| Hepatocellular carcinoma(HCC)is one of the most prevalent malignancies in China,and the five-year survival rate of HCC patients is only about 12%.The leading cause of death in most patients with HCC is high rate of recurrence and metastasis,which is a multi-step and multi-stage process involving the abnormal signal pathways and expression of multiple genes.Therefore,it is imperative to further explore the key driving genes involved in the process of HCC metastasis,and to elucidate the molecular mechanisms underlying HCC metastasis,which would contribute to the development of novel targets for the prevention and therapeutic inventions for HCC metastasis in clinic.Abundant evidence has demonstrated that the conserved kinase cascade pathway in Hippo signaling plays a potential anticancer role by regulating the transcriptional activity of its core downstream effectors,yes-associated protein(YAP)/transcriptional coactivator with PDZ binding motif(TAZ).Studies have shown that MOB2(monopolar spindle-one-binder 2),a membrane of MOB protein family,plays a role in the inhibition of NDR1/2 kinase by competing with MOB1 for the binding to a N-terminal regulatory domain of NDR1/2 kinases(but does not associate with LATS1/2 kinases)to regulate cell cycle progression and apoptosis,etc.NDR1/2 kinase can also promote YAP phosphorylation at S127 site and negatively regulate the transcriptional co-activation of YAP.These findings suggest that MOB2 may also be involved in the regulation of Hippo tumor suppression signaling pathway.Previously,we have demonstrated that MOB2 could regulate the cell cycle progression,migration,invasion and autophagy of HCC cells,but the underlying mechanism has not been fully clarified.In recent years,studies have shown that integrated Chinese and Western medicine can enhance the immunity of cancer patients,and improve the efficacy of chemoradiotherapy.The theory of "cancer toxicity" as a new understanding of the theory of tumor pathogenesis in Traditional Chinese Medicine(TCM)provides a novel idea and method for the dialectical treatment of tumor in TCM.Clearing away heat and detoxication,as one of the classical methods of HCC in TCM has achieved a better curative effect in the treatment of HCC.The mobility of cancer toxicity is the main cause of the stubborn and difficult treatment of tumors.It is of great clinical significance to develop active ingredients of TCM for the clinical treatment of HCC.Apigenin is an edible flavonoid widely enriched in natural plants,and it has been demonstrated as an anti-cancer flavonoid with clearing away heat and detoxification by modulating multiple targets and signaling pathways,however,whether apigenin might play an anti-tumor role by regulating Hippo signaling pathway has not been reported.Based on our previous findings and the study of the available literatures,this research will be carried out to elucidate the function and the mechanism of MOB2 in the regulation of the migration and invasion through modulating the Hippo signaling pathway in HCC cell lines with different migratory capacity by using the lentiviral-mediated gene silencing,and to clarify the function of MOB2 in the regulation of HCC metastasis from the HCC tissue samples and in vivo mouse animal models.Meanwhile,this study will also explore whether apigenin might affect the migration and invasion of HCC cells with different migratory capacity by regulating the Hippo pathway.The study will further reveal molecular mechanism of anti-tumor activity of apigenin,which would provide a theoretical basis for anti-tumor therapy of integrated traditional Chinese and Western medicine.The present research includes the following three chapters:Chapter 1:Effects of MOB2 on the migration,invasion and autophagy of hepatocellular carcinoma cellsObjective:To investigate the involvement of MOB2 in the pathogenesis of liver cancer and its effect on the migration,invasion and autophagy of HCC cells.Methods:In order to study the correlation between the expression of MOB2 and the growth and metastasis of HCC,we first analyzed the difference of MOB2 expression in HCC tissues and adjacent tissues by TCGA database and its correlation with disease prognosis of HCC patients.Secondly,reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and Western Blot were performed to detect the mRNA and protein expression of MOB2 in HCC tissues and their corresponding adjacent tissue samples of patients who were clinically diagnosed HCC.Finally,the subcutaneous inoculation models and the experimental metastatic models in vivo were established by using SK-HEP1 cells stably knocking down MOB2 and the control cells,to investigate the effects of MOB2 knockdown on the growth of xenograft and the metastasis in nude mice.Based on the clear involvement of MOB2 in the pathogenesis of HCC,we then evaluated the effects of MOB2 knockdown