| BACKGROUND AND OBJECTIVEColorectal cancer is a common digestive disease,the incidence and mortality rate of all kinds of malignant tumors is the third highest.In recent years,with the improvement of living standards and the trend of western change of eating habits and lifestyles,the incidence of colorectal cancer is on the rise,and has the characteristics of low age,low degree of specialization and high degree of malignancy.The main cause of death in cancer patients is metastasis,and most patients have metastasis during initial treatment or treatment.Studies have confirmed that JAM2 plays a different role in different tumors,that JAM2’s expression disordermays may be involved in the invasion and metastasis of colorectal cancer,and that abnormal expression of JAM2 may be regulated by epigenetics(methylation).However,the specific relationship between JAM2 and colorectal cancer and the molecular mechanisms of interaction have not been reported The purpose of this topic is to detect the expression of JAM2 in colorectal cancer through a series of invi and intro experiments,to analyze the relationship between JAM2 expression and clinical pathological parameters,to study the role and specific molecular mechanism of JAM2 in the occurrence and progression of colorectal cancer,and to explore the specific regulatory mechanism of JAM2 expression disorder.It is hoped that it will provide a certain basis for clinical research to treat colorectal cancer drugs.METHEODS:1.Analysis of JAM2’s expression in colorectal cancer and other malignant tumors using GEO public database.2.The expression of JAM2 and the relationship with clinical pathological parameters in colorectal cancer were detected using experimental studies such as I immunohistochemistry(IHC)3.Established JAM2 overexpression and interference colorectal cancer cell strains,SW480/JAM2、HCT116/JAM2,SW480/Lv201、HCT116/Lv201,RKO-shRNA1、RKO-shRNA2、HCT15-shRNA1、HCT15-shRNA2,through Transwell chamber invasion experiments,scratch healing experiments and 3D culture experiments to detect the proliferation and the invasive migration capacity of cells4.Using GEO public database,the enrichment of genes at low or high expression of JAM2 is analyzed by GSEA enrichment analysis,and the relevant literature is consulted to predict the signaling pathways that JAM2 may participate in5.Using the database GEO to predict the CpG island of JAM2,the methylation of CpG Island in colorectal cancer samples was detected by sequencing experiments of sulphate..6.The statistical results of the experiment were analyzed by SPSS20.0 statistical software.JAM2 was used for the expression of expression in colorectal cancer tissue and in normal mucosa tissue next to cancer using the paired sample Wilcoxon symbol rank test;The relationship between the expression level of JAM2 and the clinical pathological characteristics of colorectal cancer was analyzed by carpander and Spearman grade,and two independent samplets were tested in real-time fluorescence quantitative PCR and transwell chamber invasion experiments between two groups.The Levene variance test was carried out on the two independent sample t-tests,and if the variance was the same,the t-test under the variance-by-the-same conditions was used,and if the variance was not the same,the Satterthwaite approximation t test was used.Multi-group Transwell chamber invasion experiment using one-way ANOVA;The variance analysis of the analysis design was used in MTT experiment,scratch healing experiment and naked mouse sub-tumor experiment.P 0.05 is statistically significant.RESULTS:1.The expression of JAM2 in CRC tissue1)The expression of JAM2 in CRC in the public database In the GEO database,GSE41258 showed that the expression level of Jam2 mRNA in normal colorectal mucosa,tumor tissue and liver metastasis tissue was significantly different(P<0.001),and the expression level in colorectal cancer tissue and liver metastasis tissue was significantly lower than that in normal intestinal mucosa tissue.Oncome database analyzed the expression of JAM2 mRNA in different malignant tumors.The results also showed that the expression level of JAM2 mRNA in colorectal cancer decreased significantly.2)The expression of JAM2 in colorectal cancer and its relationship with clinical prognosisThe results of IHC showed that the expression of JAM2 in colorectal cancer was lower than that in matched normal intestinal mucosa,and the positive expression was located in cytoplasm and cell membrane,which was yellow brown.The results of Kaplan Meier survival analysis showed that the 5-year survival rate of colorectal cancer patients in the high expression group was significantly higher than that in the low expression group(P<0.05)2.The