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Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing, transactivation of gene expression, and regulation of protein translation. As a core component of the hnRNP complex in mammalian cells, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNP A2B1) participates in and coordinates various molecular events. Given its regulatory role in inflammation and cancer progression, hnRNP A2B1 has become a novel player in immune response, inflammation, and cancer development. Concomitant with these new roles, a surprising number of mechanisms deemed to regulate hnRNP A2B1 functions have been identified, including post-translational modifications, changes in subcellular localization, direct interactions with multiple DNAs, RNAs, and proteins or the formation of complexes with them, which have gradually made hnRNP A2B1 a molecular target for multiple drugs. In light of the rising interest in the intersection between cancer and inflammation, this review will focus on recent knowledge of the biological roles of hnRNP A2B1 in cancer, immune response, and inflammation.

Flail arm syndrome (FAS) is one of the atypical subtypes of amyotrophic lateral sclerosis (ALS). Mutations in hnRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare genetic cause of ALS. Herein, marked clinical heterogeneity of FAS in a pedigree with a known hnRNPA1 variant was described to raise early awareness of the ALS variant. Furtherly, a literature review of the hnRNPA1-related spectrum was made to summarize the clinical and genetic characteristics.Detailed clinical evaluation, muscle pathology, and whole-exome sequencing were performed. The sequence and co-segregation of the mutation among the family members were confirmed by Sanger sequencing.The great clinical variability was found in a FAS pedigree. Muscle pathology revealed a cluster distribution of angulated or rounded atrophic fibers, accompanied by significant multi-nucleus aggregation. Immunohistochemical staining showed that mutant hnRNPA1 proteins accumulated in muscle fiber cytoplasm. Exome sequencing identified a documented variant in hnRNPA1 gene c.1018C T (p.P340S), which co-segregated with disease in the family. Besides, highly phenotypic heterogeneity was also found in other hnRNPA1-related diseases.We described a Chinese pedigree with hnRNPA1-related FAS, which showed significant clinical variability among the intrafamilial members. FAS is a relatively milder variant of ALS, due to the highly heterogeneous clinical spectrum, early observation is of paramount importance. In addition, the highly phenotypic heterogeneity and molecular genetic mechanism of the hnRNPA1-related spectrum are still beyond fully understood. Further, the detailed molecular mechanism underlying the clinical diversity is warranted to be explored.

Long intergenic non-protein coding RNA 1833 (LINC01833) exhibits elevated expression in the non-small cell lung cancer (NSCLC) tissues, while its molecular mechanism in NSCLC progression remains elusive. Herein, the proliferation, migration, invasion as well as apoptosis of NSCLC cells were assessed. The potential N6-methyladenosine (m6A) modification site was predicted by the m6aVar tool. RNA pulldown and m6A-specific immunoprecipitation assays were used to detect the interaction between LINC01833 and methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3). RNA pull-down together with mass spectrometry were performed to assess the binding relationship between LINC01833 and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) in NSCLC. Tumor xenograft mice model was established, and the tumor size and weight were measured. The results demonstrated that LINC01833 expression was elevated in NSCLC samples. Overexpression of LINC01833 promoted proliferative, migratory, and invasive abilities and inhibited HCC827 cell apoptosis. LINC01833 knockdown inhibited tumor growth in mice. LINC01833 is further demonstrated to be modulated by METTL3, which is highly expressed in NSCLC samples. In addition, RNA pulldown and m6A-specific immunoprecipitation assays indicated that LINC01833 might form a complex with HNRNPA2B1. In conclusion, m6A transferase METTL3-induced LINC01833 m6A methylation promotes NSCLC progression through modulating HNRNPA2B1 expression. Our findings indicated that LINC01833 might be a therapeutic target for NSCLC.

Despite new combination therapies improving survival of breast cancer patients with estrogen receptor α (ER+) tumors, the molecular mechanisms for endocrine-resistant disease remain unresolved. Previously we demonstrated that expression of the RNA binding protein and N6-methyladenosine (m6A) reader HNRNPA2B1 (A2B1) is higher in LCC9 and LY2 tamoxifen (TAM)-resistant ERα breast cancer cells relative to parental TAM-sensitive MCF-7 cells. Here we report that A2B1 protein expression is higher in breast tumors than paired normal breast tissue. Modest stable overexpression of A2B1 in MCF-7 cells (MCF-7-A2B1 cells) resulted in TAM- and fulvestrant- resistance whereas knockdown of A2B1 in LCC9 and LY2 cells restored TAM and fulvestrant, endocrine-sensitivity. MCF-7-A2B1 cells gained hallmarks of TAM-resistant metastatic behavior: increased migration and invasion, clonogenicity, and soft agar colony size, which were attenuated by A2B1 knockdown in MCF-7-A2B1 and the TAM-resistant LCC9 and LY2 cells. MCF-7-A2B1, LCC9, and LY2 cells have a higher proportion of CD44+/CD24-/low cancer stem cells (CSC) compared to MCF-7 cells. MCF-7-A2B1 cells have increased ERα and reduced miR-222-3p that targets ERα. Like LCC9 cells, MCF-7-A2B1 have activated AKT and MAPK that depend on A2B1 expression and are growth inhibited by inhibitors of these pathways. These data support that targeting A2B1 could provide a complimentary therapeutic approach to reduce acquired endocrine resistance.

