The RNA-binding protein ZC3H11A interacts with the nuclear poly(A)-binding protein PABPN1 and alters polyadenylation of viral transcripts.

Nuclear mRNA metabolism is regulated by multiple proteins, which either directly bind to RNA or form multiprotein complexes. The RNA-binding protein ZC3H11A is involved in nuclear mRNA export, NF-κB signaling, and is essential during mouse embryo development. Furthermore, previous studies have shown that ZC3H11A is important for nuclear-replicating viruses. However, detailed biochemical characterization of the ZC3H11A protein has been lacking. In this study, we established the ZC3H11A protein interactome in human and mouse cells. We demonstrate that the nuclear poly(A)-binding protein PABPN1 interacts specifically with the ZC3H11A protein and controls ZC3H11A localization into nuclear speckles. We report that ZC3H11A specifically interacts with the human adenovirus type 5 (HAdV-5) capsid mRNA in a PABPN1-dependent manner. Notably, ZC3H11A uses the same zinc finger motifs to interact with PABPN1 and viral mRNA. Further, we demonstrate that the lack of ZC3H11A alters the polyadenylation of HAdV-5 capsid mRNA. Taken together, our results suggest that the ZC3H11A protein may act as a novel regulator of polyadenylation of nuclear mRNA.

lncRNA GHET1 Promotes the Progression of Triple-Negative Breast Cancer via Regulation of miR-377-3p/GRSF1 Signaling Axis.

Objective: This study is aimed at investigating the role of lncRNA GHET1 in the progression of triple-negative breast cancer (TNBC). Methods: Tumor tissues and paracancerous tissues (normal) of TNBC patients were collected. Human normal breast cells (MCF10A) and TNBC cells (MDA-MB-468 and HCC1937) were employed for in vitro analysis. The expression of lncRNA GHET1, miR-377-3p, and GRSF1 was detected by qRT-PCR. The lncRNA GHET1 and miR-377-3p were overexpressed or knocked down in the TNBC cells, respectively. To determine the specific biological activities of the TNBC cells, MTT, flow cytometry, and wound healing assay were adopted to evaluate the cellular proliferation, apoptosis, and migration abilities, respectively. MMP-9 and MMP-2 protein expression levels were detected as well by Western blot in the cells. The relationship between miR-377-3p and lncRNA GHET1, miR-377-3p, and GRSF1 was validated using dual-luciferase reporter assay. Results: lncRNA GHET1 was significantly upregulated in the TNBC patients' tissues and the TNBC cell lines. Overexpression of lncRNA GHET1 significantly increased the proliferation and migration ability, but decreased apoptosis in the TNBC cells. Additionally, overexpression of lncRNA GHET1 upregulated both MMP-9 and MMP-2 protein expression levels. Correlation analysis found that miR-377-3p had a positive relationship with GRSF1, but had a negative relationship with lncRNA GHET1. miR-377-3p mimic attenuated the effects of lncRNA GHET1 on cellular proliferation, apoptosis, and migration of the TNBC cells. Conclusion: lncRNA GHET1 promotes TNBC progression through the miR-377-3p/GRSF1 signaling axis.

Paip2A inhibits translation by competitively binding to the RNA recognition motifs of PABPC1 and promoting its dissociation from the poly(A) tail.

Eukaryotic mRNAs possess a poly(A) tail at their 3'-end, to which poly(A)-binding protein C1 (PABPC1) binds and recruits other proteins that regulate translation. Enhanced poly(A)-dependent translation, which is also PABPC1 dependent, promotes cellular and viral proliferation. PABP-interacting protein 2A (Paip2A) effectively represses poly(A)-dependent translation by causing the dissociation of PABPC1 from the poly(A) tail; however, the underlying mechanism remains unknown. This study was conducted to investigate the functional mechanisms of Paip2A action by characterizing the PABPC1-poly(A) and PABPC1-Paip2A interactions. Isothermal titration calorimetry and NMR analyses indicated that both interactions predominantly occurred at the RNA recognition motif (RRM)2-RRM3 regions of PABPC1, which have comparable affinities for poly(A) and Paip2A (dissociation constant, Kd = 1 nM). However, the Kd values of isolated RRM2 were 200 and 4 µM in their interactions with poly(A) and Paip2A, respectively; Kd values of 5 and 1 µM were observed for the interactions of isolated RRM3 with poly(A) and Paip2A, respectively. NMR analyses also revealed that Paip2A can bind to the poly(A)-binding interfaces of the RRM2 and RRM3 regions of PABPC1. Based on these results, we propose the following functional mechanism for Paip2A: Paip2A initially binds to the RRM2 region of poly(A)-bound PABPC1, and RRM2-anchored Paip2A effectively displaces the RRM3 region from poly(A), resulting in dissociation of the whole PABPC1 molecule. Together, our findings provide insight into the translation repression effect of Paip2A and may aid in the development of novel anticancer and/or antiviral drugs.

