| BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumors in china,about 110 thousand of HCC patient dead per year.Metastasis is primary cause of HCC patient's death,but the mechanism is still not well defined.Accumulating evidences shown that microRNAs have been played essential role in molecular regulation of tumors metastasis.microRNAs are small single strand non-coding RNAs(21-23 nucleotides),which transcribed from intron by RNA polymerase II,generating pri-miRNA in nucleus and processed into mature miRNA in cytoplasm.Mature mi RNA then can be loaded into RISC complex,which together can sequence specific interacting with the target mRNA 3' untranslated regions(UTRs)and silencing the gene expression by degraded the mRNA or inhibited the target mRNA translation.Numerous studies indicating that miRNA deregulation expression happened in various tumors,including HCC,it acted as oncogene or tumors suppressor and implicated in many biological processes,such as proliferation,differentiation,apoptosis,stress response and so on.It played important role in initiation and progression of tumors.miR-149 general regarded as an anti-tumor miRNA,and its expression is deregulated in multiple types of cancers,including breast cancer,colorectal cancer(CRC),gastric cancer,head and neck squamous cell carcinoma(HNSCC)and non-small-cell lung cancer(NSCLC).Chan et al.demonstrated that miR-149 targeted GIT1 to suppress integrin signaling and breast cancer metastasis.Wang et al.reported that Sp1 mediated the link between methylation of tumor suppressor miR-149 and outcome in CRC.However,to date,the mechanism of mi R-149 deregulation and its regulatory networks in HCC remain elusive.To illustrate the role of miR-149 in HCC,our group previously investigated the expression of miR-149 in HCC tissues,and found that miR-149 expression is lower in the HCC tissues than corresponding non-tumorous tissues.But the mechanism underlying is remaining unknown,this project based on priviously data in our group is aim to illustrate the role and molecular mechanism of mi R-149 that involved in HCC,and that will provide scientific evidence for biological targeting therapy in HCC.Objective: aim of this project is to investigate the role and mechanism of miR-149 in HCC.Materials and methods:(1)Real-time PCR was used to detect the relative expression of miR-149 in HCC tissues and their corresponding non-tumorous tissues.And the relative expression level of mi R-149 was employed to ananlysis overall survial rate and clinicopathologic feature.(2)Transwell assays with matrigel was performed to investigate the invasive ability of HCC cell lines with infected the control or miR-149 over-expression lentivirus.(3)Wound healing assay was carried out for detecting the effect of miR-149 on migration of HCC cell lines.(4)MTT and flow cytometry was applied to detect the effect of miR-149 on proliferation and cell cycle in HCC cell lines respectively.(5)Bioinformatics analysis was used to screen mi R-149 potential target gene.(6)Western blot was used for miR-149 target gene screening.(7)Dual luciferase assay was used to investigate regulation relationship and binding site between miR-149 and its potential target genes.And real-time PCR was carried out to investigate the effect of mi R-149 on its protential target gene in mRNA level.(8)Immunohistochemical staining(IHC)was applied to compare the difference expression of PPM1 F in the 93 pairs of HCC tissues and their corresponding non-tumorous liver tissues.The scores of immunohistochemistry was evaluated by semiquantitative analysis,which was employed for analyzing correlation between miR-149 and its target gene PPM1 F.(9)Fluorescence in situ hybridization(FISH)was used to detect the location and expression level of miR-149 in the HCC tissues and their corresponding non-tumorous liver tissues.(10)Transwell assays with Matrigel and Wound healing assay was carried out to investigate the ablility of invasion and migration in the miR-149 overexpression HCC cell with transfected PPM1 F.(11)It is reported that PPM1 F can influence expression of stress fibers.Immuno fluorescence staining was carried out to investigate wheather miR-149 can regulate stress fibers and wheather it regulates stress fibers via target gene PPM1 F in HCC cell lines.(12)It has been reported that PPM1 F can inhibit phosphorylation level of MLC2 in breast cancer,and MLC2 phosphorylating is an essential process to organize stress fiber.Western bolt was carried out to investigate effect of miR-149 on protein level of MLC2 phosphorylation in HCC.Immunofluorescence staining was performed to detect the colocalization of pMLC2 and stress fiber.