role of JAM2 in the progression of colorectal cancer1)Stable overexpression of JAM2 and establishment of stable interfering cell lineA colorectal cancer cell line with stable overexpression and interference of JAM2 was constructed by lentivirus vector and verified by Western blot.2)Effects of JAM2 overexpression and interference on the proliferation in colorectal cancer cellsThe results from MTT showed that the growth rate of JAM2 over expressed cells decreased significantly(P<0.05),and the growth rate of JAM2 disturbed cells was significantly faster than that of the control group(P<0.05).Compared with the control group,the number of clones formed by JAM2 stable overexpressed cells decreased significantly(P<0.05),and the number of clones formed by JAM2 disturbed cells was significantly higher than that of the control group(P<0.05).The subcutaneous tumorigenesis experiment in nude mice showed that compared with the control group,the tumor growth rate of JAM2 overexpression group was slower,tumor volume was smaller(P<0.05),and the positive rate of proliferation index Ki-67 was significantly lower than that of the control group(P<0.05);the tumor growth rate of JAM2 interference group was faster,tumor volume was larger(P<0.05),and the positive rate of proliferation index Ki-67 was significantly higher than that of the control group(P<0.05).3)Effects of JAM2 overexpression and interference on the migration and invasion of colorectal cancer cellsThe results of scratch test showed that compared with the control group,the migration speed of cell lines stably overexpressed with JAM2 was significantly slower,while the migration speed of cell lines disturbed by JAM2 was significantly faster than that of the control group.The results of Transwell migration assay experiment showed that the number of immigrated cells of cell lines stably overexpressing JAM2 was significantly reduced compared with the control group(P<0.05),while the number of immigrated cells of cell lines disturbed by JAM2 was significantly higher than that of the control group(P<0.05).The results of three-dimensional culture showed that compared with the control group,the number of apophysis infiltrating into the surrounding matrix of the cell lines with stable overexpression of JAM2 was significantly reduced,while the number of apophysis infiltrating into the surrounding matrix of the cell lines with JAM2 interference was significantly higher than that of the control group(P<0.05).3.The mechanism of JAM2 in the progression of colorectal cancerThe results of GSEA gene enrichment analysis showed that in GSE13067 data,NF κB and MAPK pathways were up-regulated in colorectal cancer with low expression of JAM2,and the pathway related genes were significantly enriched(P<0.05).4.The upstream regulatory mechanism of JAM2 in colorectal cancer1)Database analysis of methylation degree of CpG island of JAM2 promoter in colorectal cancerWe used the methylation chip data GSE17648 of colorectal cancer matched tissue in GEO database to analyze the methylation degree of JAM2 promoter CpG island.The results showed that the methylation degree of JAM2 CpG island in colorectal cancer tissue was significantly higher than that in normal colorectal mucosa(P<0.05).We also used large samples of non matched colorectal cancer tissue methylation chip data GSE25062 and GSE40055 to further analyze the methylation degree of JAM2 promoter CpG island.2)Expression of JAM2 in colorectal cancer cells treated with 5-Aza-CdRThe results of Western blot showed that the expression level of JAM2 in colorectal cancer cells treated with 5-Aza-CdR,a demethylation drug,increased with the increase of drug concentration,and showed a concentration dependent relationship.3)Detection of methylation degree of JAM2 CpG island in colorectal cancerThe results of BSP showed that the methylation degree of JAM2 CpG island in 5 cases of normal intestinal mucosa was 36.6%,34.3%,38.0%,35.6%and 38.4%respectively,while that in colorectal cancer was 47.7%,50.5%,40.7%,63.0%and 45.8%respectively.The methylation degree of JAM2 CpG Island in tumor tissue was significantly higher than that in normal intestinal mucosaCONCLUSION:1.JAM2 expression in colorectal cancer tissues was lower than that in para-carcinoma tissues,and that in colorectal cancer cells was lower than that in normal intestinal epithelial cells.2.JAM2 regulates the proliferation,invasion and metastasis of colorectal cancer cells by regulating the MAPK and NF B signaling pathways3.The expression of JAM2 is regulated by the methylation of CpG island,and the hypermethylation of JAM2 CpG island is the main factor of the decrease of JAM2 expression... |
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