The Molecular Mechanism Of PNO1 Affected Malignant Progression Of Hepatocellular Carcinoma Through Regulation Of Apoptosis And Autophagy

Background and ObjectiveHepatocellular carcinoma(HCC)is the fourth most common causes of cancerrelated mortality worldwide.More than 380 000 people dies of liver cancer in China each year,accounting for 51% of the global HCC-affected population.The high recurrence and metastasis rate explain the poor prognosis of HCC patients,even though the treatment of HCC is constantly updated and improved.Therefore,novel molecules should be explored to improve HCC prognosis.It is well known that the development of tumors is always related to the dysregulation of cell proliferation and programmed cell death.Apoptosis and autophagy are two kinds of programmed cell death and play crucial roles in tumor progression.Accumulating studies have shown that cancer cells usually gain apoptosis resistance resulting in cancer-cell survival and hyperproliferation.Activated apoptosis represents a potent cancer-treatment strategy.At the same time,imbalance of autophagy plays an important role in cancer-cell survival and death and is thus gaining increased attention in cancer therapy.The RNA-binding protein "partner of NOB1"(PNO1)is mainly involved in ribosome biogenesis and promotes the maturation of small ribosomal subunits.Many studies have shown that imbalance in the ribosome-biogenesis process is related to tumor progression.Some studies have demonstrated the relationship of apoptosis and autophagy with ribosome biogenesis,but the specific mechanism in tumor remains unclear.The aim of this project is to explore the clinical significance,biological function and molecular mechanism of PNO1 in HCC,and further provide a new strategy for the treatment of HCC.Methods1.We used 150 HCC tissue microarrays and 8 pairs of frozen HCC tissue samples to explore the expression of PNO1 in HCC and its impact on the prognosis of HCC patients,through immunohistochemistry staining,Kaplan-Meier analysis,Cox regression,Spearman correlation analysis,subgroup analysis and Western Blotting.2.Based on TCGA and GEO databases,bioinformatics analysis(differential expression analysis,survival analysis and GSEA analysis)was used to confirm the findings obtained from immunohistochemistry and Western Blotting.3.Explored the expression level of PNO1 in HCC Cell lines by Western Blotting and the lentiviral infection method was applied to construct HCC cell lines with PNO1 stable overexpression and decreased expression as well as cell lines in the corresponding control groups.4.HCC cells with stable over-or down-expression of PNO1 and their control cells were applied to perform cell viability assays(CCK-8 assay and colony formation assay)and cell apoptosis assays(flow cytometry assay,transmission electron microscopy assay,TUNEL apoptosis assay and Western Blotting)to investigate the effects of different expression levels of PNO1 on the cell viability and apoptosis ability of HCC cells.5.HCC cells with stable over-or down-expression of PNO1 and their control cells were applied to perform cell autophagy assays(transmission electron microscopy experiments,Western Blotting,GFP-LC3 B immunofluorescence experiments and m RFP-GFP-LC3 B autophagic flow immunofluorescence experiments)and rescue experiments to investigate the effects of different expression level of PNO1 on the autophagic capacity of HCC cells.6.Whole transcriptome sequencing analysis of PNO1 down-expressing cell lines and control cell lines were analyze the differential genes and enrich the related pathways,cell function assays,immunoblotting and rescue assays were applied to further explore the downstream molecular network of PNO1 regulating HCC progression.7.HCC xenograft models with high expression of PNO1,low expression of PNO1 and control cell lines were built to observe the effects of different levels of PNO1 on the proliferation,apoptosis and autophagy of HCC cells in vivo.And verified the altered expression levels of important molecules in related signaling pathways.Results1.Through Western Blotting,RT-PCR and immunohistochemical staining revealed that the expression levels of PNO1 in HCC tissues were significantly higher than those in their corresponding adjacent normal tissues,and high expression of PNO1 was an independent risk factor affecting overall survival(OS)and disease-free survival(DFS)in HCC,The correlation between PNO1 expression levels and clinicopathological factors was analyzed through Spearman method,and it was found that PNO1 expression levels were correlated with tumor size(defined as 3cm as the threshold of tumor size),serum alpha-fetoprotein(AFP)level(defined as7 ng/ml as the threshold of AFP)and(defined as 10% as the threshold of Ki-67positivity)were significantly positively correlated;in addition,high expression of PNO1 remained a risk factor affecting