Hnrnpk protects against osteoarthritis through targeting WWC1 mRNA and inhibiting Hippo signaling pathway.

Osteoarthritis (OA) is an age-related or post-traumatic degenerative whole joint disease characterized by the rupture of articular cartilage homeostasis, the regulatory mechanisms of which remain elusive. This study identifies the essential role of heterogeneous nuclear ribonucleoprotein K (hnRNPK) in maintaining articular cartilage homeostasis. Hnrnpk expression is markedly downregulated in human and mice OA cartilage. The deletion of Hnrnpk effectively accelerates the development of post-traumatic and age-dependent OA in mice. Mechanistically, the KH1 and KH2 domain of Hnrnpk bind and degrade the mRNA of WWC1. Hnrnpk deletion increases WWC1 expression, which in turn leads to the activation of Hippo signaling and ultimately aggravates OA. In particular, intra-articular injection of LPA and adeno-associated virus serotype 5 expressing WWC1 RNA interference ameliorates cartilage degeneration induced by Hnrnpk deletion, and intra-articular injection of adeno-associated virus serotype 5 expressing Hnrnpk protects against OA. Collectively, this study reveals the critical roles of Hnrnpk in inhibiting OA development through WWC1-dependent downregulation of Hippo signaling in chondrocytes and defines a potential target for the prevention and treatment of OA.

Heterogeneous nuclear ribonucleoprotein K promotes cap-independent translation initiation of retroviral mRNAs.

Translation initiation of the human immunodeficiency virus-type 1 (HIV-1) genomic mRNA (vRNA) is cap-dependent or mediated by an internal ribosome entry site (IRES). The HIV-1 IRES requires IRES-transacting factors (ITAFs) for function. In this study, we evaluated the role of the heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a potential ITAF for the HIV-1 IRES. In HIV-1-expressing cells, the depletion of hnRNPK reduced HIV-1 vRNA translation. Furthermore, both the depletion and overexpression of hnRNPK modulated HIV-1 IRES activity. Phosphorylations and protein arginine methyltransferase 1 (PRMT1)-induced asymmetrical dimethylation (aDMA) of hnRNPK strongly impacted the protein's ability to promote the activity of the HIV-1 IRES. We also show that hnRNPK acts as an ITAF for the human T cell lymphotropic virus-type 1 (HTLV-1) IRES, present in the 5'UTR of the viral sense mRNA, but not for the IRES present in the antisense spliced transcript encoding the HTLV-1 basic leucine zipper protein (sHBZ). This study provides evidence for a novel role of the host hnRNPK as an ITAF that stimulates IRES-mediated translation initiation for the retroviruses HIV-1 and HTLV-1.

Insight into the mechanism of AML del(9q) progression: hnRNP K targets the myeloid master regulators CEBPA (C/EBPα) and SPI1 (PU.1).

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5' and 3'UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.

Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized. RESULTS: Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs and showed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs bound to the 3' untranslated region (3'UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, these mRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO). CONCLUSIONS: This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling the subcellular localization of RNA-binding proteins to support pathways involved in cancer progression.

Epigenetic Control of Cancer Cell Proliferation and Cell Cycle Progression by HNRNPK via Promoting Exon 4 Inclusion of Histone Code Reader SPIN1.

Heterogeneous nuclear ribonucleoprotein K (HNRNPK, hnRNP K), a multifunctional RNA/DNA binding protein, mainly regulates transcription, translation and RNA splicing, and then plays oncogenic roles in many cancers. However, the related mechanisms remain largely unknown. Here, we found that HNRNPK can partially epigenetically regulate cancer cell proliferation via increasing transcription and exon 4-inclusion of SPIN1, an important oncogenic histone code reader. This exon 4 skipping event of SPIN1 generates a long non-coding RNA, followed by the downregulation of SPIN1 protein. SPIN1 is one of the most significantly co-expressed genes of HNRNPK in thirteen TCGA cancers. Our further studies revealed HNRNPK knockdown significantly inhibited cell growth and cell cycle progression in oral squamous cell carcinoma (OSCC) cells and promoted cell apoptosis. Overexpression of SPIN1 was able to partially rescue the growth inhibition triggered by HNRNPK knockdown. Moreover, CCND1 (Cyclin D1), a key cell cycle regulator and oncogene, epigenetically up-regulated by SPIN1, was also positively regulated by HNRNPK. In addition, we discovered that HNRNPK promoted SPIN1 exon 4 inclusion by interacting with an intronic splicing enhancer in intron 4. Collectively, our study suggests a novel epigenetic regulatory pathway of HNRNPK in OSCC, mediated by controlling the transcription activity and alternative splicing of SPIN1 gene.

