Development of an ARMS multiplex real-time PCR assay for the detection of HLA-B*13:01 genotype by detecting highly specific SNPs.

OBJECTIVES: HLA-B*13:01 was strongly associated with Dapsone Hypersensitivity Syndrome (DHS). This study aimed to develop and validate a rapid and economical method for HLA-B*13:01 genotyping. METHODS: Two tubes multiplex real-time PCR detection system comprising amplification refractory mutation system primers and TaqMan probes was established for HLA-B*13:01 genotyping. Sequence-based typing was applied to validate the accuracy of the assay. RESULTS: The accuracy of the assay was 100% for HLA-B*13:01 genotyping. The detection limit of the new method was 0.025 ng DNA. The positive rate of HLA-B*13:01 in the Bouyei (20%, n = 50) populations was significantly higher than that in the Uighur population (4%, n = 100), Han (4.5%, n = 200), and Tibetan (1%, n = 100) ( P  < 0.05). CONCLUSION: The proposed method is rapid and reliable for HLA-B*13:01 screening in a clinical setting.

Identification of novel lncRNA prognostic biomarkers and their associated ceRNAs in bladder urothelial carcinoma.

Bladder urothelial carcinoma (BUCA) is a common malignant tumor with a high rate of metastasis and recurrence. The lack of specific and sensitive biomarkers for the prognostic assessment makes it important to seek alternatives. Recent studies have demonstrated that long noncoding RNAs (lncRNAs) function as competitive endogenous RNAs (ceRNAs) and play an important role in BUCA prognosis. Therefore, this study aimed to establish a prognosis-related lncRNAs-microRNAs (miRNAs)-messenger RNA (mRNA) (pceRNA) network and identify novel prognostic biomarkers. Integrated weighted coexpression analysis, functional clustering, and ceRNA network were used for the prognostic assessment of BUCA. The transcriptome sequencing datasets of lncRNA, miRNA, and mRNA from The Cancer Genome Atlas database were used for the identification of key lncRNAs and construction of the lncRNAs expression signature for prognostic prediction of BUCA patients. Then, 14 differentially expressed lncRNAs (DE-lncRNAs) were identified as candidate prognostic RNAs based on the ceRNAs network and functional clustering. In the Cox regression analysis, two (AC008676.1 and ADAMTS9-AS1) of all DE-lncRNAs were significantly associated with overall survival (OS) of BUCA patients. This two DE-lncRNA signature was significantly correlated with OS and was an independent prognostic factor, which was confirmed in an independent dataset of GSE216037. Moreover, we constructed the pceRNA network that includes 2 DE-lncRNAs, 9 DE-miRNAs, and 10 DE-mRNAs. Pathway enrichment analysis showed that AC008676.1 and ADAMTS9-AS1 are involved in several cancer-related pathways such as proteoglycans in cancer and TGF-beta signaling pathway. The novel-identified DE-lncRNA prognostic signature and the pceRNA network in this study will be valuable risk predictors and diagnostic markers for BUCA.

Association Analysis of Gene Sequencing by NeoSeq Combined with Tandem Mass Spectrum and Four Neonatal Diseases.

BACKGROUND: NeoSeq is a new method of gene sequencing for newborn screening. The goal is to explore the relationship between gene sequencing by NeoSeq combined with tandem mass spectrum (TMS) and four neonatal diseases. METHODS: A total of 1,989 newborns from August 2010 to December 2021 were enrolled. The case number of congenital hypothyroidism, phenylketonuria, adrenocortical hyperplasia, and glucose-6-phosphate dehydrogenase deficiency was counted, and the results of gene sequencing by NeoSeq and TMS were analyzed. RESULTS: The proportion of male newborns was higher than that of female newborns (51.68% vs. 48.32%). The detection rate of glucose-6-phosphate dehydrogenase deficiency was higher than that of the other three diseases (0.60% vs. 0.05%, 0.05%, 0.15%). A total of 121 newborns were recalled from 1989 newborns by traditional screening technique, and TMS detected phenylketonuria, citrullinemia, glutaric acidemia type I, and 3-methylcro-tonyl-CoA carboxylase deficiency in 1 newborn each. Gene sequencing by NeoSeq of newborns with positive TMS results confirmed the presence of susceptibility genes, and 17 of 1,868 newborns with normal biochemical tests had pathogenic genes. CONCLUSIONS: The incidence of glucose-6-phosphate dehydrogenase deficiency is relatively higher in four neonatal diseases, and the detection rate of gene sequencing by NeoSeq combined with TMS is high.

