| Background:Hepatocellular carcinoma(HCC)is one of the most common cancers worldwide,tumor metastasis is the major problems leading to prognosis and survival of HCC patients.Its of great significance to deep study on the molecularmechanisms of HCC metastasis and search for the potential therapeutic targets can improve the prognosis for HCC patients.micro RNAs(mi RNAs)are ~18-24 nucleotides small non-coding RNAs which negatively regulate gene expression bydirectly binding to the 3?-untranslated region(3?-UTR)of targeted m RNAs to degrade m RNA or suppress protein translation.mi R-429 belongsto the mi R-200 family,the dysregulation ofmi R-429 associated with progression,development,prognosis,metastasis,apoptosis and drug resistance of cancers.The bioinformatics algorithm predicted that CRKL was a potential target gene of mi R-429,our group previous demonstrated that CRKL is associated with migration,invasion,proliferation and colony formation abilities of mouse hepatocarcinoma Hca-P and leukemia K562 cells.However,the exact role and underlying molecular mechanismof mi R-429-CRKL axis in HCC is still unknown.This thesis mainly studies the molecular mechanism of mi R-429 target CRKL to regulate the malignant behaviors of HCC cell.Objective:1.To clarify mi R-429 directly targeting to CRKL-3?-UTR to suppress CRKL expression;2.To reveal the negative relationship between mi R-429 and CRKL;3.To definite the influence of mi R-429 and CRKL on proliferation,colony formation,migration and invasion of Hep G2 cell malignant behaviors in vitro;4.To explore the molecular mechanism of mi R-429-CRKL axis regulate the HCC cell biological behavior.Methods:1.To construct the dual-luciferase expression vetors wild-type psi CHECK-2-CRKL-3?-UTR-WTand mutant psi CHECK-2-CRKL-3?-UTR-MUT,dual luciferase reporter experiment detected the in vitro interaction between the mi R-429 and CRKL-3?-UTR;2.mi R-429 mimics and NC,mi R-429 inhibitors and NC were transiently transfected into Hep G2 cells,q RT-PCR and Western blot detected the influence of mi R-429 on endogenous CRKL expression level;3.The si RNA targeting CRKL was designed according to the CRKLm RNA sequence,meanwhile,one with non-targeting sequence to be used as a negative control(NC),then the p GPU6/GFP/Neo-sh CRKL and p GPU6/GFP/Neo-sh NC expression vectors were stably transfected into Hep G2 cells by LipofectamineTM 2000,the monoclonal cell lines with CRKL knockdown were obtained by G418 screening selection.The expression level of CRKL was confirmed by q RT-PCR and Western blot in monoclonal cells,and the expression level change of mi R-429 after CRKL downregulation was also detected;4.The full-length sequence of CRKL was amplified and the PCDH-EF1-MCS-T2A-Puro-CRKL expression vector was constructed,the expression level of CRKL was confirmed by q RT-PCR and Western blot after Hep G2 cellstransiently transfected with PCDH-EF1-MCS-T2A-Puro and PCDH-EF1-MCS-T2A-Puro-CRKL expression vectors,meanwhile,the expression level change of mi R-429 after CRKL upregulation was also detected;5.MTT measured the influence of mi R-429 and CRKL on Hep G2 cell proliferation ability in vitro;6.Colony formation measured the influence of mi R-429 and CRKL on Hep G2 cell colony formationcapacity in vitro;7.Transwell measured the influence of mi R-429 and CRKL on Hep G2 cell migration and invasion abilities in vitro;8.Extracellular matrix adhesion and lymph node adhesion measured the influence of mi R-429 and CRKL on Hep G2 cell adhesion abilities to extracellular matrix and lymph node in vtiro;9.FITC-Phalloidin measured the influence of mi R-429 and CRKL on Hep G2 cell F-actin cytoskeleton;10.q RT-PCR and Western blot measured the influence of mi R-429 and CRKL on Raf/MEK/ERK pathway-and EMT-related molecules in Hep G2 cells;11.The influence of mi R-429,CRKL on EMT-related molecules and Hep G2 cells migration,invasion abilities were measured after blocking Raf/MEK/ERK pathway by ERK specific inhibitor PD98059.Results:1.Dual luciferase reporter demonstrated the relative luciferase activity was significantly decreased by 45.67±1.07%(P=0.004)or 37.00±0.85%(P=0.0008)in Hep G2 cells co-transfected with the psi CHECK-2-CRKL-3?