Single-cell RNA binding protein regulatory network analyses reveal oncogenic HNRNPK-MYC signalling pathway in cancer.

RNA-binding proteins (RBPs) are key players of gene expression and perturbations of RBP-RNA regulatory network have been observed in various cancer types. Here, we propose a computational method, RBPreg, to identify the RBP regulators by integration of single cell RNA-Seq (N = 233,591) and RBP binding data. Pan-cancer analyses suggest that RBP regulators exhibit cancer and cell specificity and perturbations of RBP regulatory network are involved in cancer hallmark-related functions. We prioritize an oncogenic RBP-HNRNPK, which is highly expressed in tumors and associated with poor prognosis of patients. Functional assays performed in cancer cells reveal that HNRNPK promotes cancer cell proliferation, migration, and invasion in vitro and in vivo. Mechanistic investigations further demonstrate that HNRNPK promotes tumorigenesis and progression by directly binding to MYC and perturbed the MYC targets pathway in lung cancer. Our results provide a valuable resource for characterizing RBP regulatory networks in cancer, yielding potential biomarkers for precision medicine.

hnRNP K induces HPV16 oncogene expression and promotes cervical cancerization.

PURPOSE: This study aims to explore the expression of hnRNP K in cervical carcinogenesis and to investigate the regulatory role of hnRNP K on HPV16 oncogene expression as well as biological changes in cervical cancer cells. METHODS: In total 1042 subjects, including 573 with the normal cervix and 469 with different grades of cervical lesions were enrolled in this study to explore the association between hnRNP K and HPV16 oncogene expression in cervical carcinogenesis. Additionally, the Gene Omnibus (GEO) database was used to analyze hnRNP K mRNA expression in cervical cancerization. Meanwhile, the effects of hnRNP K on cell biological functions and HPV16 oncogene expression were investigated in Siha cells. Moreover, Function analyses were conducted using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases after ChIP-seq. RESULTS: hnRNP K was highly expressed in cervical cancer and precancerous lesions, and positively correlated with HPV16 E6, but negatively correlated with HPV16 E2 and HPV16 E2/E6 ratio. hnRNP K induced cell proliferation, inhibited apoptosis and caused cell cycle arrest in the S phase, and particularly increased HPV16 E6 protein expression. CONCLUSION: This study revealed that hnRNP K overexpression has important warning significance for the malignant transformation of cervical lesions, and could be used as a potential therapeutic target for inhibiting the carcinogenicity of HPV16 and prevention of cervical carcinogenesis.

HnRNP K regulates inflammatory gene expression by mediating splicing pattern of transcriptional factors.

HnRNP K is a heterogeneous nuclear ribonucleoprotein and has been identified as an oncogene in most solid tumors via regulating gene expression or alternative splicing of genes by binding both DNA and pre-mRNA. However, how hnRNP K affects tumorigenesis and regulates the gene expression in cervical cancer (CESC) remains to be elucidated. In these data, higher expression of hnRNP K was observed in CESC and was negatively correlated with the patient survival time. We then overexpressed hnRNP K (hnRNP K-OE) and found that its overexpression promoted cell proliferation in HeLa cells (P = 0.0052). Next, global transcriptome sequencing (RNA-seq) experiments were conducted to explore gene expression and alternative splicing profiles regulated by hnRNP K. It is shown that upregulated genes by hnRNP K-OE were associated with inflammatory response and an apoptotic process of neuron cells, which involves in cancer. In addition, the alternative splicing of those genes regulated by hnRNP K-OE was associated with transcriptional regulation. Analysis of the binding features of dysregulated transcription factors (TFs) in the promoter region of the inflammatory response genes regulated by hnRNP K revealed that hnRNP K may modulate the expression level of genes related to inflammatory response by influencing the alternative splicing of TFs. Among these hnRNP K-TFs-inflammatory gene regulatory networks, quantitative reverse transcription polymerase chain reaction (RT-qPCR) experiments and gene silencing were conducted to verify the hnRNP K-IRF1-CCL5 axis. In conclusion, the hnRNP K-TFs-inflammatory gene regulatory axis provides a novel molecular mechanism for hnRNP K in promoting CESC and offers a new therapeutic target.

