The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival

NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.     

Long noncoding RNA DGCR5 involves in tumorigenesis of esophageal squamous cell carcinoma via SRSF1-mediated alternative splicing of Mcl-1

Long noncoding RNAs (lncRNAs) emerge as essential roles in the regulation of alternative splicing (AS) in various malignancies. Serine- and arginine-rich splicing factor 1 (SRSF1)-mediated AS events are the most important molecular hallmarks in cancer. Nevertheless, the biological mechanism underlying tumorigenesis of lncRNAs correlated with SRSF1 in esophageal squamous cell carcinoma (ESCC) remains elusive. In this study, we found that lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) was upregulated in ESCC clinical samples, which associated with poor prognosis. Through RNA interference and overexpression approaches, we confirmed that DGCR5 contributed to promote ESCC cell proliferation, migration, and invasion while inhibited apoptosis in vitro. Mechanistically, DGCR5 could directly bind with SRSF1 to increase its stability and thus stimulate alternative splicing events. Furthermore, we clarified that SRSF1 regulated the aberrant splicing of myeloid cell leukemia-1 (Mcl-1) and initiated a significant Mcl-1L (antiapoptotic) isoform switch, which contributed to the expression of the full length of Mcl-1. Moreover, the cell-derived xenograft (CDX) model was validated that DGCR5 could facilitate the tumorigenesis of ESCC in vivo. Collectively, our findings identified that the key biological role of lncRNA DGCR5 in alternative splicing regulation and emphasized DGCR5 as a potential biomarker and therapeutic target for ESCC.Subject terms: Tumour biomarkers, Apoptosis     

SRSF5 functions as a novel oncogenic splicing factor and is upregulated by oncogene SRSF3 in oral squamous cell carcinoma

Alternative splicing of precursor messenger RNA has been increasingly associated with tumorigenesis. The serine/arginine-rich protein (SR) family plays key roles in the regulation of pre-mRNA alternative splicing. Increasing evidence has demonstrated that the SR protein family is involved in tumorigenesis. However, the functions and mechanisms of SR proteins in tumourigenesis remain largely unknown. In the present study, we discovered that serine/arginine-rich splicing factor 5 (SRSF5) is a novel oncogenic splicing factor that is overexpressed in oral squamous cell carcinoma (OSCC) tissues and cells, being crucial for OSCC cell proliferation and tumor formation. Overexpression of SRSF5 transformed immortal rodent fibroblasts to form tumors in nude mice, while downregulation of SRSF5 in oral squamous cell lines retarded cell growth, cell cycle progression, and tumor growth. The expression of SRSF5 is controlled by an autoregulation mechanism. Serine/arginine-rich splicing factor 3 (SRSF3) has been identified as an oncogene. We found that SRSF5 is a novel target of SRSF3. SRSF3 impairs the autoregulation of SRSF5 and promotes SRSF5 overexpression in cancer cells. Altogether, the present study demonstrated that SRSF5 is a novel oncogene that is upregulated by SRSF3 in OSCC cells.     

CircPLCE1 facilitates the malignant progression of colorectal cancer by repressing the SRSF2-dependent PLCE1 pre-RNA splicing

Studies have demonstrated that circular RNAs (circRNAs) play important roles in various types of cancer; however, the mechanisms of circRNAs located in the nucleus have rarely been explored. Here, we report a novel circular RNA circPLCE1 (hsa_circ_0019230) that facilitates the malignant progression of colorectal cancer (CRC) by repressing serine/arginine-rich splicing factor 2 (SRSF2)-dependent phospholipase C epsilon 1 (PLCE1) pre-RNA splicing. Quantitative real-time polymerase chain reaction was used to determine the expression of circPLCE1 in CRC tissues and cells. Cell Counting Kit-8, Transwell and flow cytometric assays were used to assess the role of circPLE1 in CRC cell proliferation, migration and apoptosis, respectively. An animal study was conducted to test the role of circPLCE1 in vivo. Furthermore, catRAPID and RPISeq were used to predict the possible binding proteins of circPLCE1. RNA fractionation and RNA immunoprecipitation assays were used to confirm the RNA-protein interaction. In this study, we found that circPLCE1 was more significantly down-regulated in CRC tissues compared with that in adjacent normal tissues. However, circPLCE1 knockdown suppressed CRC cell proliferation, migration and invasion and increased apoptosis. Nude mouse experiments showed that ectopic expression of circPLCE1 dramatically increased tumour growth in vivo. Mechanistically, circPLCE1 directly bound to the SRSF2 protein, repressing SRSF2-dependent PLCE1 pre-RNA splicing, resulting in the progression of CRC. Individually mutating the binding sites of circPLCE1 abolished the inhibition of PLCE1 mRNA production. Our study revealed a novel molecular mechanism in the regulation of PLCE1 and suggested a new function of circular RNA.     

