Biological Function And Mechanism Research Of No-coding RNA MiR-9 And Circ-BANP In Modulating The Malignant Proliferation Of Colorectal Cancer

Background&Objective:Colorectal cancer(CRC)is one of the most common cancers in China.Sustaining proliferative signaling could promote the tumorigenesis and progression.Recently,increasing evidence has shown that non-coding RNAs(ncRNAs)play vital roles in regulating the proliferation of tumor cells.microRNA(miRNA)and circular RNA(circRNA)are two important ncRNAs.Thus,this thesis will demonstrate the biological function and mechanism of the two ncRNAs in modulating the proliferation of CRC.Methods and Results:1.Real-time PCR and in situ hybridization assays were used to determine the expression of miR-9 in 38 CRC cancerous tissues and adjacent normal tissues(n=38).The results showed that the expression of miR-9 in cancerous tissues was downregulated,compared with that in adjacent normal tissues.Kaplan-Meier survival analysis revealed that downregulated miR-9 was correlated with poor prognosis of patients with CRC(P=0.035).2.Lenti-pre-miR-9 expressing plasmid was utilized to enhance the expression of miR-9 in HCT116 and HT29.The results of CCK-8 and clone formation assays suggested that upregulation of miR-9 can significantly inhibit CRC cell proliferation.Annexin V/7-AAD kit was used to examine the apoptosis of pre-miR-9-transfected CRC cells.It was found that the proportion of apoptotic cells was significantly increased in pre-miR-9-transfected CRC cells compared with control cells((P<0.05)).3.The results showed the expression of UHRF1 significantly decreased in stable pre-miR-9-transfected cells,whereas UHRF1 was upregulated after blocking expression of miR-9 with antagonistic oligonucleotide in both cell lines.4.To further analyze the relationship between miR-9 and UHRF1,we used two computational algorithms(miRanDa and TargetScan)to predict the targets gene of miR-9.The bioinformatic methods suggested that miR-9 could target the 3’-UTR region of UHRF1.Dual luciferase reporter assay showed that,compared to miRNA control,miR-9 led to a significant relative luciferase activity reduction in the wild-type UHRF1 3’-UTR plasmid.However,luciferase activity was not reduced in the 3’-UTR with mutant binding sites.These results showed that miR-9 may restrain UHRF1 expression by targeting its 3’-UTR.5.Immunohistochemistry was performed to determine the UHRF1 expression in 38 CRC paired tissues.A total of 14 cancer lesions(36.8%)were stained positive and four adjacent normal tissues were positive(10.5%).The expression of UHRFI in cancerous tissues was upregulated,compared with that in adjacent normal tissues.Kaplan-Meier survival analysis revealed that overall 5-year survival was significantly lower in patients with high UHRFI expression as compared to patients with low UHRFI expression(P=0.002).6.Quantitative RT-PCR was used to analyze the expression of UHRF1 in 15 patients with CRC.The result showed that UHRF1 was overexpressed in CRC cancerous tissues(P<0.05).Spearman’s correlation analysis showed that the UHRF1 expression was inversely correlated with miR-9 expression in CRC(r=-0.606,P=0.0004).7.The relationship between circRNA and CRC proliferation was also analyzed.CircRNA expression profiles were analyzed using a microarray to determine differentially expressed circRNAs in 3 paired CRC cancerous and adjacent normal tissues.Base on the preliminary small sample experiment,we planned to focus on circ-BANP in the further researches.qRT-PCR was used to determine the expression of circ-BANP in 35 paired CRC cancerous and adjacent normal tissues.The results showed that the expression of circ-BANP was upregulated,compared with that in adjacent normal tissues(P<0.05).In situ hybridization results showed that circ-BANP expression level was remarkably upregulated in CRC tissues,as compared to matched normal tissues.circ-BANP was mainly localized in cytoplasm.8.Biological function of circ-BANP was also evaluated with circ-BANP siRNA.The CCK-8 result displayed that the proliferation was reduced in si-circ-BANP group,compared with negative control group(P<0.05).Additionally,colony formation was significantly reduced in siRNA group.The results of qRT-PCR and western blot showed that circ-BANP siRNA has no effect on BANP expression,compared with control group(P>0.05).9.We found that circ-BANP has three has-miR-874 binding sites by using bioinformatics software.To further analyze the relationship between circ-BANP and miR-874,dual luciferase reporter assay was performed.Compared to blank control and circ-BANP+miRNA inhibitor-NC,miR-874 mimic led to a significant relative luciferase activity reduction in the wild-type circ-BANP plasmid.Luciferase activity of circ-BANP+miR-874 inhibitor group was upregulated,compared with blank control group(P<0.05).These results suggested that miR-874 inhibitor could affect the luciferase activity via blocking the binding sites of miR-874 and circ-BANP.Luciferase activity of mut-circ-BANP+miR-874 mimic group was not significantly changed,compared with the groups of blank and out-circ-BANP+miRNA inhibitor-NC.These results suggested that miR-874 could bind with circ-BANP specifically.10.Pull-down assay was utilized to further validate the relationship between circ-BANP and miR-874.qRT-PCR result showed that circ-BANP expression was 4.32-fold higher in bio-miR-874 pull-down group than that in control group(P<0.05).To determine the binding relationship between circ-BANP and miR-874 visually,in situ hybridization co-localization assay was employed to detect the spatial relationship between circ-BANP and miR-874.The result displayed that circ-BANP and miR-874 were co-localized in CRC cells and suggested that circ-BANP could bind with miR-874.Conclusion:This thesis demonstrated that miR-9 and circ-BANP were differentially expressed between colorectal cancerous tissues and adjacent normal tissues.Additionally,the results of diverse assays suggested that ncRNAs miR-9 and circ-BANP could function as regulator of CRC cell proliferation,and could serve as novel diagnostic and therapeutic markers for CRC.