Effect Of Regulating Cellgrowthand Its Mechanisms Of "FOXM1/miRNA/Target Protein" Molecular Networks On Ovarian Cancer SkOV3Cells

EXPRESSIONAND CLINICALSIGNIFICANCE OFFOXM1IN EPITHELIALOVARIAN CARCINOMAObjective:To verify expression of FOXM1in human ovarian cancertissues and ovarian cancer cell lines. Clinical significance of FOXM1inepithelial ovarian carcinomas was analyzed.Methods:The expression of FOXM1in epithelia ovarian carcinoma(n=68), benign epithelia ovarian tumors (n=21), normal ovarian tissues(n=24), were detected by Immunocytochemistry, and human ovariansurface epithelial cells HOSEpiC, three cell lines of ovarian carcinoma(A2780, OVCAR3, SKOV3) were detected by indirect immunofluorescencetechnique and Western blot.Results: Expression of FOXM1in ovarian cancer tissues wassignificantly higher than that in benign ovarian tumors and normal ovariantissues (p <0.01).The expression of FOXM1in epithelia ovarian of stageⅢ～Ⅳ was significantly higher than that in epithelia ovarian of stageⅠ～ Ⅱ(P=0.011), and in poorly differentiated cells higher than that in highlydifferentiated cells(P=0.013). But no significant difference in the expressionof FOXM1was found in the pathological types. Expression of FOXM1inline A2780, OVCAR3and SKOV3cells were significantly higher thanHOSEpiC cells (P <0.01). Expression of FOXM1in SKOV3cells was thehighest in the three cells (P <0.05).Conclusion:Overexpression of FOXM1was detected in epithelialovarian carcinomas and three ovarian cancer cell lines, which is negativecorrelated with the cell differentiation and positive correlation with clinicalstage of epithelial ovarian carcinomas. PART TWOCONSTRUCTIONAND IDENTIFICATION OF SHORTHAIRPIN RNALENTIVIRALVECTOR TARGETINGHUMAN FOXM1GENEObjective: To construct and identify short hairpin RNA lentiviralvector targeting human FOXM1gene.Methods: shRNA sequence targeting human FOXM1gene wasdesigned and synthesized. The synthesized shRNA sequence was clonedinto lentiviral expression vector PLL3.7with U6promoter and green fluorescent protein by recombinant DNA technology. The recombinantplasmid was identified by double restriction digestion with Xba I/Nhe I andDNA sequencing. The mixture of correct recombinant plasmid andlentivirus packaging plasmidsp MD2G,pRsv-REV,pMDLg-pRRE wereco-transfected into293T cells by LipofectamineTM2000to collect andpury lentivirus virus particles. Lentiviral titer was detected by hole-by-holedilution method. Real time PCR and Western blot were used to detectFOXM1mRNAand protein respectively of SKOV3cells after infecting ourlentiviral particles96h.Results: Double restriction digestion analysis and DNA sequencingdemonstrated that the shRNA sequence targeting human FOXM1gene wassuccessfully inserted into the lentiviral vector pLL3.7. Lentiviral particleswere successfully packaged in293T cells, with a titer of1×108TU/ml.After infecting lentiviral particles96h, FOXM1mRNA expression ofSKOV3cell decreased to21.3%±2.09%, FOXM1protein decreased to17.6%±1.17%.Conclusion: A lentiviral shRNA expression vector targeting humanFOXM1gene is successfully constructed, which will provide basis tofurther reveal the molecular function of FOXM1. PART THREEEFFECTS OF GROWTH OF LENTIVIRUS-MEDIATEDFOXM1GENE SILENCING ON SKOV3CELLSObjective: To investigate the effects and possible molecularmechanisms of growth of lentiviral vector targeting FOXM1shRNA onSKOV3cells in vitro.Methods: After SKOV3cells infecting by lentiviral vector targetingFOXM1shRNA with a multiplicity of infection (MOI) of2096h,proliferation inhibition rate was detected by MTT assay, cell cyclesdetermined by flow cytometric analysis, cell apoptosis were observed byHoechst33258staining and TUNEL assay; and cell autophagy wereobserved by acridine orange staining, MDC staining, LC3-IIimmunofluorescence staining, scanning electron microscopy andtransmission electron microscopy. Changes of mRNA and protein levels ofP21, PLK1, Bax, Bcl-2, Beclin1, and LC3-II in SKOV3cells after infectingby lentiviral vector targeting FOXM1shRNA96h were detected by Realtime PCR and Western blot.Results: Lentiviral vector targeting FOXM1shRNA couldsignificantly inhibit the growth of SKOV3cells. After infected by lentiviralvector targeting FOXM1shRNA, the G1phase cells increased(P<0.01), cellautophagy activity was significantly enhanced with no obvious apoptoticmorphological changes, and expression of mRNA and protein of P21,Beclin1, LC3-II were significantly up-regulated(P <0.01), FOXM1,PLK1significantly down-regulated, but there were no change of Bax andBcl-2.Conclusion: Inhibiting FOXM1expression has a significantly effect ofinhibiting proliferation on SKOV3cells. Blocking SKOV3cells in the G1phase and inducing autophagic cell death (type II programmed cell death) byup-regulating the expression of P21, Beclin1, LC3-II, down-regulatingPLK1protein may be its molecular mechanism. PART FOURCONSTRUCTION OF EXPRESSION PROFILE OFDIFFERENTIALMIRNAIN SKOV3CELLSAFTERSILENCING FOXM1Objective: To verify expression profiles of differential miRNA inSKOV3cells after silencing FOXM1, and Determine relations of regulationbetween FOXM1and miRNA.Methods: miRNA chip technology was uesd to analyze expressionprofiles of differential