| Pancreatic cancer is highly malignant tumor,which is frequently characterized by distant metastasis,and the overall 5-year survival rate of patients is less than 10%.Given that there are numerous genes involved in the progression of pancreatic cancer,it is important for both scientific research and clinical application to further explore the key driving genes involved in the occurrence and progression of pancreatic cancer,and to reveal the underlying molecular mechanism,and to develop novel targets for prevention and treatment of pancreatic cancer.Long noncoding RNA(lncRNA),as an important regulatory element of gene expression,has been shown to be involved in regulating the expression of some related genes at various levels,including epigenetic regulation,transcriptional and post-transcriptional regulation,translation and post-translational regulation,and affect the tumor occurrence and development by interacting with other biomacromolecules.Studies have shown that lncRNA PCAT1 is abnormally expressed in a variety of malignant tumors and is closely related to the prognosis of the tumor patients.However,the mechanism of PCAT1 involved in pancreatic cancer remains to be further studied.Accumulating evidences have shown that the dysregulation of RNA m~6A level is closely related to the occurrence and development of tumors and the poor prognosis of patients.Therefore,further study of RNA m~6A modification in tumor progression may provide new targets for the treatment of malignant tumors.Although some lncRNAs themselves have been shown to occur RNA m~6A modification and play a regulatory role in the carcinogenesis and development of the tumors,so far,there is no report on the relationship between PCAT1 and RNA m~6A modification,and whether PCAT1 can affect the malignant phenotypes of pancreatic cancer cells by regulating the level of RNA m~6A modification.Celastrol is a pentacyclic triterpene extracted from Tripterygium wilfordii.Studies have shown that celastrol can exhibit potential anti-cancer activity in various cancer models in vitro and in vivo by inducing cell apoptosis,inhibiting cell cycle progression and proliferation,targeting tumor inflammatory environments,inhibiting angiogenesis,cell invasion and tumor metastasis.However,the mechanism of celastrol in pancreatic cancer has not been fully clarified,especially whether celastrol can inhibit the malignant phenotype of pancreatic cancer by regulating the level of RNA m~6A modification has not been reported.Based on our previous research combined with the review of research on the existing relevant literatures,on the one hand,current study will explore the mechanism of PCAT1 promoting the growth and metastasis of pancreatic cancer via regulating the level of RNA m~6A modification in the clinical tissue,pancreatic cancer cells and mouse animal model.On the other hand,this study was to evaluate whether celastrol regulates the proliferation,cell cycle progression and apoptosis of pancreatic cancer cells by modulating the level of RNA m~6A modification,and to further reveal the underlying molecular mechanism of celastrol against pancreatic cancer,and provide a theoretical basis for its anti-pancreatic cancer treatment.The specific content of this thesis includes the following three chapters:Chapter 1 Effects of lncRNA PCAT1 on the proliferation,migration and invasion of pancreatic cancer cellsObjective:To investigate the relationship between the expression of PC AT1 in pancreatic cancer tissues and clinicopathological parameters and the prognosis of the patients with pancreatic cancer,and to study the effects of PCAT1 on the proliferation,migration and invasion of pancreatic cancer cells,and the xenograft tumor growth and the experimental tumor metastasis in mice.Methods:RNA fluorescence in situ hybridization(RNA FISH)assay was first used to detect the expression of PCAT1 in pancreatic cancer tissue microarray.Lentiviral-mediated PCAT1 knocking down and overexpressing pancreatic cancer cell models were established,and the effects of PCAT1 on the viability,proliferation,colony formation,cell cycle progression,apoptosis,and migration and invasion of pancreatic cancer cells were investigated by CCK-8 assay,EdU assay,colony formation assay,flow cytometry,wound healing assay and Transwell assay.Furthermore,a subcutaneous xenograft tumor model and an experimental in vivo metastasis model were established