Heterogeneous Nuclear Ribonucleoprotein K Research Articles

Surface PD-1 expression in T cells is suppressed by HNRNPK through an exonic splicing silencer on exon 3.

Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear. The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells. HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well. Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.

Function and mechanism of GBP1 in the development and progression of cervical cancer

Guanylate binding protein 1 (GBP1) is the most concerned member of the GBP family, which has a series of effects such as anti-infection and anti-angiogenesis. Its role in malignant tumors including cervical cancer is still controversial. We aim to explore the effects of GBP1 on cervical cancer through bioinformatics and related experiments. In this study, we first found that GBP1 was generally expressed in cervical cancer in various online databases and was closely related to immune invasion. Secondly, we used multicolor immunofluorescence technology to verify the expression of GBP1 in cervical cancer tissues and its relationship with immune invasion, and explored its relationship with the prognosis of patients with cervical cancer. Knockdown and overexpression assays of GBP1 in vitro were used to prove GBP1 as a potential oncogene of cervical cancer, and its carcinogenicity was verified by in vivo experiment. In order to explore the potential mechanism of GBP1 in promoting cancer, RNA-seq was performed on GBP1 overexpression and knockdown expression cell lines, and GBP1 knockdown and overexpression were found to be associated with many RNA alternative splicing events, suggesting that GBP1 maybe a RNA binding protein (RBP) which affect the biological characteristics of cervical cancer cells through the alternative splicing pathway. However, the later RNA binding protein immunoprecipitation (RIP) assay proved that GBP1 was not a direct alternative splicing factor, while the co-immunoprecipitation (CoIP)-mass spectroscopy (MS) assay combined with protein protein interaction (PPI) analysis proved that 8 alternative splicing factors including Heterogeneous Nuclear Ribonucleoprotein K (HNRNPK) were interacting proteins of GBP1. Combined with the existing reports and the results of RNA-seq alternative splicing analysis, it is speculated that GBP1 may regulate the alternative splicing of CD44 protein by binding to interacting protein-HNRNPK, and thus play a role in promoting cancer in cervical cancer.

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.

LITTIP/Lgr6/HnRNPK complex regulates cementogenesis via Wnt signaling

Orthodontically induced tooth root resorption (OIRR) is a serious complication during orthodontic treatment. Stimulating cementum repair is the fundamental approach for the treatment of OIRR. Parathyroid hormone (PTH) might be a potential therapeutic agent for OIRR, but its effects still lack direct evidence, and the underlying mechanisms remain unclear. This study aims to explore the potential involvement of long noncoding RNAs (lncRNAs) in mediating the anabolic effects of intermittent PTH and contributing to cementum repair, as identifying lncRNA-disease associations can provide valuable insights for disease diagnosis and treatment. Here, we showed that intermittent PTH regulates cell proliferation and mineralization in immortalized murine cementoblast OCCM-30 via the regulation of the Wnt pathway. In vivo, daily administration of PTH is sufficient to accelerate root regeneration by locally inhibiting Wnt/β-catenin signaling. Through RNA microarray analysis, lncRNA LITTIP (LGR6 intergenic transcript under intermittent PTH) is identified as a key regulator of cementogenesis under intermittent PTH. Chromatin isolation by RNA purification (ChIRP) and RNA immunoprecipitation (RIP) assays revealed that LITTIP binds to mRNA of leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and heterogeneous nuclear ribonucleoprotein K (HnRNPK) protein. Further co-transfection experiments confirmed that LITTIP plays a structural role in the formation of the LITTIP/Lgr6/HnRNPK complex. Moreover, LITTIP is able to promote the expression of LGR6 via the RNA-binding protein HnRNPK. Collectively, our results indicate that the intermittent PTH administration accelerates root regeneration via inhibiting Wnt pathway. The lncRNA LITTIP is identified to negatively regulate cementogenesis, which activates Wnt/β-catenin signaling via high expression of LGR6 promoted by HnRNPK.

