PfAgo-based dual signal amplification biosensor for rapid and highly sensitive detection of alkaline phosphatase activity.

A Pyrococcus furiosus Argonaute (PfAgo)-based biosensor is presented for alkaline phosphatase (ALP) activity detection in which the ALP-catalyzed hydrolysis of 3'-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon mediates the fluorescent reporter cleavage as a guide sequence of PfAgo. Under the dual amplification mode of PfAgo-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied to ALP activity determination with a detection limit (LOD) of 0.0013 U L-1 (3σ) and a detection range of 0.0025 to 1 U L-1 within 90 min. The PfAgo-based method exhibits satisfactory analytic performance in the presence of potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapid analysis, high sensitivity, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications.

RNA-guided RNA silencing by an Asgard archaeal Argonaute.

Argonaute proteins are the central effectors of RNA-guided RNA silencing pathways in eukaryotes, playing crucial roles in gene repression and defense against viruses and transposons. Eukaryotic Argonautes are subdivided into two clades: AGOs generally facilitate miRNA- or siRNA-mediated silencing, while PIWIs generally facilitate piRNA-mediated silencing. It is currently unclear when and how Argonaute-based RNA silencing mechanisms arose and diverged during the emergence and early evolution of eukaryotes. Here, we show that in Asgard archaea, the closest prokaryotic relatives of eukaryotes, an evolutionary expansion of Argonaute proteins took place. In particular, a deep-branching PIWI protein (HrAgo1) encoded by the genome of the Lokiarchaeon 'Candidatus Harpocratesius repetitus' shares a common origin with eukaryotic PIWI proteins. Contrasting known prokaryotic Argonautes that use single-stranded DNA as guides and/or targets, HrAgo1 mediates RNA-guided RNA cleavage, and facilitates gene silencing when expressed in human cells and supplied with miRNA precursors. A cryo-EM structure of HrAgo1, combined with quantitative single-molecule experiments, reveals that the protein displays structural features and target-binding modes that are a mix of those of eukaryotic AGO and PIWI proteins. Thus, this deep-branching archaeal PIWI may have retained an ancestral molecular architecture that preceded the functional and mechanistic divergence of eukaryotic AGOs and PIWIs.

PIWI-Interacting RNAs: A Pivotal Regulator in Neurological Development and Disease.

PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs (sncRNAs) with 24-32 nucleotides (nt), were initially identified in the reproductive system. Unlike microRNAs (miRNAs) or small interfering RNAs (siRNAs), piRNAs normally guide P-element-induced wimpy testis protein (PIWI) families to slice extensively complementary transposon transcripts without the seed pairing. Numerous studies have shown that piRNAs are abundantly expressed in the brain, and many of them are aberrantly regulated in central neural system (CNS) disorders. However, the role of piRNAs in the related developmental and pathological processes is unclear. The elucidation of piRNAs/PIWI would greatly improve the understanding of CNS development and ultimately lead to novel strategies to treat neural diseases. In this review, we summarized the relevant structure, properties, and databases of piRNAs and their functional roles in neural development and degenerative disorders. We hope that future studies of these piRNAs will facilitate the development of RNA-based therapeutics for CNS disorders.

Argonaute-independent, Dicer-dependent antiviral defense against RNA viruses.

Antiviral RNA interference (RNAi) is conserved from yeasts to mammals. Dicer recognizes and cleaves virus-derived double-stranded RNA (dsRNA) and/or structured single-stranded RNA (ssRNA) into small-interfering RNAs, which guide effector Argonaute to homologous viral RNAs for digestion and inhibit virus replication. Thus, Argonaute is believed to be essential for antiviral RNAi. Here, we show Argonaute-independent, Dicer-dependent antiviral defense against dsRNA viruses using Cryphonectria parasitica (chestnut blight fungus), which is a model filamentous ascomycetous fungus and hosts a variety of viruses. The fungus has two dicer-like genes (dcl1 and dcl2) and four argonaute-like genes (agl1 to agl4). We prepared a suite of single to quadruple agl knockout mutants with or without dcl disruption. We tested these mutants for antiviral activities against diverse dsRNA viruses and ssRNA viruses. Although both DCL2 and AGL2 worked as antiviral players against some RNA viruses, DCL2 without argonaute was sufficient to block the replication of other RNA viruses. Overall, these results indicate the existence of a Dicer-alone defense and different degrees of susceptibility to it among RNA viruses. We discuss what determines the great difference in susceptibility to the Dicer-only defense.