by subcutaneously and tail vein injection of AsPC-1 cells with stable PCAT1 overexpression,knockdown,or indicated control,respectively,to investigate the effects of PC AT1 on the growth of the subcutaneous xenograft tumors and the experimental in vivo metastasis in nude mice.Results:The expression of PCAT1 was closely related to the TNM stage and lymph node metastasis of pancreatic cancer patients(P<0.05).Cox multivariate survival analysis showed that upregulation of PCAT1,tumor diameter,TNM stage,histological grade,lymph node metastasis and liver metastasis were independent risk factors for the prognosis of pancreatic cancer patients(P<0.05).The results from CCK-8 assay,EdU assay,colony formation assay,flow cytometry,wound healing assay and Transwell assay showed that overexpression of PCAT1 in AsPC-1 and BxPC-3 cells promoted cell proliferation and colony formation,progression of the G1/S phase,and cell migration and invasion,whereas knockdown of PCAT1 inhibited the above malignant phenotypes.The results from xenograft tumors in mice showed that the volume and weight of xenograft tumors from tumor-bearing mice inoculated with PC AT1 overexpressing AsPC-1 cells significantly increased when compared with the control group,while the volume and weight of xenograft tumors from tumor-bearing mice inoculated with PCAT1 knocking down AsPC-1 cells were obviously decreased.Compared with the indicated control group,the number of metastatic nodules in the lungs and livers in mice injected with PCAT1 overexpressing AsPC-1 cells was significantly increased,and obviously decreased in PCAT1 knockdown group.Conclusions:Our results demonstrated the upregulation of PCAT1 in pancreatic cancer tissues,which was negatively correlated with the overall survival of the pancreatic cancer patients.PCAT1 promoted the proliferation,migration and invasion of pancreatic cancer cells,and facilitated the subcutaneous xenograft tumor growth and the experimental in vivo metastasis in nude mice.Chapter 2 Mechanism of lncRNA PCAT1 regulating the migration and invasion of pancreatic cancer cellsObjective:To investigate the mechanism underlying PCAT1 in regulating the migration and invasion of pancreatic cancer cells.To further clarify that PCAT1 may promote the migration and invasion of pancreatic cancer cells by regulating the expression of E-cadherin,a key gene involved in the epithelial-mesenchymal transformation(EMT),through regulating the modification of RNA m~6A.Methods:RNA sequencing(RNA-seq)was first performed to evaluate the transcriptome changes in PCAT1 overexpressing AsPC-1 cells,and the effects of overexpression or knockdown PCAT1 on the expression of some RNA m~6A WERs(Writers,Erasers and Readers)and EMT related genes in pancreatic cancer cells were verified by RT-qPCR and Western Blot analysis.RNA dot blot assay,m~6A immunofluorescence staining and immunohistochemical staining assays were used to detect the differences in the levels of RNA m~6A modification between the pancreatic cancer tissues and the matched paracancer tissues,and between the pancreatic cancer cell lines and the normal pancreatic ductal epithelial cells.Meanwhile,the effects of PCAT1 overexpression and knockdown on the overall RNA m~6A modification in pancreatic cancer cells were detected by RNA dot blot assay and quantitative m~6A methylation assay.Moreover,S1m tagged PCAT1 was stably expressed in AsPC-1 and BxPC-3 cells and then subjected to in vivo RNA pull down assay.The pulled down proteins were analyzed by protein mass spectrometry,and then Western Blot and RNA immunoprecipitation combined with RT-qPCR(RIP-qPCR)assays were performed to validate the interaction between PCAT1 and heterogeneous nuclear ribonucleoprotein K(HNRNPK).The nuclear and cytoplasmic protein extraction from AsPC-1 and BxPC-3 cells with PCAT1 overexpression or knockdown was subjected for Western Blot assay,and the effect of PCAT1 on the distribution of HNRNPK in the cells was detected by immunofluorescence assay.RT-qPCR and Western blot analysis were performed to evaluate the effect of HNRNPK knockdown in PCAT1 overexpression AsPC-1 and BxPC-3 cells on the expression of FTO and YTHDF2,etc.Furthermore,the effect of PCAT1 on the m~6A modification in E-cadherin and Vimentin mRNA in both AsPC-1 and BxPC-3 cells was detected by methylated RNA immunoprecipitation combined with RT-qPCR(MeRIP-qPCR).The effects of FTO overexpression