| BackgroundGastric cancer is a malignant tumor that originates from the epithelial cells of the gastric mucosa,and is one of the most common causes of cancer death worldwide.This disease occurs mostly in East Asia,especially in China,South Korea and Japan.Due to the characteristics of insidious onset,easy metastasis and easy recurrence,gastric cancer patients have a very poor prognosis and a high mortality rate.The five-year survival rate of patients with gastric cancer in China is less than 30%.Although the popularity of early screening programs such as gastroscopy and advances in treatment strategies for gastric cancer have improved the survival of patients with early-stage gastric cancer,patients with advanced gastric cancer still have a high rate of metastasis and mortality.It is invasion and metastasis that are important causes of recurrence and poor prognosis in patients with advanced gastric cancer.Therefore,it is of urgent practical significance to study the molecular mechanisms of gastric cancer invasion and metastasis,and to search for key molecules in the malignant progression of gastric cancer for the treatment of gastric cancer.The role of transcription factors in regulating the expression of downstream genes is of great significance in the process of tumorigenesis and progression.Therefore,exploring the specific expression regulation patterns of transcription factors in the malignant progression of gastric cancer can provide new intervention targets and predictive indicators for the prevention and treatment of gastric cancer.STAT5(Signal Transducer and Activator of Transcription 5)is an important member of the Signal Transducer and Activator of Transcription(STAT)family,which can regulate gene expression in response to stimulation by a variety of growth factors,hormones and cytokines.STAT5A and STAT5B are two highly related isoforms of STAT5.In recent years,aberrant STAT5A signaling has been identified in hematological and solid tumors,including acute and chronic myeloid leukemia,breast,lung,prostate and head and neck squamous cell carcinoma.The primary function of STAT5A is believed to be transcriptional activation of downstream genes,such as cyclinD,BCL2,SOCS2 and MMP-2,leading to tumor growth,metastasis and chemotherapy drug resistance.Thus,STAT5A plays an important role in tumor drive.In addition,phosphorylation of STAT5A is the most reported mechanism of STAT5A activation currently.and it has been shown that STAT5 activation,rather than STAT5 protein expression,is associated with the prognosis of breast cancer.This suggests that p-STAT5A may play an important role in the malignant progression of cancer.However,the functional roles and major molecular targets of STAT5A and p-STAT5A in gastric cancer remain unclear.The Cluster of Differentiation antigen 44(CD44)is a non-kinase,single-transmembrane glycoprotein that is widely expressed on cell membranes and is one of the most common surface markers of Cancer Stem Cells(CSC),playing a key role in the communication between CSC and the microenvironment,enhancing the chemoresistance of CSC and regulating the stemness characteristics of CSC.In addition,CD44 belongs to a family of adhesion molecules that are involved in various cell-cell and cell-extracellular matrix(ECM)interactions,and play an important role in tumor development and metastasis.However,the signaling pathways upstream of CD44 activation in gastric cancer are not fully understood.The aim of this study was to investigate the effect of STAT5A on the function of gastric cancer cells in vitro and in vivo,and whether it promotes the malignant process of gastric cancer by acting as a transcription factor and transcriptionally regulating CD44 expression.Thus,targeting the STAT5A/CD44 axis may provide a new therapeutic strategy for the treatment of tumors.Objective1.To explore the effects of transcription factor STAT5A and its activation state p-STAT5A on the malignant biological behavior and the molecular mechanism of gastric cancer.2.To explore the function and mechanism of STAT5A activating the expression of CD44 through transcription and promoting the malignant progression of gastric cancer.Methods1.The expression of STAT5A mRNA in gastric cancer tissues was analyzed by TCGA database and qPCR.The expression of STAT5A and p-STAT5A in clinical samples was detected by immunohistochemical staining and Western Blot method.The correlation between the expression levels of STAT5A or p-STST5A protein and clinicopathological parameters was statistically analyzed based on the scoring criteria of immunohistochemical staining.2.To analyze the correlation between STAT5A mRNA and prognosis of gastric cancer patients using Kaplan-Meier plotter database;to further