Definition Of A New Tumor Suppressor Gene SARDH And The Mechanism For Its Defects Involved In Sporadic Colorectal Cancer

BackgroundColorectal cancer(CRC)is a common fatal tumor.80%of the CRC are sporadic colorectal cancer(sporadic colorectal cancer,sCRC).It may take many years from the colon adenoma to colon adenocarcinoma.CRC is completely curable at an early stage,but the genetic marker for current CRC screening is still not perfect.CRC is a complex disease involving multiple common and rare mutations of multiple genes.With a high degree of genetic heterogeneity of the complex disease,different patients may harbor different genetic mutations.Therefore,in order to find new candidate genes associated with CRC,more patients for different genes and mutations need to be detected.In the early stage of this study,it is indicated that there is a splice mutation in the 17th intron and 18th exon of the SARDH gene in one CRC patient but not prezent in the adjacent normal tissue through the whole exons sequencing of 3 sCRC tumor tissues.A certain frenquency of mutations were commonly found in many classic cancer-related genes in cancer patients.It is reported that the silencing of the SARDH is confirmed to be associated with the invasion of prostate cancer cells.We suspected that the SARDH is related to CRC.At present,the clinical relationship between SARDH expression or its mutation in cancerous tissues have not been researched.The mechanism for its influences on cancer cell proliferation and migration is not clear.Methods1.Through the immunohistochemistry,SARDH protein was detected in 126 CRC tissue and normal tissue without family history of CRC.The SARDH mRNA levels were analyzed by Real-time polimerase chain reaction(RT-PCR)in 28 pairs of sCRC tissues and adjacent normal tissues.The changes in SARDH mRNA levels were also analyzed in the TCGA database.The possible mutations in the coding region of SARDH gene was screened in 53 cases of sCRC and adjacent normal tissues through whole exon sequencing(3 cases)and deep exon sequencing(50 cases).2.The effect of SARDH on the proliferation of CRC cells was investigated by CCK8 cell proliferation assay.The migration of CRC cells was detected by cell migration and invasion assay.And the effect of SARDH on the tumorigenic ability in nude mice was detected.3.miRNA binding sites in the 3’-UTR region of the SARDH were predicted by Targetscan,Miranda,MiRDB and RNAhybrid.RT-PCR,western blot and dual luciferase reporter assay were used to determine wether these miRNAs could negatively regulate the expression of SARDH.4.The key coexpression genes of SARDH were identified by RNA sequencing(RNA-seq)combined with co-expression analysis,signal pathway analysis.The relationship between SARDH level and methylation status of differentially expressed genes was analyzed by UCSC xena database.The pathways and molecular mechanisms of the influence of SARDH on CRC development were revealed.Result1.According to the immunohistochemical results,it is showed that the expression of SARDH protein in 126 sCRC tissues was significantly lower than that in adjacent normal tissues.There is no correlation between SARDH expression and sex,age,CRC clinical stage or lymph node metastasis.In RT-PCR results,SARDH mRNA level in sCRC tissues was significantly decresed when compared to that in adjacent normal tissues.The results of whole exon sequencing and deep sequencing showed that there were 1 splice site mutation and 1 missense mutations in 53 sCRC tumor tissues which were both cancer tissue-specific site.2.The results of cell proliferation showed that the overexpression of SARDH significantly inhibited the proliferation ability of HT29 and SW480 cell lines(P<0.01).The inhibition of SARDH promoted the proliferation ability of SW480 and HCT116 cell lines(P<0.01).Cell cycle experiments showed that knockout of SARDH significantly improved the cells cycle phase in SW480 and HCT116 cells.The migration and invasion ability was significantly reduced in SW480 cells when SARDH was overexpressed(P<0.01).In contrast,when SARDH was inhibited in SW480 and HCT116 cells,the number of migration and invasion cells was significantly increased compared to the control group.The overexpression of SARDH in HT29 cells nude mice showed that the tumorigenic ability of HT29 cells in nude mice was significantly lower than that in the control group,and the difference was statistically significant.3.By the prediction of Targetscan,Miranda,MiRDB and RNAhybrid,it is shown that the seed regions of miR-4651,miR-4297 and miR-6508 were completely complementary to the 3’-UTR region of SARDH.The RT-PCR and western blot results showed that only miR-4651 could inhibit the expression of SARDH.The results of dual luciferase reporter assay showed that miR-4651 significantly decreased firefly luciferase activity of SARDH 3’-UTR wild-type plasmid not the mutant 3’-UTR plasmids.4.According to the RNA-seq and RT-PCR results,overexpression of SARDH down-regulated CXCL1,CX3CL1,CCL5 and CCL20 of chemokine signaling pathway,and PMSB8,PMSB9 genes in NIK/NF-kappaB signaling pathway.Through the co-expression results,CXCL1 was significantly correlated with the expression of SARDH.It is shown in RT-PCR results that there is a reverse association between CXCL1 gene and SARDH not only in 28 sCRC tumor tissues but also in their adjacent nontumor mucosa(P<0.05).The expression of CXCL1,CXCL1,CX3CL1,CCL5,CCL20,PMSB8,PMSB9,CD74 and IFNL2 were down-regulated by RNA-seq technique combined with GO analysis,differential expression gene analysis and RT-PCR.Through gene co-expression analysis,it is demonstrated that CXCL1 gene is the most relevant gene with the SARDH.The expression of CXCL1 was negatively correlated with SARDH(P<0.05)in 28 cases of sCRC tissues and adjacent normal tissues by pearson test.The results of UCSC xena database showed that the transcription level of SARDH in CRC patients was positively correlated with the methylation status of CXCL1 and CCL20 genes.Conclusion1.SARDH expression in sCRC was significantly decreased.2.The down-regulation of SARDH expression in sCRC tissue may be related to the mutations in the coding region of SARDH gene in sCRC tissue.SARDH can also be downregulated by miR-4651.3.SARDH can inhibit the proliferation,migration and invasion of CRC cells.4.SARDH may inhibit the occurrence of CRC by inhibiting chemokine signaling pathway.This inhibition may be achieved by promoting the methylation of CXCL1,and CCL20 genes.