(13)A liver-lung metastasis nude mouse model was built in this project to investigate the role of mi R-149 in HCC in vivo.Results:(1)Real-time PCR showed that miR-149 is frequently down regulated in the 95 cases of HCC tissues when comparing to their adjacent non-tumorous liver tissues,and clinic pathological analyze base on miR-149 real-time PCR value showed that miR-149 expression in the nodular HCC(NHCC)was significantly lower than solitary large HCC(SLHCC)and small HCC(SHCC).Meanwhile,clinic pathological analyze upon TNM stage showed that stage III/IV HCC tissues also have lower mi R-149 level than stage I/II HCC.In addition,survival curve revealed that when grouping the HCC tissues into High and low miR-149 expression by using median as cut-off value,lower miR-149 expression patients have a shorter surviving time than high miR-149 expression patients.(2)Transwell assays with matrigel confirmed that miR-149 inhibited invasion in both MHCC-97 H and HepG2 cell lines.(3)Wound healing assay showed that the ability of migration also be suppressed by mi R-149 overexpression in MHCC-97 H and HepG2 cells(4)MTT and flow cytometry assays demonstrated that miR-149 do not affect the proliferation and cell cycle of HCC cells respectively.(5)Bioinformatics analyzed the possible target gene in three bioinformatic website(Targetscan,PicTar,miRanda),we organized and selected 67 miR-149 target genes which have common showed on those three bioinformatic website.Finally,6 potential target genes(YWHAZ,RAP1,CD47,YWHAZ,RAP1,CD47)were picked to further investigation due to its metastasis function which was confirmed by other groups.(6)Western blot results showed that overexpression miR-149 in HepG2 and MHCC-97 H cells inhibited the protein level of PPM1 F,SP1 and PDGFRA,but not YWHAZ,RAP1 and CD47.(7)Dual luciferase assay revealed that miR-149 can interacting 3'UTR of the PPM1 F,but not SP1 and PDGFRA.Relative luciferase activities do not change by Point mutation in PPM1 F 3'UTR conserved miR-149 interaction seed region.And finally,miR-149 target gene was validated based on results above.Real-time PCR results showed that mi R-149 also inhibited the mRNA level of PPM1 F.(8)IHC staining PPM1 F showed PPM1 F is frequently up-regulated in the HCC tissues.The correlation between PPM1F(scores based on IHC semiquantitative)and miR-149(values from RT-PCR result)showed that miR-149 is inversely associated with PPM1 F in HCC tissues.(9)Fluorescence in situ hybridization results consistent with real-time PCR results,also showed that the expression of miR-149 in HCC tissues frequently lower than their corresponding non-tumorous tissues.(10)Transwell assays with Matrigel and Wound healing assay showed the inhibition of mi R-149 on invasion and migration was rescued by reintroduce PPM1 F respectively.(11)Immunofluorescence staining results observed that miR-149 overexpression induced stress fiber loss in partial MHCC-97 H and HepG2 cells,concomitantly actin filament reorganization and relocation to the cell periphery.(12)Immunofluorescence staining stress fiber showed that the loss of stress fiber can be rescued by reintroduce PPM1 F in the miR-149 overexpressed MHCC-97 H cells.Immofluorescence staining pMLC2 showed the fluorescence intensity is weaker in the MHCC-97 H cells with overexpression miR-149 than the control cells.Immunofluorescence staining colocalized pMLC2 and F-actin showed that colocalization of pMLC2 and F-actin also lower level in the MHCC-97 H cells with overexpression miR-149 than control cells.Western blot showed the consistent result with Immunofluorescence staining,miR-149 overexpression inhibits protein level of PPM1 F and pMLC2.(13)In vivo,liver-lung metastasis nude mouse model showed that the numbers and sizes of pulmonary metastasis node was significantly decreased with injected miR-149 overexpressed HepG2 cells comparing to control.Conclusion:miR-149 expression is frequently low in HCC tissues and significantly correlated with tumor TNM stages,tumorous gross type and unfavorable prognosis of HCC patients.Our data indicated that miR-149 suppressed invasion and migration of HCC cells,mechanically by directly targeted and inhibited actin-regulatory protein PPM1 F,which can promote phosphorylation of myosin light chain 2(pMLC2)at Thr18 and Ser19,an essential process in stress fiber formation,and eventually effected the invasion and migration of HCC cells.The newly identified miR-149/PPM1 F axis provided new insight into the pathogenesis of HCC and a novel potential therapeutic target for the treatment of HCC. |
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