Study On The Mechanism Of SPHK1 Gene Involved In Regulating The Development Of Colorectal Cancer Based On Bioinformatics

OBJECTIVE: Analysis of the Mechanism of SPHK1 Gene involved in regulating the Development of Colorectal Cancer based on BioinformaticsMETHODS: We acquired m RNA expression signature of colorectal cancer from TCGA database.Univariate Cox analysis and LASSO regression were used to screen out prognosis-related metabolic gene to construct prognostic model,the core gene SPHK1 was screened.The role of SPHK1 gene in colorectal cancer was analyzed by integrating multiple online databases.Then,m RNA transcription data and mi RNAs of CRC were obtained from TCGA database with high-throughput sequencing data.The circ RNA dataset was obtained from microarray data of GSE121895 dataset.The differential expression analysis of the chip expression data is mainly done through the "limma" package.Sequencing count data were analyzed for differential expression using the edge R package.After obtaining the significantly different circ RNAs,mi RNAs and m RNAs,the interactions information of mi RNA-targeted m RNAs in five databases,mi RDB,Target Scan,mi RMap,mi Randa and mi Tar Base,were utilized to build the differential mi RNA and m RNA interaction network.The interactions of mi RNAs targeting circ RNAs in the starbase database are used to construct the differential mi RNA and circ RNA interactions network.Combine the above two reciprocal networks,and screen out the reciprocal information(circ RNA negatively regulates mi RNA,mi RNA negatively regulates m RNA)that match the expression regulation trend of ce RNA network to get ce RNA regulatory network.Finally,visualization of ce RNA network using cytoscape software.q PCR was utilized to examine the expression of hsa＿circ＿0001642 in each group of CCD 841 Co N,SW480,SW1116,HCT116,LOVO,CACO2 cells,and the highest expressing SW480 cell line was selected for subsequent experiments.Immunohistochemistry was performed to detect the expression of SPHK1 gene in 40 pairs of colorectal cancer tissues and their corresponding normal tissues.Design and synthesize sh RNA-hsa＿circ＿0001642plasmid,hsa-mi R-193a-5p inhibitor plasmid,hsa-mi R-193a-5p mimics plasmid,sh RNA-SPHK1 plasmid and its blank control NC,and transfect them into SW480 cells respectively.CCK8 experiment to detect the proliferation level of SW480 cells in every group after 24,48 and 72 h;transwell assay to examine the invasion and migration capability of each group;scratch assay to examine the migration capability of each group;flow cytometry to assess the apoptosis rate of each group;q PCR assay to investigate the expression of has＿circ＿0001642,hsa＿mi R＿193a＿5p and SPHK1 expression in every group;Western Blot assay to identify SPHK1 protein expression in each group;ELISA assay to examine the S1 P content in each group.Luciferase reporter to verify the binding relationship between hsa＿circ＿0001642 and hsa-mi R-193a-5p;hsa-mi R-193a-5p and SPHK1.Design and synthesize hsa＿circ＿0001642-WT plasmid,hsa＿circ＿0001642-Mut plasmid and their control oe-vector plasmid sets,and transfect them into SW480 cells respectively.q PCR assay to detect the expression of hsa＿mi R＿193a＿5p and SPHK1 in each group.Western Blot assay was performed to identify the SPHK1 protein expression in each group;ELISA assay was performed to detect the S1 P content in each group.Ago2-RIP assay was performed to verify the binding relationship between hsa＿circ＿0001642 and hsa-mi R-193a-5p;convergent primers and divergent primers for hsa＿circ＿0001642 were also designed,and PCR amplification of circ RNA was performed on c DNA and g DNA to verify whether only c DNA could be amplified with hsa＿circ＿0001642;PCR amplification products from the loop formation validation experiments were tested for hsa＿circ＿0001642 stability using ribonuclease(RNase R)treatment.Nucleoplasmic separation and FISH experiments were performed to detect the subcellular localization of hsa＿circ＿0001642.RESULTS: A prediction model is built based on NAT2,XDH,GPX3,AKR1C4,SPHK1,ADCY5.The risk score calculated from the prognosis model is an independent prognostic element for colorectal cancer,and the survival time of patients with high score was remarkably lower than patients with