RBM3 Expression Research Articles

Cold-shock proteins accumulate in centrosomes and their expression and primary cilium morphology are regulated by hypothermia and shear stress.

Primary cilia act as cellular sensors for multiple extracellular stimuli and regulate many intracellular signaling pathways in response. Here we investigate whether the cold-shock proteins (CSPs), CIRP and RBM3, are present in the primary cilia and the physiological consequences of such a relationship. R28, an immortalized retinal precursor cell line, was stained with antibodies against CIRP, RBM3, and ciliary markers. Both CSPs were found in intimate contact with the basal body of the cilium during all stages of the cell cycle, including migrating with the centrosome during mitosis. In addition, the morphological and physiological manifestations of exposing the cells to hypothermia and shear stress were investigated. Exposure to moderately cold (32°C) temperatures, the hypothermia mimetic small molecule zr17-2, or to shear stress resulted in a significant reduction in the number and length of primary cilia. In addition, shear stress induced expression of CIRP and RBM3 in a complex pattern depending on the specific protein, flow intensity, and type of flow (laminar versus oscillatory). Flow-mediated CSP overexpression was detected by qRT-PCR and confirmed by Western blot, at least for CIRP. Furthermore, analysis of public RNA Seq databases on flow experiments confirmed an increase of CIRP and RBM3 expression following exposure to shear stress in renal cell lines. In conclusion, we found that CSPs are integral components of the centrosome and that they participate in cold and shear stress sensing.

RBM3 Promotes Anti-inflammatory Responses in Microglia and Serves as a Neuroprotective Target of Ischemic Stroke.

Ischemic stroke is a major cause of death and disability in adults. Hypothermic treatment is successful in treating neonatal cerebral ischemia, but its application is restricted in adult patients due to complex management strategies and severe adverse effects. Two homologous RNA-binding proteins, RBM3 and CIRP, are the only known cold-inducible proteins in vertebrates, and their expression levels are robustly elevated by mild to moderate hypothermia. In previous studies, we and others have demonstrated that both RBM3 and CIRP mediate the neuroprotective and neurogenic effects of hypothermia in cell and animal models. However, CIRP can also be detrimental to neurons by triggering neuroinflammatory responses, complicating its post-stroke functions. In this study, we compared the properties of the two cold-inducible RNA-binding proteins after ischemic stroke. Our results indicated that RBM3 expression was stimulated in the ischemic brain of stroke patients, while CIRP expression was not. In an experimental model, RBM3 can ameliorate ischemic-like insult by promoting neuronal survival and eliciting anti-inflammatory responses in activated microglia, while the impact of CIRP was intriguing. Collectively, our data supported the notion that RBM3 may be a more promising therapeutic target than CIRP for treating ischemic stroke. We further demonstrated that zr17-2, a small molecule initially identified to target CIRP, can specifically target RBM3 but not CIRP in microglia. zr17-2 demonstrated anti-inflammatory and neuroprotective effects after ischemic stroke both in vitro and in vivo, suggesting its potential therapeutic value.

RNA-Binding Protein RBM5 Plays an Essential Role in Acute Myeloid Leukemia By Activating the Oncogenic Protein HOXA9