Autophagy Inhibition as a Potential Therapeutic Strategy for Breast Cancer with Mitochondrial Translation Defect Caused by CBFB-Deficiency

ABSTRACT Autophagy plays a crucial role in tumor initiation and progression. However, targeting autophagy in cancer has proven challenging due to genetic or epigenetic factors that may affect the efficacy of autophagy inhibition. Therefore, identifying biomarkers is crucial for selecting patients who are likely to benefit from this treatment modality. We show that dysregulation of mitochondrial translation caused by CBFB (core-binding factor subunit beta) deficiency can sensitize the tumors to autophagy inhibition. CBFB and its binding partner HNRNPK (heterogeneous nuclear ribonucleoprotein K) interact with mRNAs encoded by the mitochondrial genome (mt-mRNAs) and maintain their translation. Specifically, CBFB enhances the binding of TUFM (Tu translation elongation factor, mitochondrial), an elongation factor for mitochondrial translation, to mt-mRNAs. CBFB deficiency, which often occurs in estrogen receptor-positive breast tumors, results in elevated autophagy and mitophagy that promote cancer cell survival. Consequently, these cells are hypersensitive to autophagy inhibition, creating a targetable vulnerability. Studies using in vivo models have shown that inhibiting autophagy selectively eliminates breast tumor cells with mitochondrial translation defects resulting from CBFB deficiency. Our results suggest that autophagy inhibition may be an effective treatment option for breast tumors carrying CBFB alterations. Abbreviations AMPK, AMP-activated protein kinase; BioID, biotinylation identification; CBFB, core-binding factor subunit beta; HCQ, hydroxychloroquine; HNRNPK, heterogeneous nuclear ribonucleoprotein K; PDX, patient-derived xenograft; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; TUFM, Tu translation elongation factor, mitochondrial; ETC, electron transport chain

Targeting the JMJD6/TGF-β axis in prostate cancer by immunotherapy: A potential treatment based on RNA splicing

Prostate cancer showcases remarkable clinical heterogeneity. Long-term treatment targeting androgen/androgen receptor-axis may drive patients to develop castration-resistant and more aggressive neuroendocrine phenotypes. The tumor immune microenvironment (TIME) presents activation of multiple progression-related programs. It is generally considered that TGFβ secreted by prostate cancer cell leads to inhibitory activity of effector T lymphocytes and contributes to inhibitory TIME by activation of TGFβ signaling pathway. However, the mechanisms regulating mRNA splicing and expression of TGFβ are poorly understood. Here we hypothesize that Jumonji domain-containing 6 (JMJD6), previously as a histone demethylase, underlies the function of aiding alternative splicing of TGFβ mRNA, which promote TGFβ production and secretion, and activate TGFβ signal pathway from upstream links, eventually forming immunosuppressive TIME and inducing therapeutic resistance. We identify that JMJD6 interacts with multiple splicing cofactors including serine/arginine enriched splicing factor 8 (SRSF8), heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) and heterogeneous nuclear ribonucleoprotein K (HNRNPK) based on mass spectrometry analysis. Moreover, the obviously positive relationships occur among JMJD6, SRSF8/HNRNPA2B1/HNRNPK and TGFβ based on database analysis. In view of limited clinical efficacies of using neutralizing antibody of TGFβ, TGFβ receptor inhibitor and TGF-β/PD-L1 coupled antibodies targeting TGFβ signal pathway and delaying tumor progression, our data provide a novel therapeutic insight on blocking TGFβ production from transcription/mRNA splicing level through targeting JMJD6. Our results suggest that targeting the JMJD6/TGF-β Axis can combine with immunotherapies targeting TGFβ signal pathway in TIME to achieve great benefits for treating prostate cancer progression. Combination with JMJD6 antagonist and immunotherapy can sensitize the anti-tumor effect produced by targeting TGFβ signal pathway, block the production of TGFβ from the origin and solve the bottleneck of immunotherapy for prostate cancer.