Regulatory mechanism of interaction between Y-box-binding protein 1 and heterogenous nuclear ribonucleoprotein K in cell division cycle 25a signal pathway and lung cancer metastasis.

The research aimed to the influences of the interaction between Y-box-binding protein 1 (YBX1) and heterogeneous nuclear ribonucleoprotein K (HNRNPK) on cell division cycle protein 25 phosphatase A (CDC25a) signal pathway and the regulatory mechanism of lung cancer (LC) metastasis. A total of 34 patients diagnosed with LC pathologically were selected as the research objects, and the expression levels of YBX1, HNRNP and CDC25a in LC non-metastasis tissues and LC metastasis tissues were detected by immunohistochemistry and Western blot (WB). High-expression stable cell lines including YBX1/A549 and HNRNPK /A549 were established in the LC A549 cell strain. The expression levels of YBX1 and HNRNP in YBX1/A549 and HNRNPK /A549 were tested by RT-PCR and WB. Besides, the number of migratory cells YBX1/A549 and HNRNPK /A549 was detected by cell migration experiment, and the influences of the interaction between YBX1 and HNRNP on the expression level of CDC25a were analyzed by co-immunoprecipitation (co-IP). The results showed that the expression level of YBX1 protein in LC metastasis tissues was higher than that in LC non-metastasis tissues (P<0.001). The expression level of HNRNPK protein in LC metastasis tissues was higher than that in LC non-metastasis tissues (P<0.01). The expression level of CDC25a protein in LC metastasis tissues was higher than that in LC non-metastasis tissues (P<0.05). Compared with the Control Group of A549 cell strain and transfected blank plasmid, mRNA levels and relative protein expression levels of YBX1 and HNRNPK in YBX1/A549 and HNRNPK/A549 cell lines were both increased (P<0.001). The number of migratory cells YBX1/A549 and HNRNPK/A549 was increased compared with A549 cells and those in Control Group (P<0.001), and cell migration rate of YBX1/A549 and HNRNPK/A549 was also enhanced compared with A549 cells and those in Control Group (P<0.001). The mRNA and protein levels of YBX1 in YBX1/A549 cell line were increased compared with those in Control Group (P<0.01), and the comparison of mRNA level and protein expression level of HNRNPK in YBX1/A549 cell line with the in Control Group showed no differences (P>0.05). The mRNA level and protein expression level of HNRNPK in HNRNPK/A549 cell line were enhanced compared with those in Control Group (P<0.01), and the comparison of YBX1 level and protein expression level in HNRNPK/A549 cell line with the in Control Group demonstrated no differences (P>0.05). YBX1 antibody adopted in co-IP was coated with magnetic beads, and numerous HNRNPK protein was abundant in YBX1/HNRNPK composite. The mRNA level and protein expression level of YBX1 and HNRNPK in YBX1/A549 and HNRNPK/A549 cell lines were enhanced compared with those in Control Group (P<0.001), and the comparison of mRNA level and protein expression level of CDC25 with those in Control Group showed no differences (P>0.05). The mRNA level and protein expression level of CDC25a in YBX1/HNRNPK/A549 were both higher than those in YBX1/A549 cell line and HNRNPK/A549 (P<0.001). With being induced by YBX1 or HNRNPK, the number of migratory cells CDC25/A549 was increased compared with that in Control Group (P<0.05). The mRNA level and protein expression level of CDC25a in YBX1/HNRNPK/A549 were both significantly higher than those in YBX1/A549 cell line and HNRNPK/A549 (P<0.001). All the above results indicated that the interaction between YBX1 and HNRNP regulated the expression of CDC25a, and further got involved in LC metastasis.

HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction.

Background: Chloride channel 3 (CLCN3) is regulated by transcription-coactivator, however, it is unclear which core transcription factor regulates CLCN3. The role of CLCN3 in lung adenocarcinoma (LUAD) is unexplored and the relationship between CLCN3 and tumor microenvironment is unknown. Methods: A 5'-biotin-labeled promoter probe of CLCN3 was used to pull down the promoter-binding transcription factor. Further study was investigated using LUAD samples, cell lines, and xenograft mice models, and the mechanism was explored. Results: CLCN3 was upregulated in human LUAD, and CLCN3 knockdown inhibited tumor proliferation and migration in vitro. Next, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was first validated as a CLCN3 promoter-binding transcription factor. Mechanistically, HNRNPK knockdown suppressed the promoter activity of CLCN3, thus regulating CLCN3 expression at the transcriptional level, and the binding motif 'GCGAGG' and binding site '-538/-248 bp' were identified. Subsequently, the RNA-seq data illustrated that the primary functions of HNRNPK were similar to those of CLCN3. The results from in vitro and in vivo trials indicated that the expression and function of CLCN3 were regulated by HNRNPK. By isolating primary cancer-associated fibroblasts (CAFs) from human LUAD, we confirmed that decreased extracellular CLCN3 secretion induced by HNRNPK knockdown inhibited CAFs activation and TGF-ß1 production, thus suppressing nuclear HNRNPK expression and LUAD progression in a feedback way. Furthermore, this phenomenon was rescued after the addition of TGF-ß1, revealing that the HNRNPK/CLCN3 axis facilitated LUAD progression through intercellular interactions. Finally, we identified that CLCN3 and HNRNPK were upregulated and correlated with poor prognosis in LUAD patients. Conclusions: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction.

Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.

Mechanistic insights into poly(C)-binding protein hnRNP K resolving i-motif DNA secondary structures.

I-motifs are four-strand noncanonical secondary structures formed by cytosine (C)-rich sequences in living cells. The structural dynamics of i-motifs play essential roles in many cellular processes, such as telomerase inhibition, DNA replication, and transcriptional regulation. In cells, the structural dynamics of the i-motif can be modulated by the interaction of poly(C)-binding proteins (PCBPs), and the interaction is closely related to human health, through modulating the transcription of oncogenes and telomere stability. Therefore, the mechanisms of how PCBPs interact with i-motif structures are fundamentally important. However, the underlying mechanisms remain elusive. I-motif structures in the promoter of the c-MYC oncogene can be unfolded by heterogeneous nuclear ribonucleoprotein K (hnRNP K), a PCBP, to activate its transcription. Here, we selected this system as an example to comprehensively study the unfolding mechanisms. We found that the promoter sequence containing 5 C-runs preferred folding into type-1245 to type-1234 i-motif structures based on their folding stability, which was further confirmed by single-molecule FRET. In addition, we first revealed that the c-MYC i-motif structure was discretely resolved by hnRNP K through two intermediate states, which were assigned to the opposite hairpin and neighboring hairpin, as further confirmed by site mutations. Furthermore, we found all three KH (hnRNP K homology) domains of hnRNP K could unfold the c-MYC i-motif structure, and KH2 and KH3 were more active than KH1. In conclusion, this study may deepen our understanding of the interactions between i-motifs and PCBPs and may be helpful for drug development.

SDCBP-AS1 destabilizes ß-catenin by regulating ubiquitination and SUMOylation of hnRNP K to suppress gastric tumorigenicity and metastasis.