verify the correlation between STAT5A protein expression and patient prognosis by plotting survival curves based on immunohistochemical staining results of clinical samples.3.Western Blot assay was performed to detect the expression levels of STAT5A in gastric cancer cell lines(AGS,HGC-27,MKN-28,MKN-45,NCI-N87,MGC-803)and immortalized gastric epithelial cell line(GES-1).Gastric cancer cell lines HGC-27,MKN-28,MKN-45 were selected to establish stable STAT5A overexpression cell lines by lentiviral infection;MKN-45 was selected to establish stable STAT5A knockdown cell line by lentiviral infection.To establish good cell models for studying the role of STAT5A in the malignant progression of gastric cancer.4.CCK-8 and EdU assays were used to detect the effects of abnormal expression of STAT5A on the proliferation of gastric cancer cells and the sensitivity of chemotherapeutic drugs.Cell scratch healing assay and Transwell migration assay were used to detect the effects of abnormal expression of STAT5A on the migration ability of gastric cancer cells.TCGA database gene correlation analysis and Western Blot assay were used to detect the effects of STAT5A on the EMT phenotype of gastric cancer cells.Tumor stem cell sphere-formation assay and plate clone formation assay were performed to detect the effect of STAT5A on the stemness characteristics of gastric cancer cells.5.The effect of STAT5A on tumor growth and metastasis in vivo were examined using subcutaneous tumor formation and caudal vein metastasis models in nude mice.6.Using TCGA database gene correlation analysis,qPCR,flow cytometry,Immunohistochemical staining,Western Blot and other techniques,we confirmed that STAT5A was closely associated with CD44 expression in gastric cancer cells and tumor tissues.7.CD44 overexpression cell lines were constructed by transient transfection technique.The effects of upregulation of STAT5A and CD44 alone or simultaneously on proliferation,chemotherapeutic drug sensitivity,migration,EMT and stemness characteristics of gastric cancer cells were examined by CCK-8,EdU,Transwell,Western Blot,tumor stem cell sphere-formation assay and plate clone formation assay.8.The CD44 knockdown gastric cancer cell lines were constructed by transfection with siRNA.CCK-8,Transwell,Western Blot and tumor stem cell sphere-forming assay were used to detect the effects of cell proliferation,migration,EMT and stemness characteristics in control,siCD44 and STAT5A overexpression combined with siCD44 groups.9.ChIP and dual luciferase reporter gene assays were performed to verify the transcriptional regulation of the CD44 gene by STAT5A.ResultsPart Ⅰ:Effect of STAT5A on the malignant biological behavior of gastric cancer1.STAT5A expression was upregulated in gastric cancer tissues compared with adjacent normal tissues,and was significantly associated with poor prognosis in gastric cancer patients.2.In vitro assays showed that STAT5A overexpression upregulated p-STAT5A and significantly enhanced the proliferation,chemoresistance and migration of gastric cancer cells;knockdown of STAT5A downregulated p-STAT5A and significantly decreased the proliferation,chemoresistance and migration of gastric cancer cells.3.Overexpression of STAT5A enhanced the EMT phenotype and stem cell characteristics of gastric cancer cells.4.Subcutaneous tumorigenesis model and caudal vein metastasis model in nude mice demonstrated that STAT5A overexpression could promote the proliferation and metastasis of gastric cancer cells in vivo.Part Ⅱ:Mechanisms of STAT5A involvement in the malignant process of gastric cancer through CD441.In gastric cancer cell lines and tumor tissues,the expression of STAT5A was positively correlated with CD44.2.In vitro experiments showed that the proliferation,chemoresistance,migration,EMT and stemness characteristics of gastric cancer cells enhanced by STAT5A overexpression could be further enhanced by CD44 overexpression.3.The proliferation,migration,EMT and stemness characteristics of the gastric cancer cells were significantly reduced after knocking down CD44 in the cell lines that overexpressed STAT5A4.ChIP and dual luciferase reporter gene assays showed that STAT5A could bind to the promoter sequence of CD44 gene and promote the transcription of CD44.Conclusions1.In vitro and in vivo experiments confirmed that STAT5A and p-STAT5A are highly expressed in gastric cancer cells and tumor tissues,and were closely associated with gastric cancer progression and clinical outcomes.2.STAT5A binds directly to the promoter region of CD44 gene,promotes CD44 transcription and contributes to the malignant process of gastric cancer by upregulating CD44 expression. |
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