Acute myeloid leukemia (AML) represents a type of malignant hematological disease that indicates poor outcomes in children and adults. HOXA9 overexpression is observed in 50-70% of human AML and positively correlates with poor patient outcomes. Leukemia subtypes with hallmark overexpression of HOXA9 include but are not limited to those carrying KMT2A gene rearrangements (KMT2A-r), NPM1c mutations, NUP98-fusions and EZH2 mutation. Accumulating genetic evidence also indicates that HOXA9 dysregulation is sufficient and necessary for leukemic transformation, and loss of function of HOXA9 and downstream pathways consequently impaired leukemia maintenance. Although HOXA9 is not an ideal direct candidate for therapeutic interventions, uncovering the novel regulators of HOXA9 might elucidate the HOXA9 regulation mechanism that could be exploited to identify novel therapeutic targets in HOXA9-driven leukemias. In this study, we performed genome-wide CRISPR/Cas9 screens in the HOXA9-endogenous reporter acute leukemia cells and unbiasedly identified that candidate genes are significantly enriched in RNA binding and RNA splicing regulation. An RBP protein, RBM5, was among the top essential genes. Initially, RBM5 was reported as a tumor suppressor gene in lung cancer. Thus far, the function of the encoding protein RBM5 in blood cancer remained largely unknown. To further test how RBM5 expression might correlate with AML development, we analyzed the public RNA-seq data from the TCGA clinical cancer sample datasets, RBM5 is highly expressed in AML, indicating a plausible role in AML oncogenesis. Functional validation by two individual sgRNAs against different exons of RBM5 confirmed the selectively survival essential of RBM5 in AML cell lines. Ectopic expression of sgRNA-resistant RBM5 cDNA ultimately rescued the cellular phenotype upon RBM5 loss. We utilized an alternative loss-of-function approach, RNA interference (RNAi), to further examine the functional requirement of RBM5, Two independent shRNAs targeted RBM5 mRNA in three AML cells lines. RBM5 loss significantly impaired cell growth, reduced colony numbers and triggered notable myeloid differentiation. We further transplant MOLM13 cells targeted with shRBM5 into NSG mice, and observed that RBM5 shRNAs-treated animals lagged progressively more behind with much less splenomegaly and reduced leukemia cell infiltration in bone marrow, spleen, and peripheral blood compared to the wild-type control, suggesting that RBM5 is required for AML progression in vivo. More importantly, Suppression of RBM5 in primary AML cells significantly reduced colony numbers and induced myeloid differentiation, as demonstrated by the maturation markers CD11b and CD14. In addition, the domain dropout CRISPR screen revealed that the DNA-binding domain C2H2 is required for the gene function. Integrative analysis further suggested that HOXA9 promises the direct transcriptional target of RBM5 as demonstrated by functional rescue assay and HOXA9 partially restore the gene transcriptional defect in RBM5 null leukemia cells. In summary, our innovative research reported that a new RNA-binding protein, RBM5, is required for the survival of AML through non-canonical transcriptional regulation via a complex regulatory mechanism. These data support RBM5 as a promising AML therapeutic target in future studies.

RBM3 Induced Immune Resistance through Modulating Tumor Microenvironment in Nasopharyngeal Carcinoma

<h3>Purpose/Objective(s)</h3> We hypothesize RBM3 could lead to immune resistance in nasopharyngeal carcinoma (NPC). In the present study, we aim to explore the role and mechanisms of RBM3 inducing immune resistance by modulating tumor microenvironment (TME) in nasopharyngeal carcinoma. <h3>Materials/Methods</h3> Bioinformatic prediction and analysis were used to explore the correlation between RBM3 expression and NPC patients' survival, TME cell characters and related cytokines level. Immunohistochemistry (IHC) were used to examine RBM3 and PD-L1 expression, as well as to quantify Treg and M2 macrophages at the tissue level. Flow cytometry (FCM) were used to quantify immune cells in different treatment settings. Multiple cytokines were detected by ELISA Assays. Gene overexpression and knockdown were used to regulate RBM3 expression. Western-blot was used to detect related proteins. <h3>Results</h3> TCGA data analysis showed NPC patients with higher RBM3 expression and more Tegs had worse prognosis than those with lower RBM3 expression and fewer Tegs. IHC assays revealed that RBM3 expression was higher in immune resistant patients than in immune sensitive patients, with more Treg and M2 macrophages in TME. However, there was no significant difference in PD-L1 expression between the two groups. CNE1/IR cell line with high RBM3 expression and the parental CNE1 cell line with low RBM3 expression were cultured and cytokines in supernatant were detected. IL-2 and IL-10 levels were significantly higher in CNE1/IR cells and IL-4 and TGF-β were slightly higher. CNE1/IR and CNE1 cell lines were co-cultured with Treg and RAW264.7 cells. Flow cytometry showed there were more Treg cells and higher M2/M1 ratio in RBM3 highly expressed CNE1/IR group than the RBM3 low expression group. Decrease RBM3 expression in CNE1/IR cell could reduce IL-2/IL-10 level in supernatant and attenuate cytokines induced Treg recruit and macrophages transformation. Increase RBM3 expression in CNE1 cell could increase IL-2/IL-10 level and boost Treg recruit and macrophages transformation. Higher p-AKT level was detected in CNE1/IR cells and was correlated with IL-10 mRNA level. P-AKT and MEK1 inhibitor suppressed IL-10 mRNA and protein in RBM3 high expressing CNE1/IR cells. These results suggested RBM3 could induce immune resistant TME through regulating cytokines secretion. We further co-cultured RBM3 low expressing CNE1 cells with Treg, M2 and Treg+M2 cells. CNE1 cells co-cultured with Treg+M2 cells had higher RBM3 level than other groups, suggesting the immune resistant TME could inversely affect tumor cells and preserve immune resistant state. <h3>Conclusion</h3> RBM3 could increase NPC cells IL-10 secretion, recruiting more Tregs and increasing M1/M2 transformation in TME, thus induce immune resistance in nasopharyngeal carcinoma.