miRNA in SKOV3cells after silencing FOXM1.Truth of difference of the differential miRNA from miRNA chip wasdetermined by real time PCR. Results: There were42differential miRNAs in SKOV3cells aftersilencing FOXM1, of which30miRNAs up-regulated and12miRNAsdown-regulated. miRNAs up-regulated more than5multiple wererespectively hsa-miR-451, hsa-miR-122, hsa-miR-636, hsa-miR-1297,hsa-miR-1301,hsa-miR-185,hsa-miR-659, of which miR-451and miR-122respectively up-regulated20multiple and95multiple. miRNAsdown-regulated more than5multiple were respectively hsa-miRPlus-E1201,hsa-miRPlus-E1074. miR-185was the real differential miRNA in SKOV3cells after silencing FOXM1showed by real time PCR.Conclusion: Expression profiles of differential miRNA in SKOV3cells after silencing FOXM1which included42differential miRNA weresuccessfully constructed. miR-185is real up-regulated miRNA in SKOV3cells after silencing FOXM1. PART FIVEEFFECT OF GROWTH OF MIR-185ON SKOV3CELLSAND PREDICTIONAND VERIFICATION OF ITSTARGET GENEObjective:To study the effect of growth when up-regulated ordown-regulated miR-185on SKOV3cells, and construct dual-luciferasereporter plasmid containing the3'UTR of target genes RAB14mRNA of miR-185. To verify target genes of miR-185from a structural point of view.Methods:①miR-185in SKOV3cells were up-regulated ordown-regulated by miR-185mimics or miR-185inhibitor: cell proliferationinhibition was detected by MTT assay, cell cycles determined by flowcytometric analysis, cell autophagy were observed by acridine orangestaining and LC3-II immunofluorescence staining, cell ultramicrostructurechanges were observed by electron microscope.②Target genes of miR-185,miR-659and miR-639were predicted by bioinformatics. Information ofmiR-185target genes RAB14was analyzed by bioinformatics.③Thewild-type and mutant of3'UTR of miR-185target genes RAB14mRNAwere cloned to dual luciferase reporter gene pmiR-RB-REPORTTM plasmidby molecular biology methods to construct recombinant reporter plasmid.Recombinant plasmids were identified by double-restriction enzymeanalysis and DNA sequencing.④Recombinant plasmids and miR-185mimics or miR-185inhibitor were co-transfected into SKOV3cells, thencells fluorescence activity was detect by dual-luciferase reporter gene assaysystem.Results:①After miR-185in SKOV3cells up-regulated by miR-185mimics, cell proliferation was inhibited (P <0.01), cell cycles were blockedin G1phase (P <0.01), no significant macrophage activity and apoptoticchanges were found. After miR-185in SKOV3cells down-regulated bymiR-185inhibitor, there were no significant changes on cell proliferation, cycle, autophagy, and apoptosis.②bioinformatics prediction showedRAB14was a candidate target gene for miR-185.there were total25candidate target gene for miR-185. CTNNBIP1, VEGFc were candidatetarget genes for miR-659, and AKT1, PKN1were candidate target genes ofmiR-639.③Results of double-restriction enzyme analysis and DNAsequencing showed recombinant plasmid containing the wild type or mutantRAB14gene3'UTR were successfully constructed.④Renilla luciferaseactivity was decreased after up-regulated miR-185in SKOV3cells, whichsuggested RAB14was the target genes of miR-185.Conclusion: up-regulated miR-185can significantly inhibit SKOV3cells proliferation, and block cell cycle in G1phase, which is related withdown-regulated RAB14by miR-185. PART SIXEFFECT OF REGULATINGTHE GROWTH OFFOXM1/MIR-185/RAB14MOLECULAR NETWORK ONXENOGRAFT TUMORS OF OVARIAN CANCER IN NUDEMICEObjective: To study effect and its possible molecular mechanism ofgrowth inhibition of inhibited FOXM1or up-regulated miR-185onxenograft tumor in nude mice. Methods:20nude mice experimental animals model with ovariancancer SKOV3cells xenograft tumor were established.Lentiviral targetingFOXM1shRNAand miR-185agomir were injected into xenograft tumor forgege treatment, then it inhibition rate of xenograft tumor wasanalyzed,morphological changes of xenograft tumor tissues and organs ofnude mice were observed by HE staining, ultrastructural changes ofxenograft tumor tissues were observed by transmission electron microscopy,expression of FOXM1, p21, PLK1, Beclin1, LC3-II and RAB14inxenograft tumor tissues were detected by Western blot.Results: nude mice experimental animals model with ovarian cancerSKOV3cells xenograft tumor were successfully established.HE stainingshowed density of SKOV3cells in tumor tissue after treatment was low. Noobvious abnormalities were found in nude mice organs. Autophagosomesand necrosis were observed by transmission electron microscopyrespectively in inhibited FOXM1group or up-regulated miR-185group.Western blot suggested that expression of protein P21, Beclin1and LC3-IIwere up-regulated after inhibited FOXM1, while RAB14, PLK1wreredown-regulated after up-regulated miR-185.Conclusion: Both inhibited FOXM1and up-regulated miR-185caninhibit ovarian cancer SKOV3xenograft tumor in nude mice, inducingautophagic cell death and cell cycle arrest by inhibited FOXM1, cell cyclearrest by up-regulated miR-185, may be its mechanism from. We drawed a molecular network map of "FOXM1/miRNA/target protein" of regulationgrowth on SKOV3cells based on our research and literature results.