DNA- and RNA-Binding Proteins Linked Transcriptional Control and Alternative Splicing Together in a Two-Layer Regulatory Network System of Chronic Myeloid Leukemia.

DNA- and RNA-binding proteins (DRBPs) typically possess multiple functions to bind both DNA and RNA and regulate gene expression from more than one level. They are controllers for post-transcriptional processes, such as splicing, polyadenylation, transportation, translation, and degradation of RNA transcripts in eukaryotic organisms, as well as regulators on the transcriptional level. Although DRBPs are reported to play critical roles in various developmental processes and diseases, it is still unclear how they work with DNAs and RNAs simultaneously and regulate genes at the transcriptional and post-transcriptional levels. To investigate the functional mechanism of DRBPs, we collected data from a variety of databases and literature and identified 118 DRBPs, which function as both transcription factors (TFs) and splicing factors (SFs), thus called DRBP-SF. Extensive investigations were conducted on four DRBP-SFs that were highly expressed in chronic myeloid leukemia (CML), heterogeneous nuclear ribonucleoprotein K (HNRNPK), heterogeneous nuclear ribonucleoprotein L (HNRNPL), non-POU domain–containing octamer–binding protein (NONO), and TAR DNA-binding protein 43 (TARDBP). By integrating and analyzing ChIP-seq, CLIP-seq, RNA-seq, and shRNA-seq data in K562 using binding and expression target analysis and Statistical Utility for RBP Functions, we discovered a two-layer regulatory network system centered on these four DRBP-SFs and proposed three possible regulatory models where DRBP-SFs can connect transcriptional and alternative splicing regulatory networks cooperatively in CML. The exploration of the identified DRBP-SFs provides new ideas for studying DRBP and regulatory networks, holding promise for further mechanistic discoveries of the two-layer gene regulatory system that may play critical roles in the occurrence and development of CML.

HNRNPK alleviates RNA toxicity by counteracting DNA damage in C9orf72 ALS

A ‘GGGGCC’ repeat expansion in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The exact mechanism resulting in these neurodegenerative diseases remains elusive, but C9 repeat RNA toxicity has been implicated as a gain-of-function mechanism. Our aim was to use a zebrafish model for C9orf72 RNA toxicity to identify modifiers of the ALS-linked phenotype. We discovered that the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (HNRNPK) reverses the toxicity of both sense and antisense repeat RNA, which is dependent on its subcellular localization and RNA recognition, and not on C9orf72 repeat RNA binding. We observed HNRNPK cytoplasmic mislocalization in C9orf72 ALS patient fibroblasts, induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem motor cortex and spinal cord, in line with a disrupted HNRNPK function in C9orf72 ALS. In C9orf72 ALS/FTD patient tissue, we discovered an increased nuclear translocation, but reduced expression of ribonucleotide reductase regulatory subunit M2 (RRM2), a downstream target of HNRNPK involved in the DNA damage response. Last but not least, we showed that increasing the expression of HNRNPK or RRM2 was sufficient to mitigate DNA damage in our C9orf72 RNA toxicity zebrafish model. Overall, our study strengthens the relevance of RNA toxicity as a pathogenic mechanism in C9orf72 ALS and demonstrates its link with an aberrant DNA damage response, opening novel therapeutic avenues for C9orf72 ALS/FTD.

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-β1 production, thus suppressing nuclear HNRNPK expression and LUAD progression in a feedback way. Furthermore, this phenomenon was rescued after the addition of TGF-β1, 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.

Mass Spectrometry and Computer Simulation Predict the Interactions of AGPS and HNRNPK in Glioma.