Hypertriglyceridemia-induced acute pancreatitis (HTGP) is characterized by the acute and excessive release of FFA produced by pancreatic lipases. However, the underlying mechanisms of this disease remain poorly understood. In this study, we describe the involvement of the RNA binding protein hnRNPA2B1 in the development of HTGP. We used palmitic acid (PA) and AR42J cells to create a model of HTGP in vitro. RT-PCR and western blot analyses revealed a decrease in the level of hnRNPA2B1 protein but not mRNA expression in PA-treated cells. Further analyses revealed that hnRNPA2B1 expression was regulated at the post-translational level by neddylation. Restoration of hnRNPA2B1 expression using the neddylation inhibitor MLN4924 protected AR42J cells from PA-induced inflammatory injury by preventing NF-κB activation and restoring fatty acid oxidation and cell proliferation. Furthermore, RNA immunoprecipitation studies demonstrated that hnRNPA2B1 orchestrates fatty acid oxidation by regulating the expression of the mitochondrial trifunctional protein-α (MTPα). Administration of MLN4924 in vivo restored hnRNPA2B1 protein expression in the pancreas of hyperlipidemic mice and ameliorated HTGP-associated inflammation and pancreatic tissue injury. In conclusion, we show that hnRNPA2B1 has a central regulatory role in preventing HTGP-induced effects on cell metabolism and viability. Furthermore, our findings indicate that pharmacological inhibitors that target neddylation may provide therapeutic benefits to HTGP patients.

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies in the world. Although increasing evidence supports the role of heterogeneous ribonucleoprotein particle A1 (HNRNP A1) in tumor progression, the function of HNRNP A1 in HCC remains unclear. Here, we focused on the role of HNRNP A1 in the development of HCC. In this study, we found HNRNP A1 participates in many aspects of HCC, such as progression and prognosis. Our results showed that HNRNP A1 is upregulated in human HCC tissues and cell lines. High expression of HNRNP A1 can promote the proliferation, migration, and invasion in HCC cells and accelerate tumor progression in mice. Moreover, we found that HNRNP A1 prevents the senescence process of HCC cells. Knocking down of HNRNP A1 promotes the expression of P16INK4, which arrests the cell cycle and then induces the senescence phenotype in HCC cells. Furthermore, we found that HNRNP A1 regulated necroptosis and mitochondrial dynamics. In summary, our study indicates that HNRNP A1 promotes the development of HCC, which suggests a potential therapeutic target for HCC.

Lung cancer in its occurrence and development of different stages exist different biological behavior changes. This paper studies the expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 in benign and malignant lung lesions and its early diagnosis value of nonsmall-cell lung cancer (NSCLC), aiming to provide reference for the early diagnosis and therapy of NSCLC. Some lung surgery specimens are selected from January 2021 to March 2022. All cases received no radiotherapy and chemotherapy before surgery, including 90 sufferers with benign lung lesions as the contrast set. hnRNP A2/B1 expressions are measured for comparison. The experimental results show that for lung cancer sufferers, the positive expression of hnRNP A2/B1 in their malignant lesion tissue is notoriously higher than that in their benign lesion tissue, and hnRNP A2/B1 is differently expressed in different differentiation and in different stages.

Transthyretin (TTR) has been proved to repress neovascularization in diabetic retinopathy environment by regulating the molecules in and downstream of the STAT-4/miR-223–3p/FBXW7 signal pathway; however, the details of its direct targets are still not well understood. The interaction between TTR and a target in nucleus of human retinal microvascular endothelial cells (hRECs), heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1, was screened by immunoprecipitation (IP) and mass spectrum (MS), and it was further confirmed by co-immunoprecipitation (co-IP). Regarding ZDOCK analysis using Discovery Studio, the interface and potential binding sites between TTR and hnRNPA2B1 were simulated; mutants were designed in these regions and five soluble ones were recombinantly expressed and prepared; the interaction between TTR and hnRNPA2B1 were disrupted by several mutated residues. In addition, for several mutated TTRs, the inhibition activities against the proliferation, migration and tube formation of hRECs were absent in vitro. Following the disruption of TTR-hnRNPA2B1, the molecules in and downstream of STAT-4/miR-223–3p/FBXW7 signal pathway, including STAT-4, miR-223–3p, FBXW7 p-Akt and Notch1 could not be regulated by TTR mutants; therefore, a TTR-hnRNPA2B1/STAT-4/miR-223–3p/FBXW7 was proposed. In conclusion, this work suggested that TTR should play a physiological role in diabetic environment by the direct binding with hnRNPA2B1, and it provided a theoretical basis for clinical diagnosis, therapy and further application.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) is crucial in the atherosclerosis. Although long noncoding RNAs (lncRNAs) are implicated in a variety of diseases, their roles in activation of VSMCs proliferation and vascular disorder diseases are not well understood. In addition, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) was reported to participate in lncRNAs-mediated function. Herein, we propose to investigate the role of lncRNA AC105942.1 and hnRNPA2/B1 in pathological VSMCs proliferation and the possible mechanisms in vitro. We have identified that lncRNA AC105942.1 was downregulated and hnRNPA2/B1 was upregulated in atherosclerotic plaques compared with normal artery tissues. Enhanced lncRNA AC105942.1 could noticeably inhibit Ang II-induced VSMCs proliferation. Further investigation suggested that lncRNA AC105942.1 could downregulate the expression of hnRNPA2/B1 and then regulate the level of CDK4 and p27. Taken together, our study indicated that lncRNA AC105942.1 downregulated hnRNPA2B1 to protect against the atherosclerosis by suppressing VSMCs proliferation. LncRNA AC105942.1 and hnRNPA2/B1 could represent potential therapeutic and diagnostic targets to atherosclerosis-related diseases.

