| Objective: Colorectal carcinoma(CRC)is the third most common tumor in the world.Every year,there are more than one million new cases and about 700,000 patients die of colorectal cancer.With the continuous improvement of surgical techniques and the application of targeted therapy and immunotherapy,the overall survival of colorectal cancer patients has been improved,but the prognosis of advanced colorectal cancer patients with metastasis is still poor.Therefore,our study purpose to identify the genes related with metastasis of CRC through bioinformatics technology,based on the sequencing data of our research group and public data sets,analyze clinical relevance and prognostic value of the gene,and explore its functions and related mechanisms through in vitro and in vivo experiments.Methods: Part 1: Screening genes related to metastasis of CRC by bioinformatics analysis and exploring their clinical and prognostic value.In the early stage,the research group established CRC cells(RKO-H and Caco2-H)with high liver metastasis ability based on RKO and Caco2 by liver metastasis model of nude mice.We sent RKO,Caco2,RKO-H and Caco2-H for transcriptome sequencing,to further study the function of these cells.Based on the transcriptome sequencing result and the data of public database,the potential genes related to CRC liver metastasis were identified by R package edge R and R package limma.We collected primary CRC samples and adjacent cancer samples from the Cancer Hospital of Liaoning Cancer Hospital.RT-q PCR and Western blot were used to verify the expression levels of SERPINA1 in CRC group and control group.IHC was used to evaluate the relationship between SERPINA1 expression and clinicopathological stages.The relationship between SERPINA1 expression and drug sensitivity was analyzed by drug-related data sets.R package Maftools was used to analyze the correlation between SERPINA1 expression and RAS mutation in colorectal cancer.Part 2: SERPINA1 overexpression lentivirus and control lentivirus were transfected into RKO and Caco2 cell lines,sh-SERPINA1 and control virus were transfected into RKO-H and Caco2-H cell lines,and the transfection efficiency was verified by RTq PCR and Western blot.MTS experiment,clone formation and EDU were used to verify the effect of SERPINA1 on the proliferation of colorectal cancer cells,and wound healing and Transwell assay were used to verify the effect of SERPINA1 on the migration and invasion of CRC cells.Based on TCGA CRC data,GSVA,GSEA and ss GSEA methods were used to predict SERPINA1-related signal pathways.Nuclear protein was extracted by nucleoplasm separation kit,and the expression of p-STAT3 was verified by Western blot.P-STAT3 inhibitor Stattic was used to inhibit the expression of p-STAT3 in RKO and Caco2 cells,and a rescue experiment was designed to verify that SERPINA1 affected the proliferation and migration of colorectal cancer through p-STAT3 pathway.Liver metastasis model of colorectal cancer was established by spleen injection in nude mice.Part 3: We use Cistrome and PROMO databases to predict transcription factors related to SERPINA1.JASPAR database analysis was used to predict related binding sites.Dual luciferase assay and Ch IP were used to verify that CEBPB combined with SERPINA1 promoter to regulate transcription.Result: Part I: First,we verified that RKO-H and Caco2-H have higher proliferation and migration abilities than RKO and Caco2 using colony formation,wound healing and Transwell assay.Based on Transcriptome sequencing results,the top 50 different expressed genes with the highest |log2FC| between RKO-H/ Caco2-H and RKO/Caco2 groups.In GSE49355 data set,the 257 differential genes were identified between primary the colorectal cancer group and colorectal cancer liver metastasis group.SERPINA1 was identified as the only overlapping gene of the three groups.Compared with normal intestinal epithelial cells,the expression of SERPINA1 in RKO and Caco2 was significantly upregulated.Compared with RKO and Caco2,the expression ofSERPINA1 in RKO-H and Caco2-H was significantly upregulated.The expression of SEPRINA1 was significantly higher in colorectal cancer tissues than that in adjacent normal tissues.Compared with healthy populations,the SERPINA1 levels of blood in patients with colorectal cancers were significantly higher than those in healthy population.The high SERPINA1 expression group had a higher rate of KRAS mutations and BRAF mutations.Part II: MTS,clone formation and EDU assay showed that overexpression of SERPINA1 promoted the proliferation of colorectal cancer cells,and knockdown of SEPRINA1 inhibited the proliferation of colorectal cancer cells.The results of wound healing and Transwell assay showed that overexpression of SERPINA1 promoted the migration and invasion of colorectal cancer cells,and knockdown of SEPRINA1 inhibited the migration and invasion of colorectal cancer cells.Meanwhile,the results of GSVA and GSEA indicated that the high expression of SERPINA1 was related to the enrichment of KEGG JAK STAT signaling pathway.The result of ss GSEA showed a positive correlation between SERPINA1 expression and KEGG JAK STAT score and a positive correlation with ST STAT3 score.Western blot results showed that overexpression of SERPINA1 was significantly increased the expression of p-JAK1,p-STAT3 and c-myc,and knockdown of SERPINA1 was significantly decreased the expression of p-JAK1,p-STAT3 and c-myc.Moreover,increased SERPINA1 could induced the higher expression of p-STAT3 in nuclear.The experimental results showed that SERPINA1 could promote the proliferation and migration of colorectal cancer through p-STAT3.Liver metastasis model of colorectal cancer in nude mice was established by spleen injection,and the area of liver metastasis of overexpressed SERPINA1 group was significantly elevated compared with the control group.Part III: We used PROMO and Cistrome databases to predict and identify CEBPB and FOXA1,as potential transcription factors associated with SERPINA1.TCGA-CRC and GSE17537 data sets suggested that CEBPB is related to poor prognosis and clinical stage of progression of CRC.The results of RT-q PCR and Western blot indicated that CEBPB had positive regulation effect on SERPINA1.Dual luciferase assay and Ch IP verified that the transcription factor CEBPB could bind to the promoter sequence ofSERPINA1 and regulate the transcription of SERPINA1 positively.The rescue experiment validated that the transcription factor CEBPB has a positive transcription regulation effect on SERPINA1 and activates STAT3 pathway to promote the proliferation and migration of colorectal cancer.Conclusion: SERPINA1 is associated with the poor prognosis and advanced clinical stages of colorectal cancer,and can promote the proliferation and metastasis of colorectal cancer.SERPINA1 can activate JAK1-STAT3 pathway and promote the nucleation of p-STAT3.Transcription factor CEBPB has a positive transcriptional regulation effect on SERPINA1,and activates STAT3 pathway to promote the proliferation and migration of colorectal cancer. |
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