The Regulatory Mechanism Of Glucose Metabolism And Diagnostic Value Of Novel LncRNAs In Hepatocellular Carcinoma

Background: Hepatocellular carcinoma(HCC)is the most common type of primary liver cancer and is the sixth most common tumor and the third leading cause of cancer-related death worldwide.The development of HCC involves many complex molecular biological processes,and is driven by different molecular pathways to produce different subtypes,including the metabolic driven type caused by metabolic changes.There is evidence that specific global metabolic reprogramming exists in HCC,suggesting that metabolism-related mechanisms play an important role in the development of HCC.Metabolic reprogramming is considered to be one of the important hallmarks of cancer,and aerobic glycolysis is the main type of metabolic reprogramming.Even when oxygen is sufficient,tumor cells are powered by glycolysis through reprogramming glucose metabolism and energy production,which is known as the Warburg effect.Dysfunction of glucose metabolism is common in HCC patients.However,abnormal glucose metabolism is an important factor affecting tumor growth rate,which seriously affects the prognosis of patients.At present,abnormal glucose metabolism has been proved to be a significant hallmark of cancer,but the research on the mechanism of glucose metabolism regulation in HCC is limited.In addition to metabolic reprogramming,HCC also has multiple and recurrent clinical characteristics.Recurrence after radical resection is a major challenge in HCC treatment and the main cause of postoperative death.Intrahepatic metastasis(IM)is considered to be an important source of multiple or recurrent liver tumors and the main source of early HCC recurrence.Intrahepatic metastasis will predict poor prognosis of HCC patients.At present,the diagnosis of HCC intrahepatic metastasis is mainly based on imaging findings or postoperative pathological findings.At this point,the tumor has advanced to a certain extent,it is difficult to intervene in the early stage of intrahepatic metastasis.Therefore,the identification of effective biomarkers for the diagnosis or identification of HCC intrahepatic metastasis and risk assessment may provide some help for the current diagnostic dilemma.Long non-coding RNAs(lnc RNAs)are an ideal biomarker.LncRNA in circulating blood can not be affected by blood ribonuclease within a certain period of time,maintain good stability and high expression level,and can meet the needs of experimental detection.In addition,lnc RNAs have a variety of biological functions,participating in the biosynthesis and function of RNA,DNA and protein and other multi-level regulation.Previous studies have shown that the tumorigenesis of HCC involves the changes of many lnc RNAs,which may play crucial roles in proliferation,metastasis and metabolism.As an important biological research resource,lnc RNA is of great research value in diseases including tumors.Therefore,the exploration of the regulatory mechanism of glucose metabolism and its diagnostic value of lnc RNA in HCC will certainly contribute to the diagnosis,treatment,prognosis judgment and metastasis risk assessment,and may provide new ideas and strategies for accurate typing and individualized treatment of HCC.Objectives:1.Differential lnc RNAs screening: LncRNA expression profiles in HCC were identified,and differentially expressed lnc RNAs with potential function or diagnostic potential in HCC were screened.2.Studies on the mechanism of lnc RNA: To clarify the influence of the screened target lnc RNA NONHSAT024276 on the biological function of HCC cells,and explore its mechanism of action.3.Diagnostic efficacy evaluation of lnc RNA: The expression levels of target lnc RNA NONHSAT053785 in HCC patients serum were detected,and its correlation with clinical characteristics of patients were analyzed,as well as the diagnostic efficacy for HCC were evaluated.Materials and Methods:1.Transcriptome expression studies: Microarray was used to detect lnc RNA and m RNA expression levels in 5 HCC tissues,5 adjacent peritumoral liver tissues(APLT)and 5distant peritumoral liver tissues(DPLT).The RNA expression profile of HCC was established.2.Bioinformatics analysis:1)Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis: GO and KEGG analysis were used to explore the potential biological functions and signaling pathways of differential m RNAs screened by microarray and proteins bind with lnc RNA NONHSAT024276.2)GO network and KEGG network analysis: GO network analysis takes the significant function of differential m RNAs as the research object to construct the network diagram of the relationship between functions.KEGG