-UTR-WT or psi CHECK-2-CRKL-3?-UTR-WT2 luciferase reporter and mi R-429 mimic compared with the negative control cells,respectively.However,the relative luciferase activity was not change in Hep G2 cells co-transfected with the psi CHECK-2-CRKL-3?-UTR-WT1 and mi R-429 mimic compared with the negative control cells.Moreover,the inhibitory effects was abolished when 3?-UTR that contained both mutant-binding sites were co-transfected with mi R-429 mimic.The above results indicated that mi R-429 directly targeting to CRKL-3?-UTR at the second binding sites;2.mi R-429 overexpression suppressed endogenous CRKL protein expression level(55.00±1.51%,P=0.0089),mi R-429 silencing increased endogenous CRKL protein expression level(44.33±1.05%,P=0.0038).While interestingly,overexpression or silencing of mi R-429 have no effect on endogenous CRKL expression at m RNA level.The above results indicated that mi R-429 directly binds to CRKL-3?-UTR to suppress its expression at the post-transcriptional protein translation level but not at transcription level;3.The monoclonal cellwith CRKLstableknockdown was obtained,Western blotand q RT-PCR showed CRKL protein and m RNA levels weredecreased by97.00±0.57%(P<0.0001)and73.33±7.26%(P=0.0005).Meanwhile,the cells with CRKL overexpression were also obtained with transiently transfected PCDH-EF1-MCS-T2A-Puro-CRKL expression plasmid,CRKL protein and m RNA levels were increased by 127.30±12.03%(P=0.0005)and25400±3329%(P=0.0016);4.MTT and colony formation results showed overexpression or silencing of mi R-429 have no effect on proliferation and colony formation capacities;CRKL downregulation significantly inhibited Hep G2 cells proliferation and colony formation abilities,comparing to Hep G2-sh NC cells,the proliferation of Hep G2-sh CRKL cells was decreased by~50.76% and 56.36% at the time interval of 72 h and 96 h(P<0.001).Meanwhile,CRKL overexpression significantly promoted Hep G2 cells proliferation and colony formation abilities,comparing to Hep G2-PCDH cells,the proliferation of Hep G2-PCDH-CRKL cells was increased by~16.99% and 39.24% at the time interval of 72 h(P<0.05)and 96 h(P<0.001),indicating CRKL only deregulated to a certain degree,which can affect Hep G2 cells proliferation and colony formation abilities;5.Transwell results showed mi R-429 overexpression inhibited the migration and invasion abilities of Hep G2 cells,the numbers of migratedand invaded Hep G2-mi R-429 cells decreased by ~48.03%(P=0.0003)and 47.56%(P=0.0045)than Hep G2-mi R-NC cells,respectively.Consistently,mi R-429 knockdown promoted the migration and invasion abilities of Hep G2 cells,the numbers of migratedand invaded Hep G2-LNA-mi R-429 cells increased by ~57.24%(P=0.0006)and 53.28%(P=0.001)than Hep G2-LNA-mi R-NC cells,respectively.CRKL knockdown inhibited the migration and invasion abilities of Hep G2 cells,the migrated and invaded numbers of Hep G2-sh CRKL cells decreased by61.73 %(P<0.0001)and 46.88%(P=0.0001)than Hep G2-sh NC cells.Consistently,CRKL overexpression promoted the migration and invasion abilities of Hep G2 cells.The relative numbers of migrated and invaded Hep G2-PCDH-CRKL cells increased by 73.55 %(P=0.0001)and 60.19%(P=0.0001)than Hep G2-PCDH cells;6.mi R-429 overexpression inhibited extracellular matrix adhesion ability of Hep G2 cells,the adhesion ability of Hep G2-mi R-429 cells adhered to FN wasdecreased by ~32.78% compared to Hep G2-mi R-NC cells(P < 0.0001);Meanwhile,mi R-429 overexpression inhibited in situlymph node(LN)adhesionpotential of Hep G2 cells,the numbers of Hep G2-mi R-429 cells adhered to LNs decreased by ~32.97% compared to Hep G2-mi R-NC cells(P=0.0005);Furthermore,mi R-429 overexpression inhibited F-actin cytoskeleton protein expression of