on the level of E-cadherin mRNA m~6A modification and E-cadherin protein in PCAT1 overexpressing pancreatic cancer cells were evaluated by MeRIP-qPCR and Western Blot analysis.In addition,RIP-qPCR assay was used to evaluate whether YTHDF2 interacted with E-cadherin mRNA in pancreatic cancer cells,and RNA stability assay and protein stability assay were applied to evaluate the effect of PCAT1 on the mRNA and protein stability of E-cadherin.Results:Data from RNA-seq analysis showed that there were 2105 differentially expressed genes in AsPC-1 cells upon PCAT1 overexpression,which included some RNA m~6A WERs and EMT related key genes,E-cadherin and Vimentin.RT-qPCR and Western Blot analysis verified that PCAT1 down-regulated FTO and up-regulated METTL3,WTAP and YTHDF2 expression in AsPC-1 and BxPC-3 cells.Results from detection of the pancreatic cancer tissues demonstrated that RNA m~6A modification level in pancreatic cancer tissues was generally higher than that in the adjacent tissues and was closely related to the poor prognosis of pancreatic cancer patients.Meanwhile,RNA m~6A modification was significantly increased in pancreatic cancer cell lines when compared with that in the normal pancreatic ductal epithelial cells.Moreover,compared with the indicated control cells,the level of RNA m~6A was significantly increased in AsPC-1 and BxPC-3 cells upon PCAT1 overexpression and decreased in PCAT1 knocking down cells.Data from RNA pull-down combined with protein spectrometry analysis,Western Blot and RIP-qPCR showed that PCAT1 could interact with HNRNPK protein,but did not affect the expression of HNRNPK in pancreatic cancer cells.Further study revealed that HNRNPK protein was enriched in the cytoplasm and decreased in the nucleus in PCAT1 overexpressing cells,while the opposite enrichment of HNRNPK protein in the cytoplasm and the nucleus was observed in PCAT1 knocking down cells as determined by Western Blot and immunofluorescence staining.RT-qPCR and Western Blot analysis showed that HNRNPK knockdown further downregulated FTO and upregulated YTHDF2 expression in PCAT1 overexpressing AsPC-1 and BxPC-3 cells when compared with those in PCAT1 overexpressing cells.Importantly,MeRIP-qPCR analysis showed that the mRNA level of E-cadherin enriched by anti-m~6A antibody in PCAT1 overexpressing pancreatic cancer cells was significantly increased,while the mRNA level of Vimentin enriched by anti-m~6A antibody was not impaired.Also,FTO overexpression partially reversed the effects of PCAT1 overexpression on the E-cadherin mRNA m~6A modification and E-cadherin protein expression in PCAT1 overexpressing pancreatic cancer cells.Furthermore,RIP-qPCR analysis revealed that the interaction of YTHDF2 with E-cadherin mRNA was increased in PCAT1 overexpressing AsPC-1 and BxPC-3 cells.In addition,data from RNA stability assay and protein stability assay showed that the degradation rate of E-cadherin mRNA was significantly accelerated in PCAT1 overexpressing AsPC-1 and BxPC-3 cells and reduced in PCAT1 knocking down cells,and no significant alteration of E-cadherin protein stability was shown in either PCAT1 overexpressing or knocking down cells.Conclusions:Our findings indicated that PCAT1 inhibited the entry of HNRNPK into the nucleus by interacting with HNRNPK protein,which resulted in up-regulation of the overall RNA m~6A modification by reducing FTO expression,especially increased E-cadherin mRNA m~6A modification,and led to increase the interaction of YTHDF2 with m~6A modified E-cadherin mRNA by up-regulating YTHDF2 expression in pancreatic cancer cells,which induced the degradation of E-cadherin mRNA and thereby down-regulated the level of E-cadherin,and promoted the EMT process and the migration and invasion of pancreatic cancer cells.Chapter 3 Mechanism underlying celastrol in inhibiting proliferation and promoting apoptosis of pancreatic cancer cells by regulating RNA m~6A modificationObjective:To determine whether celastrol regulated pancreatic cancer cell proliferation,cell cycle progression and apoptosis by regulating RNA m~6A modification.Methods:CCK-8 assay,EdU assay,colony formation assay,and flow cytometry analysis were used to investigate the effects of celastrol treatment on the viability,proliferation,cell cycle progression,and cellular apoptosis of pancreatic cancer cells.Meanwhile,a subcutaneous xenograft tumor model in nude mice was established