In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors., Suppression of terminal differentiation is essential for epidermal progenitor maintenance. Here, the authors show that intronic polyadenylation is dynamically regulated by the cooperation between CPSF and RNA-binding proteins to influence epidermal differentiation gene expression.

Funder: Howard Hughes Medical Institute, Missense variants in RNA-binding proteins (RBPs) underlie a spectrum of disease phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy. Here, we present ten independent families with a severe, progressive muscular dystrophy, reminiscent of oculopharyngeal muscular dystrophy (OPMD) but of much earlier onset, caused by heterozygous frameshift variants in the RBP hnRNPA2/B1. All disease-causing frameshift mutations abolish the native stop codon and extend the reading frame, creating novel transcripts that escape nonsense-mediated decay and are translated to produce hnRNPA2/B1 protein with the same neomorphic C-terminal sequence. In contrast to previously reported disease-causing missense variants in HNRNPA2B1, these frameshift variants do not increase the propensity of hnRNPA2 protein to fibrillize. Rather, the frameshift variants have reduced affinity for the nuclear import receptor karyopherin β2, resulting in cytoplasmic accumulation of hnRNPA2 protein in cells and in animal models that recapitulate the human pathology. Thus, we expand the phenotypes associated with HNRNPA2B1 to include an early-onset form of OPMD caused by frameshift variants that alter its nucleocytoplasmic transport dynamics.

Yin Yang 1 (YY1) regulates gene transcription in a variety of biological processes. In this study, we aim to determine the role of YY1 in vascular smooth muscle cell (VSMC) phenotypic modulation both in vivo and in vitro. Here we show that vascular injury in rodent carotid arteries induces YY1 expression along with reduced expression of smooth muscle differentiation markers in the carotids. Consistent with this finding, YY1 expression is induced in differentiated VSMCs in response to serum stimulation. To determine the underlying molecular mechanisms, we found that YY1 suppresses the transcription of CArG box-dependent SMC-specific genes including SM22α, SMα-actin and SMMHC. Interestingly, YY1 suppresses the transcriptional activity of the SM22α promoter by hindering the binding of serum response factor (SRF) to the proximal CArG box. YY1 also suppresses the transcription and the transactivation of myocardin (MYOCD), a master regulator for SMC-specific gene transcription by binding to SRF to form the MYOCD/SRF/CArG box triad (known as the ternary complex). Mechanistically, YY1 directly interacts with MYOCD to competitively displace MYOCD from SRF. This is the first evidence showing that YY1 inhibits SMC differentiation by directly targeting MYOCD. These findings provide new mechanistic insights into the regulatory mechanisms that govern SMC phenotypic modulation in the pathogenesis of vascular diseases.

hnRNPA2 is a major component of mRNA transport granules in oligodendrocytes and neurons. However, the structural details of how hnRNPA2 binds the A2 recognition element (A2RE) and if this sequence stimulates granule formation by enhancing phase separation of hnRNPA2 has not yet been studied. Using solution NMR and biophysical studies, we find that each of the two individual RRMs retain the domain structure observed in complex with RNA but are not rigidly confined (i.e. they move independently) in solution in the absence of RNA. hnRNPA2 RRMs bind the minimal rA2RE11 weakly but at least, and most likely, two hnRNPA2 molecules are able to simultaneously bind the longer 21mer myelin basic protein A2RE. Upon binding of the RNA, NMR chemical shift deviations are observed in both RRMs, suggesting both play a role in binding the A2RE11. Interestingly, addition of short A2RE RNAs or longer RNAs containing this sequence completely prevents in vitro phase separation of full-length hnRNPA2 and aggregation of the disease-associated mutants. These findings suggest that RRM interactions with specific recognition sequences alone do not account for nucleating granule formation, consistent with models where multivalent protein:RNA and protein:protein contacts form across many sites in granule proteins and long RNA transcripts.