RBM3 interacts with Raptor to regulate autophagy and protect cardiomyocytes from ischemia-reperfusion-induced injury.

Acute myocardial infarction (AMI) is a common disease with high morbidity and mortality worldwide. However, postinfarction pathogenesis remains unclear, and it is particularly important to identify new therapeutic targets. The RNA-binding motif protein RBM3 (also known as cold-inducible protein) is known to promote translation and is associated with tumor proliferation and neuroprotection. However, little is known about the biological effects of RBM3 on myocardial infarction. In the present study, we found that RBM3 expression was significantly upregulated in ischemia-reperfusion (I/R) condition and downregulation of RBM3 inhibited autophagy and promoted apoptosis in cardiomyocytes. We confirmed that RBM3 interacts with Raptor to regulate the autophagy pathway. Taken together, these findings illustrate the protective effects of RBM3 against I/R-induced myocardial apoptosis through the autophagy pathway.

Hsa_circ_0003176 Suppresses the Progression of Non-Small-Cell Lung Cancer via Regulating miR-182-5p/RBM5 Axis.

Background Non-small-cell lung cancer (NSCLC) is one of the major diseases that threaten human health, and there is still no fundamental treatment method. Emerging evidences suggested that circRNAs might be an effective target to treatment NSCLC. However, the roles and detailed mechanisms of hsa_circ_0003176 in NSCLC still not clear. Methods hsa_circ_0003176 was identified from GSE101684 and GSE112214 datasets of Gene Expression Omnibus (GEO) database. The expression of hsa_circ_0003176 was detected by RT-qPCR in NSCLC tissues, paired adjacent nontumor tissues, and cell lines. RNA fluorescence in situ hybridization and nuclear and cytoplasmic RNA fractionation analysis was used to detect the subcellular localization of hsa_circ_0003176 in H1299 and A549 cells. Dual-luciferase reporter and RNA pull-down assay were used to confirm the regulatory of miR-182-5p to hsa_circ_0003176 and RBM5. The roles of hsa_circ_0003176 in NSCLC progression was evaluated both in vitro by CCK-8 assay, colony formation assay, wound-healing assay, and matrigel transwell assay and in vivo by the subcutaneous xenograft nude mouse experiment and lung metastasis nude mouse experiment. In addition, RNA pull down and luciferase reporter assays were carried out to investigate the interaction between hsa_circ_0003176 or RBM5 and miR-182-5p. Results Our results indicated that hsa_circ_0003176 showed typical characteristic of circRNAs, which was downregulated in both NSCLC tissues and cell lines. Functionally, overexpression of hsa_circ_0003176 suppressed the proliferation, migration, and invasion of NSCLC cells in vitro and inhibited NSCLC growth and metastasis in vivo. Furthermore, we found that hsa_circ_0003176 acts as sponge of miR-182-5p to regulate RBM5 expression. Further, in vitro rescue experiments demonstrated that hsa_circ_0003176 suppressed the proliferation, migration, and invasion of NSCLC cells by regulating miR-182-5p/RBM5 axis. Conclusion We demonstrated that hsa_circ_0003176 suppressed the NSCLC progression via regulating miR-182-5p/RBM5 axis. These data indicated that hsa_circ_0003176 might be a novel molecular target for NSCLC treatment.

Abstract 2558: Determining the expression of RNA binding protein Rbm3 in tumor cells and immune cells in the tumor microenvironment in prostate cancer