on the migration,invasion and autophagy of HCC cells.We first studied the effects of MOB2 on the migration and invasion of HCC cells with different migratory capacity by using Transwell assays,3D Matrigel cell invasion assay and immunofluorescence staining for LC3B.Secondly,the expression of epithelial-mesenchymal transition(EMT)markers and autophagy related genes in SMMC-7721 cells and SK-HEP1 cells stably knocking down MOB2 and the indicated control cells were detected by RT-qPCR and Western Blot.Thirdly,rhodamine-labeled phalloidine was performed to stain the F-actin in HCC cells to explore the effect of MOB2 knockdown on the actin cytoskeleton rearrangement,and the RhoA/Rac1/Cdc42 G-LISA Activation Assay was performed to evaluate the effect of MOB2 on the activity of RhoA,Racl and Cdc42 in HCC cells with different migratory capacity.Finally,by incubation of 3-methyladenine(3MA),a pharmacological inhibitor in early autophagy studies,or chloroquine(CQ),an inhibitor in the autophagolysosome formation with HCC cells stably knocking down MOB2 and the indicated control cells,we investigated whether MOB2 could affect the migration of HCC cells through regulating autophagy.Results:TCGA dataset analysis revealed that MOB2 mRNA levels in HCC tissues was higher than those in the adjacent tissues,and was significant positive correlation with recurrence in HCC patients.The results from RT-qPCR and Western Blot analysis showed that MOB2 expression was upregulated in HCC tumor tissues compared with that in matched adjacent tissues.The results from the subcutaneous inoculation models and the tail vein metastasis models in nude mice demonstrated that MOB2 knockdown inhibited HCC xenograft growth,and pulmonary and liver metastasis in vivo.Data from Transwell assays showed that MOB2 knockdown could significantly inhibit the migration and invasion of HCC cells with different migratory capacity,which is consistent with our previous studies.Moreover,3D Matrigel cell invasion assay further confirmed that MOB2 knockdown could significantly inhibit the invasiveness of spheroidal SMMC-7721 cells through a 3D extracellular matrix.Immunofluorescence assay showed that MOB2 knockdown induced the formation of LC3B autophagy puncta in HCC cells and thereby promoted autophagy.Based on the RNA-sequencing(RNA-seq)analysis from MOB2 knockdown cells,RT-qPCR and Western Blot were performed to validate the data from RNA-seq.Compared with the indicated control cells,MOB2 knockdown obviously increased the expression of E-Cadherin and Snail,and decreased the expression of N-Cadherin in HCC cells by RT-qPCR.Meanwhile,results from Western Blot analysis showed that MOB2 knockdown significantly increased the accumulation of E-cadherin and reduced the accumulation of N-cadherin,however,there was no significant effect on Snail and Vimentin in HCC cells,when compared with indicated control group.Immunofluorescence staining results showed that HCC cells stably knocking down MOB2 displayed relatively well-developed contacts between cells,and exhibited enrichment of F-actin at the peripheral regions and a thin or minimal lamellipodia and filopodia at the leading edge.Data from the RhoA/Rac1/Cdc42 G-LISA Activation Assay showed that,compared with those in the indicated control cells,knockdown of MOB2 decreased the activity of both Racl and Cdc42,and increase the activity of RhoA in HCC cells.Results from Transwell migration assay further demonstrated that treatment with 3-Methyladenine(3MA))or chloroquine(CQ)partially abrogated the migratory ability of the HCC cells induced by MOB2 knockdown,especially in the group of cells treatment with CQ.Conclusion:MOB2 is highly expressed in HCC and is closely associated with the development,progression and metastasis of HCC.MOB2 knockdown could regulate the migration and invasion of HCC cells by regulating the activity of Rho GTPases in HCC cells,which might induce actin cytoskeleton rearrangement.MOB2 knockdown also regulated the expression of EMT markers and autophagy-related genes in HCC cells,and suppressed HCC cell migration and invasion probably via altering the autophagy.Chapter 2:MOB2 regulates the migration,invasion and autophagy of HCC cells via Hippo signaling pathwayObjective:To clarify the specific mechanism of MOB2 regulating HCC cell migration,invasion and autophagy via Hippo signaling pathway.Methods:Firstly,luciferase reporter gene assays were performed in MOB2 stable knockdown and corresponding control cells with different migratory capacity by transfection with the luciferase reporter vector which contains the promoter region of CTGF or CYR61 to evaluate the luciferase activity of the CTGF and CYR61 promoter.Secondly,we performed chromatin immunoprecipitation(ChIP)assay with anti-YAP or anti-TAZ antibody,and ChIP-qPCR assay to detect the enrichment of the amplified promoter fragments of CTGF