Following influenza A vaccination, certain individuals exhibit adverse reactions in the nervous system, which causes a problem with the safety of the influenza A vaccine. However, to the best of our knowledge, the underlying mechanism of this is unknown. The present study revealed that a monoclonal antibody (H1‑84mAb) against the H1N1 influenza virus hemagglutinin (HA) protein cross‑reacted with an antigen from brain tissue. Total brain tissue protein was immunoprecipitated with this cross‑reactive antibody, and mass spectrometry revealed that the bound antigens were heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2/B1. Subsequently, the two proteins were expressed in bacteria and it was demonstrated that H1‑84mAb bound to hnRNPA1 and hnRNPA2/B1. These two proteins were expressed in three segments and the cross‑reactivity of H1‑84mAb with the glycine (Gly)‑rich domains of hnRNPA1 (195aa‑320aa) and hnRNPA2/B1 (202aa‑349aa) was determined using ELISA blocking experiments. It was concluded that the Gly‑rich domains of these two proteins are heterophilic antigens that cross‑react with influenza virus HA. The association between the heterophilic antigen Gly‑rich domains and the safety of influenza A vaccines remains to be investigated.

Background: N6-methyladenosine (m6A) is the most prevalent RNA modification. While the role of m6A in prostate cancer remains unknown. We aim to measure the effects of m6A methylation regulatory genes during the development and progression of prostate cancer. Methods: We collected transcriptome information and gene-level alteration data from The Cancer Genome Atlas datasets. The log-rank test and Cox regression model were used to examine the prognosis value of m6A methylation regulatory genes of prostate cancer. Results: We discovered that most of m6A methylation regulators were highly expressed in aggressive prostate cancer. Univariable and multivariable Cox regression results showed that the expression of Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), Heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1) and N6-adenosine-methyltransferase non-catalytic subunit (METTL14) and copy number variant of AlkB Homolog 5 (ALKBH5) were considerably associated with a recurrence-free survival of prostate cancer. Furthermore, a high level of m6A methylation in mRNA promotes the progression of prostate cancer via regulating subcellular protein localization. Conclusion: Patients with a high level of mRNA methylation resulted from overexpression of reader proteins and methyltransferase complexes had poor survival benefits through influencing protein subcellular location in prostate cancer.

The discovery of long noncoding RNAs (lncRNAs) has increased our understanding of the development and progression of many cancers, but their contributions to non-small cell lung cancer (NSCLC) remain poorly understood. Here, we profiled lncRNA expression in NSCLC and investigated in detail the molecular function of one upregulated lncRNA, LINC01234. LINC01234 was overexpressed in NSCLC compared with normal lung tissue and correlated positively with poor prognosis. Downregulation of LINC01234 impaired cell proliferation in vitro and tumor growth in vivo. RNA pull-down/mass spectrometry experiments showed that LINC01234 interacted with the RNA-binding protein heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1), which, in turn, led to the recruitment of DiGeorge syndrome critical region gene 8 (DGCR8), a subunit of the microRNA (miRNA) microprocessor complex. Accordingly, depletion of either LINC01234 or HNRNPA2B1 reduced the processing of several miRNA precursors, including primary microRNA (pri-miR)-106b. miR-106b-5p enhanced NSCLC cell growth by downregulating cryptochrome 2 (CRY2), thereby increasing c-Myc expression. Finally, we found that activated c-Myc binds to the LINC01234 promoter to increase its transcription, creating a c-Myc-LINC01234-HNRNPA2B1-miR-106b-5p-CRY2-c-Myc positive-feedback loop. We identified numerous lncRNAs with dysregulated expression in NSCLC and demonstrated a novel oncogenic axis involving LINC01234, HNRNPA2B1, miR-106b-5p, CRY2, and c-Myc. Components of this axis may be potential novel targets for NSCLC.

Interaction of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with specific single-stranded RNA and its relation to liquid-liquid phase separation (LLPS) were studied in vitro by magnetic resonance based on site-directed spin labelling. An ensemble model of dispersed hnRNP A1 in the absence of RNA was derived from distance distributions between spin labelled sites and small angle X-ray scattering. This model revealed a compact state of the low-complexity domain and its interaction with the RNA recognition motifs. Paramagnetic relaxation enhancement NMR spectroscopy confirmed this interaction. Addition of RNA to dispersed hnRNP A1 induced liquid-droplet formation. Such LLPS depended on RNA concentration and sequence, with continuous wave EPR spectroscopy showing an influence of RNA point mutations on local protein dynamics. We propose that an interplay of sequence-specific RNA binding and LLPS contributes to regulation of specific RNA segregation during stress response., Angewandte Chemie. International Edition, 61 (40), ISSN:1433-7851, ISSN:1521-3773, ISSN:0570-0833

Some studies have suggested heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) to be a promoter in cancer development. Nonetheless, no detailed pan-cancer investigation has been reported. Thus, this study explored the possible oncogenic role of HNRNPA2B1, such as its expression levels, gene alteration, protein–protein interaction network, immune infiltration, and prognostic value in different cancer types using The Cancer Genome Atlas web platform. Many types of cancer exhibit HNRNPA2B1 overexpression, which is notably associated with poor prognosis. We also found that HNRNPA2B1 with different methylation levels causes a varied prognosis in lung adenocarcinoma (LUAD). It is noteworthy that HNRNPA2B1 levels are connected with cancer-associated fibroblasts in cancers, such as adrenocortical carcinoma, LUAD, and stomach adenocarcinoma. In addition, HNRNPA2B1 participates in the spliceosome- and cell cycle-associated pathways. Finally, HNRNPA2B1 is highly valued in the diagnosis of LUAD, lung squamous cell carcinoma, breast invasive carcinoma, esophageal carcinoma, and liver hepatocellular carcinoma. This systematic study highlighted the role of HNRNPA2B1 in pan-cancer progression.