Abstract Prostate cancer is the most common cancer among men. Currently, targeting the AR pathway, chemotherapy or immune-based therapies are major options but only provide a modest improvement in overall survival. RNA binding proteins have been shown to regulate AR expression in the progression of PCa. We and others have demonstrated that RNA binding protein RBM3 is upregulated in various solid tumors including PCa. We have also shown that RBM3 binds to 3’UTR of mRNAs of tumor-promoting factors and increases their mRNA stability and translation. However, the role of RBM3 in PCa is not well evaluated. We first analyzed the expression levels of RBM3 in the TCGA database. There was a significant increase in RBM3 expression even in cancers with a Gleason score of 6 upto Gleason 10 cancers. To confirm this we performed RT-PCR analyses of a prostate cancer cDNA panel. There was a significant increase in RBM3 expression in the PCa tissues compared to normal control. We used LNCaP and its derivative cell line C4-2B which show features of progressed disease such as metastatic capability and hormone independence. RT-PCR and western blot analyses demonstrated significantly higher RBM3 levels in C4-2B cells as compared to LNCaP cells suggesting a role for RBM3 in tumor progression. The tumor microenvironment also plays a very important role in prognosis of PCa. Specifically, tumor-associated macrophages (TAMs) have been shown to increase metastatic potential and increase tumor angiogenesis. To determine the levels of RBM3 expression and its effects on macrophage infiltration, we mined the TCGA database using TIMER2.0 software. There was a positive correlation of RBM3 expression with infiltration of both M1 and M2 macrophages. To further study the effect of interactions between PCa cells and TAMs, we used immortalized THP1 monocytes, which can be activated to M1 and M2 macrophages. We observed that just converting the THP1 cells to M1 or M2 macrophages increased RBM3 expression in both cell types. Also, when M1 and M2 macrophages were treated with conditioned media from LNCaP or C4-2B cells, there was an induction in the expression of RBM3. Similarly, when conditioned media from M0, M1, M2 activated THP1 cells were applied to LNCaP and C4-2B cells, there was an increase in RBM3 expression in the PCa cells. This suggests a positive cross-talk between the macrophages and PCa cells. We evaluated the cytokine profile in the conditioned media from M1 and M2 macrophages and determined that while M1 macrophages had increased levels of CXCL10, M2 macrophages showed higher levels of IL4 and IL10. Moreover, PCa cells have higher levels of CXCR3, the receptor for CXCL10. Together, these data suggest that crosstalk between TAMs and cancer cells in the PCA microenvironment plays a significant role in increasing RBM3 expression, which in turn enhances global translation of disease progression related genes. Citation Format: Afreen Asif Ali Sayed, David Standing, Prasad Dandawate, Shahid Umar, Roy Jensen, Rahul Parikh, John Taylor, Shrikant Anant. Determining the expression of RNA binding protein Rbm3 in tumor cells and immune cells in the tumor microenvironment in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2558.

RNA-binding proteins RBM-HuR, RBM3 and PODXL expression in urothelial carcinoma of the urinary bladder. Prognostic and clinical implications.

Aim of the studyThe clinical significance and predictive and prognostic value of HuR, RBM3, and PODXL expression in patients with urothelial bladder cancer (UBC) are not clear yet. The aim of this study was to assess HuR, RBM3 and PODXL expression in muscle invasive and non-muscle invasive UBC tissues, and to investigate the clinicopathological correlations and their predictive and prognostic impact in patients with such type of cancer.Material and methodsRBM-HuR, RBM3 and PODXL expression levels were evaluated in 70 patients with urothelial carcinoma by immunohistochemistry. The relationships between their expression, clinicopathological findings and prognostic data were analyzed.ResultsHigh RBM-HuR expression was related to muscle invasion (p = 0.008), metastasis to lymph nodes (p = 0.007), and presence of blood spread (p = 0.049). High RBM3 expression was associated with lower grade (p = 0.044), absence of distant metastasis (p = 0.025), and absence of lymph node metastasis (p = 0.018). High PODXL expression was significantly associated with advanced tumor stage (p < 0.001), larger tumor size (p = 0.050), lymphovascular invasion (p = 0.006), lymph node metastasis (p = 0.008), higher grade (p = 0.043) and distant metastasis (p = 0.002).Three-year overall survival rate was negatively associated with high expression of both RBM-HuR and PODXL while it was directly correlated with high expression of RBM3 (p = 0.008, 0.009 and 0.015 respectively). High RBM-HuR and PODXL expression and low expression of RBM3 were related to tumor recurrence (p = 0.022, 0.011 and 0.015).ConclusionsRBM-HuR and PODXL expressions are markers of poor prognosis while RBM3 is a good prognostic marker for urothelial carcinoma of the bladder.

RBM5 Acts as Tumor Suppressor in Medulloblastoma through Regulating Wnt/β-Catenin Signaling

