| Chapter 1 Screening for differential genes in colorectal cancer and identification of their upstream microRNAsObjective:Colorectal cancer(CRC)is one of the common malignancies with high mortality worldwide.With the development of treatment technology,early colorectal cancer can be cured by surgery combined with radiotherapy,but the prognosis of advanced colorectal cancer is often poor due to its high recurrence rate and high tumor drug resistance,so its pathogenesis needs to be further explored.Methods:Multiple GEO datasets(GSE75970,GSE74602,GSE89076,and GSE10950)were used to screen differential genes in colorectal cancer.Then correlation analysis between differential genes and colorectal cancer disease genes was performed to identify the differential genes with the highest association,which were identified by multiple microRNA databases(miRTarBase,TargetScan,starBase,mirDIP,miRDB,and miRSearch)to predict the corresponding miRNAs of the differential genes.Finally,the regulatory role of miRNAs on differential genes was verified by luciferase reporter system,RT-qPCR and WB assay.Results:Analysis of multiple colorectal cancer GEO datasets identified nine intersecting genes(SOX9,ABCA8,KRT80,ENC1,ITGA2,MYOM1,MAMDC2,C7,GDF15),with ITGA2 being the differential gene with the highest association with disease genes.Analysis of multiple miRNA databases identified the miRNA for IGTA2 as miR-16-5p.miR-16-5p was lowly expressed in colorectal cancer tissues and colorectal cancer cells,and the mRNA and protein level expression of IGTA2 was significantly reduced after intracellular overexpression of miR-16-5p.Conclusion:miR-16-5p is lowly expressed in colorectal cancer,and its target gene ITGA2 is highly expressed in colorectal cancer.miR-16-5p/ITGA2 is closely related to the development of colorectal cancer.Chapter 2 Role of miRNA-16-5p in colorectal cancer through regulation of target gene ITGA2Objective:In the previous chapter,we found that miR-16-5p is low expressed in colorectal cancer.Its target gene,ITGA2,is highly expressed in colorectal cancer,and ITGA2 is involved in tumor proliferation,invasion,and metastasis.In this chapter,we intend to further explore the functional role of miR-16-5p/ITGA2 in colorectal cancer.Methods:Firstly,after over expression or inhibition of miR-16-5p,its effects on proliferation,invasion,migration,and apoptosis of colorectal cancer cells were detected using EdU assay,Transwell assay,and flow cytometry.Then,the proliferation,invasion,migration,and apoptosis of colorectal cancer were observed under overexpression of miR-16-5p and simultaneous overexpression of ITGA2.Finally,the effect of miR-16-5p/ITGA2 on the tumorigenic ability of colorectal cancer cells in vivo was verified in animals.Results:Overexpression of miR-16-5p inhibited the proliferation,invasion and migration of colorectal cancer cells.It promoted their apoptosis,while inhibition of miR-16-5p promoted the proliferation,invasion and migration of colorectal cancer cells and inhibited their apoptosis.Mimic treatment of miR-16-5p followed by overexpression of ITGA2 restored the proliferation and migration ability of tumor cells.It resisted apoptosis,and overexpression of miR-16-5p or silencing of ITGA2 both inhibited the growth of transplanted tumors in nude mice.Conclusion:miR-16-5p inhibits colorectal cancer cell proliferation and migration and promotes apoptosis through the downstream target gene ITGA2.miR-16-5p low expression and ITGA2 high expression in colorectal cancer promote the development of colorectal carcinogenesis.Chapter 3 Expression and clinical significance of RHEB in colorectal cancerObjective:We clarified in the previous two chapters that ITGA2 is an essential tumor-promoting protein in colorectal cancer.Previous studies have shown that the tumor-promoting mechanism of ITGA2 is mainly the activation of the AKT/mTOR signaling pathway.The mTOR signaling pathway is thought to be highly activated for a long time as the primary pathogenesis of colorectal cancer,but the therapeutic effect of its small molecule inhibitors is poor,so further understanding of the abnormal mTOR signaling in colorectal cancer RHEB is a conserved small GTPase that activates mTOR signaling and regulates cell growth and is poorly studied in colorectal cancer.This part aims to investigate the expression of RHEB in colorectal cancer and its paracancerous tissues and its clinical significance.Methods:The GEO database and TCGA database were used to analyze the