OBJECTIVE: To identify the key hub genes in prostate cancer metastasis based on weighted gene co-expression network analysis (WGCNA) and verify the identified genes. METHODS: Whole-genome chip data GSE6919 of prostate cancer study were analyzed using principal component analysis (PCA), and the differentially expressed genes (DEGs) were analyzed using R software. WGCNA was performed to construct a gene co-expression network for screening the key genes. TCGA database was used to explore the expressions of the DEGs and their association with the prognosis. To validate the results, we designed siRNA fragments targeting the metastasis-related gene HNRNPA2B1, and observed its effect on growth, apoptosis, clone formation, migration and invasion of prostate cancer cell lines using MTT assay, flow cytometry, clone formation assay, and Transwell assay. RESULTS: PCA analysis showed obvious clustering of significant DEGs in metastatic cancer group. The modules obtained by WGCNA analysis in metastasis group involved stem cell differentiation, amino acid metabolism and immune response. Further screening of the genes identified 3 genes related with prostate cancer occurrence (BDH1, PAK4 and EXTL3) and another 3 with prostate cancer metastasis (NKTR, CTBP2 and HNRNPA2B1), which were shown to have differential expressions in TCGA database and were correlated with the patient's overall survival. In the cell experiment, PC3 and LNCap cells transfected with the siRNA fragment targeting HNRNPA2B1 showed obvious growth inhibition with increased cell apoptosis, lowered clone formation ability, and suppressed capacities for migration and invasion. CONCLUSION: We identified 3 hub genes related with the occurrence (BDH1, PAK4 and EXTL3) and another 3 with metastasis of prostate cancer (NKTR, CTBP2 and HNRNPA2B1) using WGCNA, which provides a new approach for studying the regulatory mechanisms of prostate cancer.

Epithelial-to-mesenchymal transition (EMT) is a process linked to metastasis and drug resistance with non-coding RNAs (ncRNAs) playing pivotal roles. We previously showed that miR-100 and miR-125b, embedded within the third intron of the ncRNA host gene MIR100HG, confer resistance to cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in colorectal cancer (CRC). However, whether the MIR100HG transcript itself has a role in cetuximab resistance or EMT is unknown.The correlation between MIR100HG and EMT was analyzed by curating public CRC data repositories. The biological roles of MIR100HG in EMT, metastasis and cetuximab resistance in CRC were determined both in vitro and in vivo. The expression patterns of MIR100HG, hnRNPA2B1 and TCF7L2 in CRC specimens from patients who progressed on cetuximab and patients with metastatic disease were analyzed by RNAscope and immunohistochemical staining.The expression of MIR100HG was strongly correlated with EMT markers and acted as a positive regulator of EMT. MIR100HG sustained cetuximab resistance and facilitated invasion and metastasis in CRC cells both in vitro and in vivo. hnRNPA2B1 was identified as a binding partner of MIR100HG. Mechanistically, MIR100HG maintained mRNA stability of TCF7L2, a major transcriptional coactivator of the Wnt/β-catenin signaling, by interacting with hnRNPA2B1. hnRNPA2B1 recognized the N6-methyladenosine (mMIR100HG and hnRNPA2B1 interact to control the transcriptional activity of Wnt signaling in CRC via regulation of TCF7L2 mRNA stability. Our findings identified MIR100HG as a potent EMT inducer in CRC that may contribute to cetuximab resistance and metastasis by activation of a MIR100HG/hnRNPA2B1/TCF7L2 feedback loop.

Long non-coding RNAs (lncRNAs) play key roles in Angiotensin II (AngII) signaling but their role in chondrogenic transformation of vascular smooth muscle cells (VSMCs) is unknown. We describe a novel AngII-induced lncRNA Alivec (Angiotensin II-induced lncRNA in VSMCs eliciting chondrogenic phenotype) implicated in VSMC chondrogenesis. In rat VSMCs, Alivec and the nearby gene Acan, a chondrogenic marker, were induced by growth factors AngII and PDGF and the inflammatory cytokine TNF-α. AngII co-regulated Alivec and Acan through the activation of AngII type1 receptor signaling and Sox9, a master transcriptional regulator of chondrogenesis. Alivec knockdown with GapmeR antisense-oligonucleotides attenuated the expression of AngII-induced chondrogenic marker genes, including Acan, and inhibited the chondrogenic phenotype of VSMCs. Conversely, Alivec overexpression upregulated these genes and promoted chondrogenic transformation. RNA-pulldown coupled to mass-spectrometry identified Tropomyosin-3-alpha and hnRNPA2B1 proteins as Alivec-binding proteins in VSMCs. Furthermore, male rats with AngII-driven hypertension showed increased aortic expression of Alivec and Acan. A putative human ortholog ALIVEC, was induced by AngII in human VSMCs, and this locus was found to harbor the quantitative trait loci affecting blood pressure. Together, these findings suggest that AngII-regulated lncRNA Alivec functions, at least in part, to mediate the AngII-induced chondrogenic transformation of VSMCs implicated in vascular dysfunction and hypertension.

