| BackgroundHepatocellular carcinoma(HCC)is an incurable malignant tumor of the digestive system,with high incidence and low survival rate among the highest in the world.Although with the development of chemotherapy and immune targeted therapy,the efficacy of drug therapy is still not ideal and drug resistance is obvious.Therefore,it is urgent to explore the causes of drug resistance in HCC.More and more studies have shown that,Epigenetic regulation may play an important role in the pathogenesis and drug resistance of HCC.Endoplasmic reticulum stress(ERS)is a kind of subcellular organelle pathological state in which cells are stimulated by various physical and chemical factors such as ischemia,hypoxia and oxidative stress,which is characterized by imbalance of calcium homeostasis and excessive protein synthesis.It is an extremely important signal response pathway.Endoplasmic reticulum(ER)is the synthesis site of secretory and membrane proteins.Hepatoma cells are prone to produce ERS under inflammatory stimulation due to its high secretory function.The existing literature reports and the previous study of our group have found that the expression of NAT10 in chromatin,transcriptome and protein in hepatoma cells increased after ERS,which suggests that NAT10 may play an important role in endoplasmic reticulum stress-mediated hepatoma cells.NAT10(N-acetyltransferase)is a nuclear protein that functions as a lysine acetyltransferase and is the only reported modification enzyme of RNA ac4C.It is involved in a variety of cellular processes.It has been reported that NAT10-mediated acetylation plays an important role in gastric cancer,breast cancer,multiple myeloma,acute myeloid leukemia and other cancers.The relationship between NAT10-mediated RNA acetylation modification and endoplasmic reticulum stress-related drug resistance in HCC has not been reported.In recent years,the study of epigenetics has become a research hotspot of scholars at home and abroad.N4-acetylcytidine(ac4C),N4 position acetylcytosine,the total amount of acetylation modification is much lower than that of m6A methylation.ac4C modification was first reported on t RNA and r RNA.With the rise of ac RIP-seq technology,in 2018,Arango D et al.proposed that there is also a large amount of ac4C on m RNA,and ac4C modification is crucial for the fate decision of m RNA,suggesting that RNA ac4C modification may play an important role in regulating cell life activities.Studies have found that this modification is closely related to the occurrence,development,prognosis,metastasis,epithelial-mesenchymal transition,and drug resistance of tumors.The continuous activation of endoplasmic reticulum stress is closely related to tumor drug resistance.Previous studies mostly focused on a gene,a protein or a signaling pathway.However,the role of specific molecular modifications such as ac4C modification in tumor endoplasmic reticulum stress is rarely reported.Sorafenib has been used as the first-line systemic treatment for HCC for more than a decade.However,sorafenib is highly resistant and has been phased out in recent years.Lenvatinib is a multi-receptor tyrosine kinase inhibitor used for the treatment of patients with advanced HCC in the past three years,but the clinical benefit of the drug is still limited,and the specific mechanism is unclear.Studies have shown that the activation of EGFR limits the response of HCC to lenvatinib.From the perspective of biological modification,this study explored the new mechanism of ERS-mediated drug resistance in HCC from a new perspective,and explored the mechanism of NAT10-mediated RNA acetylation ac4C modification in endoplasmic reticulum stress-induced metastasis of hepatoma cells and lenvatinib resistance.In addition,it is necessary to further clarify the related target genes and signaling pathways that regulate the modification of RNA acetylation ac4C and their effects on apoptosis of liver cancer and the possible molecular mechanism,and change the biological characteristics of tumor cells,which will become a new idea for the clinical treatment of HCC.Objective(1)The correlation between NAT10 and ERS marker proteins in liver cancer patients in the public database was analyzed,and the differential expression of NAT10 in different hepatoma cell lines and survival prognosis of hepatoma patients was analyzed.