Ether lipids are overexpressed in malignant tumor and play an important role in tumor process. Glioma is the most common malignant central nervous system tumor, and the content of ether lipids is higher than that of normal tissues. Alkylglycerone phosphate synthase (AGPS) is a key enzyme in the synthesis of ether esters and plays a vital role in maintaining the morphology and pathogenic properties of tumor cells. The cell proliferation and the content of tumor-related lipid such as monoalkylglycerol ether (MAGe), lysophosphatidic acid ether (LPAe), lysophosphatidylcholine ether (LPCe), lysophosphatidylethanolamine ether (LPEe), phosphatidyl inositol (PI), phosphatidylcholine (PC), and phosphatidylserine (PS) were suppressed after AGPS silencing in U251, H4, and TJ905 cells; however, heterogeneous nuclear ribonucleoprotein K (HNRNPK) could reverse the above phenomenon such as cellar proliferation and ether lipid secretion. We found that HNRNPK was the target protein of AGPS by coimmunoprecipitation and mass spectrometry assay and verified by western blot assay in U251 cells. It confirmed that AGPS and HNRNPK are coexpressed in the cellular nucleus by a confocal laser microscope. The main protein-protein interaction mechanism between AGPS and HNRNPK is hydrogen bond, conjugation bond, hydrophobic bond, and electrostatic force by computer simulation prediction.

Inhibition of circRNA circVPS33B Reduces Warburg Effect and Tumor Growth Through Regulating the miR-873-5p/HNRNPK Axis in Infiltrative Gastric Cancer.

BackgroundCircular RNA VPS33B (circVPS33B) has been revealed to be upregulated in gastric cancer (GC) tissues. However, the role of circVPS33B in infiltrative GC is indistinct.MethodsExpression of circVPS33B was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, migration, and invasion of infiltrative GC cells (XGC-1) were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), plate clone, wound-healing, or transwell assays. Protein levels were detected by Western blotting. Measurements of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were executed using an XF96 extracellular flux analyzer. Glucose uptake and lactate production were analyzed by glycolysis assay. The regulatory mechanism of circVPS33B had been explored by bioinformatics analysis, dual-luciferase reporter assay, and/or RNA pull-down assay. In vivo tumorigenesis assay was executed to verify the oncogenicity of circVPS33B.ResultsCircVPS33B was upregulated in infiltrative GC tissues and cells. CircVPS33B silencing decreased tumor growth in vivo and inhibited proliferation, migration, invasion, EMT, and Warburg effect of infiltrative GC cells in vitro. Mechanically, circVPS33B regulated heterogeneous nuclear ribonucleoprotein K (HNRNPK) expression via sponging miR-873-5p. Furthermore, miR-873-5p inhibitor offset circVPS33B knockdown-mediated effects on malignant behaviors and Warburg effect of infiltrative GC cells. HNRNPK overexpression reversed the inhibitory impact of miR-873-5p mimic on malignant behaviors and Warburg effect of infiltrative GC cells.ConclusionCircVPS33B accelerated Warburg effect and tumor growth through regulating the miR-873-5p/HNRNPK axis in infiltrative GC, manifesting that circVPS33B might be a potential target for infiltrative GC treatment.

Comparative proteomics on resting and activated human basophilic leukemic KU812 upon stimulation with PMA plus A23187

Basophil activation has been implicated in allergy reaction and immune modulation. Signaling transduction, mediator release and morphology change of basophils have been largely deciphered. This study examined the feasibility of using proteomic strategy to investigate the whole cell proteomic changes in KU812 basophils following activation with phorbol myristate acetate (PMA) plus ionophore (A23187). The two-dimensional electrophoresis (2D) coupled with matrix-assisted laser desorption/ionization-quadrupole/time-of-flight (MALDI-Q-TOF) was selected for studying comparative proteome differences; and cytokine mRNA expressions were monitored. The differentially displayed proteins were further validated by 1D- and 2D-Western blotting for protein expressions. IL-4, IL-5, and IL-13 mRNAs in KU812 were significantly induced; a phenomenon similar to the allergy reaction. The protein expressions of heterogeneous nuclear ribonucleoprotein K (HNRPK) and α-enolase (ENO1) were shown a good correlation according to the results from the 2D, and 2D-Western blots. Since the majority of the identified proteins were associated with immunological, inflammatory disorders and tumor genesis, this study in defining the proteomic changes in basophilic cells that occur in response to treatment with PMA plus A23187 can serve as biomarkers for characterization of in vitro immuno-stimulation.