BACKGROUND: Gastric cancer (GC) is among the most malignant tumors, yet the pathogenesis is not fully understood, especially the lack of detailed information about the mechanisms underlying long non-coding RNA (lncRNA)-mediated post-translational modifications. Here, the molecular mechanisms and clinical significance of the novel lncRNA syndecan-binding protein 2-antisense RNA 1 (SDCBP2-AS1) in the tumorigenesis and progression of GC were investigated. METHODS: The expression levels of SDCBP2-AS1 in 132 pairs of GC and adjacent normal tissues were compared, and the biological functions were assessed in vitro and in vivo. RNA pull-down and immunoprecipitation assays were conducted to clarify the interactions of SDCBP2-AS1 and heterogeneous nuclear ribonucleoprotein (hnRNP) K. RNA-sequencing, immunoprecipitation, immunofluorescence, and luciferase analyses were performed to investigate the functions of SDCBP2-AS1. RESULTS: SDCBP2-AS1 was significantly downregulated in GC tissues and predictive of poor patient prognosis. Silencing of SDCBP2-AS1 promoted the proliferation and migration of GC cells both in vitro and in vivo. Mechanically, SDCBP2-AS1 physically bound to hnRNP K to repress SUMOylation of hnRNP K and facilitated ubiquitination of hnRNP K and ß-catenin, thereby promoting the degradation of ß-catenin in the cytoplasm. Silencing of SDCBP2-AS1 caused SUMOylation of hnRNP K and stabilized ß-catenin activity, which altered transcription of downstream genes, resulting in tumorigenesis and metastasis of GC. Moreover, the knockdown of hnRNP K partially abrogated the effects of SDCBP2-AS1. CONCLUSIONS: SDCBP2-AS1 interacts with hnRNP K to suppress tumorigenesis and metastasis of GC and regulates post-transcriptional modifications of hnRNP K to destabilize ß-catenin. These findings suggest SDCBP2-AS1 as a potential target for the treatment of GC.

The SYNGAP1 3'UTR Variant in ALS Patients Causes Aberrant SYNGAP1 Splicing and Dendritic Spine Loss by Recruiting HNRNPK.

Fused in sarcoma (FUS) is a pathogenic RNA-binding protein in amyotrophic lateral sclerosis (ALS). We previously reported that FUS stabilizes Synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains spine maturation. To elucidate the pathologic roles of this mechanism in ALS patients, we identified the SYNGAP1 3'UTR variant rs149438267 in seven (four males and three females) out of 807 ALS patients at the FUS binding site from a multicenter cohort in Japan. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, increased isoform α1 levels, and decreased isoform γ levels, which caused dendritic spine loss. Moreover, the SYNGAP1 variant excessively recruited FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK), and antisense oligonucleotides (ASOs) blocking HNRNPK altered aberrant splicing and ameliorated dendritic spine loss. These data suggest that excessive recruitment of RNA-binding proteins, especially HNRNPK, as well as changes in SYNGAP1 isoforms, are crucial for spine formation in motor neurons.SIGNIFICANCE STATEMENT It is not yet known which RNAs cause the pathogenesis of amyotrophic lateral sclerosis (ALS). We previously reported that Fused in sarcoma (FUS), a pathogenic RNA-binding protein in ALS, stabilizes synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains dendritic spine maturation. To elucidate whether this mechanism is crucial for ALS, we identified the SYNGAP1 3'UTR variant rs149438267 at the FUS binding site. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, which caused dendritic spine loss along with excessive recruitment of FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK). Our findings that dendritic spine loss is because of excess recruitment of RNA-binding proteins provide a basis for the future exploration of ALS-related RNA-binding proteins.

CircRFX3 Up-regulates Its Host Gene RFX3 to Facilitate Tumorigenesis and Progression of Glioma.

