| Objective: Colorectal cancer(CRC)is a disease in which malignant cells form in the tissues of the colon and rectum.CRC is the third frequently diagnosed malignancies and the second leading cause of cancer-related deaths.According to the estimation for CRC,there are approximately 1,800,977 new cases and 861,663 deaths in 2018 around worldwide.In clinical setting,the therapeutic regimens for CRC patients include surgical resection,targeted therapy,chemotherapy,radiotherapy and immunotherapy.Despite dramatic improvements that leading to longer overall patient survival has been made in recent decades,the 5-year survival ratio(approximately57%)for CRC is still unsatisfactory in China.Consequently,it is urgent to identify novel therapeutic strategies or targets for CRC treatment.Most recently,The microRNA is receiving significant attention given its influence on a host of human diseases including cancer.Among these miRNAs,miR-425-5p is located on human chromosome 3 and has been found to frequently expressed in various types of cancer,including gastric,cervical,and hepatocellular carcinoma.While little are known about its functional role and underlying mechanisms in CRC.Here,we investigated the effect of miR-425-5p on the biological behavior of CRC cells and elucidated its underlying molecular mechanism.Methods: In current study,we collected 30 paired peripheral blood samples from colorectal cancer patients and healthy subjects.Meanwhile,24 paired colorectal cancer and para-cancer tissues were collected for the following experiments.Quantitative Real-time PCR(qRT-PCR)was performed to detect the relative expression of mi R-425-5p in peripheral blood of CRC patients and health control.The levels of mi R-425-5p and CTNND1 in colorectal cancer tissues were determined by qRT-PCR.In vitro experiments,two CRC cell lines,LOVO and SW480,were used for in vitro experiments.The relative expression of miR-425-5p in cells were qRT-PCR.miR-425-5p mimic and miR-425-5p inhibitor were used to manipulated theexpression levels of miR-425-5p in LOVO and SW480 cells.Cell viability was detected by cell counting kit-8(CCK-8).While cell apoptosis and cell cycle distribution were quantified using flow cytometry following AnnexinV-FITC/Propidium staining(PI)or PI staining,respectively.Cell migration and invasion abilities of CRC cells were measured using wound healing assay and Transwell invasion assay,respectively.The relative expression levels epithelial-mesenchymal transition(EMT)-related genes and ?-catenin ? c-myc ? Cyclin D1 ? MMP7 and CTNND1 in cells were detected using qRT-PCR,western blotting,and Immunofluorescence(IF).The dual-luciferase reporter assay was used to determine the targeting relationship between miR-425-5p and CTNND1.A CRC xenograft tumor model was used to validate the anti-tumor activity of miR-425-5p inhibitor in vivo.Results: Our results showed the serum level of miR-425-5p in patients with CRC was significantly higher than that in healthy controls(P<0.05),and the expression level of miR-425-5p in colon cancer tissues was significantly higher than that in adjacent control tissues(P<0.01).The expression of CTNND1 in CRC tissues was significantly lower than that in adjacent tissues(P < 0 05).The miR-425-5p mimic could effectively up-regulate miR-425-5p levels in CRC cells(P<0.001),while miR-425-5p inhibitor reduce its level(P<0.001).Moreover,up-regulation miR-425-5p using miR-425-5p mimic significantly increased the CRC cell viability when compared with control(P<0.001).Meanwhile,knockdown mi R-425-5p obviously suppressed cell viability in comparison of control(P<0.001).Additionally,up-regulation of miR-425-5p also significantly enhanced the cell cycle progression,as supported by the decreased of cells in G0/G1 phase(P<0.001),as well as the increased of cells in S phase(P<0.05)and G2/M phase(P<0.01).Consistently,our results also demonstrated that the migratory(P<0.01)and invasive(P<0.01)activities of CRC cells are markedly suppressed by miR-425-5p knockdown.Moreover,our findings also revealed that up-regulation of miR-425-5p could significantly promote EMT process in CRC cells,as evidenced by the increase of mesenchymal markers(Fibronectin,N-cadherin,and Vimentin)(P<0.001),as well as the decrease of epithelial marker(E-cadherin)(P<0.001).Similarly,IF results also confirmed that up-regulation of miR-425-5p increased fibronectin level in cells,while mi R-425-5p knockdown effectively reduced fibronectin.Moreover,up-regulation of mi R-425-5p also enhanced the distribution of ?-catenin in nuclear(P<0.001),while reduced its levels in cytoplasm(P<0.001).Western blotting results also showed that up-regulation of miR-425-5p could obviously rise c-myc,Cyclin D1,and MMP-7 levels(P<0.001),while miR-425-5p knockdown revered these effects.We found that CTNND1 was a predicted potential target of miR-425-5p using miR-code database.Consistently,CRC cells co-transfected with miR-425-5p mimics and CTNND1-wt showed obvious decrease in luciferase activity,confirming the target relationship between miR-425-5p and CTNND1(P<0.001).Meanwhile,the co-transfection of miR-425-5p and CTNND1-mut showed little change on the luciferase activity,which suggesting that CTNND1 was the target gene of miR-425-5p.Moreover,a negative correlation between miR-425-5p and CTNND1 m RNA and protein levels were also found(P<0.001).Our results demonstrated that miR-425-5p controlled the cell viability,cell cycle,migration,invasion,and EMT process through negatively regulating CTNND1 in CRC cells.In addition,our results showed that knockdown CTNND1 in miR-425-5p inhibitor transfected cells could significantly promote cell proliferation(P<0.01),cell cycle progression(P<0.001),migration(P<0.05),invasion(P<0.01),and EMT process(P<0.001).Consistent with in vitro observation,miR-425-5p knockdown could suppress tumor growth and metastasis in vivo,as evidenced by the reduced tumor volume(P<0.001)and decreased metastatic nodule(P<0.01).Conclusion: Our results suggest that miR-425-5 was over-expressed,while CTNND1 expression was decreased in CRC tissues.Knockdown miR-425-5p significantly down-regulates the expression of ?-catenin and c-myc,Cyclin D1 and MMP-7 in the nucleus,and inhibits CRC cell proliferation,cycle,metastasis and EMT.Further studies revealed that miR-425-5p promoted CRC development and progression through directly targeting CTNND1 and reducing its levels.Therefore,our study provided a theoretical basis to support miR-425-5p as a potential target for CRC. |
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