Introduction: RBM5 acts as a tumor suppressor gene in lung and breast cancers; however, its role in the pathogenesis of medulloblastoma (MB) remains unclear. We previously identified 4 RBM5 mutations in whole exome sequencing analysis of 40 MB patients. This study examined the role of RBM5 in MB progression. Methods: The expression patterns of RBM5 in tissues of 40 MB patients were analyzed using immunohistochemistry. Associations between RBM5 expression and overall survival (OS) were evaluated using Kaplan-Meier analysis. The RBM5 role in Daoy cells’ proliferation, migration, and Wnt/β-catenin signaling was analyzed after RBM5 knockdown and overexpression. Results: The expression level of RBM5 mRNA and protein was significantly lower in MB than that in adjacent normal control tissues, and low RBM5 expression was significantly associated with reduced OS (p = 0.034). RBM5 knockdown induced Daoy and ONS-76 cells proliferation, while RBM5 overexpression repressed cell proliferation and migration in vitro (all p < 0.05). β-Catenin, LEF1, and cyclin D1 mRNA levels were upregulated, while DKK1 expression was downregulated in Daoy cells following RBM5 knockdown. Conclusion: RBM5 may function as a tumor suppressor in MB by regulating Wnt/β-catenin signaling, and its reduced expression is associated with lower OS.

RNA Binding Protein RBM3 Modulates Novel LncRNAs to Increase Tumor Progression in Colon Cancer Cells

RNA binding proteins play a significant role in regulating gene expression, and dysregulation in their expression may skew cells to various pathophysiological conditions. In previous studies, we demonstrated that RNA binding protein RBM3 acts as a protooncogene, with its expression. Increasing in a stage dependent manner in colon cancers. To get a better understanding of how RBM3 works, we took an unbiased approach and performed total RNA‐sequencing (RNAseq) and RNA‐immunoprecipitation‐coupled sequencing (RIPSeq) to identify transcripts targeted by the protein. We identified several genes involved in cell movement and angiogenesis, including VEGF, ZEB1, TWIST1 and Slug mRNAs. We validated these results by Real time RT‐PCR analyses. Coupled with this, we observed that RBM3 overexpression increased stemness, migration and invasion in vitro. A second discovery from the RNA seq and RNA‐IP studies was that RBM3 overexpression increased the levels of multiple long noncoding RNAs (lncRNAs), a class of regulatory RNAs that play important roles in tumorigenesis. Of these, we chose four lncRNAs, two known (HOTAIR, TUG1) and two novel lncRNAs based on their ability to interact with VEGF, ZEB1, TWIST1 and Slug mRNAs using the RNAfold program. Knocking down the lncRNAs using a combination of specific siRNAs and locked nucleic acid (LNA) oligonucleotides showed reduced levels of the four mRNAs and their protein expression. The consequence of this was reduced cell migration and invasion in vitro, in scratch plate and Boyden chamber assays. We also confirmed these studies in vivo. We generated transgenic mice overexpressing RBM3. LncRNA levels were higher in the colons of the transgenic animals. We also generated intestine specific RBM3 knockout mice. Here, lncRNA levels were lower. We further treated the wild type and RBM3−/− mice with azoxymethane (AOM)‐dextran sodium sulfate (DSS) to induce colitis‐associated colorectal tumorigenesis. There was significantly lower number of tumors in colons of RBM3−/− mice, coupled with lower levels of the four lncRNAs and mRNAs. In addition, RBM3 expression significantly increased tumor xenograft growth as compared to vector control cells. RBM3 overexpressing xenograft tumors also showed increased expression of lncRNAs along with EMT and angiogenic markers. Intratumoral knockdown of the lncRNAs decreased the tumor size and volume of tumor xenograft along with a decrease in Slug and VEGF protein expression. We used IntaRNA to analyze lncRNA‐mRNA interaction in silico. The lncRNAs formed a bridge between stem‐loop structures in the 5’ untranslated and 3’ untranslated regions of the four mRNAs (VEGF, ZEB1, TWIST1 and Slug) resulting in a closed circle formation. The interaction results in the formation of kissing loop interaction between lncRNA‐mRNA that contributes to the increased translation of the mRNA. Based on these results, we conclude that RNA binding protein RBM3 enhances tumor progression by modulating novel lncRNAs.

LncRNA LINP1 promotes proliferation and inhibits apoptosis of gastric cancer cells by repressing RBM5.