Changes in mRNA stability play an important role in the adaptation of yeast cells to iron deprivation.

Eukaryotic cells rely on iron as an indispensable cofactor for multiple biological functions including mitochondrial respiration and protein synthesis. The budding yeast Saccharomyces cerevisiae utilizes both transcriptional and posttranscriptional mechanisms to couple mRNA levels to the requirements of iron deprivation. Thus, in response to iron deficiency, transcription factors Aft1 and Aft2 activate the expression of genes implicated in iron acquisition and mobilization, whereas two mRNA-binding proteins, Cth1 and Cth2, posttranscriptionally control iron metabolism. By using a genome-wide approach, we describe here a global stabilization of mRNAs, including transcripts encoding ribosomal proteins (RPs), when iron bioavailability diminishes. mRNA decay assays indicate that the mRNA-binding protein Pub1 contributes to RP transcript stabilization during adaptation to iron limitation. In fact, Pub1 becomes critical for growth and translational repression in low-iron conditions. Remarkably, we observe that pub1Δ cells also exhibit an increase in the transcription of RP genes that evidences the crosstalk between transcription and degradation mechanisms to maintain the appropriate mRNA balance under iron deficiency conditions.

The flip-flop configuration of the PABP-dimer leads to switching of the translation function.

Poly(A)-binding protein (PABP) is a translation initiation factor that interacts with the poly(A) tail of mRNAs. PABP bound to poly(A) stimulates translation by interacting with the eukaryotic initiation factor 4G (eIF4G), which brings the 3' end of an mRNA close to its 5' m7G cap structure through consecutive interactions of the 3'-poly(A)-PABP-eIF4G-eIF4E-5' m7G cap. PABP is a highly abundant translation factor present in considerably larger quantities than mRNA and eIF4G in cells. However, it has not been elucidated how eIF4G, present in limited cellular concentrations, is not sequestered by mRNA-free PABP, present at high cellular concentrations, but associates with PABP complexed with the poly(A) tail of an mRNA. Here, we report that RNA-free PABPs dimerize with a head-to-head type configuration of PABP, which interferes in the interaction between PABP and eIF4G. We identified the domains of PABP responsible for PABP-PABP interaction. Poly(A) RNA was shown to convert the PABP-PABP complex into a poly(A)-PABP complex, with a head-to-tail-type configuration of PABP that facilitates the interaction between PABP and eIF4G. Lastly, we showed that the transition from the PABP dimer to the poly(A)-PABP complex is necessary for the translational activation function.

Discovery of a novel role of tumor suppressor PDCD4 in stimulation of translation termination.

Programmed cell death 4 protein (PDCD4) regulates many vital cell processes, although is classified as a tumor suppressor because it inhibits neoplastic transformation and tumor growth. For example, PCDC4 has been implicated in the regulation of transcription and mRNA translation. PDCD4 is known to inhibit translation initiation by binding to eukaryotic initiation factor 4A and elongation of oncogenic c- and A-myb mRNAs. Additionally, PDCD4 has been shown to interact with poly(A)-binding protein (PABP), which affects translation termination, although the significance of this interaction is not fully understood. Considering the interaction between PABP and PDCD4, we hypothesized that PDCD4 may also be involved in translation termination. Using in vitro translation systems, we revealed that PDCD4 directly activates translation termination. PDCD4 stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the PABP, which also stimulates peptide release, PDCD4 activity in translation termination increases. PDCD4 regulates translation termination by facilitating the binding of release factors to the ribosome, increasing the GTPase activity of eRF3, and dissociating eRF3 from the posttermination complex. Using a toe-printing assay, we determined the first stage at which PDCD4 functions-binding of release factors to the A-site of the ribosome. However, preventing binding of eRF3 with PABP, PDCD4 suppresses subsequent rounds of translation termination. Based on these data, we assumed that human PDCD4 controls protein synthesis during translation termination. The described mechanism of the activity of PDCD4 in translation termination provides a new insight into its functioning during suppression of protein biosynthesis.

