| The anti-colorectal cancer research is one of the most important tasks in our research work as the incidence,development and metastasis of colorectal cancer severely threaten human health.In the previous research work,the author has screened out several genes that promote the development and metastasis of colorectal cancer through various bioinformatics analysis methods by which obtained good results in the follow-up experiments.Since the successful cure of tumors is the ultimate goal of our research,this study comprehensively analyzed the gene methylation level of colorectal cancer genes,the differentiation degree of tissue,the time of metastasis,the signal network module,the key molecular events and the targeting small molecule compounds from another perspective,to screen out and study various types of molecules which inhibit the growth and metastasis of colorectal cancer from molecular markers,cell signaling network and small molecule compounds perspective.1.Screen of suppressor molecular markers in colorectal cancerIn order to screen out the genes that inhibit the development and metastasis of colorectal cancer,in this study,we screened out the MBD1 which down-regulate the expression level and up-regulate methylation in colorectal cancers only in the late stage,and progressively increase methylation levels at each stage of colorectal cancer development by comparing the early and late colorectal cancer expression profile data and normal colorectal data..Survival analysis confirmed that MBD1 has high survival rate at high expression and low survival rate at low expression(P=0.0227).Through GSEA enrichment analysis based on MBD1 expression values,the gene consistent with MBD1 high expression was found to be mainly located at the chromosome 17p13 and 17p12,and eight cancer suppressor genes(HIC1,VPS53,RPA1,ALOX15B,TP53,PFN1,MYBBP1A,GABARAP)were found in the 17p13.The transcription factor SP1 was found to be highly enriched by further enrichment analysis of transcription factors in this region.Because SP1 is able to bind to MBD1 and the GC content of the SP1 binding region is high,it further demonstrated that it is related to MBD1 function.Therefore,the molecular mechanism of MBD1 tumor suppressor may be through the combination of transcription factor SP1 to regulate the expression of 8 tumor suppressor genes.After the interference of expression of MBD1 in colorectal cancer cells,CCK8 proliferation assay showed that the growth rate of cancer cells in the interfering group was greater than that of the control group.The plate colony formation assay and Transwell invasion assay showed that the cloning ability of the interfering group and invasion ability obviously increased.It was further confirmed that MBD1 can inhibit the growth and invasion of colorectal cancer.Therefore,MBD1 is a tumor suppressor molecular marker for colorectal cancer.Because the degree of differentiation is closely related to the degree of malignancy of colorectal cancer,we screened out molecular markers of colorectal cancer specific to differentiation from another perspective in this study.We obtained IL22RA1 gene through intersection of tissue-specific genes of colorectal cancer and genes with diminished expression as diminished differentiation screened out by comparing the expression profile data of colorectal cancers of different differentiation states.Therefore,the expression of IL22RA1 gene was sustainable declined in highly differentiated,moderately differentiated,poorly differentiated,and undifferentiated tissues of colorectal cancer.Gene expression analysis showed that the IL22RA1 expression levels in colorectal cancer and normal tissues were higher than other tissues and cells,which have significant tissue specificity.The survival analysis revealed that the IL22RA1 gene expression was associated with the overall survival rate of the patients(P=0.0224).The survival rate was high when IL22RA1 expression was high,and the survival rate was low when the expression was low.The ROC curve analysis found that IL22RA1 expression has good specificity and sensitivity to grade 2 colorectal cancer.Therefore,IL22RA1 is a differentiation-specific molecular marker for colorectal cancer.2.Construction of signaling network of colorectal cancer cellsThe time of tumor metastasis is an important factor in metastasis research.We screened out the genes that are positively and negatively correlated with the metastasis time of colorectal cancer and performed network construction based on the tumor metastasis time information and expression information in the colorectal cancer expression profile data in this study.The analysis of the network module found that many genes belong to a certain organelle or cell component and numerous metastasis promotion genes belong to the ribosome component,and the metastasis suppression gene belongs to the proteasome,mitochondrial ribosome,mitochondrial ATP synthase and splicing factor.The survival analysis of these genes that constitute the organelle components found that the combined survival curves that constitutes the ribosome module was shown to promote metastasis(P=0.0022939),and the combined survival curves that constitute proteasome(P=7e-07),mitochondrial ribosome(P=0.0001157),mitochondrial ATP synthase(P=0.0001936)and splicing factor(P=1.36e-05)modules was shown to inhibit metastasis.In order to further clarify the key molecular events that occur