A genome landscape of SRSF3-regulated splicing events and gene expression in human osteosarcoma U2OS cells

Alternative RNA splicing is an essential process to yield proteomic diversity in eukaryotic cells, and aberrant splicing is often associated with numerous human diseases and cancers. We recently described serine/arginine-rich splicing factor 3 (SRSF3 or SRp20) being a proto-oncogene. However, the SRSF3-regulated splicing events responsible for its oncogenic activities remain largely unknown. By global profiling of the SRSF3-regulated splicing events in human osteosarcoma U2OS cells, we found that SRSF3 regulates the expression of 60 genes including ERRFI1, ANXA1 and TGFB2, and 182 splicing events in 164 genes, including EP300, PUS3, CLINT1, PKP4, KIF23, CHK1, SMC2, CKLF, MAP4, MBNL1, MELK, DDX5, PABPC1, MAP4K4, Sp1 and SRSF1, which are primarily associated with cell proliferation or cell cycle. Two SRSF3-binding motifs, CCAGC(G)C and A(G)CAGCA, are enriched to the alternative exons. An SRSF3-binding site in the EP300 exon 14 is essential for exon 14 inclusion. We found that the expression of SRSF1 and SRSF3 are mutually dependent and coexpressed in normal and tumor tissues/cells. SRSF3 also significantly regulates the expression of at least 20 miRNAs, including a subset of oncogenic or tumor suppressive miRNAs. These data indicate that SRSF3 affects a global change of gene expression to maintain cell homeostasis.     

Hepatocyte Growth Factor Enhances Alternative Splicing of the Kruppel-like Factor 6 (KLF6) Tumor Suppressor to Promote Growth through SRSF1

Alternative splicing of the Krüppel-like factor 6 (KLF6) tumor suppressor into an antagonistic splice variant 1 (SV1) is a pathogenic event in several cancers including hepatocellular carcinoma (HCC) because elevated SV1 is associated with increased tumor metastasis and mortality. Ras activation is one factor that can enhance KLF6 splicing in cancer cells, however pathways driving KLF6 splicing are unknown. Splice site selection is regulated by splice factors that include serine/arginine-rich (SR) proteins such as SRSF1 (ASF-SF2), which in turn is controlled by phosphoinositide 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) signaling pathway. Because signaling pathways downstream of the liver mitogen hepatocyte growth factor (HGF) include Akt, we explored whether HGF induces KLF6 alternative splicing. In HepG2 cells, HGF (25 ng/mL) significantly increases the ratio of SV1/KLF6 full by 40% through phosphorylation of Akt and subsequent downregulation of two splicing regulators, SRSF3 (SRp20) and SRSF1. Decreased SRSF3 levels regulate SRSF1 levels by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD), which stimulates cell growth by decreasing p21 levels. Enhanced cell replication through increased KLF6 alternative splicing is a novel growth-promoting pathway of HGF that could contribute to the molecule's mitogenic activity in physiologic liver growth and hepatocellular carcinoma. Mol Cancer Res; 10(9); 1216-27. ?2012 AACR.     

Phosphorylation of SRSF1 is modulated by replicational stress

DNA ligase I-deficient 46BR.1G1 cells show a delay in the maturation of replicative intermediates resulting in the accumulation of single- and double-stranded DNA breaks. As a consequence the ataxia telangiectasia mutated protein kinase (ATM) is constitutively phosphorylated at a basal level. Here, we use 46BR.1G1 cells as a model system to study the cell response to chronic replication-dependent DNA damage. Starting from a proteomic approach, we demonstrate that the phosphorylation level of factors controlling constitutive and alternative splicing is affected by the damage elicited by DNA ligase I deficiency. In particular, we show that SRSF1 is hyperphosphorylated in 46BR.1G1 cells compared to control fibroblasts. This hyperphosphorylation can be partially prevented by inhibiting ATM activity with caffeine. Notably, hyperphosphorylation of SRSF1 affects the subnuclear distribution of the protein and the alternative splicing pattern of target genes. We also unveil a modulation of SRSF1 phosphorylation after exposure of MRC-5V1 control fibroblasts to different exogenous sources of DNA damage. Altogether, our observations indicate that a relevant aspect of the cell response to DNA damage involves the post-translational regulation of splicing factor SRSF1 which is associated with a shift in the alternative splicing program of target genes to control cell survival or cell death.     

Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage

Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation.     

Ubiquitination Regulates Expression of the Serine/Arginine-rich Splicing Factor 1 (SRSF1) in Normal and Systemic Lupus Erythematosus (SLE) T Cells

