| The incidence rate and mortality rate of cancer worldwide are increasing with the aging of population.By 2017,cancer has become the second leading cause of human death,next only to the cardiovascular and cerebrovascular diseases.At the same time,the burden of cancer in China has been increasing in recent years,and the incidence and mortality rate of cancer in China are the highest in the world,among which the incidence rate accounts for 21.1% and the mortality rate accounts for 23.9% in the world.Therefore,it is urgently needed to develop new cancer treatment to solve the current problems.The proposal of new treatment of cancer relies on the in-depth exploration of cancer pathogenesis.Currently,the difficulties of cancer treatment lies in the metastasis of lesions and the emergence of drug resistance to chemotherapy.Epithelial-mesenchymal transition(EMT)could promote tumor metastasis and drug resistance,and the occurrence of EMT is a hallmark event of tumor metastasis.This study established a method to study cell heterogeneity using tumor cell lines,and combined with multi-omics sequencing technology to explore the specific mechanism of RUNX2 promoting cell migration.It is found that the expression of RUNX2 is positively correlated with tumor metastasis,and negatively correlated with the survival of various cancer patients.There are different splicing forms of TEAD1 in Wnt-high and-low cells,which can be regulated by ESRP1 and MBNL1,and it is found that different splicing isoforms of TEAD1 have different functions in Wnt-high cells.Through single-cell sequencing,we found two groups of cells with different activity of Wnt signaling pathway and EMT in SW480 cells.SW480 cells were infected with a lentivirus-based reporter system,which can recognize Wnt signaling pathway activity by expressing green fluorescent protein(GFP).Then the cells were isolated by flow cytometry based on GFP signal,which were named as Wnt-high cells and Wnt-low cells,respectively.The expression of downstream genes in Wnt,AXIN2 and LEF1 was detected by q PCR on m RNA level,validating that Wnt-high cells had high Wnt activity,and Wnt-low cells had low Wnt activity.Through Transwell assay,it was found that Wnt-high cells had high cell migration ability,while Wnt-low cells had low cell migration ability.Then,m RNA-seq and ATAC-seq sequencing were performed on Wnt-high cells and Wnt-low cells,recognizing two important differential genes:RUNX2 and TEAD1,where RUNX2 had differently expression levels,and TEAD1 had different splicing isoforms between Wnt-high cells and Wnt-low cells.In Wnt-high cells,using sh RNA to interfere with the expression of RUNX2 could reduce cell migration ability in vitro and vivo,while in Wnt-low cells,overexpression of RUNX2 could promote cell migration in vitro and vivo.The Wnt signaling pathway could regulate RUNX2 expression in SW480 cells.The expression of RUNX2 was promoted with the activation of the intracellular Wnt signaling pathway by CHIR99021 in Wnt-low cells.whereas the expression of RUNX2 was inhibited with the silenced expression of β-catenin induced by sh RNA.To further study the mechanism of RUNX2 in promoting the migration ability of SW480 cells,a variety of omics sequencing technologies were applied,including the detection of ATAC-seq and m RNA-seq in Wnt-low and Wnt-high cells,detection of Ch IP-seq based on RUNX2 protein in Wnt-high cells,as well as m RNA-seq in Wnt-high cells with RUNX2 interfered.Based on the analysis of the above sequencing results,genes directly regulated by RUNX2 need to meet the following conditions: have peaks in Ch IP-seq data;in ATAC-seq data,the peak intensity in Wnt-high cells is higher than that in Wnt-low cells;the m RNA expression level in Wnt-high cells is higher than that in Wnt-low;in Wnt-high cells,when the expression of RUNX2 is down-regulated,the gene expression is decreased.A total of 401 genes directly regulated by RUNX2 were found through analysis,among which 210 were EMT-related genes,including PSG1,EPAS1,and MPZL2.Through clinical data analysis of colorectal cancer,it was found that the expression of RUNX2 in clinical stage IV was significantly higher than that in clinical stage II,and the expression of RUNX2 was higher in patients with tumor metastasis than in patients without metastasis.At the same time,in colorectal cancer patients with higher RUNX2 expression,the survival rate was lower.The similar situation was also found in glioma,renal cancer,bladder cancer and gastric cancer,where the expression of RUNX2 is negatively correlated with the survival rate of patients.By comparing the multi-omics data of Wnt-high cells and Wnt-low cells,it was found that the splicing form of exons 5-6-7 of TEAD1 was significantly different between the two cell groups,and this difference also existed between stem cells and differentiated cells.Exon 5 and exon 6 of TEAD1 are a pair of mutually exclusive exons with different functions,and exon 7 is a tiny exon only encoding 4 amino acids,and it has different combinations in different cell types.By analyzing the protein sequences of the TEAD family,it was found that for the positions corresponding to TEAD1 exon5-6-7 in other TEAD family,the sequences of different TEAD family members can be composed of different combinations of exons 5,6,and 7 of TEAD1.Two splicing-regulatory genes ESRP1 and MBNL1 previously known to regulate EMT could regulate alternative splicing of TEAD1.In Wnt-low cells,all isoforms of TEAD1 could promote cell migration,while only TEAD1-468 could promote cell migration in Wnt-high cells.It was speculated by m RNA-seq analysis that,in Wnt-high cells,TEAD1-468 could promote cell migration through increasing the expression of EMT associated genes expression such as ZEB1,KRT81,LCN2,SPARC,CX3CL1 and TLE1.And TEAD1-4578 could inhibit cell migration through increasing IL24 expression and decreasing CLU,CTGF,FSCN1,and MPP7 expression.In conclusion,this study explored the feasibility of studying tumor heterogeneity by using SW480 tumor cell lines as research objects,and explored the application scenarios of combining multi-omics approaches,and found the mechanism of RUNX2 promoting EMT.Through the in-depth study of TEAD1,it was found that different splicing isoforms of TEAD1 have different functions in Wnt-high cells.This study promotes the exploration of tumor heterogeneity,and provides a new theoretical basis for the diagnosis and treatment of tumor metastasis,and offers a potential therapeutic target. |
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