DNA Methylation Mediates Sperm Quality via piwil1 and piwil2 Regulation in Japanese Flounder (Paralichthys olivaceus).

DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific piwi gene expression during spermatogenesis of Japanese flounder (Paralichthys olivaceus), the expression profiles of piwil1 (piwi-like 1) and piwil2 (piwi-like 2) genes in the gonads of female, male, and sex-reversed pseudo-male P. olivaceus were analyzed, and the dynamic of DNA methylation was investigated. As a result, piwil1 and piwil2 genes were highly expressed in the testis of both male and pseudo-male P. olivaceus, with significant variation among male individuals. The DNA methylation levels in the promoter regions of both piwil1 and piwil2 were negatively correlated with their expression levels, which may contribute to the transcriptional regulation of piwi genes during spermatogenesis. There was also sperm quality variation among male P. olivaceus, and the sperm curvilinear velocity was positively correlated with the expression of both piwil1 and piwil2 genes. These results indicated that the DNA methylation in piwil1 and piwil2 promoter regions may affect the initiation of piwi gene transcription, thereby regulating gene expression and further affecting the spermatogenesis process and gamete quality in P. olivaceus.

Integrative transcriptomic and proteomic profiling of the effects of cell confluency on gene expression.

In this study we examine the impact of cell confluency on gene expression. We focused on Argonaute (AGO) protein dynamics and associated gene and protein expression in HEK293, A375, and SHSY5Y cell lines. As a consequence of cell confluency, AGO2 protein translocates into the nucleus. Therefore, we generated transcriptomic data using RNA sequencing to compare gene expression in subconfluent versus confluent cells, which highlighted significant alterations in gene regulation patterns directly corresponding to changes in cell density. Our study also encompasses miRNA profiling data obtained through small RNA sequencing, revealing miRNA expressional changes dependent on cellular confluency, as well as cellular localization. Finally, we derived proteomic data from mass spectrometry analyses following AGO1-4 immunoprecipitation, providing a comprehensive view of AGO interactome in both nuclear and cytoplasmic compartments under varying confluency. These datasets offer a detailed exploration of the cellular and molecular dynamics, influenced by cell confluency, presenting a valuable resource for further research in cellular biology, particularly in understanding the basic mechanisms of cell density in cancer cells.

Structure and Stability of Ago2 MID-Nucleotide Complexes: All-in-One (Drop) His6-SUMO Tag Removal, Nucleotide Binding, and Crystal Growth.

The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5'-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His6-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2'-3' linked α-L-threofuranosyl thymidine-3'-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (Kd) of MID-nucleotide interactions for nucleoside 5'-monophosphates and nucleoside 3',5'-bisphosphates. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Crystallization of Ago2 MID-nucleotide complexes Basic Protocol 2: Measurement of dissociation constant Kd between Ago2 MID and nucleotides.

Nuclear miR-451a activates KDM7A and leads to cetuximab resistance in head and neck squamous cell carcinoma.

Cetuximab resistance has been a major challenge for head and neck squamous cell carcinoma (HNSCC) patients receiving targeted therapy. However, the mechanism that causes cetuximab resistance, especially microRNA (miRNA) regulation, remains unclear. Growing evidence suggests that miRNAs may act as "nuclear activating miRNAs" for targeting promoter regions or enhancers related to target genes. This study elucidates a novel mechanism underlying cetuximab resistance in HNSCC involving the nuclear activation of KDM7A transcription via miR-451a. Herein, small RNA sequencing, quantitative real-time polymerase chain reaction (qRT‒PCR) and fluorescence in situ hybridization (FISH) results provided compelling evidence of miR-451a nuclear enrichment in response to cetuximab treatment. Chromatin isolation via RNA purification, microarray analysis, and bioinformatic analysis revealed that miR-451a interacts with an enhancer region in KDM7A, activating its expression and further facilitating cetuximab resistance. It has also been demonstrated that the activation of KDM7A by nuclear miR-451a is induced by cetuximab treatment and is AGO2 dependent. Logistic regression analyses of 87 HNSCC samples indicated the significance of miR-451a and KDM7A in the development of cetuximab resistance. These discoveries support the potential of miR-451a and KDM7A as valuable biomarkers for cetuximab resistance and emphasize the function of nuclear-activating miRNAs.

Nucleic acid mediated activation of a short prokaryotic Argonaute immune system.