the prognosis of hepatocellular carcinoma in the high-risk subgroup of patients with tumor diameter ≥3 cm,high serum AFP levels and high Ki-67 positivity.2.The analysis results of TCGA and GEO database showed that the expression level of PNO1 in HCC tissues was significantly higher than its corresponding adjacent normal tissues,and patients with high expression of PNO1 had poor prognosis.3.PNO1 was expressed at a higher level in Hep3 B cell line and at a lower level in HLE cell line.On this basis,Hep3 B and HLE cells were used to establish stable PNO1 downregulation(sh-PNO1)and PNO1 upregulation(PNO1)cell lines respectively.4.Cell viability assay and colony formation assay confirmed that HLE PNO1 group had stronger proliferation ability than the control group and on the contrary,cell viability decreased in Hep3 B sh-PNO1 group compared with the control group;flow cytometry assay,transmission electron microscopy assay(TEM),TUNEL apoptosis assay and Western Blotting assay confirmed that HLE PNO1 cells had weaker apoptosis ability than the control group and on the contrary,Hep3 B shPNO1 had stronger apoptosis ability than the control group.5.TEM experiments,Western Blotting experiments,GFP-LC3 B immunofluorescence experiments and m RFP-GFP-LC3 B autophagic flow immunofluorescence experiments confirmed that HLE PNO1 cells were more autophagic than control group,conversely,Hep3 B sh-PNO1 cells were less autophagic than controls.6.Designed rescue experiments that treat HLE PNO1 cells and Hep3 B sh-PNO1 cells with autophagy inhibitor(3-methyladenine,3-MA)and autophagy activator(rapamycin,Rapa),respectively.When Hep3 B sh-PNO1 cells were treated with rapamycin to activate autophagy,the rate of apoptosis was reduced and cell viability was promoted.Meanwhile,when HLE PNO1 cells were treated with 3-MA to inhibit autophagy,the apoptosis rate was increased and cell viability was inhibited.7.RNA-seq analysis of Hep3 B cells stably down-expressing PNO1 and its control cells revealed that the differentially expressed genes were mainly enriched in the MAPK signaling pathway,and Western Blotting experiments confirmed that the protein level of p-Erk was higher in the PNO1 overexpression group.8.After treatment of PNO1 overexpressed cells with MEK/Erk inhibitor PD98059,pErk was significantly inhibited as verified by Western Blotting.Meanwhile,the expression of autophagy-related proteins Atg5,Atg7,Beclin1,and LC3 B II was inhibited and the expression levels of apoptosis-related proteins Bax and activated Caspase3 were increased in PNO1 overexpressing cells.PNO1 overexpressing cells treated with PD98059 showed a significant decrease in GFP-LC3 B aggregate vesicles compared with untreated PNO1 overexpressing cells,indicating a decrease in the level of autophagy.Meanwhile,TUNEL immunofluorescence assay,apoptosis flow assay and CCK-8 proliferation assay suggested increased apoptosis in PD98059-treated PNO1 overexpression cells compared with untreated PNO1 overexpression cells.9.The results of animal experiments showed that the tumor volume was larger in the PNO1 overexpression group compared with the control group,and conversely the tumor volume was smaller in the PNO1 down-expression group.As in the in vitro experiments,the PNO1 overexpression group showed higher expression levels of p-Erk,along with enhanced autophagy and weaker apoptosis.ConclusionsPNO1 as a nuclear protein is involved in ribosome assembly.PNO1 is reportedly responsible for the cleavage of 18 S mediated by interaction with Nob1,and both proteins form complexes with the 19 S.Currently,there is no research to explain the specific regulatory mechanism of PNO1 in the progression of HCC.In the previous work,we have used immunohistochemical staining analysis with a large number of samples,as well as TCGA and GEO database analysis to initially clarify that high expression of PNO1 could be an independent risk factor for predicting the prognosis of HCC,and high expression of PNO1 is closely related to tumor size,serum AFP level and Ki-67 positive rate.A series of function experiments have confirmed that high expression of PNO1 can promote autophagy and proliferation of HCC.The whole transcriptome sequencing revealed that PNO1 regulates the malignant phenotype transformation of HCC through MAPK signaling pathway,and the transplantation tumor model confirmed that high expression of PNO1 promotes the progression of HCC.Combining the analysis of large database of molecular biology experiments,biological samples,cell biology experiments and transplantation tumor model,our study has for the first time confirmed the role and molecular mechanism of PNO1 in the malignant phenotypic transformation of HCC,and further explored the role of MAPK signaling pathway influenced by PNO1 on the proliferation of HCC,which is crucial to enrich the clinical treatment means or therapeutic targets.