network analysis takes the significant pathway of differential m RNAs as the research object to construct the signal transduction relationship between the pathways.3)Co-expression network analysis: By constructing the lnc RNA-m RNA co-expression regulatory network,the function of unknown lnc RNA was predicted,the potential regulatory relationship between lnc RNA and m RNAs was explored,and lnc RNA with important regulatory functions in the network and their possible mechanisms of action were identified.4)Prediction of RNA structure and protein coding potential: Predicting the secondary structure of NONHSAT024276 by RNAfold;The protein coding potential of NONHSAT024276 was analyzed by Genome Browser,Coding-Potential Assessment Tool(CPAT)and ATGpr.The region of NONHSAT024276 bound with polypyrimidine tract-binding protein 1(PTBP1)was predicted by cat RAPID.5)Public database analysis: PTBP1 and pyruvate kinase M2(PKM2)expression data in HCC were investigated from the Cancer Genome Atlas(TCGA)and the International Cancer Genome Consortium(ICGC)databases.3.Cell function experiments:1)Cell counting kit-8 and colony formation assay: To detect the proliferation of HCC cells.2)Transwell and scratch wound-healing assays: To detect the metastasis and invasion ability of HCC cells.3)Flow cytometry: To detect the apoptosis rate and cell cycle distribution of HCC cells.4.Molecular biology experiments:1)Quantitative real-time PCR(q RT-PCR): The target lnc RNAs(NONHSAT024276,NONHSAT053785,ENST00000514608.1,NONHSAT138156,NONHSAT124357.2,ENST00000560295.1)were detected by q RT-PCR in 20 HCC tissues,20 APLT and 20 DPLT,to verify the expression trend of microarray;The expression levels of NONHSAT024276,PTBP1,pyruvate kinase M1(PKM1)and PKM2 were detected in 18 HCC tissues and 18 paired adjacent liver tissues;The expression levels of NONHSAT024276,PTBP1,PKM1,PKM2,U6,and glyceraldehyde-3-phosphate dehydrogenase(GAPDH)were determined in HCC cells.2)Nuclear and cytoplasmic separation and RNA fluorescence in situ hybridization(FISH): To determine the distribution of NONHSAT024276 in HCC cells.3)Rapid amplification of c DNA end(RACE): Full length of NONHSAT024276 was confirmed.4)Construction of stable transfected cell lines: NONHSAT024276 was stably overexpressed in HCC cells to study its effect on the biological behavior of HCC cells and its intermolecular regulatory relationship.5)Transfection of plasmid and si RNA: Overexpression of PTBP1 and PKM2 was achieved by transfecting plasmid into HCC cells;The low expression of NONHSAT024276 and PTBP1 was achieved by transfection of si RNA into HCC cells.6)Western Blot(WB): The protein expression levels of PTBP1,PKM1,PKM2 and GAPDH were determined.7)RNA pull-down: Proteins binding to NONHSAT024276 was isolated in HCC cells.8)Silver staining: The RNA Pull-down enriched proteins were subjected to SDS-PAGE,and then silver staining was performed to realize the visualization of NONHSAT024276 binding proteins.9)Mass spectrometry: The proteins enriched in RNA pull-down were analyzed by mass spectrometry,and the proteins binding with NONHSAT024276 were identified in detail.10)RNA immunoprecipitation(RIP): To verify that PTBP1 can be combined with NONHSAT024276.11)Droplet digital polymerase chain reaction(dd PCR): lnc RNA NONHSAT053785 expression levels were determined in serum of 112 HCC patients,96 chronic hepatitis B(CHB)patients and 99 healthy controls(HC).5.Glucose,Lactate,ATP detection: Automatic biochemical analyzer was used to detect the levels of glucose and lactate in cell culture medium;The expression level of ATP in the cells was determined by ATP detection kit.6.Statistical methods: Comparison between the two groups of data was performed using Student’s t or Mann-Whitney U test.Data from two or more groups were compared using one-way ANOVA analysis or Kruskal-Wallis H test.The chi-square test was used to compare the variables expressed in frequency.Spearman correlation analysis was used for the correlation between the two variables.Univariate and multivariate logistic regression analyses were used to identify risk factors for intrahepatic metastasis.The receiver operating characteristic(ROC)curve was used to evaluate the diagnostic performance of NONHSAT053785.Results:1.Screening of differentially expressed lnc RNAs in HCC:1)A large number of lnc RNAs and m RNAs were dysregulated in HCC: A total of 719 lnc RNAs and 3438 m RNAs were identified as differentially expressed in HCC by microarray.2)Differentially expressed m RNAs were mainly involved in metabolic related biological processes: The results of GO and KEGG analysis showed that 337 GO terms and53 KEGG pathways were significantly