A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P)+ hydrolysis, was recently identified in bacteria. We report the crystal structure of SPARTA heterodimer in the absence of guide-RNA/target-ssDNA (2.66 Å) and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide-RNA/target-ssDNA at nominal 3.15-3.35 Å resolution. The crystal structure provides a high-resolution view of SPARTA, revealing the APAZ domain as equivalent to the N, L1, and L2 regions of long pAgos and the MID domain containing a unique insertion (insert57). Cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix αN) domains that reconfigure for nucleic-acid binding and decrypts regions/residues that reorganize to expose a positively charged pocket for higher-order assembly. The TIR domains amass in a parallel-strands arrangement for catalysis. We visualize SPARTA before and after RNA/ssDNA binding and uncover the basis of its active assembly leading to abortive infection.

HEPPAR1 and PIWIL2 as Panel Markers for Hepatocellular Carcinoma.

OBJECTIVE: The aim of this study was to evaluate the expression profiles of PIWI-like protein- 2 (PIWIL2), and HepPar1 and their immunohistochemical (IHC) characteristics in Hepatocellular Carcinoma (HCC), and determine their correlation with clinicopathological parameters of this type of cancer to determine their diagnostic value in combination. METHODS: Seventy-five patients with HCC were assessed for the expression of PIWIL2 in serum and tissue using real-time polymerase chain reaction (RT-PCR) and IHC was performed for PIWIL2 and HepPar1 was performed on all patients. RESULTS: A statistically significantly higher level of PIWIL2 was found in HCC compared to controls (p≤0.001). Both HepPar1 and PIWIL2 were detected in 84% of HCC cases, the diagnostic and prognostic factors for PIWIL2 were found to be significant in liver tumour tissue samples and non-tumorous sections p<0.001, and the same was observed for serum samples and results of healthy serum controls (p<0.001) when compared to AFP. CONCLUSION: Our results affirm the hypothesis that reactivation of PIWI expression in various caner types is crucial for cancer development, and that a possible panel maybe used for these markers HCC diagnosis.

m6A modification inhibits miRNAs' intracellular function, favoring their extracellular export for intercellular communication.

Epitranscriptomics represents a further layer of gene expression regulation. Specifically, N6-methyladenosine (m6A) regulates RNA maturation, stability, degradation, and translation. Regarding microRNAs (miRNAs), while it has been reported that m6A impacts their biogenesis, the functional effects on mature miRNAs remain unclear. Here, we show that m6A modification on specific miRNAs weakens their coupling to AGO2, impairs their function on target mRNAs, determines their delivery into extracellular vesicles (EVs), and provides functional information to receiving cells. Mechanistically, the intracellular functional impairment is caused by m6A-mediated inhibition of AGO2/miRNA interaction, the EV loading is favored by m6A-mediated recognition by the RNA-binding protein (RBP) hnRNPA2B1, and the EV-miRNA function in the receiving cell requires their FTO-mediated demethylation. Consequently, cells express specific miRNAs that do not impact endogenous transcripts but provide regulatory information for cell-to-cell communication. This highlights that a further level of complexity should be considered when relating cellular dynamics to specific miRNAs.

Plasmid targeting and destruction by the DdmDE bacterial defence system.

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.

The Evolution and Characterization of the RNA Interference Pathways in Lophotrochozoa.

In animals, three main RNA interference mechanisms have been described so far, which respectively maturate three types of small noncoding RNAs (sncRNAs): miRNAs, piRNAs, and endo-siRNAs. The diversification of these mechanisms is deeply linked with the evolution of the Argonaute gene superfamily since each type of sncRNA is typically loaded by a specific Argonaute homolog. Moreover, other protein families play pivotal roles in the maturation of sncRNAs, like the DICER ribonuclease family, whose DICER1 and DICER2 paralogs maturate respectively miRNAs and endo-siRNAs. Within Metazoa, the distribution of these families has been only studied in major groups, and there are very few data for clades like Lophotrochozoa. Thus, we here inferred the evolutionary history of the animal Argonaute and DICER families including 43 lophotrochozoan species. Phylogenetic analyses along with newly sequenced sncRNA libraries suggested that in all Trochozoa, the proteins related to the endo-siRNA pathway have been lost, a part of them in some phyla (i.e. Nemertea, Bryozoa, Entoprocta), while all of them in all the others. On the contrary, early diverging phyla, Platyhelminthes and Syndermata, showed a complete endo-siRNA pathway. On the other hand, miRNAs were revealed the most conserved and ubiquitous mechanism of the metazoan RNA interference machinery, confirming their pivotal role in animal cell regulation.

tRF-33-P4R8YP9LON4VDP inhibits gastric cancer progression via modulating STAT3 signaling pathway in an AGO2-dependent manner.