(2)To investigate the association of NAT10 with endoplasmic reticulum stress and clinicopathological features in HCC patients;(3)To confirm that NAT10 enhanced endoplasmic reticulum stress signal in hepatoma cells,and to clarify the effect of NAT10 on metastasis,cell cycle arrest,apoptosis and lenvatinib sensitivity in HCC cells under endoplasmic reticulum stress;(4)To investigate the role of NAT10-mediated RNA acetylation ac4C and its signaling pathway in ER stress-mediated lenvatinib resistance in HCC cells.Methods(1)The GEO database(GSE62043)was used to study the expression of NAT10 in tumor tissues and adjacent tissues of HCC patients(n=50),and the difference of NAT10expression between tumor tissues and adjacent tissues was analyzed.The expression of NAT10 in liver cancer patients(n=336)and healthy patients(n=50)in the TCGA database was analyzed,and the expression difference of NAT10 between liver cancer patients and healthy patients was compared.Kaplan-Meier survival analysis was used to analyze the relationship between NAT10 expression and clinical prognosis of HCC patients in TCGA database,and the differences in prognosis were analyzed.(2)Immunohistochemistry was used to detect the expression of NAT10,GRP78,ATF-6,IRE-1 and PERK in slides of HCC tissues.The correlation between the expression of NAT10 and the clinicopathological features of HCC patients was analyzed.(3)The expression of NAT10 in normal hepatocyte LO-2 and four HCC cell lines Huh-7,Hep3B,MHCC-97h and Sk-hep-1 was detected,and huh-7 and hep3B hepatoma cell lines with higher expression of NAT10 were selected for subsequent experiments.NAT10-si RNA was used to knock down the gene in the cell lines,and immunofluorescence quantitative technology was used to detect the knockout effect.(4)Hepatoma cells were treated with five concentrations of lenvatinib and Remodelin hydrobromide,a NAT10 inhibitor,for 24 and 48h.CCK-8 was used to detect the inhibition rate,and IC50 values of lenvatinib and Remodelin were calculated.(5)The expressions of NAT10,GRP78,ATF-6,IRE-1 and PERK were detected by Western Blotting.Remodelin,a NAT10 inhibitor,was used to inhibit the expression of NAT10 in hepatoma cells(select the appropriate concentration that can significantly inhibit the level of NAT10 without affecting cell growth).The apoptosis of hepatoma cells under ERS after NAT10 inhibition was observed.Western Blotting was used to detect the expressions of GRP78,ATF-6,IRE-1,PERK,bax,bak and bcl-2.Cell cycle analysis was used to analyze the inhibitory effect of NAT10 on the proliferation of hepatocellular carcinoma cells under endoplasmic reticulum stress.Western Blotting was used to detect the expression of cyclin CDK2,cylin A and PCNA.After adding si RNA and Remodelin,CCK-8 assay was used to detect the effect of different concentrations of lenvatinib on HCC cells under endoplasmic reticulum stress,and flow cytometry was used to detect apoptosis.(6)To evaluate the RNA content of NAT10-mediated ac4C modification in HCC under endoplasmic reticulum stress,liquid chromatography tandem mass spectrometry was used to detect the total amount of ac4C modification in normal hepatocellular carcinoma cells and hepatoma cells under endoplasmic reticulum before and after NAT10 inhibition.ac RIP-sep sequencing and m RNA-sep were used to analyze the target genes of NAT10.Apparent experiment verification;The dual luciferase reporter gene assay was used to explore whether NAT10 regulated the expression level of downstream target genes by mediating ac4C modification.(7)Animal experiments The effect of NAT10 on tumorigenicity in 3-4 weeks old Balb/c nude mice was analyzed.Twelve mice were randomly divided into two groups,the normal group and the NAT10 inhibition group.Huh-7 cells(2×10~6/100μl,6 mice per group)were collected,suspended in precooled PBS,and injected subcutaneously into the lateral right thigh.Remodelin solution(5 mg/kg)was intraperitoneally injected into mice in NAT10 inhibitor group every two days when the tumor volume reached100mm3,while the mice in normal group were injected with the same amount of PBS every two days.After tumor formation,the maximum diameter and the perpendicular short diameter of the tumor were measured by caliper every two days,and the tumor volume was calculated and weighed.After 21 days,the mice were sacrificed,and the tumors were removed for HE staining,immunohistochemical staining(NAT10,HSP90AA1,GRP78),WB and q PCR(NAT10,HSP90AA1,GRP78).Fourteen mice were randomly divided into two groups with 10 mice in each group to observe the effect of NAT10 on tumor drug resistance.Each mouse was intraperitoneally injected with huh-7 cells(2×106/100μl)in the right thigh.Lenvatinib(10mg/kg)was injected intraperitoneally every Tuesday,Thursday,and Saturday after the tumor size reached100mm3.After 14 days,the mice were killed to observe the tumor size.Results(1)The GEO(GSE 7186)database found that the expression level of NAT10 in HCC tissues was significantly higher than that in adjacent tissues.TCGA database showed that the expression level of NAT10 in HCC patients was significantly higher than that in normal healthy people.Kaplan-Meier survival analysis was used to analyze the relationship between NAT10 expression and clinical prognosis of HCC patients using TCGA STAD database.The results showed that OS(overall survival),PPS(post progression survival)and DSS(disease specific survival)of HCC patients with high NAT10 expression were significantly lower than those of HCC patients with low NAT10 expression,suggesting that overexpression of NAT10 in HCC population is associated with poor prognosis.