Comprehensive expression-based isoform biomarkers predictive of drug responses based on isoform co-expression networks and clinical data.

Drug sensitivity biomarkers are molecular features informative for drug response. Previous studies have identified many candidate drug-sensitivity signatures at the transcript level based on significant p values. However, the potential of sensitivity biomarkers has not been sufficiently understood because these investigations have focused on individual biomarkers and have not been carried out at the systems level. In this study, we applied a meta-analytical framework to compute co-expression between isoform pairs in two large datasets of RNA-seq profiles of breast cancer cell lines. We then built hallmark-related direct (HRD) networks by integrating a breast cancer specific isoform co-expression (BCIC) network and hallmark-related isoforms. Next, we explored the associations between isoform biomarkers and the functional clusters of the HRD network. The crucial isoform-based biomarkers for drugs were identified by functional clusters analysis and elucidated by combining isoform expression profiles with clinical information for breast cancer in The Cancer Genome Atlas.

Screening candidate microRNA-mRNA network for predicting the response to chemoresistance in osteosarcoma by bioinformatics analysis.

The search for biomarkers is important for providing more targeted treatments for osteosarcoma patients with chemoresistance. In this study, differentially expressed microRNAs (miRNAs) were identified from miRNA expression profiles. And the target messenger RNAs (mRNAs) of miRNA were obtained from two websites in public domains. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway by these miRNA targets suggests that they may have potential links to osteosarcoma chemoresistance. In the protein-protein interaction (PPI) network, we screened three subnetworks and 10 hub RNAs, and analyzed through KEGG pathway and searched the PubMed database, indicating that they were significantly associated with drug resistance. Then we found 12 key mRNAs by analyzing the mRNA expression profile. Survival analyses showed that most of the 10 hub mRNAs and 12 key mRNAs had a significant influence on the prognosis of patients with chemoresistance osteosarcoma. A miRNA-mRNA network is constructed by integrating mRNAs and miRNAs information. The network biomarkers in this study have an advantage over traditional single-molecule biomarkers in terms of predictive power. And the mRNAs in this network biomarkers are supported by survival analysis or by existing theories. These results will contribute to the choice of chemotherapy before treatment and the prediction of patient prognosis.

Comprehensive Analysis of the Canonical and Non-canonical Wnt Signaling Pathways in Gastric Cancer.

BACKGROUND: Previous studies showed that dysregulation of Wnt signaling by gene mutation and abnormal gene expression is one of the causative factors for gastric cancer (GC). So far, a systematic and comprehensive analysis of gene mutation, gene expression, and DNA methylation profiles of the Wnt pathway associated with gastric carcinogenesis, however, has not yet been reported. AIMS: To this end, we investigated all the above-mentioned genetic alterations associated with the canonical and non-canonical Wnt pathways in GC tumors, in order to understand the molecular mechanism underlying gastric carcinogenesis. METHODS: The information on gene mutations and expression was obtained from data resources, such as TCGA, GSEA, and TCGA-STAD, and was analyzed with the cBioPortal platform. We also performed in vitro analysis on DDK2 gene, a Wnt inhibitor, to characterize its role in GC tumor cells. RESULTS: We found that gene mutations of 43 Wnt genes and abnormal expression of 13 Wnt genes occurred at a high frequency in GC tumors, and gene amplification and deletion are the major mutation types. Clusters of DNA methylation associated with Wnt signaling genes and GC tumors were also revealed, and a significant increase in ß-catenin activity was found in the hypermethylated group of GC tumors. In addition, overexpression of DKK2 gene significantly inhibited multiple biological processes of the GC cells, including their growth, clonal forming, migration, and invasion ability, and induced apoptosis of the GC cells. CONCLUSIONS: Our current study suggested that gene mutation, abnormal gene expression, and altered DNA methylation profiles associated with the Wnt signaling may play an important role in gastric carcinogenesis, and DKK2 gene may act as a tumor suppressor in gastric cells.