A Quantitative Proteomic Approach for the Identification of DNA Guanine Quadruplex-Binding Proteins.

DNA sequences of high guanine (G) content have the potential to form G quadruplex (G4) structures. A more complete understanding about the biological functions of G4 DNA requires the investigation about how these structures are recognized by proteins. Here, we conducted exhaustive quantitative proteomic experiments to profile the interaction proteomes of G4 structures by employing different sequences of G4 DNA derived from the human telomere and the promoters of c-MYC and c-KIT genes. Our results led to the identification of a number of candidate G4-interacting proteins, many of which were discovered here for the first time. These included three proteins that can bind to all three DNA G4 structures and 78 other proteins that can bind selectively to one or two of the three DNA G4 structure(s). We also validated that GRSF1 can bind directly and selectively toward G4 structure derived from the c-MYC promoter. Our quantitative proteomic screening also led to the identification of a number of candidate "antireader" proteins of G4 DNA. Together, we uncovered a number of cellular proteins that exhibit general and selective recognitions of G4 folding patterns, which underscore the complexity of G4 DNA in biology and the importance of understanding fully the G4-interaction proteome.

Translation Initiation Regulated by RNA-Binding Protein in Mammals: The Modulation of Translation Initiation Complex by Trans-Acting Factors.

Protein synthesis is tightly regulated at each step of translation. In particular, the formation of the basic cap-binding complex, eukaryotic initiation factor 4F (eIF4F) complex, on the 5' cap structure of mRNA is positioned as the rate-limiting step, and various cis-elements on mRNA contribute to fine-tune spatiotemporal protein expression. The cis-element on mRNAs is recognized and bound to the trans-acting factors, which enable the regulation of the translation rate or mRNA stability. In this review, we focus on the molecular mechanism of how the assembly of the eIF4F complex is regulated on the cap structure of mRNAs. We also summarize the fine-tuned regulation of translation initiation by various trans-acting factors through cis-elements on mRNAs.

Network-based approach to identify prognosis-related genes in tamoxifen-treated patients with estrogen receptor-positive breast cancer.

Tamoxifen is an estrogen receptor (ER) antagonist that is most commonly used for the treatment of ER-positive breast cancer. However, tamoxifen resistance remains a major cause of cancer recurrence and progression. Here, we aimed to identify hub genes implicated in the progression and prognosis of ER-positive breast cancer following tamoxifen treatment. Microarray data (GSE9893) for 155 tamoxifen-treated primary ER-positive breast cancer samples were obtained from the Gene Expression Omnibus database. In total, 1706 differentially expressed genes (DEGs), including 859 up-regulated and 847 down-regulated genes, were identified between relapse and relapse-free samples. Weighted correlation network analysis clustered genes into 13 modules, among which the tan and blue modules were the most significantly related to prognosis. From these two modules, we further identified and validated two prognosis-related hub genes (G-rich RNA sequence binding factor 1 (GRSF1) and microtubule-associated protein τ (MAPT)) via survival analysis based on several publicly available datasets. High expression of GRSF1 predicted poor prognosis, whereas MAPT indicated favorable outcomes in ER-positive breast cancer. Using breast cancer cell lines and tissue samples, we confirmed that GRSF1 was significantly up-regulated and MAPT was down-regulated in the tamoxifen-resistant group compared with the tamoxifen-sensitive group. The prognostic value of GRSF1 and MAPT was also verified in 48 tamoxifen-treated ER-positive breast cancer patients in our hospital. Gene set enrichment analysis (GSEA) suggested that GRSF1 was potentially involved in RNA degradation and cell cycle pathways, while MAPT was strongly linked to immune-related signaling pathways. Taken together, our findings established novel prognostic biomarkers to predict tamoxifen sensitivity, which may facilitate individualized management of breast cancer.

GRSF1 promotes tumorigenesis and EMT-mediated metastasis through PI3K/AKT pathway in gastric cancer.

