| Objective:Hepatocellular carcinoma(HCC)is one of the most common malignancies in the world.In China,HCC causes the second highest number of deaths among all malignant tumors,after lung cancer.The best clinical treatment option for HCC is radical surgery,but because of the high malignancy and rapid disease progression,many cases are already lost the chance to surgery at the time of detection.Even if surgery is possible,the postoperative recurrence and metastasis rates are very high.Even if surgery is possible,the postoperative recurrence and metastasis rates are very high.In such a situation,a comprehensive treatment process including chemotherapy and targeted therapy becomes an irreplaceable and critical part.However,chemotherapy for HCC is ineffective due to its insensitivity to most chemotherapeutic agents.Sorafenib,a tyrosine kinase inhibitor,is currently the first-line agent for the treatment of unresectable or distantly metastatic HCC,with inhibitory effects on tumor cell proliferation and anti-angiogenesis,but resistance to the drug may still occur.The etiology and mechanisms of drug resistance in HCC are not fully understood,therefore,a deeper understanding of the genetics and epigenetic mechanisms behind the onset and progression of HCC and drug resistance is needed to develop more effective therapeutic intervention strategies.The etiology and mechanisms of drug resistance in HCC are not fully understood,and thus we need to gain a deeper understanding of the mechanisms of HCC onset and progression,as well as the genetic and epigenetic features behind drug resistance,in order to develop more effective therapeutic intervention strategies.In the present study,we found significant upregulation of DUXAP8 in a patientderived tumor xenograft(PDX)model after sorafenib-based treatment,which usually predicts a poorer prognosis for patients.In HCC,DUXAP8 maintains its expression upregulation by increasing m6 A methylation-mediated RNA stability.In vivo and in vitro,DUXAP8 levels positively correlated with HCC proliferation,stem cell signature maintenance,and sorafenib resistance.In mechanistic studies,we found that DUXAP8 competitively binds mi R-584-5p through a ce RNA mechanism,thus acting as a molecular sponge for mi R-584-5p to regulate MAPK1 expression,which in turn activates the MAPK/ERK pathway.Our findings could provide ideas for finding new prognostic indicators and therapeutic targets for HCC patients.Methods:(1)A patient-derived xenograft(PDX)model treated with sorafenib was constructed,and lnc RNA-seq was applied to identify the differentially expressed lnc RNAs between P4-PDX treated with saline or sorafenib.(2)The expression of DUXAP8 in HCC tissues and normal tissues adjacent to the tumor was verified by RT-q PCR,and the expression of DUXAP8 was correlated with clinicopathological parameters to investigate the relationship between DUXAP8 expression and patient prognosis.(3)The expression of DUXAP8 in seven hepatocellular carcinoma cell lines(MHCC-97 H,Huh7,HCC-LM3,Bel-7405,SNU-449,SK-Hep-1,SNU-182)and human liver immortalized cell line(THLE-3)was verified by RT-q PCR,and the experimental hepatocellular carcinoma cell lines were screened.(4)To construct HCC cell models with low and overexpression of DUXAP8,and to investigate the effect of DUXAP8 on the stem cell characteristics of HCC cells by sphere-forming assay,Western Blot and RT-q PCR;to investigate the role of DUXAP8 in the proliferation and resistance to sorafenib of HCC cells by colony formation assay and CCK-8 assay.(5)To construct transplantation tumor models and lung metastasis models in nude mice with normal and low expression of DUXAP8,and to investigate the role of DUXAP8 in HCC cell proliferation and resistance to sorafenib by detecting subcutaneous transplantation tumor size and growth curve and immunohistochemistry.(6)To detect the m6 A enrichment of DUXAP8 in HCC cells and human normal hepatocytes by methylation RNA immunoprecipitation q PCR(Me RIP-q PCR),and the TCGA(The Cancer Genome Atlas)database was applied to analyze the difference in the expression of methyltransferase-like protein 3(Mettl3)in normal liver tissues and HCC tissues and its The correlation between the expression of Mettl3 and DUXAP8 in normal liver tissues and HCC tissues was analyzed using the TCGA Genome Atlas database.The HCC cell models with low expression and overexpression of Mettl3 were constructed,and the expression and m6 A enrichment of DUXAP8 were detected by RTq PCR and Me RIP-q PCR;the effect of Mettl3 on the stability of DUXAP8 was investigated by radiolysin D assay.(7)To detect the localization of DUXAP8 in HCC cells by RT-q PCR,micro RNAseq was performed on HCC cells with low DUXAP8 expression to screen out differentially expressed micro RNAs;combined with the bioinformatics database Encyclopedia of RNA Interactomes(ENCORI,previously known as star Base v2.0)to predict the micro RNAs that can bind to DUXAP8 target;explore the relationship between DUXAP8 and mi R-584-5p by dual luciferase assay,RNA immunoprecipitation(RIP),and RNA pull-down assay.mi R-584-5p.(8)To investigate the role of DUXAP8 and mi R-584-5p in the maintenance of stem cell characteristics,proliferation and resistance to sorafenib in HCC cells by sphereforming assay,Western Blot,RT-q PCR,and CCK-8 assay.