PAIP1 binds to pre-mRNA and regulates alternative splicing of cancer pathway genes including VEGFA.

BACKGROUND: Poly (A) binding protein interacting protein 1 (PAIP1) has been shown to causally contribute to the development and progression of cancer. However, the mechanisms of the PAIP1 regulation in tumor cells remain poorly understood. RESULTS: Here, we used a recently developed UV cross-linking and RNA immunoprecipitation method (iRIP-seq) to map the direct and indirect interaction sites between PAIP1 and RNA on a transcriptome-wide level in HeLa cells. We found that PAIP1 not only binds to 3'UTRs, but also to pre-mRNAs/mRNAs with a strong bias towards the coding region and intron. PAIP1 binding sites are enriched in splicing enhancer consensus GA-rich motifs. RNA-seq analysis revealed that PAIP1 selectively modulates the alternative splicing of genes in some cancer hallmarks including cell migration, the mTOR signaling pathway and the HIF-1 signaling pathway. PAIP1-regulated alternative splicing events were strongly associated with PAIP1 binding, demonstrating that the binding may promote selection of the nearby splice sites. Deletion of a PAIP1 binding site containing seven repeats of GA motif reduced the PAIP1-mediated suppression of the exon 6 inclusion in a VEGFA mRNA isoform. Proteomic analysis of the PAIP1-interacted proteins revealed the enrichment of the spliceosome components and splicing factors. CONCLUSIONS: These findings suggest that PAIP1 is both a polyadenylation and alternative splicing regulator, that may play a large role in RNA processing via its role in alternative splicing regulation.

HMGB1 prefers to interact with structural RNAs and regulates rRNA methylation modification and translation in HeLa cells.

BACKGROUND: High-mobility group B1 (HMGB1) is both a DNA binding nuclear factor modulating transcription and a crucial cytokine that mediates the response to both infectious and noninfectious inflammation such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. HMGB1 has been proposed to control ribosome biogenesis, similar as the other members of a class of HMGB proteins. RESULTS: Here, we report that HMGB1 selectively promotes transcription of genes involved in the regulation of transcription, osteoclast differentiation and apoptotic process. Improved RNA immunoprecipitation by UV cross-linking and deep sequencing (iRIP-seq) experiment revealed that HMGB1 selectively bound to mRNAs functioning not only in signal transduction and gene expression, but also in axon guidance, focal adhesion, and extracellular matrix organization. Importantly, HMGB1-bound reads were strongly enriched in specific structured RNAs, including the domain II of 28S rRNA, H/ACA box snoRNAs including snoRNA63 and scaRNAs. RTL-P experiment showed that overexpression of HMGB1 led to a decreased methylation modification of 28S rRNA at position Am2388, Cm2409, and Gm2411. We further showed that HMGB1 overexpression increased ribosome RNA expression levels and enhanced protein synthesis. CONCLUSION: Taken together, our results support a model in which HMGB1 binds to multiple RNA species in human cancer cells, which could at least partially contribute to HMGB1-modulated rRNA modification, protein synthesis function of ribosomes, and differential gene expression including rRNA genes. These findings provide additional mechanistic clues to HMGB1 functions in cancers and cell differentiation.

Comparative study on seasonal hair follicle cycling by analysis of the transcriptomes from cashmere and milk goats.

Guard hair and cashmere undercoat are developed from primary and secondary hair follicle, respectively. Little is known about the gene expression differences between primary and secondary hair follicle cycling. In this study, we obtained RNA-seq data from cashmere and milk goats grown at four different seasons. We studied the differentially expressed genes (DEGs) during the yearly hair follicle cycling, and between cashmere and milk goats. WNT, NOTCH, MAPK, BMP, TGFß and Hedgehog signaling pathways were involved in hair follicle cycling in both cashmere and milk goat. However, Milk goat DEGs between different months were significantly more than cashmere goat DEGs, with the largest difference being identified in December. Some expression dynamics were confirmed by quantitative PCR and western blot, and immunohistochemistry. This study offers new information sources related to hair follicle cycling in milk and cashmere goats, which could be applicable to improve the wool production and quality.

RBM4 modulates the proliferation and expression of inflammatory factors via the alternative splicing of regulatory factors in HeLa cells.