It is vital to identify effective therapeutic targets and explore the underlying mechanisms to curb the progression of Gastric cancer (GC) and improve the prognosis of GC patients. Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the RNA-binding protein family. The present study showed that GRSF1 knockdown suppressed GC cells proliferation, migration and invasion in vitro, while GRSF1 overexpression enhanced the proliferation, migration and invasion of GC cells. Meanwhile, knockdown of GRSF1 inhibited tumor growth and tumor metastasis in vivo. Furthermore, we demonstrated that GRSF1 induced epithelial-mesenchymal transition (EMT) and activated PI3K/AKT pathway in vitro and in vivo through gain and loss of function. In conclusion, we demonstrated that GRSF1 promotes tumorigenesis and EMT-mediated metastasis through PI3K/AKT pathway in GC. Our study for the first time identified the functions of GRSF1 serving as an oncogene in GC, which may be a potential effective therapeutic target and malignant indicator in GC.

MKRN3-mediated ubiquitination of Poly(A)-binding proteins modulates the stability and translation of GNRH1 mRNA in mammalian puberty.

The family of Poly(A)-binding proteins (PABPs) regulates the stability and translation of messenger RNAs (mRNAs). Here we reported that the three members of PABPs, including PABPC1, PABPC3 and PABPC4, were identified as novel substrates for MKRN3, whose deletion or loss-of-function mutations were genetically associated with human central precocious puberty (CPP). MKRN3-mediated ubiquitination was found to attenuate the binding of PABPs to the poly(A) tails of mRNA, which led to shortened poly(A) tail-length of GNRH1 mRNA and compromised the formation of translation initiation complex (TIC). Recently, we have shown that MKRN3 epigenetically regulates the transcription of GNRH1 through conjugating poly-Ub chains onto methyl-DNA bind protein 3 (MBD3). Therefore, MKRN3-mediated ubiquitin signalling could control both transcriptional and post-transcriptional switches of mammalian puberty initiation. While identifying MKRN3 as a novel tissue-specific translational regulator, our work also provided new mechanistic insights into the etiology of MKRN3 dysfunction-associated human CPP.

Gene variants of coagulation related proteins that interact with SARS-CoV-2.

Thrombosis is a recognized complication of Coronavirus disease of 2019 (COVID-19) and is often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms, by which some of these variants may contribute to disease, are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches.

Cytoplasmic cleavage of IMPA1 3' UTR is necessary for maintaining axon integrity.

The 3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3' UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3' UTR and stable 3' UTR-derived fragments of unknown function. Cleavage of the long 3' UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3' UTR-derived RNAs.

Mitochondrial ubiquitin ligase alleviates Alzheimer's disease pathology via blocking the toxic amyloid-ß oligomer generation.

Mitochondrial pathophysiology is implicated in the development of Alzheimer's disease (AD). An integrative database of gene dysregulation suggests that the mitochondrial ubiquitin ligase MITOL/MARCH5, a fine-tuner of mitochondrial dynamics and functions, is downregulated in patients with AD. Here, we report that the perturbation of mitochondrial dynamics by MITOL deletion triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. Notably, MITOL deletion in the brain enhanced the seeding effect of Aß fibrils, but not the spontaneous formation of Aß fibrils and plaques, leading to excessive secondary generation of toxic and dispersible Aß oligomers. Consistent with this, MITOL-deficient mice with Aß etiology exhibited worsening cognitive decline depending on Aß oligomers rather than Aß plaques themselves. Our findings suggest that alteration in mitochondrial morphology might be a key factor in AD due to directing the production of Aß form, oligomers or plaques, responsible for disease development.

Nuclear SR-protein mediated mRNA quality control is continued in cytoplasmic nonsense-mediated decay.

One important task of eukaryotic cells is to translate only mRNAs that were correctly processed to prevent the production of truncated proteins, found in neurodegenerative diseases and cancer. Nuclear quality control of splicing requires the SR-like proteins Gbp2 and Hrb1 in S. cerevisiae, where they promote the degradation of faulty pre-mRNAs. Here we show that Gbp2 and Hrb1 also function in nonsense mediated decay (NMD) of spliced premature termination codon (PTC)-containing mRNAs. Our data support a model in which they are in a complex with the Upf-proteins and help to transmit the Upf1-mediated PTC recognition to the transcripts ends. Most importantly they appear to promote translation repression of spliced transcripts that contain a PTC and to finally facilitate degradation of the RNA, presumably by supporting the recruitment of the degradation factors. Therefore, they seem to control mRNA quality beyond the nuclear border and may thus be global surveillance factors. Identification of SR-proteins as general cellular surveillance factors in yeast will help to understand the complex human system in which many diseases with defects in SR-proteins or NMD are known, but the proteins were not yet recognized as general RNA surveillance factors.

Poly(A)-Binding Protein Regulates the Efficiency of Translation Termination.

Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3' end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3' UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD).

A novel miRNA identified in GRSF1 complex drives the metastasis via the PIK3R3/AKT/NF-κB and TIMP3/MMP9 pathways in cervical cancer cells.

microRNAs (miRNAs) play an important role in carcinogenesis. Typically, miRNAs downregulate the target expression by binding to the 3' UTR of mRNAs. However, recent studies have demonstrated that miRNAs can upregulate target gene expression, but its mechanism is not fully understood. We previously found that G-rich RNA sequence binding protein (GRSF1) mediates upregulation of miR-346 on hTERT gene. To explore whether GRSF1 mediate other miRNA's upregulation on their target genes, we obtained profile of GRSF1-bound miRNAs by Flag-GRSF1-RIP-deep sequencing and found 12 novel miRNAs, named miR-G. In this study, we focused on miR-G-10, which is highly expressed in cervical cancer tissues and cell lines and serum from patients with metastatic cervical cancer. miR-G-10 in cervical cancer cells significantly promoted migration/invasion and anoikis resistance in vitro and lung metastasis in vivo. Furthermore, miR-G-10 bound to the 3' UTR of PIK3R3 and upregulated its expression to activate the AKT/NF-κB signal pathway in a GRSF1-dependent manner, whereas miR-G-10 suppressed TIMP3 in the AGO2 complex to modulate the MMP9 signaling pathway in cervical cancer cells. Taken together, our findings may provide a new insight into the upregulation mechanism mediated by miRNAs and a potential biomarker for cervical cancer.

Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.

The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.

O-GlcNAcylation of core components of the translation initiation machinery regulates protein synthesis.

Protein synthesis is essential for cell growth, proliferation, and survival. Protein synthesis is a tightly regulated process that involves multiple mechanisms. Deregulation of protein synthesis is considered as a key factor in the development and progression of a number of diseases, such as cancer. Here we show that the dynamic modification of proteins by O-linked ß-N-acetyl-glucosamine (O-GlcNAcylation) regulates translation initiation by modifying core initiation factors eIF4A and eIF4G, respectively. Mechanistically, site-specific O-GlcNAcylation of eIF4A on Ser322/323 disrupts the formation of the translation initiation complex by perturbing its interaction with eIF4G. In addition, O-GlcNAcylation inhibits the duplex unwinding activity of eIF4A, leading to impaired protein synthesis, and decreased cell proliferation. In contrast, site-specific O-GlcNAcylation of eIF4G on Ser61 promotes its interaction with poly(A)-binding protein (PABP) and poly(A) mRNA. Depletion of eIF4G O-GlcNAcylation results in inhibition of protein synthesis, cell proliferation, and soft agar colony formation. The differential glycosylation of eIF4A and eIF4G appears to be regulated in the initiation complex to fine-tune protein synthesis. Our study thus expands the current understanding of protein synthesis, and adds another dimension of complexity to translational control of cellular proteins.

miR-192-5p Silencing by Genetic Aberrations Is a Key Event in Hepatocellular Carcinomas with Cancer Stem Cell Features.

Various cancer stem cell (CSC) biomarkers have been identified for hepatocellular carcinoma (HCC), but little is known about the implications of heterogeneity and shared molecular networks within the CSC population. Through miRNA profile analysis in an HCC cohort (n = 241) for five groups of CSC+ HCC tissues, i.e., EpCAM+, CD90+, CD133+, CD44+, and CD24+ HCC, we identified a 14-miRNA signature commonly altered among these five groups of CSC+ HCC. miR-192-5p, the top-ranked CSC miRNA, was liver-abundant and -specific and markedly downregulated in all five groups of CSC+ HCC from two independent cohorts (n = 613). Suppressing miR-192-5p in HCC cells significantly increased multiple CSC populations and CSC-related features through targeting PABPC4. Both TP53 mutation and hypermethylation of the mir-192 promoter impeded transcriptional activation of miR-192-5p in HCC cell lines and primary CSC+ HCC. This study reveals the circuit from hypermethylation of the mir-192 promoter through the increase in PABPC4 as a shared genetic regulatory pathway in various groups of primary CSC+ HCC. This circuit may be the driver that steers liver cells toward hepatic CSC cells, leading to hepatic carcinogenesis. SIGNIFICANCE: miR-192-5p and its regulatory pathway is significantly abolished in multiple groups of HCC expressing high levels of CSC markers, which may represent a key event for hepatic carcinogenesis.