FGD5-AS1 facilitates glioblastoma progression by activation of Wnt/β-catenin signaling via regulating miR-129-5p/HNRNPK axis

AimsAccumulating evidence elucidates the biological significance of long non-coding RNA (lncRNAs) in tumorigenesis and development. FGD5 antisense RNA 1 (FGD5-AS1) was previously revealed as an oncogene in several types of malignancies. However, the roles of FGD5-AS1 in glioblastoma (GBM) and its potential molecular mechanisms remain unclear. Materials and methodsThe expression of FGD5-AS1, miR-129-5p, and heterogeneous nuclear ribonucleoprotein K (HNRNPK) mRNA were measured by qRT-PCR. Cell proliferation, invasion and apoptosis were determined by MTT, colony formation, transwell and flow cytometry assays. The protein levels of Ki-67, HNRNPK and Wnt signaling-associated genes were examined by western blot assay. The possible action mechanism of FGD5-AS1 was detected by bioinformatic tools, luciferase reporter, RIP and TOP/FOP Flash reporter assays. A nude mouse xenograft model was built to analyze the function of FGD5-AS1 in vivo. Key findingsFGD5-AS1 expression was increased in GBM tumor tissues and cells. Knockdown of FGD5-AS1 inhibited cell proliferation and invasion in vitro, and slowed tumor growth in vivo. Mechanistically, FGD5-AS1 served as a sponge of miR-129-5p to relieve its suppression on HNRNPK. Moreover, down-regulation of HNRNPK repressed cell proliferation and invasion, while enhanced apoptosis. Additionally, si-FGD5-AS1-mediated suppression of cell proliferation and invasion was obviously reversed by the decrease of miR-129-5p or restoration of HNRNPK. Furthermore, FGD5-AS1 promoted cell growth and invasion by stimulating Wnt/β-catenin signaling via regulation of miR-129-5p/HNRNPK. SignificanceFGD5-AS1 promoted GBM progression at least partly by regulating miR-129-5p/HNRNPK to activate Wnt/β-catenin signaling, suggesting the potential of FGD5-AS1 as a candidate target to improve GBM therapy.

Circular RNA (circ-0075804) promotes the proliferation of retinoblastoma via combining heterogeneous nuclear ribonucleoprotein K (HNRNPK) to improve the stability of E2F transcription factor 3 E2F3.

It is growingly recognized that messenger RNAs (mRNAs) are important regulators of various cancers. However, there are few reporters about the function of E2F3 in retinoblastoma (RB), which needs more exploration. In addition, the circRNA circ-0075804 was derived from the E2F3 host gene. The purpose of the study is to figure out the role and molecular regulation mechanism of E2F3 and circ-0075804 in RB. The role of E2F3 in RB was determined through E2F3 silencing and loss of expression was evaluated by real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, CCK-8, colony formation, and 5-ethynyl-2'-deoxyuridine assays. The interactions between E2F3 and circ-0075804 were validated through loss and gain function of circ-0075804. Besides, the role of circ-0075804 in RB was determined by several functional assays. And the binding ability between heterogeneous nuclear ribonucleoprotein K and circ-0075804 was verified by RNA pull-down, Western blot, and RT-qPCR assays. The expression of E2F3 was upregulated in RB cell lines. Furthermore, knockdown of E2F3 inhibited cell proliferation and induced cell apoptosis in RB. And circ-0075804 positively regulated the expression of E2F3. Moreover, circ-0075804 facilitated cell proliferation and suppressed cell apoptosis. Besides, HNRNPK could bind with circ-0075804 in RB. Finally, knockdown of E2F3 partly rescued the promoting role of circ-0075804 overexpression in RB. Overall, circ-0075804 promotes the proliferation of RB via combining HNRNPK to improve the stability of E2F3, which brings new light for treating RB.