Regulatory factors function by modulating a variety of cascade mechanisms in cells. RBM4 is a multifunctional RNA-binding protein in post-transcriptional gene regulation. Cytoplasmic RBM4 interacts with Ago2 to regulate inflammatory responses by affecting mRNA decay and cap-dependent translation. However, it is unclear whether RBM4 functions in inflammation regulation by its splicing factor role. Here, the cell biology, gene expression profile and alternative splicing pattern of HeLa cells with RBM4 overexpression (RBM-OE) were compared with the control. The results showed that RBM4-OE inhibited proliferation. RBM4-OE extensively affects the transcriptional level of genes involved in cell surface receptor signalling pathway, inflammatory responses and the response to lipopolysaccharide. RBM4 broadly regulated the alternative splicing of hundreds of genes with functions of protein binding, helicase activity, DNA binding and transcription co-activator. RBM4-regulated splicing of these genes plays an important role in apoptotic process and gene transcription regulation. As an example, exon inclusion of TNIP1 mediated by RBM4 affects the expression of its targets in inflammatory pathways. These results indicated that RBM4 can mediate the inflammatory response via splicing regulation, which adds to the understanding of the critical role of RBM4 in cancer complicated by inflammation. In conclusion, this study indicated a mechanism in which the dysregulation of alternative splicing can influence cellular biology and lead to various immune-related diseases.

Tristetraprolin specifically regulates the expression and alternative splicing of immune response genes in HeLa cells.

BACKGROUND: Tristetraprolin (TTP) is an RNA binding protein that plays a critical role in regulating proinflammatory immune responses by destabilizing target mRNAs via binding to their AU-rich elements (AREs) in the 3'-UTRs of mRNAs. A recent CLIP-seq study revealed that TTP-binding sites are enriched in the intronic regions of RNA. TTP is also a nuclear protein that exhibits putative DNA-binding activity. These features suggested that TTP might regulate gene transcription and/or alternative splicing of pre-mRNAs in the absence of stimulation. RESULTS: To elucidate the regulatory pattern of TTP, we cloned and overexpressed the human TTP-encoding gene, ZFP36, in HeLa cells in the absence of inflammatory stimuli. The transcriptomes of the control and ZFP36-overexpressing cells were sequenced and subjected to analysis and validation. Upon ZFP36 overexpression, the expression of genes associated with innate immunity, including those in the type I interferon signaling pathway and viral response, were specifically upregulated, implying a transcriptional regulatory mechanism associated with the predicted DNA binding activity of TTP. TTP preferentially regulated the alternative splicing of genes involved in the positive regulation of the I-κB/NF-κB cascade and the TRIF-dependent toll-like receptor, MAPK, TNF, and T cell receptor signaling pathways. CONCLUSIONS: Our findings indicated that TTP may regulate the immune response via the regulation of alternative splicing and potentially transcription, which greatly expands the current understanding of the mechanisms of TTP-mediated gene regulation.

CRKL regulates alternative splicing of cancer-related genes in cervical cancer samples and HeLa cell.

BACKGROUND: Aberrant spliced isoforms are specifically associated with cancer progression and metastasis. The cytoplasmic adaptor CRKL (v-crk avian sarcoma virus CT10 oncogene homolog-like) is a CRK like proto-oncogene, which encodes a SH2 and SH3 (src homology) domain-containing adaptor protein. CRKL is tightly linked to leukemia via its binding partners BCR-ABL and TEL-ABL, upregulated in multiple types of human cancers, and induce cancer cell proliferation and invasion. However, it remains unclear whether signaling adaptors such as CRKL could regulate alternative splicing. METHODS: We analyzed the expression level of CRKL in 305 cervical cancer tissue samples available in TCGA database, and then selected two groups of cancer samples with CRKL differentially expressed to analyzed potential CRKL-regulated alternative splicing events (ASEs). CRKL was knocked down by shRNA to further study CRKL-regulated alternative splicing and the activity of SR protein kinases in HeLa cells using RNA-Seq and Western blot techniques. We validated 43 CRKL-regulated ASEs detected by RNA-seq in HeLa cells, using RT-qPCR analysis of HeLa cell samples and using RNA-seq data of the two group of clinical cervical samples. RESULTS: The expression of CRKL was mostly up-regulated in stage I cervical cancer samples. Knock-down of CRKL led to a reduced cell proliferation. CRKL-regulated alternative splicing of a large number of genes were enriched in cancer-related functional pathways, among which DNA repair and G2/M mitotic cell cycle, GnRH signaling were shared among the top 10 enriched GO terms and KEGG pathways by results from clinical samples and HeLa cell model. We showed that CRKL-regulated ASEs revealed by computational analysis using ABLas software in HeLa cell were highly validated by RT-qPCR, and also validated by cervical cancer clinical samples. CONCLUSIONS: This is the first report of CRKL-regulation of the alternative splicing of a number of genes critical in tumorigenesis and cancer progression, which is consistent with CRKL reported role as a signaling adaptor and a kinase. Our results underline that the signaling adaptor CRKL might integrate the external and intrinsic cellular signals and coordinate the dynamic activation of cellular signaling pathways including alternative splicing regulation.