It has been demonstrated that tRNA-derived small RNAs (tsRNAs) perform essential functions in the pathophysiology of cancer. In this study, we focused on the possible mechanisms of tRF-33-P4R8YP9LON4VDP (tRF-33) underlying the development of gastric malignancy. In total, 454 tissue samples with different gastric mucosal lesions were collected. The tRF-33 expression level in different cohorts was determined, and its value for diagnostic efficiency and prognosis evaluation were assessed. Cell proliferation assays, Transwell assay, flow cytometry, and xenotransplantation model were used to evaluate its effect on gastric cancer cells. The molecular mechanism was verified by fluorescence in situ hybridization, dual luciferase assay, Western blot, and RNA binding protein immunoprecipitation. The results showed that the expression of tRF-33 exhibited a gradual modification from normal control samples to gastritis tissues, early and latent stage of gastric cancer tissues. Consequently, tRF-33 holds significant potential as a predictive and diagnostic biomarker for gastric malignancy. Over-expression of tRF-33 inhibited gastric cancer cell progression and metastatic viability, and induced cell apoptosis. Tumorigenicity in nude mice showed the suppressive characteristics of tRF-33. Mechanistic investigation revealed that tRF-33 exerted silencing on STAT3 mRNA via binding to AGO2. In conclusion, tRF-33 exhibited values in diagnosing gastric cancer and evaluating its prognosis, and suppressed tumor cell viability by inhibiting STAT3 signaling pathway. The schematic mechanisms underlying tRF-33 regulating gastric cancer occurrence. tRF-33 binds to AGO2 proteins and then negatively regulates STAT3 expression through targeting its 3'UTR. The downregulated expression of STAT3 results in the decrease of STAT3 and p-STAT3 and further blocks the transcription of the downstream genes and finally inhibits the gastric cancer occurrence. MMP-9, matrix metalloproteinase-9; Bcl-2, B-cell lymphoma-2; STAT3, signal transducer and activator of transcription 3; UTR, untranslated region.

Identification of a novel mutation in chibby family member 2 in a non-obstructive azoospermic patient.

Azoospermia constitutes a significant factor in male infertility, defined by the absence of spermatozoa in the ejaculate, afflicting 15% of infertile men. However, a subset of azoospermic cases remains unattributed to known genetic variants. Prior investigations have identified the chibby family member 2 (CBY2) as prominently and specifically expressed in the testes of both humans and mice, implicating its potential involvement in spermatogenesis. In this study, we conducted whole exome sequencing (WES) on an infertile family to uncover novel genetic factors contributing to azoospermia. Our analysis revealed a homozygous c .355 C>A variant of CBY2 in a non-obstructive azoospermic patient. This deleterious variant significantly diminished the protein expression of CBY2 both in vivo and in vitro, leading to a pronounced disruption of spermatogenesis at the early round spermatid stage post-meiosis. This disruption was characterized by a nearly complete loss of elongating and elongated spermatids. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation assays demonstrated the interaction between CBY2 and Piwi-like protein 1 (PIWIL1). Immunofluorescence staining further confirmed the co-localization of CBY2 and PIWIL1 in the testes during the spermatogenic process in both humans and mice. Additionally, diminished PIWIL1 expression was observed in the testicular tissue from the affected patient. Our findings suggest that the homozygous c .355 C>A variant of CBY2 compromises CBY2 function, contributing to defective spermatogenesis at the round spermiogenic stage and implicating its role in the pathogenesis of azoospermia.

Prokaryotic Argonaute nuclease cooperates with co-encoded RNase to acquire guide RNAs and target invader DNA.

Argonautes are an evolutionary conserved family of programmable nucleases that identify target nucleic acids using small guide oligonucleotides. In contrast to eukaryotic Argonautes (eAgos) that act on RNA, most studied prokaryotic Argonautes (pAgos) recognize DNA targets. Similarly to eAgos, pAgos can protect prokaryotic cells from invaders, but the biogenesis of guide oligonucleotides that confer them specificity to their targets remains poorly understood. Here, we have identified a new group of RNA-guided pAgo nucleases and demonstrated that a representative pAgo from this group, AmAgo from the mesophilic bacterium Alteromonas macleodii, binds guide RNAs of varying lengths for specific DNA targeting. Unlike most pAgos and eAgos, AmAgo is strictly specific to hydroxylated RNA guides containing a 5'-adenosine. AmAgo and related pAgos are co-encoded with a conserved RNA endonuclease from the HEPN superfamily (Ago-associated protein, Agap-HEPN). In vitro, Agap cleaves RNA between guanine and adenine nucleotides producing hydroxylated 5'-A guide oligonucleotides bound by AmAgo. In vivo, Agap cooperates with AmAgo in acquiring guide RNAs and counteracting bacteriophage infection. The AmAgo-Agap pair represents the first example of a pAgo system that autonomously produces RNA guides for DNA targeting and antiviral defense, which holds promise for programmable DNA targeting in biotechnology.