enriched.The biological processes related to metabolism are abundant,including ATP binding(GO: 0005524),RNA binding(GO:0003723),Cell cycle(hsa04110),glycolysis/gluconeogenesis(hsa00010),and pyruvate metabolism(hsa00620).3)LncRNA NONHSAT024276 and NONHSAT053785 may play potentially important roles in HCC: LncRNA NONHSAT024276 and NONHSAT053785 are at the core of lnc RNA-m RNA co-expression network,and both are associated with 15 m RNAs,suggesting that they may play important roles in HCC.In addition,NONHSAT024276 was closely related to PTBP1,an important molecule of Warburg effect,suggesting that NONHSAT024276 may be involved in aerobic glycolysis of HCC cells.4)Microarray data has high reliability: The expression trend of 5/6 lnc RNAs(NONHSAT024276,NONHSAT053785,ENST00000514608.1,NONHSAT138156,NONHSAT124357.2)was consistent with that of microarray after q RT-PCR.Only the expression of ENST00000560295.1 showed no significant difference.2.Mechanism of lnc RNA NONHSAT024276 regulating glucose metabolism in HCC:1)The expression of lnc RNA NONHSAT024276 was decreased in HCC cell lines and HCC tissues,and its expression level was correlated with tumor volume and aspartate transaminase(AST)level.2)NONHSAT024276 was distributed in both cytoplasm and nucleus of HCC cells,with a total length of 3701 nt and no protein-coding potential.3)Overexpression of NONHSAT024276 inhibited the proliferation and metastasis of HCC cells,increased the apoptosis rate of HCC cells,and decreased the proportion of S-phase cells.4)NONHSAT024276 combined with PTBP1 and negatively regulated PTBP1,and the two formed a feedback loop to regulate each other.5)Overexpression of NONHSAT024276 increased the PKM1/PKM2 ratio and hindered aerobic glycolysis of HCC cells.6)PTBP1 can regulate the expression of PKM1 and PKM2,and reduce the PKM1/PKM2 ratio7)NONHSAT024276 inhibited HCC cell proliferation and glycolysis through the PTBP1/PKM axis.3.Diagnostic value of lnc RNA NONHSAT053785 in HCC:1)The expression level of lnc RNA NONHSAT053785 in HCC patients serum was significantly increased.Its expression level was higher in HCC patients with intrahepatic metastasis than that without.2)The serum expression level of lnc RNA NONHSAT053785 was correlated with the clinical characteristics of patients,including intrahepatic metastasis,Child-Pugh classification and carcinoembryonic antigen(CEA),and was positively correlated with multiple liver enzymology indexes such as aspartate transaminase(AST)and alkaline phosphatase(ALP).3)Increased expression of serum NONHSAT053785 is an independent risk factor for intrahepatic metastasis in HCC patients.4)Serum NONHSAT053785 had a certain ability to differentiate intrahepatic metastasis of HCC(AUC: 0.678)and a good diagnostic efficacy for HCC(AUC: 0.801).In addition,NONHSAT053785 showed good diagnostic potential(AUC: 0.771)in HCC patients with negative alpha-fetoprotein(AFP < 15ng/ml).Conclusion:1.A large number of lnc RNAs and m RNAs have significant expression differences in HCC.The differentially expressed m RNAs were closely related to variety metabolic biological processes,suggesting that the tumorigenesis and development of HCC may be related to metabolic changes caused by molecular changes at the transcriptome level.2.LncRNA NONHSAT024276 and NONHSAT053785 are at the core of lnc RNA-m RNA co-expression network,and both are associated with 15 m RNAs,suggesting that they may play important roles in the development of HCC.In addition,NONHSAT024276 was closely related to PTBP1,an important molecule of Warburg effect,suggesting that NONHSAT024276 may be involved in aerobic glycolysis of HCC cells.3.LncRNA NONHSAT024276 can bind to PTBP1 and form a feedback loop while negatively regulating PTBP1.NONHSAT024276 inhibits the proliferation and aerobic glycolysis of HCC cells by increasing the PKM1/PKM2 ratio through this feedback loop.4.The expression of lnc RNA NONHSAT053785 was up-regulated in the serum of HCC patients,and was correlated with many clinical features of patients such as intrahepatic metastasis,especially with many liver enzymology indexes in peripheral blood.5.Elevated serum lnc RNA NONHSAT053785 expression is an independent risk factor for intrahepatic metastasis in HCC patients,which has a certain ability to differentiate intrahepatic metastasis and has a good diagnostic value in HCC,especially in HCC with negative AFP.In summary,the tumorigenesis and development of HCC may be related to metabolic disorders caused by changes in transcriptome level.lnc RNA plays an important role in the regulation of glucose metabolism in HCC,and is of great value for the diagnosis of HCC and the risk assessment of intrahepatic metastasis.