While dysregulation of RNA splicing has been recognized as an emerging target for cancer therapy, the functional significance of RNA splicing and individual splicing factors in brain tumors is poorly understood. Here, we identify SON as a master regulator that activates PTBP1-mediated oncogenic splicing while suppressing RBFOX2-mediated non-oncogenic neuronal splicing in glioblastoma multiforme (GBM). SON is overexpressed in GBM patients and SON knockdown causes failure in intron removal from the PTBP1 transcript, resulting in PTBP1 downregulation and inhibition of its downstream oncogenic splicing. Furthermore, SON forms a complex with hnRNP A2B1 and antagonizes RBFOX2, which leads to skipping of RBFOX2-targeted cassette exons, including the PTBP2 neuronal exon. SON knockdown inhibits proliferation and clonogenicity of GBM cells in vitro and significantly suppresses tumor growth in orthotopic xenografts in vivo. Collectively, our study reveals that SON-mediated RNA splicing is a GBM vulnerability, implicating SON as a potential therapeutic target in brain tumors., Splicing factor PTBP1 is reported to promote oncogenic functions in glioblastoma (GBM). Here the authors show splicing factor SON upregulates PTBP1 expression while supresses its paralog PTBP2 through alternative splicing and the inhibition of SON reduces GBM stemness and growth.

The aim of this study was to elucidate the correlation between m6A modification and the tumor immune microenvironment (TIME) in prostate cancer (PCa) and to identify the m6A regulation patterns suitable for immune checkpoint inhibitors (ICIs) therapy. We evaluated the m6A regulation patterns of PCa based on 24 m6A regulators and correlated these modification patterns with TIME characteristics. Three distinct m6A regulation patterns were determined in PCa. The m6A regulators cluster with the best prognosis had significantly increased METTL14 and ZC3H13 expression and was characterized by low mutation rate, tumor heterogeneity, and neoantigens. The m6A regulators cluster with a poor prognosis had markedly high KIAA1429 and HNRNPA2B1 expression and was characterized by high intratumor heterogeneity and Th2 cell infiltration, while low Th17 cell infiltration and Macrophages M1/M2. The m6Ascore was constructed to quantify the m6A modification pattern of individual PCa patients based on m6A-associated genes. We found that the low-m6Ascore group with poor prognosis had a higher immunotherapeutic response rate than the high-m6Ascore group. The low-m6Ascore group was more likely to benefit from ICIs therapy. This study was determined that immunotherapy is more effective in low-m6Ascore PCa patients with poor prognosis.

Patients with bladder cancer (BCa) with clinical lymph node (LN) metastasis have an extremely poor prognosis. VEGF-C has been demonstrated to play vital roles in LN metastasis in BCa. However, approximately 20% of BCa with LN metastasis exhibits low VEGF-C expression, suggesting a VEGF-C-independent mechanism for LN metastasis of BCa. Herein, we demonstrate that BCa cell-secreted exosome-mediated lymphangiogenesis promoted LN metastasis in BCa in a VEGF-C-independent manner. We identified an exosomal long noncoding RNA (lncRNA), termed lymph node metastasis-associated transcript 2 (LNMAT2), that stimulated human lymphatic endothelial cell (HLEC) tube formation and migration in vitro and enhanced tumor lymphangiogenesis and LN metastasis in vivo. Mechanistically, LNMAT2 was loaded to BCa cell-secreted exosomes by directly interacting with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1). Subsequently, exosomal LNMAT2 was internalized by HLECs and epigenetically upregulated prospero homeobox 1 (PROX1) expression by recruitment of hnRNPA2B1 and increasing the H3K4 trimethylation level in the PROX1 promoter, ultimately resulting in lymphangiogenesis and lymphatic metastasis. Therefore, our findings highlight a VEGF-C-independent mechanism of exosomal lncRNA-mediated LN metastasis and identify LNMAT2 as a therapeutic target for LN metastasis in BCa.