Hep G2 cells,mi R-429 overexpression resulted in obviously decreased of F-actin microfilament compared to Hep G2-mi R-NC cells;CRKL knockdown inhibited extracellular matrix adhesion ability of Hep G2 cells,the adhesion ability of Hep G2-sh CRKLcells adhered to FN wasdecreased by ~40.19% compared to Hep G2-sh NC cells(P<0.0001);Meanwhile,CRKL knockdown also inhibited in situ LN adhesionpotential of Hep G2 cells,the numbers of Hep G2-sh CRKL cellsadhered to LNs decreased by ~73.13% compared to Hep G2-sh NC cells(P<0.0001);Furthermore,CRKLknockdowninhibited F-actin cytoskeleton protein expression of Hep G2 cells.CRKL downregulation resulted in obviously decreased of F-actin microfilament compared to Hep G2-sh NC cells,further verified that mi R-429 and CRKL have effect on Hep G2 cell migration and invasion abilities;7.CRKL deregulation affected the expression level of mi R-429 in Hep G2 cells,following CRKL knockdown,the endogenous mi R-429 expression level in Hep G2-sh CRKL cells increased by 102.80±20.0%(P=0.0069)than Hep G2-sh NC cells;Consistently,CRKL overexpression resulted in decreased endogenous mi R-429 expression level in Hep G2 cells.The transientlyoverexpression of CRKL in Hep G2-PCDH-CRKL cells led to a decrease of mi R-429 by 92.40±3.2%(P=0.0009)than Hep G2-PCDH cells.The above results indicated that CRKL could also regulate mi R-429 expression;8.Rescue experiment results showed that on account of PCDH-CRKLwithout its 3?-UTR,mi R-429 could not downregulate exogenous CRKL expression by directly targeting its 3?-UTR.Consistently,CRKL overexprssion markedly counteracted the inhibition effect of mi R-429 on Hep G2 cellsmigration andinvasion.The above results once again proved that mi R-429 only directly targeting CRKL-3?-UTR to downregulate its expression;9.CRKL knockdown or mi R-429 overexpression resulted in the same downregulation of Raf,p-Raf,p-MEK and p-ERK2 protein expression level in Hep G2 cells,consistently,CRKL overexpression or mi R-429 silencing increased the expression of Raf,p-Raf,p-MEK and p-ERK2,while there were no changed of Ras and ERK1/2.CRKL knockdown or mi R-429 overexpression inhibited EMT by increasing the epithelial marker E-cadherin expression,and decreasing the mesenchymal marker N-cadherin and Vimentin expression.While,CRKL overexpression or mi R-429 silencing promoted EMT by decreasing E-cadherin expression,and increasing N-cadherin and Vimentin expression.Furthermore,CRKL knockdown or overexpression decreased or increased the m RNA expression level of c-Jun,while mi R-429 overexpression and silencing have no influence on c-Jun expression.The result consistent with re-expression or silencing of mi R-429 could not significantly inhibit Hep G2 cells proliferation and colony formation abilities;10.The expression level of E-cadherin increased and N-cadherin,Vimentin decreased after blocking Raf/MEK/ERK pathway by ERK inhibitor PD98059,indicating Raf/MEK/ERK pathway regulate EMT.Furthermore,PD98059 exacerbated the effect of mi R-429 overexpression on the cellmigration and invasion in Hep G2 cells,PD98059 weakened the effect of CRKL overexpression on Hep G2 cellsmigration and invasion.The results indicated that mi R-429-CRKL axis regulated Hep G2 cell migration and invasion by Raf/MEK/ERK-EMT pathway.Conclusion:1.mi R-429 directly targeting CRKL-3?-UTR to suppress its expression at the post-transcriptional protein translation level;2.CRKL negatively regulated endogenous mi R-429 expression;3.mi R-429 affected Hep G2 cells migration andinvasion abilities in vitro,but have no effect on Hep G2 cells proliferation and colony formation abilities in vitro;4.CRKL affected Hep G2 cells migration andinvasion abilities in vitro,but CRKL only deregulated to a certain degree,which can affect Hep G2 cells proliferationand colony formation abilities in vitro;5.mi R-429 regulated Hep G2 cells migration and invasion by targeting CRKL via inhibiting Raf/MEK/ERK-EMT pathway. |
PDF Full Text Request