at each stage of the development of colorectal cancer,in this study,we compared genes that are differentially expressed in colorectal cancer at each stage to screen out genes that show persistent activation and persistent suppression during the progression of colorectal cancer.Based on the enrichment situation,we obtained genes that are constantly activated and delocalized at each stage of extracellular,and constantly suppressed and delocalized at each stage of mitochondria.The enrichment analysis and network construction of genes that deviated from continuous direction at each stage found that the key molecular event occurred between stage Ⅱ and stage Ⅲ of colorectal cancer.The chemotaxis of inflammatory cells was mainly in the extracellular,and aerobic respiration was mainly in the mitochondria.The network module analysis found that VEGFA,CCL7-CCR3 complexes are the key nodes in the extracellular,and MAPKI is the key node in the mitochondria.The analysis of hub network found that FN1→SPARC→COL1A1→MMP2 may be the key signal transduction chain in the extracellular;and CAV1→MAPK3→RAF1→NR3C1→MAPK1→ESR1 may be the key signal transduction chain in the mitochondria.3.Screen of small molecular compounds targeted to colorectal cancer for interventionThe study about small molecule compounds for the targeted treatment of colorectal cancer is less reported,though the study about traditional drug target has been much reported.In this study,the differentially expressed genes in colorectal cancer were transformed into 6 small molecule compounds related to the inhibition of colorectal cancer.The targeted protein analysis revealed that the non-receptor tyrosine kinase TNK2 was the common target of the above 3 small molecule compounds.The expression analysis found that TNK2 shows continued active at all stages of colorectal cancer development.The analysis of small molecule compounds bound to TNK2 in the PDB database by chemical informatics revealed that one of the small molecule compounds(PDB ID:4EWH)had good kinase specificity,and the analysis of its target protein revealed that there are 21 target proteins with kinase activity in 122 target proteins,including TNK2.The enrichment analysis of 122 target protein-involved human diseases revealed the highest correlation with colorectal cancer(47 target proteins),which further validates that TNK2 is a key drug target for colorectal cancer.Since small-molecule compounds for a single target are prone to drug resistance,in this study,we firstly selected two reliable metastasis-associated proteins PRL-3 and CILC4 as drug targets from previous studies of the research group,and then selected two reliable proteins,THBS2 and BGN as drug targets from the above-mentioned development of colorectal cancer.We constructed a pharmacophore model based on the above four target structures,and searched small molecule compounds conforming to the pharmacophore model and purchasable from the compound database.Then though the analysis of molecular docking and scoring,five small-molecule compounds were obtained for each target structure and three of them(Macrocarpal I,sildenafil,and neoandrographolide)were found to bind two targets at the same time.The inhibitory efficiency of these three small molecules compounds through CCK8 proliferation assay based on the concentration gradient detection of colorectal cancer cells revealed that Macrocarpal I has the highest inhibition efficiency and the lowest IC50 value.The time gradient CCK8 proliferation assay of Macrocarpal I found that it shows significantly inhibition to colorectal cancer cells,but not obvious to normal colorectal cells,indicating that Macrocarpal I has certain specificity.The concentration gradient Macrocarpal I plate colony assay and Transwell invasion assay found that the number of cancer cell colony formation was significantly reduced and invasion significantly weaken as concentration increase.It was further verified that Macrocarpal I has the ability to inhibit colorectal cancer.Macrocarpal I extracted from Eucalyptus is a promising natural small molecule compounds.Conclusion:1.Based on gene methylation/expression levels,we screened out a tumor suppressor molecular marker MBD1 associated with metastasis of colorectal cancer,suggesting that MBD1 may act as a suppressor of colorectal cancer by controlling the expression of eight tumor suppressor genes in combination with transcription factor SP1.Based on tissue-specific genes,we screened out the molecular marker IL22RA1 associated with the degree of differentiation of colorectal cancer tissue.2.We constructed cell signal regulatory networks related to metastasis of colorectal cancer,identified cell components that affect the time of metastasis,clarified the molecular events that play a leading role in the development of colorectal cancer,and obtained signal network modules and signal transmission chain.3.We screened out the drug targets TNK2 relate to metastasis of colorectal cancer,proposing that the small molecule compounds targeted to TNK2 may have potential inhibitory effects of colorectal cancer.We find the small molecule compounds binding to the selected target(PRL-3,CLIC4,BGN,and THBS2).Macrocarpal I is proposed to be a natural product effective to inhibit growth and metastasis of colorectal cancer as small molecule compounds through analysis and verification. |
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