Identification of Genetic Polymorphisms of CYP2W1 in the Three Main Chinese Ethnicities: Han, Tibetan, and Uighur.

CYP2W1 is an orphan member of the cytochrome P450 superfamily. Recently, CYP2W1 has gained great research interest because of its unknown enzymatic function and tumor-specific expression property. This study aims to investigate the genetic polymorphisms of the CYP2W1 gene in Chinese populations and explore the functions of the detected variants. All of the nine exons and exon-intron junction regions of the CYP2W1 gene were sequenced in 150 Chinese subjects, including 50 Han Chinese, 50 Tibetans, and 50 Uighurs. A total of 26 genetic variants were identified in this study, and 19 polymorphisms were detected in each population. Frequency comparison between populations showed that nine variants exhibited significantly different allelic distributions. A total of 12 different haplotypes were inferred from 150 samples by using the genotype data of nine exonic variants found in this study. CYP2W1*1A, *1B, *2, *4, and *6 were detected as the main alleles/haplotypes. Moreover, one, three, and two ethnically specific haplotypes were observed in the Han, Tibetan, and Uighur samples, respectively. Then, the effects of four detected missense mutations (Ala181Thr, Gly376Ser, Val432Ile, and Pro488Leu) on the CYP2W1 protein function were predicted using three in silico tools: Polymorphism Phenotyping v2, Sorts Intolerant from Tolerant, and MutationTaster. The results showed that Gly376Ser and Pro488Leu may have deleterious effects. In summary, this study showed that the genetic pattern of CYP2W1 is interethnically different among the three Chinese populations, and this finding can extend our understanding of population genetics of CYP2W1 in the Chinese population.

Identification of MGMT promoter methylation sites correlating with gene expression and IDH1 mutation in gliomas.

O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation was reported to be an independent prognostic and predictive factor in glioma patients who received temozolomide treatment. However, the predictive value of MGMT methylation was recently questioned by several large clinical studies. The purpose of this study is to identify MGMT gene promoter CpG sites or region whose methylation were closely correlated with its gene expression to elucidate this contradictory clinical observations. The methylation status for all CpG dinucleotides in MGMT promoter and first exon region were determined in 42 Chinese glioma patients, which were then correlated with MGMT gene expression, IDH1 mutation, and tumor grade. In whole 87 CpG dinucleotides analyzed, three distinct CpG regions covering 28 CpG dinucleotides were significantly correlated with MGMT gene expression; 10 CpG dinucleotides were significantly correlated with glioma classification (p < 0.05). Isocitrate dehydrogenase 1 (IDH1) mutation and MGMT gene hypermethylation significantly co-existed, but not for MGMT gene expression. The validation cohort of gliomas treated with standard of care and comparison of the CpGs we identified with the current CpGs used in clinical setting will be very important for gliomas individual medicine in the future.

Diagnostic significance of suppressor of cytokine signalling 3 (SOCS3) methylation and its correlation with IDH1 mutation in Chinese glioma patients.

CONTEXT: Epigenetic alterations in suppressor of cytokine signalling 3 (SOCS3) have been suggested as a potential biomarker in glioma. OBJECTIVE: To investigate whether SOCS3 methylation could act as a biomarker for glioma grading and prognosis. MATERIALS AND METHODS: Glioma samples were evaluated by pyrosequencing and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: SOCS3 promoter methylation was more frequent in the WHO III and (anaplastic) oligoastrocytomas group. SOCS3 methylation status was significantly inversely correlated with mRNA expression level. Mutant IDH1 caused marked increase in SOCS3 methylation. CONCLUSION: SOCS3 methylation is a potential biomarker for grading and prognosis in human glioma.

The allele frequency of CYP2A6*4 in four ethnic groups of China.

The CYP2A6*4 allele, characterized as the whole deletion of this gene, is closely associated with nicotine dependence, cancer susceptibility, and drug responsiveness. The frequency of this molecular variant differs across populations. Although genetic polymorphisms of CYP2A6*4 and its functional results have been reported in Chinese Han population, the allele frequency of CYP2A6*4 was largely unknown in other Chinese ethnic population. In this study, we investigated the allele frequency of CYP2A6*4 in four main ethnic groups of China based on our newly developed quantitative real-time PCR assay. The frequencies of the CYP2A6*4 allele were 7.9%, 15%, 0% and 2% in Han (N=120), Uighur (N=100), Bouyei (N=100) and Tibetan (N=100) (P<0.0001), respectively. This work greatly expanded our understanding of the distribution of CYP2A6*4 in Chinese population and provided more information of different ethnic population's smoking behavior and also in disease susceptibility and drug response.