and CYR61 in MOB2 stable knockdown and corresponding control SMMC-7721 and SK-HEP1 cells with CTGF and CYR61 promoter-specific primers.Finally,the expression and activity of the core components of the Hippo signaling in MOB2 stable knockdown and corresponding control SMMC-7721 cells were detected by Western Blot and immunoprecipitation.We then evaluated the effects of YAP/TAZ on the migration,invasion and autophagy of HCC cells in the presence of MOB2 regulating the transcriptional activity of YAP/TAZ.Total RNA was first extracted from YAP knockdown,TAZ knockdown and the control SMMC-7721 cells,and RNA-seq was employed to analyze the expression profiles in the cells,and RT-qPCR and Western Blot were performed to validate the data from RNA-seq,and to further elucidate the effects of YAP knockdown and TAZ knockdown on the expression of EMT markers and autophagy-related genes in HCC cells.Subsequently,Transwell assay and autophagy flow assay were used to investigate the effects of YAP knockdown,TAZ knockdown and TAZ knockout on the migration,invasion and autophagy of HCC cells with different migratory capacity.Furthermore,treatment with the autophagy inhibitors in YAP knocking down,TAZ knocking down or TAZ knockout HCC cells was given to cell motility assays to investigate whether YAP knockdown,TAZ knockdown and TAZ knockout affects the migration and invasion of HCC cells via regulating autophagy.In addition,RNA dot blot and Western Blot assays were performed to study the modification of RNA m6A and the expression of RNA m6A regulators,respectively,in MOB2 knocking down,YAP knocking down and TAZ knocking down HCC cells.Methylated RNA immunoprecipitation(Me-RIP)-qPCR assay was performed to evaluate Snail mRNA enrichment by anti-m6A antibody in MOB2 knocking down cells.Results:The results from the luciferase reporter gene assay showed that knockdown of MOB2 markedly down-regulated the luciferase activity of the CTGF and CYR61 promoter,respectively,in comparison with those in the indicated control cells.Moreover,results from ChIP-qPCR assay demonstrated that the enrichment of the amplified promoter fragments of CTGF and CYR61,which are well-known downstream targets of YAP/TAZ,in MOB2 knocking down cells was significantly reduced.These results suggested that MOB2 could regulate the expression of the CTGF and CYR61 in HCC cells by regulating the transcriptional activity of YAP/TAZ.Results from Western Blot analysis showed that MOB2 knockdown in HCC cells obviously increased the phosphorylation of MOB1,LATS1,YAP and TAZ,when compared with indicated control cells.Moreover,results from co-immunoprecipitation followed by Western Blot analysis demonstrated that MOB2 may interact with NDR1/2 but not LATS1/2.It was revealed that MOB1 was enriched in NDR1/2 precipitates but reduced in LATS1 precipitates in MOB2-silencing cells.In addition,Western Blot analysis also demonstrated that phosphorylation at Thr444/442 of NDR1/2(pT444/442)was increased in MOB2 silencing cells.Differential expression of genes analysis from RNA-seq in YAP knocking down and TAZ knocking down HCC cells followed by RT-qPCR and Western Blot analysis showed that YAP knockdown and TAZ knockdown downregulated N-cadherin,Vimentin and SQSTM1/p62,upregulated E-cadherin,β-catenin,LC3BⅡ and ULK1,when compared with the control group.Meanwhile,data from Transwell cell migration and invasion assay showed that YAP knockdown,TAZ knockdown and TAZ knockout dramatically decreased the migration and invasion of HCC cells.Moreover,the results from autophagic flux assay also showed that YAP knockdown,TAZ knockdown and TAZ knockout increased autophagic flux in HCC cells.Furthermore,Data from Transwell assays demonstrated that treatment with 3MA or CQ partially abrogated the migratory ability of the HCC cells induced by YAP knockdown,TAZ knockdown and TAZ knockout.In addition,Western Blot assay showed that treatment with 3MA or CQ obviously increased the amount of N-cadherin,Vimentin,LC3BⅡ and SQSTM1/p62,and downregulated E-cadherin in HCC cells induced by YAP knockdown,TAZ knockdown or TAZ knockout,especially in the group of cells treatment with CQ.The results from RNA dot blot assay showed that MOB2 knockdown,YAP knockdown and TAZ knockdown could decrease the levels of RNA m6A in HCC cells.Western Blot analysis showed that MOB2 knockdown,YAP knockdown and TAZ knockdown could up-regulate FTO,ALKBH5,YTHDC1 and YTHDF3,and down-regulate WTAP in HCC cells,while YAP knockdown could also up-regulate IGF2BP2.Then Me-RIP-qPCR assays revealed that knockdown of MOB2 could significantly promote m6A modification of Snail mRNA in SMMC-7721 and SK-HEP1 cells compared with that in control group.Conclusion:On the one hand,silencing of MOB2 may promote the interaction of MOB1 with NDR1/2,activate the NDR1/2 Kinase,increase the phosphorylation of YAP,and result in the activation of Hippo