IL-34 Promotes The Proliferation And Epithelial-mesenchymal Transition Of Gastric Cancer Cells

Background and Objectives Gastric cancer is one of the most aggressive cancers,with approximately 1 million new cases diagnosed worldwide each year.According to WHO statistics in 2020,about480,000 cases were diagnosed newly in China.Due to the atypical clinical symptoms and lack of extensive initial screening for early gastric cancer,most gastric cancer patients are already at the advanced stage.Therefore,despite aggressive and effective comprehensive treatment programs（surgery,radiotherapy and immunotherapy,etc.）,the overall clinical prognosis of gastric cancer patients is not optimistic,which face a series of physical or psychological problems including postoperative recurrence,cancer pain,cachexia,anxiety and depression,etc.Epithelial-mesenchymal transition（EMT）refers to the biological process that epithelial cells are transformed into mesenchymal cells with mesenchymal properties through a complex series of procedures in response to relevant factors.The protein molecules associated with EMT transformation include E-calmodulin,N-calmodulin and the waveform protein Vimentin.Through EMT,epithelial cells lose their polarity and acquire a higher migration and invasive capacity,leading to carcinogenesis.It has been shown that EMT plays an important role in the invasion and metastasis of gastric cancer cells,and gastric cancer cells acquire a stronger metastatic ability through EMT.Clarifying the regulatory mechanism of EMT in gastric cancer development is of great practical significance in attempting to understand the occurrence,development and metastasis of gastric cancer.IL-34 is a newly discovered cytokine in 2008.As a second ligand protein of CSF,IL-34 binds to its functional receptor and plays a role in regulating cellular functions（differentiation,proliferation,adhesion）,metabolism,angiogenesis,inflammation and immune response.IL-34 binding to the receptor triggers multiple intracellular pathways that affect tumorigenesis and development by mediating cellular processes such as cell proliferation and cell cycle.IL-34 can be secreted extracellularly and bind to CSF-1receptors in concert with macrophage colony-stimulating factors such as M-CSF or CSF-1 to regulate the biological properties of immune-related cells.Besides,IL-34 has been shown to be closely associated with the development of neurological diseases,autoimmune diseases and infectious diseases,etc.In recent years,IL-34 plays a pro-cancer role in a variety of tumors,including thyroid,colorectal,and liver cancers.However,it remains unclear whether IL-34 affects the development of gastric cancer.After infected with Helicobacter pylori,the expression of IL-34 is increased in early gastric cance.Therefore,this study aimed to elucidate the relationship between IL-34 and the clinicopathological characteristics in gastric cancer patients and whether IL-34 plays a regulatory role in the proliferation of gastric cancer cells and the EMT process.Methods.1.The expression levels of IL-34 mRNA and protein were detected in three gastric cancer cell lines（AGS,HGC-27 and MKN-45）and gastric normal epithelial cell line（GES-1）using qRT-PCR and Western blot methods,respectively.The expression levels of IL-34 protein in 60 paired gastric cancer tissues and normal paracancerous tissues were detected using immunohistochemical staining,and the correlation between IL-34 expression levels and each clinicopathological feature was analyzed using SPSS software（version 22.0）.2.AGS cell lines with stable knockdown and overexpression of IL-34 were established successfully after lentiviral infection and treatment with puromycin,which were validated by qRT-PCR and Western blot methods.In vitro,CCK-8,clone formation assay,cell scratch assay and transwell system were used to detect the proliferation,clone formation,migration,and invasion ability of the above cell lines,respectively.Further,the effect of IL-34 on the expression levels of EMT-related proteins in AGS cells was detected by Western blot method.In vivo,the effect of IL-34 expression on the growth of gastric cancer transplant tumors was assessed using a subcutaneous transplant tumor assay in nude mice.Results.1.Compared with GES-1 cells,the expression levels of IL-34 protein and mRNA were increased in three gastric cancer cell lines.Besides,compared with paired paraneoplastic normal tissues,the expression levels of IL-34 protein were increased in60 gastric cancer tissues.Moreover,the expression of IL-34 was associated with tumor size,T-stage,N-stage,TNM-stage and differentiation degree.2.Western blot and qRT-PCR results demonstrated the successful establishment of AGS cell lines with stable knockdown or overexpression of IL-34.Knockdown of IL-34 expression inhibited cell proliferation and clone formation,while overexpression of IL-34 promoted proliferation and clone formation of AGS cells.In vivo,overexpression of IL-34 promoted the growth of subcutaneous transplanted tumors in nude mice.Moreover,knockdown of IL-34 expression inhibited cell migration and invasion ability,while overexpression of IL-34 promoted migration and invasion ability of AGS cells.And knockdown of IL-34 expression promoted expression of E-cadherin,but inhibited the expression of Vimentin and N-cadherin in AGS cells.On the contrary,overexpression of IL-34 inhibited expression of E-cadherin but promoted the expression of Vimentin and N-cadherin in AGS cells.Conclusion.The expression of IL-34 is increased in gastric cancer tissues and cell lines compared to normal gastric tissues and cell lines,respectively.Moreover,IL-34 promotes the proliferation,clone formation,migration and invasion ability of gastric cancer cells by regulating the expression of EMT-related protein.IL-34,as a potential molecular marker,could be a new option for diagnosis and targeted therapy in gastric cancer.