Our previous study demonstrated that heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) is a key gene that facilitates metastasis of hepatocellular carcinoma (HCC). However, the molecular mechanisms behind this relationship are not fully understood. In our study, we utilized long-noncoding RNA (lncRNA) microarrays to identify a HNRNPAB-regulated lncRNA named lnc-ELF209. Our findings from chromatin immunoprecipitation assays indicate that HNRNPAB represses lnc-ELF209 transcription by directly binding to its promoter region. We also analyzed clinical samples from HCC patients and cell lines with quantitative real-time polymerase chain reactions, RNA in situ hybridization and immunohistochemistry, and found that there is a negative relationship between HNRNPAB and lnc-ELF209 expression. Up/downregulation assays and rescue assays indicate that lnc-ELF209 inhibits cell migration, invasion and epithelial-mesenchymal transition regulated by HNRNPAB. This suggests a new regulatory mechanism for HNRNPAB-promoted HCC progression. RNA pull-down and LC-MS/MS were used to determine triosephosphate isomerase, heat shock protein 90-beta and vimentin may be involved in the tumor-suppressed function of lnc-ELF209. Furthermore, we found lnc-ELF209 could stabilize TPI protein expression. We also found that lnc-ELF209 overexpression in HCCLM3 cell resulted in a lower rate of lung metastatic, which suggested a less aggressive HCC phenotype. Collectively, these findings offer new insights into the regulatory mechanisms that underlie HNRNPAB cancer-promoting activities and demonstrate that lnc-ELF209 is a HNRNPAB-regulated lncRNA that may play an important role in the inhibition of HCC progression.

Extracellular vesicle (EV)–cargo miRNAs mediate intercellular communications. How cells selectively sort the miRNAs into EVs remains unclear. Lee et al. demonstrate a novel mechanism by which functional miRNAs are selectively sorted into EVs via caveolin-1/hnRNPA2B1 complex. This process is regulated by stimulation-associated posttranslational modifications of both cav-1 and hnRNPA2B1., Emerging evidence suggests that extracellular vesicle (EV)–containing miRNAs mediate intercellular communications in response to noxious stimuli. It remains unclear how a cell selectively sorts the cellular miRNAs into EVs. We report that caveolin-1 (cav-1) is essential for sorting of selected miRNAs into microvesicles (MVs), a main type of EVs generated by outward budding of the plasma membrane. We found that cav-1 tyrosine 14 (Y14)–phosphorylation leads to interactions between cav-1 and hnRNPA2B1, an RNA-binding protein. The cav-1/hnRNPA2B1 complex subsequently traffics together into MVs. Oxidative stress induces O-GlcNAcylation of hnRNPA2B1, resulting in a robustly altered hnRNPA2B1-bound miRNA repertoire. Notably, cav-1 pY14 also promotes hnRNPA2B1 O-GlcNAcylation. Functionally, macrophages serve as the principal recipient of epithelial MVs in the lung. MV-containing cav-1/hnRNPA2B1 complex-bound miR-17/93 activate tissue macrophages. Collectively, cav-1 is the first identified membranous protein that directly guides RNA-binding protein into EVs. Our work delineates a novel mechanism by which oxidative stress compels epithelial cells to package and secrete specific miRNAs and elicits an innate immune response.

Hepatocellular carcinoma (HCC) is generally accompanied by high mortality and low cure rate. CCAAT enhancer-binding proteins (CEBPs) are transcriptional regulators that play a key role in maintaining liver function. Altered expression of C/EBPα and C/EBPβ occurs in many tumours including HCC. saRNAs are small double-stranded RNAs that enhance target gene expression at the transcriptional level. In this report, we activate CEPBA with saRNAs and suppress CEBPB with siRNAs in cells that represent three different degrees of HCC. We performed functional assays to investigate the effects of enhancing C/EBPα and its downstream targets, p21 and albumin across these lines. We also used Mass-spectrometry (MS) subsequent to a ChIP pull-down assay to characterise the components of the protein complex involved in regulating saRNA function. Putative saRNA interacting protein candidates that were identified by MS were knocked-down with siRNAs to investigate its impact on saRNA activity. We confirmed CEBPA-saRNA decreased proliferation and migration in the differentiated lines (HepG3/Hep3B). The undifferentiated line (PLCPRF5) showed saRNA-induced increase in CEBPA but with no loss in proliferation. This effect was reversed when CEBPB was suppressed with CEBPB-siRNA. When interrogating saRNA mode of action; three saRNA interacting proteins, CTR9, HnRNPA2/B1 and DDX5 were identified by MS. Targeted knock-down of these two proteins (by siRNA) abrogated saRNA activity. This study provides insight into how different HCC lines are affected by CEBPA-saRNAs and that endogenous abundance of CEBPB and saRNA accessory proteins may dictate efficacy of CEBPA-saRNA when used in a therapeutic context.