Development of an optimized RPA-PfAgo detection system for MTHFR C677T polymorphism genotyping.

This study introduces an efficient RPA-PfAgo detection system for the MTHFR C677T polymorphism, proposing a potential strategy to simplify the genotyping process. By optimizing recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) nucleases, we achieved DNA amplification at a constant temperature. The assay was fine-tuned through meticulous primer and guide DNA selection, with optimal conditions established at 2.0 µL of MgAc, a reaction temperature of 42 °C, and a 10-minute reaction time for RPA. Further optimization of the PfAgo cleavage assay revealed the ideal concentrations of MnCl2, guide DNA, molecular beacon probes, the PfAgo enzyme, and the RPA product to maximize sensitivity and specificity. Clinical validation of 20 samples showed 100% concordance with Sanger sequencing, confirming the method's precision. The RPA-PfAgo system is a promising tool for on-site genotyping, with broad applications in personalized medicine and disease prevention.

Phyllostachys edulis argonaute genes function in the shoot architecture.

Argonaute (AGO) proteins are the core components of the RNA-induced silencing complexes (RISC) in the cytoplasm and nucleus, and are necessary for the development of plant shoot meristem, which gives rise to the above-ground plant body. In this study, we identified 23 Phyllostachys edulis AGO genes (PhAGOs) that were distributed unequally on the 14 unmapped scaffolds. Gene collinearity and phylogeny analysis showed that the innovation of PhAGO genes was mainly due to dispersed duplication and whole-genome duplication, which resulted in the enlarged PhAGO family. PhAGO genes were expressed in a temporal-spatial expression pattern, and they encoded proteins differently localized in the cytoplasm and/or nucleus. Overexpression of the PhAGO2 and PhAGO4 genes increased the number of tillers or leaves in Oryza sativa and affected the shoot architecture of Arabidopsis thaliana. These results provided insight into the fact that PhAGO genes play important roles in plant development.

Pseudopregnant mice generated from Piwil1 deficiency sterile mice.

Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there is an urgent need to find a suitable replacement for vasectomized mice in the production of transgenic mice. In this study, we generated C57BL/6J mice (Piwil1 D633A-INS99, Piwil1mt/mt) with a 99-base insertion in the Miwi (Piwil1) gene using CRISPR/Cas9 technology and showed that Piwil1mt/+ heterozygous mice were normally fertile and that homozygous Piwil1mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1mt/mt males produced no Piwil1mt/mt genotype offspring, and the number of offspring did not differ significantly from that of pseudopregnant mice following mating and breeding with ligated males. The CRISPR‒Cas9 system is available for generating Miwi-modified mice, and provides a powerful resource to replace ligated males in assisted reproduction research.

The Search for and Functional Analysis of Genetic Variants in microRNA-Binding Sites using Massively Parallel Reporter Assay.

A large-scale search for the genetic variants with a bias in the representation of alleles in transcriptome data (AE SNPs) and the binding sites in microRNA 3'-UTRs was performed and their functional significance was assessed using massively parallel reporter assay (MPRA). Of the 629,559 associated "SNP-gene" pairs (eQTLs) discovered in the human liver tissue according to the GTEx Analysis V8 data, 4394 polymorphic positions in the 3'-UTRs of the genes, which represent the eQTLs for these genes were selected. The TargetScanHuman 7.0 algorithm and PolymiRTS database were searched for the potential microRNA-binding sites. Of the predicted microRNA sites affected by eQTL-SNPs, we selected 51 sites with the best evidence of functionality according to Ago2-CLIP-seq, CLEAR-CLIP, and eCLIP-seq for RNA-binding proteins. For MPRA, a library of the plasmids carrying the main and alternative alleles for each AE SNP (in total, 102 constructs) was created. Allele-specific expression for 6 SNPs was detected by transfection of the HepG2 cell line with the constructed plasmid library and sequencing of target DNA and RNA sequences using the Illumina (MiSeq) platform.