difference in RHEB expression in colorectal cancer and its paraneoplastic tissues.Then 83 colorectal cancer and adjacent paraneoplastic tissue specimens were collected for immunohistochemistry(IHC)validation.Finally,the correlation between RHEB and the clinicopathological features of colorectal cancer was analyzed using statistics.Results:Based on the GEO database and TCGA database,we found that RHEB was highly expressed in colorectal cancer.Meanwhile,we found strong positivity for RHEB in colorectal cancer tissues and weak positivity for RHEB in paracancerous tissues based on clinical tissue samples.There was a positive correlation between RHEB and the important cancer Ki-67.Based on patient clinical data,we found that RHEB was more expressed in poorly differentiated colorectal cancer tissues than in highly differentiated colorectal cancer tissues.RHEB expression was stronger in colorectal cancer tissues with lymph node metastasis than in cancer tissues without lymph node metastasis.In addition,RHEB expression increased with the progressive increase of Dukes’ stage.Conclusion:RHEB was highly expressed in colorectal cancer tissues and correlated with patients’ tumor differentiation,Dukes’ stage,and lymph node metastasis.Chapter 4 RHEB regulates the mTOR signaling pathway in colorectal cancerObjective:The mTOR(mammalian target of rapamycin)signaling pathway regulates various tumor biological functions,including proliferation,metastasis,survival,and angiogenesis.And is a key signal for colorectal carcinogenesis and development.Our previous study showed that RHEB is highly expressed in colorectal cancer and correlates with the clinicopathology of colorectal cancer.This chapter aims to explore and validate that RHEB can regulate the mTOR signaling pathway in colorectal cancer.Methods:RT-qPCR and protein blot analysis was used to detect mRNA and protein levels of RHEB and mTOR signaling pathway related genes in colorectal cancer and its paracancerous tissues and to analyze their correlation.RT-qPCR and Western Blot were used to analyze the changes of mTOR signaling pathway-related protein expression after siRNA knockdown of RHEB in HCT116 cells transfected with RHEB.Results:The expression levels of RHEB and mTOR-related genes in colorectal cancer tissues were significantly higher than those in paracancerous tissues.The expression of RHEB and mTOR-related genes were positively correlated.In in vitro cell experiments,we found that silencing RHEB expression mTOR signaling pathway-related gene expression levels were significantly reduced.Conclusion:In colorectal cancer,RHEB activates the mTOR signaling pathway.Chapter 5 Study of RHEB regulation of colorectal cancer cell functionObjective:The above studies demonstrated that RHEB is highly expressed in colorectal cancer tissues and activates the mTOR signaling pathway.This chapter aims to clarify the effect of RHEB on the cell biology of colorectal cancer and further explore the impact of RHEB on the function of mTOR inhibitors.Methods:The Double-layer agarose assay and the clone formation assay were used to study the effect of RHEB on colorectal cancer cell colony formation ability and stemness.The impact of RHEB on the proliferation of colorectal cancer cells was examined using CCK-8.The impact of RHEB on the colorectal cancer cell cycle and apoptosis was detected using flow cytometry.Finally,a subcutaneous tumor-forming model in nude mice was constructed to analyze the effect of RHEB on the growth and proliferation of colorectal cancer cells in vivo.Results:The Clone formation assay showed that silencing RHEB significantly decreased colorectal cancer cell colony formation ability.The double-layer agarose assay showed that silencing RHEB significantly reduced the malignancy of colorectal cancer cells.CCK-8 assay showed that silencing RHEB expression significantly reduced the proliferation ability of colorectal cancer cells.Flow cytometry assay revealed that silencing RHEB increased the number of G0/G1 phase cells,decreased the number of G2/M and S phase cells in colorectal cancer cells,and significantly increased the apoptosis rate.Through animal experiments,we also found that downregulation of RHEB expression inhibited colorectal cancer cell growth in vivo.Conclusion;RHEB promotes the proliferation and stemness of colorectal cancer cells,and inhibition of RHEB promotes apoptosis of colorectal cancer cells.23 figures,1 table,113 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