low risk score.The SPHK1 gene is highly expressed in the colorectum and is related to prognosis in colorectal cancer,while SPHK1 is involved in activating the EMT pathway and is a core gene among prognosis-related metabolic genes.SPHK1 is participated in the modulation of most essential biological processes and signaling pathways during cancer development.For example,apoptosis,angiogenesis,glycolysis,hypoxia,IL6 JAK＿STAT3 signaling,KRAS signaling pathway activation,NOTCH signaling,P53 pathway,etc.The SPHK1 gene and its co-expressed genes are mainly involved in sphingolipid family-related synthesis and metabolism,and in the regulation of sphingolipid-related signaling pathways.The expression level of SPHK1 was remarkably correlated with 22 immune cells infiltration level,the highest association is between SPHK1 gene expression and macrophages.The infiltration level of immune cells is remarkably higher in the high SPHK1 gene expression group than in the low group.Eight mi RNAs were negatively regulated corresponding to high SPHK1 gene expression,mi RNA,hsa-mi R-330-3p,hsa-mi R-6511b-3p,hsa-mi R-484,hsa-mi R-193a-5p,hsa-mi R-125b-5p,hsa-mi R-361-3p,hsa-mi R-146b-3p,and hsa-mi R-378a-5p.Eight mi RNAs negatively regulated with SPHK1 were identified and the corresponding highly expressed circ RNA for hsa-mi R-193a-5p was hsa＿circ＿0001642,and the corresponding highly expressed circ RNA for hsa-mi R-361-3p was hsa＿circ＿0001167.We found for the first time that hsa＿circ＿0001642 was highly expressed in CRC and that hsa-mi R-6511b-3p was lowly expressed in CRC.The ce RNA network of hsa＿circ＿0001642/hsa-mi R-193a-5p/SPHK1 was successfully constructed.hsa＿circ＿0001642 is over-expressed in human CRC cell lines: SW480,SW1116,HCT116,LOVO,CACO2,but the highest level of hsa＿circ＿0001642 expression was examined in SW480 cells.Knockdown of hsa＿circ＿0001642 and SPHK1 suppressed the malignant phenotype of SW480 cells,such as a decrease in the number of invasive and migrating cells and a decrease in cell migration ability;a remarkable decline in cell viability and an increase in apoptosis.Knockdown of hsa-mi R-193a-5p promoted the malignant phenotype of SW480 cells,such as a significant increase in the number of invading and migrating cells and an increase in cell migration ability;a remarkable increase in cell viability and decline in the apoptosis rate.The results of Ago2-RIP assay showed a binding relationship between hsa＿circ＿0001642 and hsa-mi R-193a-5p.The luciferase reporter gene plasmid assay found that hsa-mi R-193a-5p and hsa＿circ＿0001642 and SPHK1 were bound.After hsa＿circ＿0001642-WT transfection of SW480 cells,hsa-mi R-193a-5p expression was decreased,m RNA and protein expression of SPHK1 in cells was significantly increased,and S1 P content was increased.hsa＿circ＿0001642-MUT transfection of W480 cells,hsa-mi R-193a-5p expression increased,SPHK1 m RNA and protein expression in the cells were significantly decreased,and S1 P content decreased.hsami R-193a-5p-WT transfected SW480 cells,SPHK1 m RNA and protein expression decreased,and S1 P content decreased.hsa-mi R-193a-5p-MUT transfected SW480 cells,SPHK1 m RNA and protein expression increased and S1 P content increased.The loop formation validation assay confirmed that hsa＿circ＿0001642 is a loop RNA formed by reverse shearing,which is resistant to RNase R digestion and is mainly enriched in the cytoplasm of SW480 cells.CONCLUSION: In this study,we found that hsa＿circ＿0001642 was highly expressed in colorectal cancer and deregulated its negative effect on SPHK1 by binding to hsa-mi R-193a-5p,and subsequently the highly expressed SPHK1 was involved in regulating anti-apoptosis as well as cell proliferation,migration and invasion of colorectal cancer cells by catalyzing S1 P production,a lipid metabolic pathway that promotes progression of colorectal cancer,leading to poorer patient prognosis.Therefore,hsa＿circ＿0001642/hsa-mi R-193a-5p/SPHK1/S1 P axis plays a key role in the progression of colorectal cancer.hsa＿circ＿0001642,hsa-mi R-193a-5p,and SPHK1 can be potential targets for colorectal cancer diagnosis and treatment.SPHK1 gene can be involved in regulating the process of colorectal cancer development through the hsa＿circ＿0001642/hsa-miR-193a-5p/SPHK1/S1 P axis.