Recent studies have revealed that long non-coding RNAs (lncRNAs) play a crucial role in tumor progression. Gastric cancer (GC) is one of the common types of malignancies worldwide. This study aimed to identify the exact function of lncRNA LINP1 in the progression of GC. LINP1 expression in paired cancer tissues and adjacent normal tissues of GC patients was detected by Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). The effect of LINP1 silence on proliferation and apoptosis of GC cells was detected. Meanwhile, the underlying mechanism of LINP1 function was explored by RT-qPCR and Western blot assay. Furthermore, tumor formation assay was performed to examine the ability of LINP1 in tumor formation in vivo. LINP1 expression was remarkably up-regulated in GC tissues compared with adjacent normal tissues. The growth ability of GC cells was significantly inhibited after silencing of LINP1 in vitro. Besides, the apoptosis of GC cells was markedly induced after silencing of LINP1. The silence of LINP1 significantly up-regulated the expression of RBM5 in GC cells. Meanwhile, RBM5 expression in GC tissues was remarkably lower than that of the adjacent normal tissues. Furthermore, tumor formation assay showed that knockdown of LINP1 markedly inhibited tumor formation in vivo. These results suggested that LINP1 could down-regulate RBM5. Meanwhile, LINP1 remarkably promoted growth ability and suppressed apoptosis of GC in vitro and in vivo. Our findings might provide a novel regulator and therapeutic strategy for GC patients.

LINC00096 Promotes the Proliferation and Invasion by Sponging miR-383-5p and Regulating RBM3 Expression in Triple-Negative Breast Cancer.

BackgroundRecent studies revealed that long non-coding RNAs (lncRNA) play crucial roles in cancer initiation and progression. However, the function and underlying mechanism of lncRNAs in triple-negative breast cancer (TNBC) are little investigated.MethodsqRT-PCR was used to investigate LINC00096 expression in TNBC tissues and cells. Function assays were used to test the effects of LINC00096 on TNBC cells progression. In addition, luciferase reporter and qRT-PCR assays were used to determine the underlying mechanism of LINC00096 on TNBC progression.ResultsIn our present study, we identify LINC00096 as one of the most upregulated lncRNA in TNBC progression by using microarray screening. High LINC00096 expression was obviously related to advanced tumor stage, metastasis, poor prognosis of patients. Loss-of-function assays showed that LINC00096 suppression reduced TNBC cells proliferation and invasive abilities in vitro. Mechanistically, we demonstrated that LINC00096 directly interacted with miR-383-5p, subsequently acted as a miRNA sponge to increase RBM3 expression.ConclusionIn the present study, we indicated that LINC00096 might promote the proliferation and invasion through regulating the miR-383-5p/RBM3 pathway in TNBC, which providing a novel therapeutic target for cancer treatment.

The RNA-Binding Protein RBM3 Promotes Neural Stem Cell (NSC) Proliferation Under Hypoxia.

Neural stem cells (NSCs) reside physiologically in a hypoxic niche to maintain self-renewal and multipotency. Whereas mild hypoxia is known to promote NSC proliferation, severe hypoxia in pathological conditions exerts the reverse effect. The multi-functional RNA-binding protein RBM3 is abundant in NSCs and can be regulated by hypoxic exposure. Although RBM3 has been shown to accelerate cell growth in many cell types, whether and how it affects NSC proliferation in hypoxic environment remains largely unknown. In this study, we tested how RBM3 regulates cell proliferation under hypoxia in C17.2 mouse NSC cell line and in primary mouse NSCs from both the forebrain of postnatal day 0 (P0) mice and the subgranular zone (SGZ) of adult mice. Our results demonstrated that RBM3 expression was highly sensitive to hypoxia, and NSCs were arrested in G0/G1 phase by 5, 2.5, and 1% O2 treatment. When we overexpressed RBM3, hypoxia-induced cell cycle arrest in G0/G1 phase was relieved and more cell transit into S phase was observed. Furthermore, cell viability under hypoxia was also increased by RBM3. In contrast, in RBM3-depleted primary NSCs, less BrdU-incorporated cells were detected, indicating exacerbated cell cycle arrest in G1 to S phase transition. Instead, overexpressed RBM3 significantly increased proliferation ratio in primary NSCs. Our findings indicate RBM3 as a potential target to maintain the proliferation capacity of NSCs under hypoxia, which can be important in NSC-based therapies of acute brain injury and chronic neurodegenerative diseases.

Post-TTM Rebound Pyrexia after Ischemia-Reperfusion Injury Results in Sterile Inflammation and Apoptosis in Cardiomyocytes.

