Dysregulation Of MiR-144-5p/RNF187 Axis Contributes To The Progression Of Colorectal Cancer

Background:Colorectal cancer（CRC）is one of the most common and lethal malignancies.With the change of lifestyle,the incidence of CRC has gradually increased,becoming a public problem seriously endangering human health and bringing a serious economic burden to the society.Current treatments for CRC include surgery,radiotherapy,chemotherapy,targeted therapy,and combination therapy.Although the 5-year survival rate of CRC patients has improved significantly with the great progress of these treatments,the prognosis of advanced CRC remains extremely poor.Therefore,it is crucial to elucidate the precise molecular regulatory mechanisms in the occurrence and development of CRC,which is of great significance for the development of new CRC diagnosis,prevention and treatment strategies.RING finger protein 187（RNF187）belongs to the E3 ligase family containing the RING domain,and it has been recently reported that it is involved in the occurrence and development of various malignant tumors.However,the role of RNF187 in CRC pathogenesis and progression remains unclear.Objective:To study the expression and regulatory mechanism of RNF187 in CRC and its effect on the biological behavior of CRC cells.Methods:The expression levels of RNF187 m RNA and miR-144-5p in CRC specimens and paired adjacent colon tissues were analyzed by qRT-PCR.The protein level of RNF187 was determined in CRC specimens and normal colon tissues by western blot.The distribution of RNF187 in cells,and the expression in CRC tumor tissue and normal tissue were analyzed by immunohistochemical staining.RNF187 m RNA expression and protein level in NCM460 cells and CRC cell lines CaCO₂,SW480,H29and HCT-116 were detected by western blot.CaCO₂ and SW480 cells were transfected with RNF187 si RNA or negative control si RNA,respectively,at a final concentration of 100 n M.24 hours after transfection,cell proliferative capacity was examined by CCK8 assay at24 hours,48 hours and 72 hours,respectively.CaCO₂ and SW480 cells were transfected with RNF187 si RNA or negative control si RNA for 48hours before colony formation experiments were performed.Ten days after Colony-forming assay,the number of cell monoclonal was quantified.To establish a CRC xenograft mouse model,a mixture of CaCO₂ cells（2×10⁶）and Matrigel was injected subcutaneously into the left buttocks of mice.Seven days after injection,mice were injected intratumorally with RNF187 si RNA（1 nmol per mouse）or an equivalent amount of negative control si RNA every 3 days for 21 days.Twenty-eight days after inoculation,mice were sacrificed and xenograft tumors were dissected for weight measurement,by measuring tumor volume every 4days.CaCO₂ and SW480 cells were transfected with RNF187 si RNA or negative control si RNA for 24 hours.CRC cell migration ability was detected by cell scratch assay and Transwell assay.Cell invasion ability was determined by Transwell Matrigel invasion assay.The pmir GLO vector（emptyvector）,pmir GLO-RNF187-3’UTR-wtor pmir GLO-RNF187-3’UTR-mut were co-transfected into HEK293T cells with miR-144-5p or negative control miRNA,respectively.Cells were collected to detect luciferase activity.CaCO₂ and SW480 cells were transfected with miR-144-5p or negative control miRNA,and 48 hours later,RNF187 protein and m RNA levels in cells were detected by western blot and q PCR.After transfection of miR-144-5p or negative control miRNA,the proliferation,migration and invasion abilities of CRC cells were detected by CCK8,colony formation,cell scratch assay,Transwell assay and CRC xenograft mouse model.CaCO₂ cells were infected with RNF187 expressing lentiviral vector or empty vector,and after transfection with miR-144-5p,the proliferation,migration and invasion ability of CRC cells were detected by CCK8,colony formation,cell scratch assay and Transwell assay.Results:RNF187 was broadly distributed in the nucleus and cytoplasm of CRC cells,and the protein level and m RNA expression of RNF187 in CRC tissues were significantly higher than those in normal tissues.The m RNA expression as well as the protein level of RNF187 were significantly increased in CRC cell lines compared with normal colon mucosal cell line cells.Results from an analysis of data from the TCGA database showed that patients with higher RNF187 expression had shorter overall survival compared with patients with lower RNF187 expression.In CaCO₂ and SW480 cells,knockdown of RNF187 significantly reduced the viability of CRC cells and the ability to form clones in vitro in a time-dependent manner.RNF187 si RNA treatment reduced tumor weight and tumor volume in tumor-bearing mice.The cell scratch test results showed that the scratch repair rate of CaCO₂ or SW480 cells transfected with RNF187 si RNA was significantly lower than that of CRC cells transfected with negative control si RNA.Meanwhile,Transwell experiments found that transfection of RNF187 si RNA significantly reduced the number of migrating and invasive CaCO₂ or SW480 cells.By bioinformatics analysis,RNF187 was found to be a direct target of miR-144-5p.In HEK-293T cells transfected with RNF187-3’UTR-wild-type（wt）but not RNF187-3’UTR-mutant（mut）,miR-144-5p transfection resulted in a marked decrease in luciferase activity.Furthermore,miR-144-5p transfection reduced RNF187 protein and m RNA levels in CaCO₂ and SW480 cells.Moreover,the expression of miR-144-5p was down-regulated in CRC tissues,which was negatively correlated with the expression of RNF187.Overexpression of miR-144-5p significantly inhibited the proliferation of CRC cells in vivo and in vitro.Cell scratch test results showed that CaCO₂ or SW480 cells transfected with miR-144-5p mimic had lower wound repair rates compared to cells transfected with negative control miRNA.Meanwhile,miR-144-5p significantly reduced the number of migrating and invasive CaCO₂ or SW480 cells as assessed by Transwell assay.RNF187expression in CaCO₂ cells stably expressing RNF187 was much higher than in cells infected with empty vector virus.In the in vitro experiments,the proliferation of RNF187-overexpressed CaCO₂ cells was significantly higher than that of cells infected with empty vector virus particles when co-transfected with miR-144-5p.Furthermore,overexpression of RNF187 significantly blocked the inhibition of the weight and volume of tumor xenografts by miR-144-5p.Further research showed that the migration and proliferation ability of RNF187-overexpressed CaCO₂ cells was significantly higher than that of cells infected with empty vector virus particles when co-transfected with miR-144-5p.Conclusion:In conclusion,miR-144-5p directly targets and inhibits the expression of RNF-187,and the abnormally regulated miR-144-5p/RNF187 axis promotes tumor growth and metastasis in CRC,and indicates poor prognosis of CRC patients.