(9)RNA-seq of HCC cells with low DUXAP8 expression,screening of target genes and KEGG enrichment analysis of differentially expressed genes regulated by DUXAP8;verification of mi R-584-5p with MAPK1 by dual luciferase reporter gene,Western Blot,RT-q PCR.(10)To explore the role of DUXAP8 and MAPK1 in the maintenance of stem cell characteristics,proliferation and resistance to sorafenib in HCC cells by sphere-forming assay,Western Blot,RT-q PCR,and CCK-8 assay;and to perform mechanistic analysis by Western Blot.Results:(1)DUXAP8 was significantly upregulated in lnc RNAs differentially expressed between P4-PDX treated with saline or sorafenib;DUXAP8 was significantly highly expressed in HCC tissues compared with normal tissues adjacent to the tumor,and high levels of DUXAP8 correlated with tumor TNM stage,tumor size,microvascular invasion,and distant metastasis,and high DUXAP8 expression in HCC patients with high DUXAP8 expression tend to have poorer prognosis.(2)The expression of DUXAP8 in seven hepatocellular carcinoma cell lines(MHCC-97 H,Huh7,HCC-LM3,Bel-7405,SNU-449,SK-Hep-1,SNU-182)was significantly higher than its expression in human liver immortalized cell line(THLE-3);among the seven hepatocellular carcinoma cell lines,DUXAP8 was significantly higher in Huh7 and Among the seven hepatocellular carcinoma cell lines,the expression of DUXAP8 was relatively high in Huh7 and SNU-449 cell lines and relatively low in SK-Hep-1 and HCC-LM3 cell lines.(3)In vitro experiments confirmed that DUXAP8 knockdown reduced the stem cell characteristics of HCC cells,inhibited the proliferation ability of HCC cells,and enhanced the sensitivity of HCC cells to sorafenib;while DUXAP8 overexpression promoted the stem cell characteristics and proliferation ability of HCC cells,and also promoted the resistance of HCC cells to sorafenib;in vivo experiments confirmed that DUXAP8 knockdown In vivo experiments confirmed that DUXAP8 knockdown inhibited the proliferation ability of HCC cells,enhanced the sensitivity of HCC cells to sorafenib,and suppressed the lung metastasis ability of HCC cells.(4)Me RIP-q PCR results showed that the m6 A enrichment of DUXAP8 in HCC cell lines was significantly higher than that in normal hepatocyte lines;by searching the TCGA database,it was found that the expression level of Mettl3 in HCC tissues was significantly higher than that in the corresponding normal liver tissues,and there was a significant positive correlation between the expression levels of Mettl3 and DUXAP8;in silencing Mettl3 in HCC cells,the expression level of DUXAP8 was significantly reduced,and the m6 A enrichment of DUXAP8 also appeared to be reduced,as well as reducing the stability of DUXAP8;in HCC cells overexpressing Mettl3,the expression level of DUXAP8 was correspondingly increased,and the m6 A enrichment of DUXAP8 also appeared to be increased,as well as increasing the stability.(5)The localization of DUXAP8 in HCC cells is mainly in the cytoplasm.mi R-584-5p is a micro RNA-seq combined with the bioinformatics database Encyclopedia of RNA Interactomes(ENCORI,previously known as star Base v2.0)screened The mi R-584-5p was confirmed to be a micro RNA targeting DUXAP8 by RT-q PCR,dualluciferase reporter gene,RNA immunoprecipitation,and RNA pull-down assay;RNA interference technology and cotransfection technology confirmed that mi R-584-5p could reverse DUXAP8 low expression / overexpression in the maintenance of stem cell characteristics and resistance to sorafenib in HCC cells.(6)MAPK1 was the target gene screened by RNA-seq in HCC cells with DUXAP8 knockdown,and MAPK/ERK pathway was the predominant relevant signaling pathway for KEGG enrichment analysis;mi R-584-5p was confirmed to directly target MAPK1 in HCC cells by RT-q PCR,dual luciferase reporter gene,RNA interference technology and co-transfection techniques confirmed the ability of MAPK1 to reverse the role of mi R-584-5p low expression/overexpression in the maintenance of stem cell characteristics and resistance to sorafenib in HCC cells.(7)Western Blot results showed that DUXAP8 knockdown inhibited the phosphorylation level of ERK/CREB,but had no effect on the phosphorylation level of JNK/P-38,while overexpression of MAPK1 could eliminate the inhibitory effect of DUXAP8 knockdown on the phosphorylation level of ERK/CREB;overexpression of DUXAP8 could promote the phosphorylation level of ERK/CREB phosphorylation levels,and knockdown of MAPK1 eliminated the promoting effect of DUXAP8 overexpression on ERK/CREB phosphorylation levels.Conclusions:(1)DUXAP8 was highly expressed in HCC tissues and positively correlated with the malignancy of HCC;overexpression of DUXAP8 promoted the stem cell characteristics and proliferation of HCC cells and promoted the resistance of HCC cells to sorafenib.(2)Mettl3-mediated m6 A modification plays an important role in maintaining the high expression as well as stability of DUXAP8 in HCC cells.(3)DUXAP8 competitively binds mi R-584-5p through a ce RNA mechanism,thereby targeting MAPK1 and activating the MAPK/ERK pathway to regulate the malignant phenotype of HCC and resistance to sorafenib. |
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