Introduction Fever is frequently observed after acute ischemic events and is associated with poor outcome and higher mortality. Targeted temperature management (TTM) is recommended for neuroprotection in comatose cardiac arrest survivors, but pyrexia after rewarming is proven to be detrimental in clinical trials. However, the cellular mechanisms and kinetics of post-TTM rebound pyrexia remain to be elucidated. Therefore, we investigated the effects of cooling and post-TTM pyrexia on the inflammatory response and apoptosis in a cardiomyocyte ischemia-reperfusion (IR) injury model. Methods HL-1 cardiomyocytes were divided into the following groups to investigate the effect of oxygen-glucose deprivation/reperfusion (OGD/R), hypothermia (33.5°C), and pyrexia (40°C): normoxia controls maintained at 37°C and warmed to 40°C, OGD/R groups maintained at 37°C and cooled to 33.5°C for 24 h with rewarming to 37°C, and OGD/R pyrexia groups further warmed from 37 to 40°C. Caspase-3 and RBM3 were assessed by Western blot and TNF-α, IL-6, IL-1β, SOCS3, iNOS, and RBM3 transcriptions by RT-qPCR. Results OGD-induced oxidative stress (iNOS) in cardiomyocytes was attenuated post-TTM by cooling. Cytokine transcriptions were suppressed by OGD, while reperfusion induced significant TNF-α transcription that was exacerbated by cooling. Significant inductions of TNF-α, IL-6, IL-1β, and SOCS3 were observed in noncooled, but not in cooled and rewarmed, OGD/R-injured cardiomyocytes. Further warming to pyrexia induced a sterile inflammatory response in OGD/R-injured groups that was attenuated by previous cooling, but no inflammation was observed in pyrexic normoxia groups. Moreover, cytoprotective RBM3 expression was induced by cooling but suppressed by pyrexia, correlating with apoptotic caspase-3 activation. Conclusion Our findings show that maintaining a period of post-TTM “therapeutic normothermia” is effective in preventing secondary apoptosis-driven myocardial cell death, thus minimizing the infarct area and further release of mediators of the innate sterile inflammatory response after acute IR injury.

Long noncoding RNA PCAT-1 accelerates the metastasis of pancreatic cancer by repressing RBM5.

The role of long noncoding RNAs (lncRNAs) is vital in tumor progression. Our study aims to identify the role of PCAT-1 in the metastasis of pancreatic cancer. Real time-quantitative polymerase chain reaction (RT-qPCR) was used to measure PCAT-1 expression in 50 pancreatic cancer patients' tissues. Furthermore, to identify the function of PCAT-1 in pancreatic cancer in vitro wound healing assay and transwell assay were conducted. Besides, RT-qPCR and Western blot assay were performed to explore the underlying mechanism. The expression level of PCAT-1 was significantly upregulated in pancreatic cancer samples compared with adjacent tissues. Moreover, cell migration and cell invasion were inhibited via knockdown of PCAT-1 in pancreatic cancer cells. Moreover, the mRNA and protein expression of RBM5 was upregulated via knockdown of PCAT-1 in pancreatic cancer cells. Furthermore, the RBM5 expression level was negatively related to the PCAT-1 expression level in pancreatic cancer tissues. This study suggests that PCAT-1 acts as an oncogene in pancreatic cancer and promotes cell metastasis via inhibiting RBM5, which might be a novel therapeutic strategy in pancreatic cancer.

Long noncoding RNA AFAP1-AS1 accelerates the proliferation and metastasis of prostate cancer via inhibiting RBM5 expression.

Recently, the role of long noncoding RNA (lncRNAs) in tumor progression has caught many attentions. In this research, lncRNA AFAP1-AS1 was studied to identify how it functioned in the progression of prostate cancer. LncRNA AFAP1-AS1 expression was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) in both prostate cancer cells and tissue samples. In addition, to identify the function of AFAP1-AS1 on prostate cancer in vitro, cell proliferation, transwell assay, and Matrigel assay were conducted. Furthermore, by performing qRT-PCR and Western blot assay, the underlying mechanism was explored. The expression level of AFAP1-AS1 was significantly higher in prostate cancer samples than that in corresponding ones. Additionally, the cell proliferation, migration, and invasion capacities were inhibited after AFAP1-AS1 was knocked down in prostate cancer cells. Moreover, the mRNA and protein expressions of RBM5 were upregulated after AFAP1-AS1 was knocked down. Furthermore, the RBM5 expression level was negatively related to AFAP1-AS1 expression level in prostate cancer samples. AFAP1-AS1 acts as an oncogene in prostate cancer by enhancing cell metastasis and proliferation via suppressing RBM5, which might be a novel therapeutic strategy in treatment for prostate cancer.

Role of RNA-binding protein 5 in the diagnosis and chemotherapeutic response of lung cancer.