signaling pathway;on the other hand,MOB2 knockdown can inhibit the transcriptional activity of YAP/TAZ via activating the Hippo signal pathway in HCC cells,and then regulate the expression of downstream EMT markers and autophagy related gene,and thereby inhibit HCC cell migration and invasion and promote autophagy.Moreover,silencing of YAP or TAZ can inhibit EMT process,and then inhibit HCC cell migration and invasion via autophagy induction.In addition,MOB2 and YAP/TAZ might presumably be attributed at least in part to regulate the expression of EMT markers by regulating the mRNA m6A modification.Chapter 3:Apigenin regulates the migration,invasion and autophagy of hepatocellular carcinoma cells by downregulating YAPObjective:To investigate the mechanism of apigenin regulating the migration,invasion and autophagy of HCC cells.Methods:Firstly,cell counting kit-8(CCK-8)assay,wound healing assay and Transwell cell migration and invasion assays were performed to assess the effect of apigenin on cell viability,migration and invasion of SMMC-7721 cells with low metastatic potential,and SK-HEP1 cells with high metastatic potential.Rhodamine-labelled phalloidin was performed to stain the actin cytoskeleton in SMMC-7721 and SK-HEP1 cells in the presence or absence of apigenin,and to evaluate the effect of apigenin on the assembly of the actin cytoskeleton in HCC cells.Secondly,RNA-seq was employed to compare the transcriptional expression profiles in SMMC-7721 cells in the presence or absence of apigenin,and differentially expressed genes were subjected to Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Then RT-qPCR and Western Blot assays were performed to verify the expression of some differentially expressed genes from RNA-seq analysis.Thirdly,We integrated both data from RNA-seq analysis for apigenin treatment and YAP knockdown to establish the gene set overlaps,and RT-qPCR and Western blotting were conducted to validate the impact of apigenin on the expression of some EMT markers and autophagy-related genes in both SMMC-7721 and SK-HEP1 cells,and autophagy flow assay was also performed to detect the effects of apigenin on autophagy of HCC cells.Finally,we generated YAP overexpressing SMMC-7721 and SK-HEP1 cells by infection with adenovirus encoding YAP or control,and Transwell assays,RT-qPCR and Western blotting analysis were conducted to evaluate whether apigenin-induced reduction of the migration and invasion and promotion of autophagy of HCC cells accounted for the regulating YAP expression.Results:The results from CCK-8 assay showed that incubation with different concentrations of apigenin for 48 h decreased the cell viability of both SMMC-7721 and SK-HEP1 cells with different migratory capacity in a dose-dependent manner.Meanwhile,data from cell scratch assay,Trans well assays and immunofluorescence assay demonstrated that treatment with apigenin for HCC cells decreased the cell migratory and invasive ability,and resulted in the actin cytoskeleton rearrangement in HCC cells.RNA-seq analysis showed that a total of 713 up-regulated genes and 956 down-regulated genes were differentially expressed in apigenin-treated cells in comparison with the controls,and KEGG analysis showed Hippo signaling pathways highly altered in apigenin-treated cells.RT-qPCR or Western blotting analysis demonstrated significant reduction of CTGF,CYR61 and YAP(but not TAZ)showed significant reduction in apigenin-treated cells.A total of 785 genes from the gene set overlaps in apigenin treated and YAP knocking down cells were demonstrated by the Venn diagram,including YAP,CTGF,CYR61,some EMT markers and autophagy-related genes.Data from RT-qPCR or Western blotting analysis revealed consistent significant up-regulation of E-Cadherin,β-catenin,ULK1 and LC3BⅡ,and down-regulate CTGF,CYR61,YAP,N-Cadherin,Vimentin and SQSTM1/p62 in apigenin-treated HCC cells compared to the control cells.Moreover,our findings further showed that treatment with apigenin increased autophagic flux in both SMMC-7721 and SK-HEP1 cells.Furthermore,treatment with apigenin partially abrogated the migratory and invasive capacity of both SMMC-7721 and SK-HEP1 cells induced by YAP overexpression.Our results further demonstrated that treatment with apigenin partially exhibited the reverse effects on the expression of EMT markers and autophagy-related genes induced by YAP overexpression in YAP overexpressing SMMC-7721 cells,and induced synergistic effects for inhibition of the cell migratory and invasive ability in stable YAP knockdown SMMC-7721 cells.Conclusion:Apigenin inhibited the migration and invasion and increased autophagy of HCC cells by down-regulating YAP,which is a core downstream effector of Hippo signaling,and subsequently modulating the expression of EMT markers and autophagy-related genes. |
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