MiR-34a Downregulates NOTCH1and Suppresses Proliferation In Colorectal Cancer

Part ⅠHsa-miR-34a target prediction and analysisObjective: To predict and analyze miR-34a target, we uesbioinformatic approaches to explore more regulatory effect of miR-34a atpost-transcriptional level. Method: we predict miR-34a target site throughmiRGen software. Hsa-miR-34a was used as search term and chosen theintersection of the three algorithms: miRanda， PicTar， TargetScans.Candidate targets were related to cell proliferation. Result: Eight geneswere screened as candidate targets through above methods. NOTCH1waschosen for thorough investigation as result of important role of NOTCH1incell proliferation. We found that3′UTR of NOTCH1contains a binding siteof miR-34a, simultaneously the sequence is highly conserved in all fourgenomes and integrate free energy is lower. Conclusion: miR-34a has a lotof potential regulatory targets and NOTCH1may be one of targets. Part ⅡmiR-34a targeting regulated NOTCH1gene and affectedproliferation of SW480cellsObjective: To analyze miR-34a target regulate expression of NOTCH1and affect proliferation of SW480cells．Method: NOTCH1waspredicted as the specific target gene of miR-34a by bioinformationmethod. The dual luciferase vector which contain3’-UTR of Notch1was constructed, and was regared as the binding sites with miR-34a.miR-34a affect luciferase activity of3’-UTR of Notch1,which weredetected by using the dual luciferase assay system.The expression levelof Notch1protein was affected by miR-34a which were detected byusing western blot. Proliferation of SW480cells which was affected bymiR-34a were measured by MTT assay and flow cytometry.Result:3’-UTR of Notch1which was contained in the dual luciferaserecombinant vector was successfully constructed and vertified byenzyme digestion and gene sequence methods. The results of luciferaseassays revealed that miR-34a could negative regulate the luciferaseactivity from the reporter construct containing the NOTCH13’-UTRsignificantly. Protein of NOTCH1was found to be negative regulatedby miR-34a． miR-34a inhibited proliferation of SW480cell.Conclusion: Theses results suggested that miR-34a negative targectedregulates the expression of NOTCH1and inhibit the proliferation of SW480cells． Part ⅢmiR-34a affect cell proliferation of SW480cells through targetingregulate NOTCH1and its molecular mechanismsObjective: To analyze the effect of miR-34a on the proliferation of SW480colorectal cancer cells and to investigate the probable mechanisms.Method: pLenti6-NOTCH1-shRNA plasmid and pLenti6plasmid wererespectively transfected into SW480cells, and the empty vector andpNL-NICD vector were transfected respectively into SW480-miR-34a cells.After transient transfection, the cells were cultured for three days to detectthe expression of NOTCH1and p53through Western blot. Result:Expression of NOTCH1and p53were detected by Western blot in order tointerference efficency. pLenti6-NOTCH1-shRNA significantly inhibited theexpression of NOTCH1(F=24.13，p=0.000), the inhibitory rate were43.94%. The marked increase of p53protein level were observed in thecorresponding groups (F=6.65， p=0.04), and the expression of p53increase by82.3%. These results show that expression level of p53wasnegatively correlated with NOTCH1. Compared with untreated SW480-miR-34a cells, Expression of NOTCH1was enhanced and p53wasdecreased which were observed in SW480-miR-34a cells transfected withby pNL-NICD (P<0.01). Conclusion：The proliferation inhibition effectof miR-34a could be regared through miR-34a mediated increase of p53bytargeting inhibit NOTCH1 Part ⅣEffect of miR-34a on proliferation of SW480Cell Transplantedto Nude MiceObjective:To investigate the effect of miR-34a on proliferation ofcolorectal cancer SW480cells transplanted to nude mice and the relevantmechanism． Method: SW480cells， SW480-miR-34a cells andSW480-control cells were injected subcutaneous of nude mice in order toobserve growth of xenograft tumor．The tumor growth chart was depictedby surveying tumor diameter． The nude mice were killed after the tumorcell were transplanted45days; and to weigh all xenograft tumors in orderto cipher the tumor inhibition rate.The morphology of tumor was observedthrough HE staining assay.The expressions of NOTCH1protein in tumortissues of various groups were measured by Western blot andimmunohistochemical methods.Results: Comparing with SW480cells group, the tumors weight of nude mice of SW480-miR-34a cell groupdecreased significantly（F=1598,p＜0.01），tumor inhibition rate was73.60％．whereas the sizes and weights of tumors in SW480-control cellgroup showed no significant difference（p＞0.82）.Comparing with theother two groups, the dimension of cells，the ratio of nucleus to cytoplasmand mitosis figures in tumor xenografts in SW480-miR-34a cell groupdecreased more significantly.The expression of NOTCH1protein inSW480-miR-34acell group decreased more significantly than that ofSW480cell group through western blot and immunohistochemica（lp＜0.01），whereas that in SW480-control cell group showed no significantdifference （p＞0.05）.Conclusion: miR-34a could inhibited theproliferation of SW480cells in nude mice significantly, which might beinterrelated with down-regulating the expression of NOTCH1protein bylatent mechanism．...