Lung cancer remains one of the leading causes of cancer-associated mortality in the world. Lung carcinogenesis is frequently associated with deletions or the loss of heterozygosity at the critical chromosomal region 3p21.3, where RNA-binding protein 5 (RBM5) is localized. RBM5 regulates cell growth, cell cycle progression and apoptosis in cell homeostasis. In the lungs, altered RBM5 protein expression leads to alterations in cell growth and apoptosis, with subsequent lung pathogenesis and varied responses to treatment in patients with lung cancer. Detection of RBM5 expression may be a tumor marker for diagnosis, prediction and treatment response in lung cancer, and may be developed as a potential therapeutic target for drug resistant lung cancer. This review discusses the most recent progress on the role of RBM5 in lung cancer.

Reduced RBM3 expression is associated with aggressive tumor features in esophageal cancer but not significantly linked to patient outcome

BackgroundRBM3 expression has been suggested as prognostic marker in several cancer types. The purpose of this study was to assess the prevalence and clinical significance of altered RBM3 expression in esophageal cancer.MethodsRBM3 protein expression was measured by immunohistochemistry using tissue microarrays containing samples from 359 esophageal adenocarcinoma (EAC) and 254 esophageal squamous cell cancer (ESCC) patients with oncological follow-up data.ResultsWhile nuclear RBM3 expression was always high in benign esophageal epithelium, high RBM3 expression was only detectable in 66.4% of interpretable EACs and 59.3% of ESCCs. Decreased RBM3 expression was linked to a subset of EACs with advanced UICC stage and presence of distant metastasis (P = 0.0031 and P = 0.0024). In ESCC, decreased RBM3 expression was associated with advanced UICC stage, high tumor stage, and positive lymph node status (P = 0.0213, P = 0.0061, and P = 0.0192). However, RBM3 expression was largely unrelated to survival of patients with esophageal cancer (EAC: P = 0.212 and ESCC: P = 0.5992).ConclusionsIn summary, the present study shows that decreased RBM3 expression is associated with unfavourable esophageal cancer phenotype, but not significantly linked to patient prognosis.

RBM3 expression is upregulated by NF-κB p65 activity, protecting cells from apoptosis, during mild hypothermia.

The RNA-binding protein RBM3, a cold shock protein whose expression is elevated under hypothermic conditions, plays an important role in cell survival; however, little is known about the mechanism underlying the mild hypothermia-mediated regulation of RBM3 expression and apoptosis. Here we show that the transcription factor NF-κB p65 is phosphorylated at Ser276 and activates RBM3 gene transcription via binding to a particular element within the promoter region in response to induced hypothermia, elevating the protein expression, and suppressing apoptosis. Treatment with caffeic acid phenethyl ester (CAPE), a potent and specific inhibitor that suppresses the translocation of NF-κB p65 from the cytoplasm to the nucleus, resulted in decreased levels of RBM3 mRNA and protein and increased incidence of apoptosis despite cells were cultured under hypothermic conditions. Overexpression of RBM3 abolished the induction of apoptosis in cells treated with CAPE, indicating that NF-κB p65-upregulated RBM3 expression is necessary for the suppression of apoptosis. In addition, experiments with cells overexpressing RBM3 supported the finding demonstrating that the mild hypothermia-mediated higher expression of RBM3 suppressed the induction of apoptosis. Conversely, experiments with cells deficient in RBM3 supported the finding demonstrating that the CAPE-mediated loss of RBM3 induced apoptosis. These results suggest that NF-κB p65 is a critical mediator of mild hypothermia, to which cells are exposed as an extracellular environment, and a central inducer of RBM3 expression, which is responsible for preventing cells from apoptosis. Moreover, CAPE may have a potential for the application to a therapeutic agent for the treatment of cancers.

Belinostat effects on expression of RBM5 tumor suppressor gene and inhibits prostate cancer cell line (PC3) proliferation

Prostate cancer as the second most prevalent cancer and the sixth most common cause of cancer death among men appears with increasing mortality incidence rate amongst Asian population. Although, surgery, chemotherapy and radiotherapy are investigated as potent therapeutic strategies for the treatment of localized prostate cancer, advanced prostate cancer does not completely respond to these methods. According to the ambiguous molecular mechanism of belinostat, the present study was conducted to determine the effect of belinostat on an advanced prostate cancer cell line (PC3). The Flowcytometry assay was done in a triplicate repeat to measure the viability and the percent of apoptotic cells. Real-time PCR was done to evaluate mRNA expression changes in RBM5 tumor suppressor gene and growth inhibition of PC3. The results indicated that PC3 that treated with 1μM belinostat showed significant increase in expression of RBM5 mRNA and also a significant increase in apoptosis. In conclusion, this study demonstrated that belinostat increases RBM5 expression and this may be responsible for some part of belinostat apoptosis inducing effect.