T cells from patients with systemic lupus erythematosus (SLE) exhibit reduced expression of the critical T cell receptor (TCR)-associated CD3ζ signaling chain and are poor producers of the vital cytokine IL-2. By oligonucleotide pulldown and mass spectrometry discovery approaches, we identified the splicing regulator serine/arginine-rich splicing factor (SRSF) 1 or splicing factor 2/alternative splicing factor (SF2/ASF) to be important in the expression of CD3ζ chain. Importantly, increases in the expression of SRSF1 rescued IL-2 production in T cells from patients with SLE. In this study, we investigated the regulation of SRSF1 expression in resting and activated human T cells. We found that T cell stimulation induced a rapid and significant increase in mRNA expression of SRSF1; however, protein expression levels did not correlate with this increase. Co-engagement of CD28 induced a similar mRNA induction and reduction in protein levels. Proteasomal but not lysosomal degradation was involved in this down-regulation as evidenced by blocking with specific inhibitors MG132 and bafilomycin, respectively. Immunoprecipitation studies showed increased ubiquitination of SRSF1 in activated T cells. Interestingly, T cells from patients with SLE showed increased ubiquitination of SRSF1 when compared with those from healthy individuals. Our results demonstrate a novel mechanism of regulation of the splicing factor SRSF1 in human T cells and a potential molecular mechanism that controls its expression in SLE.     

lncRNA OSER1-AS1 acts as a ceRNA to promote tumorigenesis in hepatocellular carcinoma by regulating miR-372-3p/Rab23 axis

Long non-coding RNAs (lncRNAs) are crucial regulators of tumorigenesis and progression in human cancer, including hepatocellular carcinoma (HCC). However, the role of most lncRNAs that are dysregulated in HCC remains to be elucidated. Here, we investigated the role of OSER1-AS1 in the progression of HCC. The results of database and qRT-PCR analysis demonstrated that OSER1-AS1 was highly expressed in HCC tissues and the high expression of OSER1-AS1 was closely associated with larger tumor size, advanced tumor stages, lower disease free survival and overall survival of HCC patients. OSER1-AS1 knockdown significantly inhibited the proliferation, invasion and migration of HCC cells, and induced the apoptosis. In addition, the dual luciferase reporter assay directly demonstrated that OSER1-AS1 functioned as a molecular sponge for miR-372-3p to promote Rab23 expression. Moreover, the results of immunohistochemistry and western blot analysis showed that Rab23 was highly expressed in HCC tissues, and the high expression of Rab23 was closely associated with the poor overall survival of HCC patients. Immunofluorescence assay also found the subcellular localization of Rab23 in HCC cells. Rab23 was obviously downregulated in cells that were transfected with miR-372-3p mimics. MiR-372-3p mimics significantly inhibited the proliferation and invasion of HCC cells). Rab23 restoration partially reversed miR-372-3p-induced tumor suppressive effects on HCC cells. In conclusion, we found that OSER1-AS1 acted as a ceRNA to sponge miR-372-3p, thereby positively regulating the Rab23 expression and ultimately acting as a tumor suppressor gene in HCC progression.     

Transcriptome analysis of alternative splicing events regulated by SRSF10 reveals position-dependent splicing modulation

Splicing factor SRSF10 is known to function as a sequence-specific splicing activator. Here, we used RNA-seq coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Motif analysis revealed that SRSF10 binding to cassette exons was associated with exon inclusion, whereas the binding of SRSF10 within downstream constitutive exons was associated with exon exclusion. This positional effect was further demonstrated by the mutagenesis of potential SRSF10 binding motifs in two minigene constructs. Functionally, many of SRSF10-verified alternative exons are linked to pathways of stress and apoptosis. Consistent with this observation, cells depleted of SRSF10 expression were far more susceptible to endoplasmic reticulum stress-induced apoptosis than control cells. Importantly, reconstituted SRSF10 in knockout cells recovered wild-type splicing patterns and considerably rescued the stress-related defects. Together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions.     

MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion

CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. In this study, we silenced CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) and examined the invasiveness and proliferation of CCR1-silenced HCCLM3 cells and the matrix metalloproteinase (MMP) activity. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of HCCLM3 cells, but had only a minor effect on the cellular proliferation rate. Moreover, CCR1 knockdown significantly reduced the secretion of MMP-2. Together, these findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment.     

Rock2 regulates Cdc25A through ubiquitin proteasome system in hepatocellular carcinoma cells

Rho-associated coiled-coil containing protein kinase 2 (Rock2) belongs to a family of serine/threonine kinases which are actived via interaction with Rho GTPases. Recently, overexpression of Rock2 has been demonstrated in human hepatocellular carcinoma (HCC), but the potential role of Rock2 in tumorigenesis remains unclear. Cdc25A acts as a key checkpoint during the G1/S phase and has also been found to be overexpressed in HCC. Here, we report that Rock2 regulates cell cycle progression via ubiquitination of Cdc25A in HCC. In HCC tissues, Rock2 and Cdc25A were aberrantly upregulated and revealed a significantly positive correlation. Knockdown of Rock2 inhibited HCC cell growth and promoted cell-cycle arrest at the G1/S phase via regulation of Cdc25A. When cells were exposed to DNA damage, Rock2 increased cell survival by regulating Cdc25A. Co-immunoprecipitation and immunofluorescence analyses indicated that Rock2 regulated Cdc25A via direct binding. Furthermore, knockdown of Rock2 activated Cdc25A ubiquitination and promoted its degradation. Our results defined a role for Rock2 in modulation of Cdc25A ubiquitination, indicating a novel mechanism of Cdc25A regulation and a potential function for Rock2 in the development of HCC.