| Part One Research in the specific expression profile of microRNAs and their Clinical significance in serum samples from patients with colorectal cancerBackground:Colorectal cancer(CRCs)is the third most common cancer following lung cancer and breast cancer in the world,especially in developed countries.The 5-year survival rate reaches to 60%?95%.However,the 5-year survival rate of the advanced CRC is only 35%,which is a serious threat to human health.The pathogenesis of CRC is complex,which is characterized by multiple gene mutation and signal network regulation disorder,and obvious genetic variation among individuals.Accumulated studies have shown that micro RNAs(miRNAs)can regulate the expression of target genes at the posttranscriptional level by fully or incompletely pairing with their 3'-UTR of targeted mRNAs,which participate in the regulation of cell proliferation,differentiation,apoptosis,and metabolism and so on.A miRN A can regulate the expression of multiple genes,and several miRNAs can also regulate one gene finely.The expression of miRNAs is highly conserved with space-and time-dependent specificity.Accordingly,different diseases may have different miRNAs specific expression profiles,and the same disease may have different organ-or tissue-specific expression profiles of miRNAs.MiRNAs is closely related to the occurrence and development of human tumors and the discovery of circulating exosomal miRNAs plays a new role in the regulation of tumor gene expression.Tumor-derived exosomal miRNAs are expected to become new circulating tumor markers and therapeutic targets due to their effects on angiogenesis,tumor cell growth,and invasion and so on.Accumulating studies have demonstrated that miRNAs mediated by exosome play a key role in regulating intercellular communication and many other pathophysiological processes.These findings will provide potential biological targets for the diagnosis,treatment and prognosis evaluation of CRC.Objective:To study the specific expression profile of miRNAs in serum exosomes of patients with CRC.Methods:3 patients with CRC and 3 healthy controls were randomly selected to screen the specific expression profiles of miRNA in serum exosomes of CRC patients by miRNA Microarray microarray.77 patients with CRC and 20 healthy controls were selected for clinical validation of serum exosomal samples by qRT-PCR method.We evaluate the relationship between differential expressed miRNAs and the status of vascular invasion,lymph node metastasis,liver metastasis and TNM-staging of patients with CRC.The potential targeted genes of differentially expressed miRNAs were screened based on the prediction in three databases of Targetscan,microRNA.ORG,and miRDBA databases.The potential signal pathways regulated by the differentially expressed serum exosomal miRNAs were performed by the pathway enriched analysis.Results:There were 39 abnormal expressions of miRNAs in serum exosomes of CRC patients,29 of which were down-regulated and 10 miRNAs were up-regulated.After verification by qRT-PCR,5 serum exosomal miRNAs(miR-638,miR-5787,miR-8075,miR-6869-5p and miR-548c-5p)were significantly down regulated,and 2 serum exosomal miRNAs were upregulated(miR-486-5p and miR-3180-5p).Patients with low levels of miR-638 miR-8075 and miR-548c-5p were at higher risk of developing vascular infiltration,lymph nodemetastasis and liver metastasis.In addition,patients with low level of miR-638 and miR-8075 in serum exosomes were at later stage of TNM.Bioinformatics analysis suggested that miR-5787 miR-8075 miR-6869-5p and iniR-548c-5p may be involved in the regulation of glucose metabolism in CRC by regulating the key target gene HIF-1,PGK1 and so on.TLR4 is one of the targeted genes of miR-6869-5p.Pathway enrichment analysis showed that these differentially expressed miRNAs may influence the development of CRC by regulating tumor-related signal transduction pathways such as WNT-MAPK.Conclusion:There are specific miRNAs profiles in serum exosomes of CRC.The abnormal expression of miRNAs may influence the occurrence,development and metabolism of CRC by regulating its signal transduction pathways,but the specific molecular mechanism needs to be further investigated.Part Two Effect of miR-6869-5p on colorectal cancer targeting TLR4/NF-?B and its clinical significanceBackground:CRC is one of the most fatal cancers in the world.Its pathogenesis has been extensively studied at molecular levels.MiRNAs are a kind of small non-coding RNAs that targetedly regulate the expression of mRNAs and participate in many physiology and pathological process such as cell growth,differentiation,apoptosis and so on.At present,many studies have found that there are abnormally expressed miRNAs in many tumors,including CRC.Many studies have shown that miRNAs can be used as biomarkers for the diagnosis and prognosis estimation of tumors,and to judge the growth and invasion of tumor,and tumor metastasis and response to chemotherapeutic agents.Studies have shown the presence of dysregulated miRNAs in tissues and body fluids(such as serum,plasma,and fecal samples)in patients with CRC.Recently,some studies have shown that miRNAs could exert effect on anticancer drugs,which can be regarded as predictor of successful treatment.Exosome is a biofilm structure with diameter in 40-100nm.It is widely distributed in saliva,plasma,milk and other body fluids.It contains a variety of bioactive substances,such as mRNA,miRNAs,cytokines,and so on.Exosome mediates cell communication and participates in the regulation of immune response,antigen presentation,immune response,cell differentiation,cell metabolism,tumor invasion and so on,by directly fusing with the target cell membrane and membrane protein binding.Exosome-mediated miRNA is closely related to the development of CRC.These miRNAs and their potential regulatory mechanisms will provide a theoretical supplement for the study of the development mechanism of CRC and provide novel biomarkers for the diagnosis and targetment of CRC.Objective:To study the expression,regulation and clinical significance of miR-6869-5p in CRC.Methods:The expression of miR-6869-5p in serum exocrine of CRC and tumor tissues was detected by qRT-PCR in 142 patients with CRC and 40 healthy controls.The relationship between the expression level and TNM stage,lymph node metastasis and liver metastasis in CRC patients was estimated.Bioinformatics software was used to predict the possible target genes of miR-6869-5p.We used luciferase reporter assay to confirm that TLR4 was a targeted gene of miR-6869-5p.The relationship between miR-6869-5p and TLR4 was further analyzed in vitro in CRC cells.Cell proliferation was detected by CCK-8 and EdU.The expression of IL-6 and TNF-a was detected by ELISA,and the activation of NF-?B mediated by LPS was analyzed by immunofluorescence assay.The effect of miR-6869-5p on tumor growth was observed in a subcutaneous tumor bearing model.Survivval survival analysis was used to evaluate the effect of serum exosomal miR-6869-5p on survival rate of CRC patients.Results:The expression of miR-6869-5p in serum exosomes and tumor tissues of CRC patients was significantly down-regulated.The expression level of miR-6869-5p was negatively correlated with TNM stage,lymph node and liver metastasis in CRC patients.TLR4 was the targeted gene of miR-6869-5p.MiR-6869-5p regulated the proliferation,apoptosis and activation of CRC cells by regulating TLR4/NF-?B signaling pathway.MiR-6869-5p could inhibit the growth of tumor cells in vivo.Additionally,the 3-year survival rate of CRC patients with low level of miR-6869-5pin serum exosomes was decreased.Conclusion:MiR-6869-5p can be used as a tumor suppressor to inhibit the growth of CRC cells by regulating TLR4/NF-?B signaling network,and the expression level of miR-6869-5p in serum exosomes can be used as a predictor of prognosis in CRC patients. |
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