| Background:Esophageal cancer is one of the most common malignant tumors of digestive tract system,with the characteristics of high incidence,strong invasive ability and poor prognosis.Esophageal cancer is mainly divided into two subtypes: esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC).Although the incidence of adenocarcinoma in developed regions gradually increases,ESCC is still the most common histological subtype of esophageal cancer in the world.In China,the incidence of esophageal cancer ranks sixth among all malignant tumors,and forth for mortality,and it seems both rankings are in increasing.It generally does not result in obvious symptoms in early stage of esophageal cancer,which leads to delayed diagnosis of patients.It results in unresectability and more than half of patients will have distant metastases.At present,a great deal of progress has been made in early screening,early diagnosis and comprehensive therapy of esophageal cancer.However,the prognosis of patients with advanced esophageal cancer is still unfavorable.In addition,chemotherapy is an important treatment of esophageal cancer,but that the multi-drug resistance occurrs in the course of treatment impacts the outcome seriously.In view of this,it is imperative to search for novel reliable biomarkers and therapeutic targets for early diagnosis and effective treatment of esophageal cancer.With three sushi repeat domains(CCP domains)and one HYR domain,SRPX2 is a novel chondroitin sulfate proteoglycan that was first found in leukemia cells.At present,it has been found that SRPX2 is overexpressed in various kinds of malignant tumor tissues,which is correlated with the poor prognosis of patients.SRPX2 involves in the biological processes such as cell proliferation,adhesion,migration and invasion of tumor cells,and promotes angiogenesis.These results indicate that SRPX2 plays an important role in tumor progression.However,it is still unknown whether SRPX2 has the same functions in esophageal cancer.In addition,although it has been reported that SRPX2 may be related to the chemoresistance of tumor cells,the research is still very limited.The role of SRPX2 in tumor drug resistance remains to be further explored.In this study,CRISPR / Cas9 technology was used to silence the SRPX2 gene in esophageal cancer cell line KYSE450.We constructed a stable SRPX2 knock-out esophageal cancer cell line to explore the biological features and functions of SRPX2 in esophageal cancer cells in vitro.The expression of SRPX2 protein in esophageal cancer tissues was verified by immunohistochemical staining,and the relationship between SRPX2 expression and clinicopathological features and prognosis of patients was analyzed.Objective:The main purpose of this study was to explore the effects of SRPX2 gene silencing on biological characteristics and cell function of esophageal cancer cell line KYSE450,and examine the expression of SRPX2 in esophageal cancer samples and its relationship with the clinical outcome foe the esophageal cancer patients.Materials and methods:1 The expression of SRPX2 in four different ESCC cell lines was examined by Western blot before cell line selection for further gene silencing experiment.SRPX2 gene knockout esophageal cancer cell line was established by using CRISPR/Cas9 gene editing technology and the results were verified by DNA sequencing,RT-PCR and Western blot.2 Analyses were performed by using flow cytometer to clarify the cell cycle and apoptosis in control cells and SRPX2 knockout cells.The Seahorse XFe96 Analyzer was used to examine the mitochondrial function.To study cell biological behavior influence of the gene silencing,a series of experiments such as cell proliferation assay,Transwell cell migration assay,cell scratch assay and cell sphere experiment were performed.3 The sensitivities of cells to chemotherapeutic drugs including cisplatin,paclitaxel and docetaxel were analyzed by IncuCyte ZOOM and colony formation assay.Radiosensitivity influence of the cells was analysed with a irradiation experiment.4 Whole transcriptome sequencing was conduceted to investigate and analyze the changes of gene expression and related signaling pathways in SRPX2 knockout cells.5 A total of 138 paraffin specimens of esophageal cancers from Anyang Tumor Hospital were detected by immunohistochemistry to investigate SRPX2 expression in esophageal cancer tissues and analyze the relationship between SRPX2 expression and clinicopathological characteristics and prognosis of esophageal cancer patients.Results:1 After SRPX2 gene knowckout,the KYSE450 cells showed S phase arrest and increased apoptosis cell proportion.The proliferation ability of cells was impaired.In addition,mitochondrial damage,such as significantly reduced reserve respiratory,was observed in the cells.2 SRPX2 gene knockout significantly reduced the sphere formation and migration capacity in vitro in the KYSE450 cells.Correspondingly,it enhanced the chemosensitivity and radiosensitivity of KYSE450 cells,which were embodied in the inhibited growth curves and depressed cloning efficiency of SRPX2-KO cells treated with chemotherapy or radiation.3 The results of whole transcriptome sequencing showed a total of 2562 up-regulated genes and 2640 down-regulated genes.These differentially expressed genes are involved in multiple biological processes and signaling pathways.4 The immunohistochemical staining results showed that SRPX2 protein was highly expressed in esophageal cancer tissues.SRPX2 expression was positively correlated with clinical stage,tumor differentiation,lymph node metastasis and distant metastasis of esophageal cancer patients.Compared with SRPX2 weakly positive or negative patients,the overall survival of patients with SRPX2 strongly positive was shorter.Moreover,SRPX2 overexpression was an independent risk factor for survival in esophageal cancer patients.Conclusions:1 SRPX2 is involved in the biological functions and characteristics of KYSE450 cells such as cell cycle,apoptosis,proliferation and migration,and it plays an crucial role in chemoresistance and radioresistance in the cell line.2 SRPX2 expression in esophageal cancer is up-regulated.SRPX2 overexpression predicts unfavorable outcomes in esophageal cancer patients and can be regarded as an independent prognostic biomarker. |
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