Expression Of IL-6ST In Colorectal Cancer And Its Effects On Proliferation,invasion And Migration Of Colorectal Cancer Cells

Objective: Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The onset of CRC is often hidden and prone to early metastasis,and its prognosis is closely related to the disease stage.Therefore,how to diagnose early and improve the survival rate of colorectal cancer patients is the focus of current research.Signal transduction and activator of transcription(STAT)protein,which exists in the cytoplasmic protein,is activated by tyrosine phosphorylation to form a dimer,and binds to the target gene promoter region after entering the nucleus to regulate gene expression.STAT is both a cell signal transduction protein and a nuclear transcription factor,which can regulate the transcription of various genes,including genes related to tumor proliferation and migration.Janus kinase(Janus kinase,JAK)-STAT signaling pathway can participate in immune function,and cell proliferation and differentiation processes.The continuously activated JAK-STAT signaling pathway is related to the occurrence and development of tumors and regulates the malignant transformation of tumors.The current study found that JAK-STAT signaling pathway plays a role in the occurrence and development of hematological malignancies and solid tumors such as breast cancer,prostate cancer,and lung cancer.In CRC,the regulation mechanism of JAK-STAT signaling pathway is not yet clear,and further investigation is needed.JAK-STAT signaling pathway can regulate the transcription of more than 50 cytokines.Studies have found that cytokines can affect tumor cell immune microenvironment,leading to tumor proliferation and migration.At present,researchers mainly discuss the interaction and relationship between JAK-STAT signaling pathway and cytokines.JAK-STAT signaling pathway may be used as a therapeutic target for tumors.Improve patient quality of life.,Studies have found that tumor necrosis factor α(TNFα),interleukin-6(interleukins-6,IL-6)and interferon γ(interferons,IFNFγ)γ can upregulate STAT expression,IFNs and IL-6 Acting on cells,JAK1 and JAK2 can bind to receptor subunit γ chain(γc)and IL6 ST,mediate cell signal transduction,and promote STAT expression.Studies have shown that IL6 ST is closely related to gastric cancer,hepatocellular carcinoma,osteosarcoma,and breast cancer,and may be used as a biomarker for some solid tumors.CXCL9 plays an important role in the development of gastric cancer,breast cancer,hepatocellular carcinoma,and cervical adenocarcinoma.The method of RT2 Profiler PCR Array was used to evaluate the expression of JAK-STAT signaling pathway protein and pathway-related cytokine genes in colorectal cancer,and to screen differentially expressed genes in order to find new biomarkers for CRC and explore further.The regulatory mechanism of JAK-STAT signaling pathway in the development of colorectal cancer.Methods: We collected tumor and normal fresh tissue samples from patients with CRC,and analyzed the 84 target genes(GOI)in JAK-STAT signaling pathway by RT2 Profiler PCR Array experiment,and obtained 14 differential genes.The GO,KEGG,and TCGA databases were used for bioinformatics analysis of the selected differential genes.Then use the CRC patient tissues to detect the five differential genes CXCL9,IL6 ST,A2M,GHR and FAS by real-time fluorescence quantitative RT-PCR.Five different genes were found to be consistent with the RT2 Profiler PCR Array results.Based on the current literature and previous experimental results,we intend to further explore the mechanism of action of IL6 ST and CXCL9 in colorectal cancer.We used Western blot technology to detect IL6 ST and CXCL9 protein expression;collected CRC patient tissue sections for H & E staining and immunohistochemical detection to evaluate the pathological and clinical characteristics of patients.According to the follow-up information of patients,the single factor survival analysis curves of IL6 ST and CXCL9 were drawn.Finally,we used the CRC cell line to detect the expression levels of STAT3 and p-STAT3 using Western blot after transfection of the overexpressing IL6 ST plasmid,and to detect changes in cell cycle,cell proliferation,cell migration,invasion,adhesion rate and apoptosis.Results: 1.RT2 Profiler PCR Array was used to detect the changes of JAK-STAT signaling pathway proteins in 3 pairs of CRC patients.The results showed that 14 differential genes were screened,of which only CXCL9 expression was up-regulated;GHR,FCER2,NR3C1,IL6 ST,A2M,FAS,PIAS2,SMAD2,PTPRC,STAT4,INSR,SMAD4 and B2 M expression were all down-regulated.2.In this study,bioinformatics analysis of the 14 differential genes screened through GO,KEGG,and TCGA databases showed that CXCL9,IL6 ST,A2M,GHR,FAS were consistent with our screening results.3.Real-time fluorescence quantitative RT-PCR detection showed that the results of CXCL9,IL6 ST,A2M,GHR,FAS,RT2 Profiler PCR Array and database were consistent.Through Western blot detection,we found that the expression of IL6 ST protein in CRC tissue was significantly down-regulated,while the expression of CXCL9 protein was significantly up-regulated,which is consistent with the mRNA expression results we previously tested.4.H & E staining and immunohistochemical analysis were performed on 30 cases of CRC and 30 normal tissue samples of high,medium and low differentiation.The results showed that 1)In CRC tissues,the expression of IL6 ST was lower than that in normal tissues,and as the degree of differentiation decreased,the high expression of IL6 ST decreased.2)In CRC tissue,CXCL9 expression is higher than normal tissue.And as the degree of differentiation decreases,the high expression of CXCL9 increases.3)The expression of IL6 ST and CXCL9 showed a negative correlation.5)IL6ST and CXCL9 are related to the survival of patients.5.Western blot analysis showed that there was no significant difference in the expression of STAT3 in HT29 cells overexpressing IL6 ST,while the expression of p-STAT3 was significantly increased compared with the control group.6.The cytology results of this study showed that 1)Overexpression of IL6 ST significantly increased the proliferation of HT29 cells,but there was no significant difference in cell cycle.2)The apoptosis of HT29 cells overexpressing IL6 ST is significantly reduced.3)Transwell cell migration experiment results show that overexpression of IL6 ST can significantly improve the migration ability of HT29 cells.4)Adhesion experiment results show that the adhesion rate of HT29 cells overexpressing IL6 ST is significantly reduced.Conclusion:1.In CRC tissue,the expression of CXCL9 is up-regulated,and the expressions of GHR,IL6 ST,A2M and FAS are down-regulated.2.IL6 ST and CXCL9 have a negative correlation,and are closely related to the clinical characteristics of colorectal cancer;patients with high expression of IL6 ST had poor prognosis,while those with high expression of CXCL9 had better prognosis.3.In HT29 cells,overexpression of IL6 ST up-regulates p-STAT3 expression.4.Overexpression of IL6 ST enhances HT29 cell proliferation,increases migration ability,reduces adhesion rate,and reduces apoptosis.