by subcutaneous injection of AsPC-1 cells,and the tumor-bearing mice were randomly divided into 3 groups:a solvent control group,a low-dose group of celastrol adminstraton(1.0 mg/kg),and a high-dose group of celastrol adminstraton(3.0 mg/kg),to investigate the effects of celastrol adminstraton on the growth of the subcutaneous xenograft tumors in nude mice.Moreover,RNA dot blot assay and total RNA m~6A modification quantitative assay were applied to study the effect of celastrol on the overal RNA m~6A modification in pancreatic cancer cells.Transcriptomic differences between celastrol-treated cells and the control cells were analyzed by RNA-seq.Furthermore,EdU assay and flow cytometry analysis were used to study the effect of Claspin on the proliferation and apoptosis of pancreatic cancer cells,and the rescue assay was further performed to evaluate the effect of Claspin overexpression on the proliferation and apoptosis in celastrol-treated AsPC-1 and BxPC-3 cells.Finally,the regulatory mechanism of celastrol in regulating the proliferation and apoptosis of pancreatic cancer cells was evaluated by RIP-qPCR assay,MeRIP-qPCR assay,RNA stability and protein stability assay.Results:Celastrol inhibited the viability and proliferation of AsPC-1 and BxPC-3 cells in a dose-dependent manner,and induced G2/M phase arrest and cellular apoptosis.Results from tumor xenograft model indicated that celastrol administration repressed xenograft tumor growth in vivo when compared to the solvent control group.Immunohistochemical staining for Ki-67 and TUNEL apoptosis assay further confirmed that celastrol administration obviously decreased cell proliferation and increased cellular apoptosis in xenograft tumor tissues.Results from RNA-seq analysis,RT-qPCR and Western Blot analysis showed that celastrol treatment significantly downregulated the mRNA and protein levels of Bcl-2,Claspin,METTL3,and YTHDF3 in pancreatic cancer cells.Meanwhile,data from EdU assay and flow cytometry analysis showed that silencing of Claspin resulted in the inhibition of cell proliferation and cell cycle progression,and the promotion of apoptosis in both AsPC-1 and BxPC-3 cells,whereas overexpressing of Claspin partially reversed the inhibition of cell proliferation and cell cycle progression,and the promotion of apoptosis caused by celastrol treatment in both AsPC-1 and BxPC-3 cells.Moreover,RNA dot blot assay and quantitative m~6A methylation assay showed that the overal RNA m~6A level was significantly reduced in celastrol-treated AsPC-1 and BxPC-3 cells when compared with that in the control cells.MeRIP-qPCR analysis showed that celastrol treatment decreased the level of Claspin and Bcl-2 mRNA m~6A modification in both AsPC-1 and BxPC-3 cells.Importantly,Western Blot analysis showed that overexpression of METTL3 partially reversed the inhibition of Claspin and Bcl-2 in celastrol-treated AsPC-1 and BxPC-3 cells.Furthermore,data from RNA stability assay and protein stability assay showed that the degradation rate of Claspin and Bcl-2 mRNA were significantly accelerated in celastrol-treated AsPC-1 and BxPC-3 cells,but no significant alteration of Claspin and Bcl-2 protein stability were shown in celastrol-treated cells.RIP-qPCR analysis uncovered that the level of Claspin and Bcl-2 mRNA enriched by anti-YTHDF3 antibody was significantly decreased in celastrol-treated AsPC-1 and BxPC-3 cells.Furthermore,we demonstrated that overexpression of YTHDF3 partially reversed celastrol-induced down-regulation of Claspin and Bcl-2,and abrogated the suppression of cell proliferation and cell cycle progression,and the induction of apoptosis in celastrol-treated pancreatic cancer cells when compared to those in the indicated vector control cells.Conclusions:The findings present here suggested that celastrol treatment reduced the level of total RNA m~6A modification in pancreatic cancer cells by down-regulating METTL3 expression,especially decreased Claspin and Bcl-2 mRNA m~6A modification,which induced the Claspin and Bcl-2 mRNA decay.Our study further highlighted a novel mechanism underlying celastrol-induced cellular proliferation inhibition and apoptosis in pancreatic cancer cells by downregulating Claspin and Bcl-2 expression,which was due to the down-regulation of YTHDF3 induced by celastrol treatment and thereby reduced the interaction of YTHDF3 with Claspin and Bcl-2 mRNA. |
PDF Full Text Request