Tissue-specific selection of optimal reference genes for expression analysis of anti-cancer drug-related genes in tumor samples using quantitative real-time RT-PCR.

Gene transcription analysis in clinical tumor samples can help with diagnosis, prognosis, and treatment of cancers. We aimed to identify the optimal reference genes for reliable expression analysis in various tumor samples by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Using a one-step TaqMan-based qRT-PCR, 5 commonly used reference genes (ACTB, GAPDH, RPLPO, GUSB, and TFRC) and 10 anticancer drug-related genes (TYMS, RRM1, TUBB3, STMN1, TOP2A, EGFR, VEGFR2, HER2, ERCC1, and BRCA1) were analyzed in 327 tissue samples from lung, rectal, colon, gastric, esophageal, and breast tumors. According to the expression stability assessments obtained by using three programs (geNorm, NormFinder, and BestKeeper) and a comprehensive ranking method, the optimal reference genes for lung, gastric, esophageal, and breast tumors were RPLPO, GAPDH, ACTB, and ACTB, respectively. For rectal tumors, a combination of the 3 most stable genes (GUSB, ACTB, and RPLPO) was suitable for qRT-PCR, whereas for colon tumors, a combination of the 4 most stable genes (GAPDH, ACTB, GUSB, and RPLPO) was optimal for qRT-PCR. Based on the expression data of target genes normalized against selected reference genes, the principal component analysis revealed 4 expression patterns in 6 different tissues. One pattern was observed in gastric, rectal, and colon tumor tissues, which are gastrointestinal tumors. Expressions in the breast, lung, and esophageal tissues were separately represented as one pattern. Our results could facilitate the practice of personalized cancer medicine based on the gene expression profile of the patients.

Genetic alterations of PIK3CA and tumor response in patients with locally advanced cervical squamous cell carcinoma treated with cisplatin-based concurrent chemoradiotherapy.

OBJECTIVE: The objective of this study was to investigate the predictive value of common genetic alterations of PI3K/AKT/mTOR and Ras/Raf/MAPK pathways in patients with locally advanced cervical squamous cell carcinoma (LACSCC) treated with cisplatin-based concurrent chemoradiotherapy (CCRT). METHODS: Patients with LACSCC, treated at a single institution with CCRT were eligible for this retrospective study. A total of sixty pre-treatment tumor biopsies were retrieved. Somatic mutations were detected by pyrosequencing and CNV was determined by quantitative realtime PCR. The association of genetic alterations with clinicopathological characteristics and treatment response were analyzed. RESULTS: Patients without genetic alterations (mutations or amplification) of PIK3CA had a significantly higher response rate than patients with these alterations (p=0.006). In the logistic regression analysis, PIK3CA genetic alterations retained an independent factor in predicting response to CCRT. CONCLUSIONS: Somatic mutations and copy number amplification of PIK3CA were associated with response to CCRT in patients with cervical squamous cell carcinoma.

A multiplex allele-specific real-time PCR assay for screening of ESR1 mutations in metastatic breast cancer.

BACKGROUND: Acquired resistance to endocrine-based therapies occurs in virtually all estrogen receptor-α (ERα, encoded by ESR1) positive breast cancer patients. The underlying molecular mechanism is attributed to the activating mutations in ESR1. These mutations provide an exciting opportunity for the development of new antagonists that specifically inhibit the mutant proteins. Therefore, accurate detection of ESR1 mutations is of critical importance in clinical practice. MATERIALS AND METHODS: We carried out a single tube, multiplex allele-specific real-time PCR assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N, and D538G). RESULTS: The assay was found to be highly specific and sensitive. With this assay, as low as 1% mutant DNA template in wild type DNA could be detected. Fifteen DNA samples were prepared from archived formalin-fixed paraffin-embedded metastatic breast cancer biopsies. They were further screened with this assay, and three samples were identified as ESR1 mutant. The results were validated with pyrosequencing and complete concordance was observed between the two assays. CONCLUSION: The multiplex allele-specific real-time PCR assay provides a rapid and reliable diagnostic tool for accurate detection of ESR1 mutations. This procedure may be used in the clinical treatment of breast cancer.

