Exploring The Role Of GINS1 Gene In Colorectal Cancer Based On Bioinformatics Analysis And Experiments

Background and Objective:Colorectal cancer（CRC）is one of the malignant tumors with a very high mortality rate in the world^[1].At present,many risk factors including obesity,smoking habits and lack of physical exercise have increased the chance and risk of colorectal cancer^[2].With the development of surgical technology,surgical treatment has made great progress for patients with early CRC.However,despite the development of therapies such as laparoscopic surgery,radiotherapy,palliative chemotherapy and immunotherapy,the treatment of patients with advanced colorectal cancer is still not ideal^[3].Therefore,the determination of colorectal cancer-related targeted markers will make a great contribution to the early diagnosis of colorectal cancer patients and the improvement of the prognosis of colorectal cancer patients.At present,several biomarkers for the early diagnosis of colorectal cancer have been identified,but the clinical outcome and prognosis of colorectal cancer patients are still very poor^[4].Therefore,this article aims to identify potential diagnostic markers of colorectal cancer through bioinformatics analysis.For the reliability of the results,the selected biomarkers need to be further verified by molecular experiments.Bioinformatics analysis has become an important research method for cancer.It can reveal the etiology and pathogenesis of colorectal cancer and new biomarkers related to monitoring disease progression^[5,6].Previous researchers used bioinformatics tools to identify TIMP1,SERPINE1,CCND1,COL1A2 and SST as prognostic markers of colorectal cancer^[7].Bioinformatics analysis showed that Circ DDX17 is dysregulated in colorectal cancer.The author verified that it plays a tumor suppressor effect in colorectal cancer through follow-up experiments^[8].The purpose of this study is to mine potential biomarkers for the early diagnosis of colorectal cancer,and to further verify the results of data mining through subsequent molecular experiments.The results show that GINS1 is a reliable biomarker for the diagnosis and treatment of colorectal cancer.The expression of GINS1 is related to the invasion and migration of colorectal cancer and the progression of cancer.Methods:1.Mining potential diagnostic markers of colorectal cancer in public databases.Download the transcriptome data of 47 colorectal cancer tissues and 28 adjacent tissues in the GSE24514 dataset and GSE110223 dataset in the GEO database（http://www.ncbi.nlm.nih.gov/geo/）^[9].And through R（limma software package）^[10]to analysze the genes that are differentially expressed in colorectal cancer.The functional enrichment analysis of differentially expressed genes in colorectal cancer was carried out through the DAVID database^[11].Next,the protein-protein interaction network（PPI）of differentially expressed genes was constructed through the STRING database^[12]and visualized with Cytoscape software^[13].Then,with the help of the MCODE plug-in^[14]in Cytoscope to detect the densely acting areas in the PPI network.2.Analyze the expression and prognostic value of GINS1 in 33 common cancers through the online analysis platform of GEPIA^[15].10 genes closely related to GINS1 were unearthed from the STRING database.Analyze the mutations of GINS1 in colorectal cancer through the c Bioportal^[16]database.3.Experimental verification and analysis of the expression of GINS1 in colorectal cancer.Collected surgical specimens of cancer tissues and adjacent normal tissues from 76 patients with colorectal cancer in our hospital,and detected the protein and m RNA expression levels of GINS1 in cancer tissues and adjacent tissues by immunohistochemistry,western blot analysis,and Quantitative Real-time PCR.At the same time,Western blot analysis was used to detect the expression level of GINS1 in colorectal cancer cell lines SW480,HCT116,HT29,Lo Vo cells and normal colorectal organelle NCM460 cell line.4.Verify the effect of GINS1 expression level on the proliferation of colorectal cancer cells.The SW480 and HCT116 cell lines were transfected with Lipofectamine 3000 to construct colorectal cancer cells with high and low expression of GINS1.CCK-8 and colony formation assay were used to determine the proliferation ability of colorectal cancer cells after different treatments.5.Verify the effect of GINS1 expression level on the invasion and migration of colorectal cancer cells.The transwell experiment was used to compare the invasion and migration abilities of colorectal cancer cells after different treatments.Results:1.In the GSE24514 data set,there are 788 differentially expressed genes.In the GSE110223 data set,there are 604 differentially expressed genes.There are 389genes differentially expressed in the two data sets at the same time.The results of the DAVID database show that these 389 genes mainly affect the cell cycle and cell proliferation.Among these 389 genes,GINS1,TPX2,CDK1,TOP2A,KPNA2,PRC1,UBE2C,ASPM,RRM2,FOXM1,CEP55,CKAP2,MCM7,RFC3 and CCNB1 are particularly closely related.2.The results of GEPIA show that GINS1 is overexpressed in 20 cancer types including colon adenocarcinoma and rectal adenocarcinoma.At the same time,the overexpression of GINS1 is closely related to the poor prognosis of several cancer patients.STRING database results show that GINS1 has the closest relationship with GINS2,GINS3,GINS4,MCM2,MCM3,MCM4,MCM5,MCM6,MCM7 and CDC45.Previous reports found that these genes form a CMG（CDC45–MCM–GINS）complex,which is the core of eukaryotic chromosome replication.The lack of any of these components will affect eukaryotic chromosome replication^[17].The c Bioportal database results show that GINS1 has a genomics change rate of 20%in CRC.The most common type of mutation is increased m RNA expression.3.The results of immunohistochemistry,western blot analysis,and real-time fluorescent quantitative PCR technology showed that the m RNA and protein expression levels of GINS1 in colorectal cancer were higher than those in normal tissues adjacent to cancer.GINS1 expression is highest in HCT116 and SW480 cell lines.4.The results of CCK-8 and plate clone formation experiments show that low expression of GINS1 inhibits the proliferation of colorectal cancer cells;overexpression of GINS1 can promote the proliferation of colorectal cancer cells.5.Transwell experiment results show that low expression of GINS1 inhibits the invasion and migration of colorectal cancer cells;overexpression of GINS1 can promote the invasion and migration of colorectal cancer cells.Conclusions:Bioinformatics screening and molecular experiments proved that GINS1 is highly expressed in colorectal cancer,and the overexpression of GINS1 is related to the proliferation,invasion and migration of colorectal cancer cells.Therefore,GINS1 can be regarded as a diagnostic marker for colorectal cancer.