Microrna-21(MiR-21) Promotes Growth, Invasion And Metastasis, In Colorectal Cancer Cells By Regulating PTEN/AKT/PI3K/mtor Signal Pathway

PARTⅠCLINICAL SIGNIFICANCE OF MIR-21ANDRELATED PROTEINS OF PTEN/PI3K／AKT SIGNALPATHWAY IN COLORECTAL CANCERObjective: To investigate the expression level and clinical significance ofmiR-21and related proteins of PTEN/PI3K／AKT signal pathway inpatients with colorectal cancer(CRC).Methods:The level of miR-21was detected with real time fluorescencequantitive RT-PCR(FQ-PCR), the related proteins of PTEN/PI3K／AKTsignal pathway were evaluated by immunohistochemistry(IHC).Therelationship between miR-21and PTEN/PI3K／AKT signal pathway,theirassosication with clinicopathological parameters was also investigated.Results: Compared with normal tissue of adjacent cancer, the level ofmiR-21, PI3K and AKT was significantly higher in CRC（p<0.05）,thelevel of PTEN was significantly lower in CRC p<0.05）. The expression of miR-21,PTEN,PI3K and Akt had no relationship with age, sex and tumorlocation，was associated with invasion depth, differentiation degree,TNMstage and lymph node metastasis of CRC（p<0.05）. The expression ofmiR-21was positively related with PI3K and Akt （p<0.05）, negativelywith PTEN（p<0.05）. The expression of PI3K was positively related withand Akt （p<0.05）. The expression of PTEN was negatively related withPI3K and Akt （p<0.05）.Conclusion: miR-21can activate the P13K／Akt signal pathway throughinhibition of PTEN, promote initiation and development of CRC.PART ⅡTHE EXPRESSION AND FUNCTION RESEARCH OFMICRORNA-21（MIR-21） IN COLORECTAL CANCERObjective: To explore the expression of microRNA-21(miR-21) incolorectal cancer(CRC) and its target gene, and to investigate the effect ofmiR-21inhibitor on proteins of PI3K/PTEN/AKT signal pathway.Methods The anti-sense oligonucleotide（ASO）technology was employedto suppress the expression of miR-21in HCT116cells. The changes inPTEN, PI3K,Akt, MMP-9,mTOR protein expressions were detected byWestern blotting. The potential target mRNA prediction was performed byBioinformatics. Dual luciferase report gene assay was used to identify thetarget gene of miR-21． Results: The level of miR-21was the highest in HCT116cell, lowest inSW480cell. The suppression of miR-21can lead to the increment of PTENprotein, the downregulation of PI3K,Akt, MMP-9and mTOR. PTEN wasidentified as one of target genes of miR-21in CRC cell．Conclusion: PTEN, as one of target genes of miR-21in CRC cell, canregulate the process of CRC.miR-21was overexpressed in CRC,andpromoted cancer cell invasion and metastasis by inhibiting PTEN．PART Ⅲ THE EFFECT OF MICRORNA-21DOWN-REGULATION ON BIOLOGICAL BEHAVIOUR INCOLORECTAL CELLSObjective: To observe the effects of MicroRNA-21(miR-21)downregulation on biological behavior, such as proliferation, apoptosis, cellcycle and invasion and migration of colon cancer HCT116cells bytransfected with miR-21inhibitor through LipofectamineTM2000.Methods:The influence on the proliferation, colony formation，apoptosis,cell cycle,invasion and migration in CRC lines were evaluated by methylthiazolyl tetrazolium (MTT),colony formation assay,flowcytometry(FCM),Transwell invasion and migration test, respectively; Thechange of PTEN expression and activity after miR-21depression in CRCcell line HCT116were evaluated with Western blotting and luciferase reporter assay.Results: Compared with negative control(NC)group and blankcontrol(MOCK)group, the expression level of miR-21significantlydecreased(p<0.05)after transfected with Anti-miRTMmiR-21Inhibitor(IN).MTT assay showed that down-regulation of miR-21producedan obvious inhibitive effect on the proliferation of HCT116cellline(p<0.05);the colony formation ability decreased after miR-21downregulation(p<0.05）.FCM showed that miR-21depression promotedapoptosis(p<0.05）, increased G0／G1stage(p<0.05）,declined S stage ofcell line HCT116（p<0.05）.Invasion and migration test showed that abilityof cell invasion migration were significantly inhibited（p<0.05）; theWestern blotting and luciferase reporter assay also showed that PTENexpression （p<0.05）and activity （p<0.05）increased remarkably aftermiR-21inhibition.Conclusion: miR-21may regulate the biological behavior throughinhibition of PTEN, promote initiation and development of CRC, PTENmay be a target gene in CRC.