Differential regulation of myosin X movements by its cargos, DCC and neogenin.

Myosin X (Myo X), also known as MYO10, is an unconventional actin-based motor protein that plays an important role in filopodium formation. Its intra-filopodia movement, an event tightly associated with the function of Myo X, has been extensively studied. However, how the motor activity of Myo X and the direction of its movements are regulated remains largely unknown. In our previous study, we demonstrated that DCC (for 'deleted in colorectal carcinoma') and neogenin (neogenin 1, NEO1 or NGN), a family of immunoglobin-domain-containing transmembrane receptors for netrins, interact with Myo X and that DCC is a cargo of Myo X to be delivered to the neurites of cultured neurons. Here, we provide evidence for DCC and neogenin as regulators of Myo X. DCC promotes movement of Myo X along basal actin filaments and enhances Myo-X-mediated basal filopodium elongation. By contrast, neogenin appears to suppress Myo X movement on the basal side, but increases its movement towards the apical and dorsal side of a cell, promoting dorsal filopodium formation and growth. Further studies have demonstrated that DCC, but not neogenin, enhances integrin-mediated tyrosine phosphorylation of focal adhesion kinase and basal F-actin reorganization, providing a cellular mechanism underlying their distinct effects on Myo X. These results thus demonstrate differential regulatory roles on Myo X activity by its cargo proteins, DCC and neogenin, revealing different cellular functions of DCC and neogenin.

Myosin X regulates netrin receptors and functions in axonal path-finding.

Netrins regulate axon path-finding during development, but the underlying mechanisms are not well understood. Here, we provide evidence for the involvement of the unconventional myosin X (Myo X) in netrin-1 function. We find that Myo X interacts with the netrin receptor deleted in colorectal cancer (DCC) and neogenin, a DCC-related protein. Expression of Myo X redistributes DCC to the cell periphery or to the tips of neurites, whereas its silencing prevents DCC distribution in neurites. Moreover, expression of DCC, but not neogenin, stimulates Myo X-mediated formation and elongation of filopodia, suggesting that Myo X function may be differentially regulated by DCC and neogenin. The involvement of Myo X in netrin-1 function was further supported by the effects of inhibiting Myo X function in neurons. Cortical explants derived from mouse embryos expressing a motor-less Myo X exhibit reduced neurite outgrowth in response to netrin-1 and chick commissural neurons expressing the motor-less Myo X, or in which Myo X is silenced using microRNA (miRNA), show impaired axon projection in vivo. Taken together, these results identify a novel role for Myo X in regulating netrin-1 function.

Single tube genotyping of CYP2A6 gene deletion based on copy number determination by quantitative real-time PCR.

The CYP2A6*4 allele, characterized as the whole deletion of this gene, is closely associated with nicotine dependence, cancer susceptibility, and drug responsiveness. It has long been a significant challenge for pharmacogenetics scientists to develop a reliable method to detect this molecular variant due to its high homology with its homologous genes CYP2A6 and CYP2A3 in the clinical setting. Here, we introduce a quantitative real-time PCR assay that specifically amplifies CYP2A6 by designing a specific set of primers and the probe, which effectively prevent the amplification of the CYP2A7 and CYP2A13 alleles. CYP2A6 gene copy numbers were normalized to albumin (ALB) which was co-amplified simultaneously in a single-tube duplex reaction and at a setting as the internal reference gene. The established assay was validated with a selection of previously genotyped DNA samples, which harbored none, one or two CYP2A6 gene copies. The results were in complete concordance with previously published data and no overlap between the three groups was observed. Further analysis of a cohort of 120 samples revealed high specificity and sensitivity of this assay as demonstrated by the agreement of determined gene copy numbers in all of the cases. In conclusion, this novel assay allows reliable and sensitive detection of the CYP2A6 gene deletion, which will be useful for pharmacogenetics studies and routine clinical settings.

Single tube genotyping of TYMS 1494del6 polymorphism in the Chinese Han population by duplex scorpion primers.