The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA-profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2), and LC3-dependent recruitment of Factor-associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.

Downregulation of HNRNPK in human cancer cells inhibits lung metastasis.

BackgroundLung cancer frequently occurs in the clinic, leading to poor prognosis and high mortality. Markers for early diagnosis of lung cancer are scarce, and further potential therapeutic targets are also urgently needed.MethodWe established a new mouse model in which the specific gene HNRNPK (heterogeneous nuclear ribonucleoprotein K) was downregulated after administration of doxycycline. The lung metastatic nodules were investigated using bioluminescence imaging, micro‐CT, and autopsy quantification.ResultsCompared with the short hairpin negative control group, less lung metastatic nodules were formed in the short hairpin RNA group.ConclusionDownregulation of HNRNPK in cancer cells can inhibit lung metastasis.

Estrogen receptor α (ERα) indirectly induces transcription of human renal organic anion transporter 1 (OAT1).

Organic anion transporter 1 (OAT1) is a polyspecific transport protein located in the basolateral membrane of renal proximal tubule cells. OAT1 plays a pivotal role in drug clearance. Adverse drug reactions (ADR) are observed more frequently in women than in men, especially ADR are higher in women for drugs which are known interactors of OAT1. Sex‐dependent expression of Oat1 has been observed in rodents with a tendency to male‐dominant expression. This study aims at elucidating the transcriptional regulation of human OAT1 and tests the effect of estrogen receptor α (ERα). Promoter activation of OAT1 was assessed by luciferase assays carried out by Opossum kidney (OK) cells, transiently transfected with promoter constructs of human OAT1 and expression vectors for ERα and exposed to 100 nmol/L 17β‐estradiol. Furthermore, a transcription factor array and proteomic analysis was performed to identify estrogen‐induced transcription factors. Human OAT1 was significantly activated by ligand activated ERα. However, activation occurred without a direct interaction of ERα with the OAT1 promoter. Our data rather show an activation of the transcription factors CCAAT‐box‐binding transcription factor (CBF) and heterogeneous nuclear ribonucleoprotein K (HNRNPK) by ERα, which in turn bind and initiate OAT1 promoter activity. Herewith, we provide novel evidence of estrogen‐dependent, transcriptional regulation of polyspecific drug transporters including the estrogen‐induced transcription factors CBF and HNRNPK.

MiR-1249-3p accelerates the malignancy phenotype of hepatocellular carcinoma by directly targeting HNRNPK.

BackgroundmicroRNAs (miRNAs) have been implicated to play crucial roles in carcinogenesis. miR‐1249‐3p was reported to be abnormally expressed in multiple human cancers. However, its biological role and the associated underlying mechanisms in hepatocellular carcinoma (HCC) remain largely unknown.Methods miR‐1249‐3p expression level in HCC cell lines and normal cell line was measured by quantitative real‐time PCR. Role of miR‐1249‐3p on HCC cell proliferation, colony formation, and invasion was examined by cell counting kit‐8 assay, colony formation assay, and transwell invasion assay, respectively. Luciferase activity reporter assay and western blot were performed to validate whether heterogeneous nuclear ribonucleoprotein K (HNRNPK) was a direct target of miR‐1249‐3p. Effect of miR‐1249‐3p on overall survival of HCC patients was analyzed at KM Plotter website.ResultsWe found miR‐1249‐3p expression level was increased, while HNRNPK expression level was decreased in HCC cell lines compared with normal cell line. Knockdown miR‐1249‐3p expression inhibits HCC cell proliferation, colony formation, and cell invasion through regulating HNRNPK in vitro. We also showed high miR‐1249‐3p expression was a predictor for poor overall survival of HCC patients.ConclusionsThese findings about miR‐1249‐3p/HNRNPK pair provide a novel therapeutic method for HCC patients.