BACKGROUND: Glioma is classified as one of the most common types of primary brain tumors. The high expression of CircRFX3 has been found in glioma. However, its functional roles in glioma and underlying mechanism remain unknown. PURPOSE: Our study aimed to explore the function and specific mechanism of circRFX3 in glioma. METHODS: RT-qPCR or western blot was applied to examine the expression of RNAs or proteins. Functional assays were carried out to evaluate the influence of circRFX3, RFX3 and PROX1 on glioma cells. In vivo experiments were done to ascertain the impact of circRFX3 on glioma growth. Moreover, mechanism assays were conducted to investigate the molecular relation among circRFX3, RFX3, HNRNPK and PROX1. RESULTS: CircRFX3 was highly expressed in glioma cells. CircRFX3 knockdown led to the suppression of glioma cell and tumor growth. CircRFX3 overexpression resulted in the opposite outcomes. Mechanism analyses suggested that circRFX3 recruited HNRNPK to enhance RFX3 mRNA stability, thereby facilitating glioma cell malignant behaviors. RFX3 was also unveiled to affect glioma cells via stimulating PROX1 transcription. CONCLUSION: CircRFX3, as a tumor promoter, could recruit HNRNPK to stabilize RFX3 mRNA in glioma cells. Additionally, RFX3 could promote PROX1 transcription to promote glioma progression.

Heterogeneous nuclear ribonucleoprotein K promotes the progression of lung cancer by inhibiting the p53-dependent signaling pathway.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein. Reportedly, hnRNPK is overexpressed in many human tumors, and such overexpression is associated with poor prognosis, implicating the role of hnRNPK as an oncogene during tumorigenesis. In this study, hnRNPK expression in lung cancer tissues was investigated. METHODS: Briefly, hnRNPK was knocked down in lung cancer cell lines, and effects of knockdown on the cell proliferation, migration, and cell cycle were assessed using a cell counting kit-8 (CCK-8) assay, colony formation assay, transwell assay and flow cytometry. The effects of hnRNPK knockdown on the p53-dependent signaling pathway were examined using western blotting. Finally, the effect of hnRNPK knockdown on tumor growth was verified in vivo using a lung cancer xenograft mouse model. RESULTS: hnRNPK knockdown inhibited the cell proliferation, migration and cell cycle. In addition to phenotypic changes, hnRNPK knockdown upregulated expressions of pCHK1, pCHK2, and p53，p21，cyclin D1, thereby mediating the DNA damage response (DDR). The regulatory function of hnRNPK during p53/p21/cyclin D1 signaling in hnRNPK-knockdown A549 cells was confirmed by suppressed the protein expression of associated signaling pathways, which inhibited DDR. CONCLUSION: hnRNPK plays a crucial role in the progression of lung cancer, ultimately affecting survival rate. Inhibition of progression of lung cancer cells induced by hnRNPK-knockdown is dependent on activation of p53 by the p53/p21/cyclin D1 pathway.

Co-expression of nuclear heterogeneous nuclear ribonucleic protein K and estrogen receptor α in endometrial cancer.

Heterogeneous nuclear ribonucleic protein K (hnRNPK) regulates the expression of various genes, but has contradictory roles as a tumor promoter and a tumor suppressor. We recently reported that the expression of hnRNPK is negatively associated with malignant behavior of breast cancer where it was induced by estrogen, and bound to estrogen receptor α (ERα) in the nucleus of breast cancer cells. However, the significance of hnRNPK in endometrial cancer, also an estrogen-dependent cancer, remains unclear. In this study, we first examined the localization of hnRNPK and ERα in normal endometrium and endometrial cancer. hnRNPK and ERα immunoreactivity was detected in the nuclei of endometrial glandular and carcinoma cells. In normal endometria, hnRNPK labeling index/immuno-intensity was significantly higher in the proliferative phase than in the secretory phase. In endometrial cancer tissues, hnRNPK labeling index/immuno-intensity was significantly higher in the adjacent non-malignant glandular cells compared to that in carcinoma cells. Immunohistochemistry results for ERα were identical to that of hnRNPK both in normal endometrium and endometrial cancer. In normal and cancerous tissues, the median value of the hnRNPK labeling index was significantly higher in the ERα-high group. Intratumoral estrogen, but not androgen, measured using liquid chromatography-tandem mass spectrometry, was significantly positively correlated with the hnRNPK labeling index in endometrial cancer tissues. Database analysis revealed that the hnRNPK high expression group had a significantly better prognosis for both overall and disease-free survival. These results suggest that hnRNPK interacts with ERα to regulate endometrial changes during the menstrual cycle and suppress the malignant behavior of endometrial cancer.