(2)Immunohistochemical results showed that NAT10 negative expression in 8 cases,low expression in 35 cases,high expression in 57 cases,GRP78 negative expression in2 cases,low expression in 19 cases,high expression in 79 cases,ATF-6 negative expression in 3 cases,low expression in 24 cases,high expression in 73 cases,IRE-1negative expression in 4 cases,low expression in 25 cases.There were 71 cases with high expression of NAT10,4 cases with negative expression of PERK,40 cases with low expression of PERK,and 56 cases with high expression of PERK.Spearman rank correlation analysis showed that NAT10 and endoplasmic reticulum stress marker protein GRP78(rs=0.454,P<0.001).ATF-6(rs=0.288,P=0.002);IRE-1(rs=0.431,P<0.001);PERK(rs=0.394,P<0.001)was positively correlated.Multivariate analysis showed that the expression of NAT10 was significantly correlated with the history of hepatitis and liver cirrhosis,but not with other factors such as tumor size,differentiation degree,AFP,ALT,AST.(3)The results showed that NAT10 expression was the highest in hep3B cells,and Huh-7 cells also showed significant high expression.Two si RNA-2710 and si RNA-3049 of NAT10 were used to generate NAT10 knockdown in Huh-7 and Hep3B cells(Figure2B).The inhibition rates of Huh-7 and Hep3B were 64.4%and 73.1%and 34.6%and32.8%,respectively,as confirmed by immunofluorescence assay.WB showed that the inhibition effect was more significant with the increase of inhibitor concentration.(4)Remodelin,a NAT10 inhibitor,was inhibited by lenvatinib in Huh-7 and Hep3B cells in a time-and dose-dependent manner.The 48h IC50 values of lenvatinib in Huh-7 and Hep3B cells were 9.327μM and 14.630μM,respectively.The IC50 values of Remodelin in the two cell lines at 48h were 29.2μM and 27.92μM,respectively.(5)Western blotting showed that Remodelin reduced the expression of GRP78,IRE1,ATF-6 and PERK in Huh-7 and hep3B cells,and NAT10 knockdown also inhibited the expression of GRP78,IRE1,ATF-6 and PERK in huh-7 cells.transwell assay showed that NAT10 knockdown significantly inhibited the migration and invasion of hepatocellular carcinoma cells under ERS condition.Scratch assay showed that NAT10knockdown significantly inhibited the migration of Huh-7 and Hep3b cells under ERS condition.In Huh-7 cells,the percentage of apoptotic cells in NAT10 knockout group was significantly higher than that in untreated control group(TM)and si NC group,and quantitative data showed that there was a significant difference between si RNA-NAT10group and control group.Similar results were observed after treatment with Remodelin,a NAT10 inhibitor,which induced a dose-dependent increase in the apoptosis rate of Huh-7 and Hep3B hepatoma cells.Remodelin treatment increased the expression of apoptotic proteins Bax and Bak and decreased the expression of anti-apoptotic protein Bcl-2 after NAT10 knockdown.Cell cycle analysis showed that the cell cycle distribution of Hep3B and Huh-7 were different,and Remodelin treatment inhibited the S phase of Hep3B cell cycle.NAT10-si RNA and Remodelin hydrobromide treatment inhibited Huh-7,showing only a mild S-phase arrest,and WB results showed that CDK2,cylin A,and PCNA were decreased in Huh-7 and Hep3B.CCK-8 assay confirmed that the inhibition rate of lenvatinib on Huh-7 and hep3B cells was gradually increased with the concentration of NAT10 inhibition.Flow cytometry also showed that the apoptosis rate of lenvatinib on Hu H-7 and Hep3B cells was increased after NAT10-sirna or Remodelin inhibition.