The heterogeneous nuclear ribonucleoprotein hnRNP A1 is a nucleocytoplasmic-shuttling RNA-binding protein that plays an important role in nucleic acid metabolism and gene expression regulation. The function of hnRNP A1 is determined in part by its specific location within the cell. Although some work has been done to elucidate the signaling pathways that regulate the cellular localization of hnRNP A1, the precise mechanism(s), including physiological and pathophysiological conditions that alter hnRNP A1 localization, are not known. We previously conducted an unbiased RNAi-based kinome-wide screen to identify kinases that regulate hnRNP A1 localization during hypertonic stress. One of the hits from this screen is AMPK-related protein kinase 5 (ARK5). Here, we validate ARK5 as the kinase responsible for controlling hnRNP A1 subcellular localization in response to hypertonic stress. We find using immunoprecipitation and in vitro kinase assay methods that ARK5 directly interacts with and phosphorylates hnRNP A1 on serine residues within the F-peptide region. We further show that the M9 motif of hnRNP A1 is essential for the ARK5-hnRNP A1 interaction and subsequent phosphorylation. In addition, the silencing of ARK5 increases the expression of antiapoptotic protein Bcl-xL and consequently delays caspase activation during hypertonic stress. Our results indicate that ARK5 phosphorylates hnRNP A1 and regulates its subcellular localization during hypertonic stress.

Objective To investigate the interaction between Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and its biological functions. Methods The interaction between WASH/FAM21, the core member of WASH complex, and hnRNP A1 was identified by mass spectrometry and co-immunoprecipitation. Telomere lengths of shWASH and shScramble cells were measured by Real-time qPCR. RNA-seq was used to generate the transcription profiles of shWASH and shScramble cells. Results Mass spectrometry results indicated that a variety of hnRNP family members, including hnRNP A1, interact with FAM21-d219N. The interaction between endogenous WASH/FAM21 and hnRNP A1 was confirmed by co-immunoprecipitation results. Depletion of WASH did not affect the relative length of telomeres, but it significantly increased the exon skipping and led to the variation of transcriptional expression of hundreds of genes, including some NF-κB target genes. Conclusion WASH in the nucleus interacts with hnRNP A1 to participate in the regulation of alternative splicing and gene transcription.

MicroRNAs are dysregulated in breast cancer. Heterogeneous Nuclear Ribonucleoprotein A2/B1 (HNRNPA2/B1) is a reader of the N(6)-methyladenosine (m6A) mark in primary-miRNAs (pri-miRNAs) and promotes DROSHA processing to precursor-miRNAs (pre-miRNAs). We examined the expression of writers, readers, and erasers of m6A and report that HNRNPA2/B1 expression is higher in tamoxifen-resistant LCC9 breast cancer cells as compared to parental, tamoxifen-sensitive MCF-7 cells. To examine how increased expression of HNRNPA2/B1 affects miRNA expression, HNRNPA2/B1 was transiently overexpressed (~5.4-fold) in MCF-7 cells for whole genome miRNA profiling (miRNA-seq). 148 and 88 miRNAs were up- and down-regulated, respectively, 48 h after transfection and 177 and 172 up- and down-regulated, respectively, 72 h after transfection. MetaCore Enrichment analysis identified progesterone receptor action and transforming growth factor β (TGFβ) signaling via miRNA in breast cancer as pathways downstream of the upregulated miRNAs and TGFβ signaling via SMADs and Notch signaling as pathways of the downregulated miRNAs. GO biological processes for mRNA targets of HNRNPA2/B1-regulated miRNAs included response to estradiol and cell-substrate adhesion. qPCR confirmed HNRNPA2B1 downregulation of miR-29a-3p, miR-29b-3p, and miR-222 and upregulation of miR-1266-5p, miR-1268a, miR-671-3p. Transient overexpression of HNRNPA2/B1 reduced MCF-7 sensitivity to 4-hydroxytamoxifen and fulvestrant, suggesting a role for HNRNPA2/B1 in endocrine-resistance.

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Multiple myeloma (MM) is an incurable disease caused by the infiltration of malignant plasma B cells into bone marrow, whose pathogenesis remains largely unknown. Long non‑coding RNAs (lncRNAs) have emerged as important factors in pathogenesis. Our previous study validated that lncRNA ST3 β‑galactoside α‑2,3‑sialyltransferase 6 antisense RNA 1 (ST3GAL6‑AS1) was upregulated markedly in MM. Therefore, the aim of the study was to investigate the molecular mechanisms of ST3GAL6‑AS1 in MM cells. ST3GAL6‑AS1 expression levels in MM cells was detected using reverse transcription‑quantitative PCR. ST3GAL6‑AS1 antisense oligonucleotides and small interfering RNAs were transfected into MM cells to downregulate expression. In vitro assays were performed to investigate the functional role of ST3GAL6‑AS1 in MM cells. RNA pull‑down, RNA immunoprecipitation and comprehensive identification of RNA‑binding proteins using mass spectrometry assays were used to determine the mechanism of ST3GAL6‑AS1‑mediated regulation of underlying targets. It was reported that knockdown of ST3GAL6‑AS1 suppressed the adhesion, migration and invasion ability of MM cells in vitro. Expression of ST3GAL6 was significantly reduced when ST3GAL6‑AS1 was knock downed in MM cells. Moreover, mechanistic investigation showed that ST3GAL6‑AS1 could suppress ST3GAL6 mRNA degradation via interacting with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1). The present results suggested that upregulated lncRNA ST3GAL6‑AS1 promotes adhesion and invasion of MM cells by binding with hnRNPA2B1 to regulate ST3GAL6 expression.