The cancer-testis lncRNA lnc-CTHCC promotes hepatocellular carcinogenesis by binding hnRNP K and activating YAP1 transcription.

Cancer-testis (CT) genes participate in the initiation and progression of cancer, but the role of CT-associated long non-coding RNAs (CT-lncRNAs) in hepatocellular carcinoma (HCC) is still elusive. Here, we discovered a conserved CT-lncRNA, named lnc-CTHCC, which was highly expressed in the testes and HCC. A lnc-CTHCC-knockout (KO) mouse model further confirmed that the global loss of lnc-CTHCC inhibited the occurrence and development of HCC. In vitro and in vivo assays also showed that lnc-CTHCC promoted HCC growth and metastasis. Mechanistically, lnc-CTHCC bound to heterogeneous nuclear ribonucleoprotein K (hnRNP K), which was recruited to the YAP1 promoter for its activation. Additionally, the N6-methyladenosine (m6A) modification was mediated by N6-adenosine-methyltransferase 70-kDa subunit (METTL3) and recognized by insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)/IGF2BP3, which maintained lnc-CTHCC stability and increased its expression in HCC. Together, our results show that lnc-CTHCC directly binds to hnRNP K and promotes hepatocellular carcinogenesis and progression by activating YAP1 transcription, suggesting that lnc-CTHCC is a potential biomarker and therapeutic target of HCC.

Long Noncoding RNA LINC00941 Promotes Cell Proliferation and Invasion by Interacting with hnRNPK in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a prevalent carcinoma of the head, neck and mouth. Recently studies involving the role of long noncoding RNAs (lncRNAs) that play key regulatory roles in altering gene expression has been reported in the context of promoting tumorigenesis. However, the functions of lncRNAs in the context of oral squamous cell carcinoma have not been extensively described. In this study, we report a never identified before lncRNA, LINC00941, which was highly expressed in OSCC tissues and cells. Expression of LINC00941 promoted cell proliferation, migration, invasion, and metastasis of OSCC cells In Vitro by inducing epithelial-mesenchymal transition (EMT) and activating the Wnt/ß-catenin signaling cascade. In silico analyses revealed heterogeneous nuclear ribonucleoprotein K (hnRNPK) to be a strong positive regulator of LINC00941 activity. Experimental verification of this association revealed a direct interaction of LINC00941 and hnRNPK to induce cell growth and invasion by activating EMT in OSCC cells. Therefore, our study reports that LINC00941 promotes progression of OSCC by its interaction with hnRNPK, and it may present a promising strategy for diagnosis and treatment of OSCC.

hnRNPK/Beclin1 signaling regulates autophagy to promote imatinib resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia cells.

This study sought to clarify the role of heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a regulator of imatinib resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). Expression of hnRNPK was assessed in Ph+ ALL leukemia cells in vitro and in vivo, and imatinib susceptibility was assessed via CCK-8 assay. In cells in which hnRNPK levels had or had not been modulated, LC3Ⅰ/Ⅱ and mTOR/p-ERK/Beclin1 levels were assessed via Western blotting, while electron microscopy was used to evaluate autophagic vacuole formation. Interactions between hnRNPK and Beclin1 were assessed through an RNA binding protein immunoprecipitation assay. Imatinib-resistant Ph+ ALL cell lines and patient bone marrow samples exhibited significant hnRNPK overexpression. Knockdown of hnRNPK increased the imatinib sensitivity of these tumor cells and decreased in vivo tumor burden in a xenograft model system as evidenced by a reduction in tumor volume. Levels of LC3Ⅰ/Ⅱ and Beclin1, but not p-ERK and mTOR, were consistent with the regulatory activity of hnRNPK. Electron microscopy revealed that imatinib-resistant cells harbored significantly more autophagic vacuoles relative to wild-type cells, while hnRNPK knockdown reduced the number of these vacuoles. In an RNA-binding protein immunoprecipitation assay, anti-hnRNPK was able to precipitate the Beclin1 mRNA. These results suggest that the hnRNPK/Beclin1 signaling pathway may play a role in shaping imatinib resistance in Ph+ ALL cells.