(6)The results of liquid chromatography tandem mass spectrometry showed that the content of total RNA ac4C in hepatoma cells after ERS was significantly higher than that in the control group,and the content of total RNA ac4C in NAT10 knockout group in hepatoma cells under ERS was lower than that in the stress group.0.05,1.5 fold difference);GO pathway analysis showed that the differentially expressed genes were mainly enriched in cell cycle and extracellular matrix pathways.ac RIP-sep sequencing analysis showed that the overall ac4C modification level of ERS Huh-7 cells was significantly higher than that of non-stressed Huh-7 cells,and genes were enriched in ERS and cell cycle pathways.The overall level of ac4C modification was significantly reduced in sir NA-NAT10-transfected ERS Huh-7 cells,and GOkeep analysis showed enrichment of genes in cell cycle pathways and m RNA processing and metabolic regulation pathways.Comprehensive analysis of ac RIP-Seq and m RNA-Seq data showed that the ac4C modification in the CDS coding region of HSP90AA1 gene was down-regulated by NAT10 knockdown.Through the cross analysis of ac RIPseq and RNAseq before and after the establishment of ERS model and after NAT10 interference ERS model,It was found that HSP90AA1 gene acetylation and expression were significantly correlated with NAT10 gene expression.ac RIP-PCR further verified the correlation between NAT10 and HSP90AA1 gene expression.After knocking down the expression of NAT10 gene,the expression of HSP90AA1 ac4C modified was decreased.Compared with si-NAT10 IP,the ac4C peak of HSP90AA1 in ERS IP group was significantly increased.(7)HSP90AA1 wild type and mutant reporter genes were constructed by dual luciferase reporter gene assay.For the HSP90AA1 mutant,the ac4C cosequence CAC was replaced by GAG,which abolished the ac4C modification.The luciferase activity of NAT10 and wild-type HSP90AA1 reporters was enhanced,whereas NAT10 suppressed the luciferase activity of mutant HSP90AA1 reporter expression.The results showed that NAT10 could bind to HSP90AA1 and promote the translation of HSP90AA1.Therefore,NAT10 may modify the CDS coding region of HSP90AA1 through ac4C to positively regulate HSP90AA1 expression.(8)Western blot analysis of liver cancer cell lines huh-7 and Hep3B in vitro.In the ERS state,HSP90AA1 protein expression was increased.Knockdown of NAT10 reduced the expression of HSP90AA1 protein.Taken together,HSP90AA1 may be a downstream regulatory target of NAT10-mediated m RNA ac4C modification.Wound healing assay showed that HSP90AA1 knockdown significantly inhibited the migration of HCC cells,while transwell assay showed that HSP90AA1 knockdown inhibited the migration and invasion of HCC cells.The results of flow cytometry staining showed that the apoptosis rates of Huh-7 and Hep3B hepatoma cells with target gene HSP90AA1 knockdown under ERS conditions were significantly higher than those in the control group.Both q RT-PCR and WB showed that the expression of HSP90AA1 was increased in Huh-7and Hep3B cells after ER stress.Similarly,after HSP90AA1 knockdown,the cell cycle also showed S phase cell arrest.WB showed that the apoptotic proteins bas and bak were decreased,and the anti-apoptotic protein bcl-2 was increased.The expression of cyclin CDK2,cyclin A and PCNA showed a downward trend.(9)In the subcutaneous tumor formation experiment in nude mice,the size and weight of subcutaneous transplanted tumors in NAT10 knockout mice and HSP90AA1knockout mice were significantly smaller than those in the control group.Tumor growth in lenvatinib treated nude mice was significantly slower than that in control group,and Remodelin treated nude mice also showed slower tumor growth than lenvatinib group.The tumor volume of nude mice injected with si RNA-HSP90AA1 was smaller than that of lenvatinib group.Conclusions(1)The expression of NAT10 was significantly up-regulated in HCC patients.In HCC patients,the expression of NAT10 is positively correlated with ERS marker proteins,and the expression level of NAT10 is related to the prognosis of HCC.(2)NAT10 was highly expressed in HCC tissues,which was significantly correlated with endoplasmic reticulum stress-related markers GRP78,ATF-6,IRE-1 and PERK,and was also associated with clinical pathological features such as history of hepatitis and liver cirrhosis.(3)Inhibition of NAT10 by si RNA or inhibitor increased cell cycle arrest and apoptosis in S phase of HCC cells under ERS condition.NAT10 may trigger apoptotic signals by activating Bax/Bcl-2 axis and enhancing endoplasmic reticulum stress signaling to enhance the apoptosis of hepatocellular carcinoma cells.NAT10 enhanced the sensitivity of lenvatinib to hepatocellular carcinoma cells under ERS.(4)The level of NAT10-mediated RNA acetylation ac4C was increased in HCC cells under ER stress.(5)HSP90AA1 is the downstream regulatory target of NAT10-mediated RNA ac4C modification,and NAT10 can promote the expression of HSP90AA1 gene in HCC under ERS.(6)HSP90AA1 may play a role in promoting metastasis,cell cycle arrest,apoptosis resistance and lenvatinib resistance in ERS hepatocellular carcinoma... |
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