was significantly lower in the knockout strain of S100A12 than in the non-knockout strain.The results of transwell assay showed that S100A12 significantly enhanced both invasion and migration of glioma cells,which was approximately 3-fold enhanced compared with the mutant strain of S100A12.HAX-1:It was found that the expression of HAX-1 in glioma cells was significantly higher than that in HA cells,and HAX-1 had the highest expression in U87 and U251 glioma cells among the five glioma cells studied in this paper.WB and q PCR analysis revealed that HAX-1 was overexpressed in glioma cells,and the expression of HAX-1 in cells was not associated with other factors such as age and gender.The results of transwell assay showed that HAX-1 gene increased the invasion and migration of glioma cells,in addition to affecting the colony formation ability of glioma cells,and the cell invasion ability of HAX-1 overexpressing cells was about 3 times higher than that of HAX-1silenced cells.Conclusion: S100A12 plays a vital role in glioma progression,and may be an important regulatory molecule for biological behaviors of glioma cell lines.HAX-1 protein also promotes the growth and proliferation of glioma cells and are independent prognostic factors for glioma.