BACKGROUND: The development of pharmacogenomics has created an urgent need for robust molecular characterization. And it has become a challenge to develop suitable detecting methods for routine clinical use. AIM: The aim of the current study is to develop a simple and reliable TYMS 1494del6 polymorphism genotyping assay by duplex scorpion primers in the Chinese Han population. METHOD: We evaluated the performance of the duplex scorpion primer assay in the genotyping of TYMS 1494del6 polymorphism and screened 54 DNA samples of the Chinese Han population. The results were further validated by pyrosequencing. RESULTS: The duplex scorpion primer assay showed high specificity and accuracy for genotyping TYMS 1494del6 polymorphism. Complete concordance was observed between the duplex scorpion primer assay and pyrosequencing. The frequency of the TYMS +6 bp allele was 34% and the -6 bp allele was 66% in 54 Chinese Han population DNA samples. CONCLUSION: The duplex scorpion primer assay provides a rapid, reliable and high-throughput method to genotype TYMS 1494del6 polymorphism in the Chinese Han population.

Reprogrammed IDO-Induced Immunosuppressive Microenvironment Synergizes with Immunogenic Magnetothermodynamics for Improved Cancer Therapy.

Indoleamine 2,3-dioxygenase (IDO), highly expressed in hepatocellular carcinoma (HCC), plays a pivotal role in creating an immune-suppressive tumor microenvironment. Inhibiting IDO activity has emerged as a promising immunotherapeutic strategy; however, the delivery of IDO inhibitors to the tumor site is constrained, limiting their therapeutic efficacy. In this study, we developed a magnetic vortex nanodelivery system for the targeted delivery of the IDO inhibitor NLG919, integrated with magnetic hyperthermia therapy to reverse the immune-suppressive microenvironment of liver cancer and inhibit tumor growth. This system comprises thermoresponsive polyethylenimine-coated ferrimagnetic vortex-domain iron oxide nanorings (PI-FVIOs) loaded with NLG919 (NLG919/PI-FVIOs). Under thermal effects, NLG919 can be precisely released from the delivery system, counteracting IDO-mediated immune suppression and synergizing with NLG919/PI-FVIOs-mediated magnetothermodynamic (MTD) therapy-induced immunogenic cell death (ICD), resulting in effective HCC suppression. In vivo studies demonstrate that this combination therapy significantly inhibits tumor growth and metastasis by enhancing the accumulation of cytotoxic T lymphocytes and suppressing regulatory T cells within the tumor. Overall, our findings reveal that NLG919/PI-FVIOs can induce a potent antitumor immune response by disrupting the IDO pathway and activating the ICD, offering a promising therapeutic avenue for HCC treatment.

LINC00540 promotes sorafenib resistance and functions as a ceRNA for miR-4677-3p to regulate AKR1C2 in hepatocellular carcinoma.

Sorafenib resistance is one of the main causes of poor prognosis in patients with advanced hepatocellular carcinoma (HCC). Long noncoding RNAs (lncRNAs) function as suppressors or oncogenic factors during tumor progression and drug resistance. Here, to identify therapeutic targets for HCC, the biological mechanisms of abnormally expressed lncRNAs were examined in sorafenib-resistant HCC cells. Specifically, we established sorafenib-resistant HCC cell lines (Huh7-S and SMMC7721-S), which displayed an epithelial-mesenchymal transition (EMT) phenotype. Transcriptome sequencing (RNA-Seq) was performed to established differential lncRNA expression profiles for sorafenib-resistant cells. Through this analysis, we identified LINC00540 as significantly up-regulated in sorafenib-resistant cells and a candidate lncRNA for further mechanistic investigation. Functionally, LINC00540 knockdown promoted sorafenib sensitivity and suppressed migration, invasion, EMT and the activation of PI3K/AKT signaling pathway in sorafenib-resistant HCC cells, whereas overexpression of LINC00540 resulted in the opposite effects in parental cells. LINC00540 functions as a competing endogenous RNA (ceRNA) by competitively binding to miR-4677-3p , thereby promoting AKR1C2 expression. This is the first study that demonstrates a role for LINC00540 in enhancing sorafenib resistance, migration and invasion of HCC cells through the LINC00540/miR-4677-3p/AKR1C2 axis, suggesting that LINC00540 may represent a potential therapeutic target and prognosis biomarker for HCC.