SERPINA3 is a key modulator of HNRNP-K transcriptional activity against oxidative stress in HCC

Most studies about serpin peptidase inhibitor, clade A member 3 (SERPINA3) has been limited to its inhibitory functions and mechanisms. Herein, we report a novel role of SERPINA3 in transcriptional regulation of HCC progression-related genes. Among 19 selected genes through HCC cell isolation system based on telomere length, microarray analyses, and cell-based studies, SERPINA3 was the strongest determinant of increases in telomere length, HCC cell proliferation, survival, migration, and invasion. We also found that SERPINA3 strongly interacted with heterogeneous nuclear ribonucleoprotein K (HNRNP-K) under H2O2 exposure, and the oxidation-elicited SERPINA3-HNRNP-K complex enhanced the promoter activities and transcript levels of a telomere-relating gene (POT1) and HCC-promoting genes (UHRF1 and HIST2H2BE). Intriguingly, the inhibition of SERPINA3 oxidation rendered the transcriptional activity of the SERPINA3-HNRNP-K complex suppressed. Moreover, the co-immunoprecipitated HNRNP-K with SERPINA3 quantitatively correlated with not only the level of SERPINA3 oxidation but also the level of POT1, UHRF1, and HIST2H2BE transcripts and telomere length in HCC tissues. Therefore, the upregulated transcriptional activity of HNRNP-K mediated by SERPINA3 promotes HCC cell survival and proliferation and could be an indicator of poor prognosis for HCC patients.

Therapeutic potential of targeting SNORD126 in hepatocellular carcinoma

Introduction: SNORD126 is a C/D box small nucleolar RNAs (snoRNAs) which has been identified as a hepatocellular carcinoma (HCC) oncogene in our previous study. Targeting non-coding RNAs are promising therapeutic strategy in cancer, while a few studies focus on targeting snoRNAs. We investigated the therapeutic potential of targeting SNORD126 in HCC. Method: We investigated the localization of SNORD126 in cells. Small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) were used to knock down SNORD126 expression. To examine ASOs function, HCC cells were transfected with ASOs and analyzed in proliferation, colony formation, invasion assays and in vivo xenografts assays. To identify SNORD126-binding proteins, we performed RNA pull-down followed by mass spectrometry in HepG2 cells. To identify functional regions in SNORD126, we constructed mutants in SNORD126 conserved box and investigated its effects on binding protein, 2-O-methylation, activating Akt and proliferation in HCC cells. Result: We found that siRNA failed to down-regulate SNORD126 expression due to its nucleolar localization. ASOs targeting SNORD126 successfully knocked down SNORD126 expression and suppressed the proliferation and invasion of HCC cells. Intratumoral injections of SNORD126 ASOs reduced the growth of established HCC xenografts. SNORD126 ASOs decreased the levels of phosphorylated Akt and p70S6K both in vitro and in vivo. We identified several SNORD126-binding proteins, including DHX9, DDX1, ILF3, PRPF8, and HNRNPK. Two nucleotide bases mutation in C’ box (C’2) decreased SNORD126 binding to pre-mRNA processing factor 8 (PRPF8) and heterogeneous nuclear ribonucleoprotein K (HNRNPK) and impaired its ribose 2’-O-methylation ability. The phosphorylation of Akt and p70S6K was decreased in the Huh7 cells with SNORD126 C’2 overexpression. This mutant also abolished the proliferation induced by wild type SNORD126 in HCC both in vitro and in vivo. Conclusion: Our results indicate that targeting SNORD126 is a promising candidate for the therapies against HCC.