Iron metabolism protein transferrin receptor 1 involves in cervical cancer progression by affecting gene expression and alternative splicing in HeLa cells.

BACKGROUND: Transferrin receptor 1 (TfR1), encoded by TFRC, is a key regulator of iron homeostasis and plays important roles in many diseases, including cancers. OBJECTIVE: To decipher the underlying molecular functions of TfR1 based on its influence on transcriptome profile in cancer cells. METHODS: In this study, we first identified the expression pattern and prognostic influence of TFRC in cervical cancer patients from TCGA database. To explore the regulatory outcomes of TfR1 from the view of whole transcriptome profile, we generated TFRC knockdown (TFRC-KD) HeLa cells and negative control (NC) cells using short hairpin RNA (shRNA) method. Unbiased transcriptome sequencing (RNA-seq) experiment was used to analyze the global expression level and alternative splicing (AS) changes between TFRC-KD and NC cells. RESULTS: We found TFRC was consistently elevated in cervical cancer samples and tightly associated with prognosis of patients. Differential expression analysis revealed that 629 differentially expressed genes (DEGs) were identified between TFRC-KD and NC. Functional enrichment analysis of these DEGs revealed that TFRC-KD extensively disturbed cell physiology related pathways, including immunity, cell metabolism and gene expression. Moreover, dysregulated AS profile also indicated that TfR1 has important roles in the AS regulation. Hundreds of TfR1-regulated AS genes were involved in DNA repair, cell death, transcription and viral reproduction pathways, which were tightly associated with cancer cell progression. CONCLUSIONS: In summary, we for the first time explored the molecular functions of TfR1 at transcriptional and post-transcriptional levels. These results demonstrate TfR1 participates in the progression of cervical cancer by affecting the expression and AS levels of genes in cancer associated pathways, which greatly extends our understanding of TfR1 functions besides iron homeostasis and provide novel options in cancer treatment by targeting TfR1.

Transcriptome analysis reveals the potential biological function of FSCN1 in HeLa cervical cancer cells.

Fascin actin-bundling protein 1 (FSCN1), an actin-bundling protein associated with cell migration and invasion, is highly expressed in various tumor tissues. FSCN1 has also been reported to be a marker of increased invasive potential in cervical cancers. However, the functions of FSCN1 are still not fully understood in cervical cancers. Here, the gene expression profile of HeLa cells transfected with FSCN1 shRNA (shFSCN1) was compared with that of cells transfected with empty vector (shCtrl). The results showed that shFSCN1 extensively affected the transcription level of 5,043 genes in HeLa cells. In particular, Gene Ontology (GO) analysis showed that a large number of upregulated genes were annotated with terms including transcription regulation and DNA binding. The downregulated genes were enriched in some cancer pathways, including angiogenesis and cell adhesion. qPCR validation confirmed that FSCN1 knockdown significantly affected the expression of selected genes in HeLa cells either negatively or positively. Expression analysis in TCGA (The Cancer Genome Atlas) revealed that FSCN1 had negative correlations with several transcription factors and a positive correlation with an angiogenic factor (angiopoietin like 4, ANGPTL4) in cervical tumor tissue. In particular, validation by Western blotting showed that FSCN1 knockdown decreased the protein level of ANGPTL4. Our results demonstrated that FSCN1 is not only an actin-binding protein but also a transcriptional regulator and an angiogenic factor in cervical cancer. Thus, our study provides important insights for further study on the regulatory mechanism of FSCN1 in cervical cancer.

Attenuated Duck Hepatitis A Virus Infection Is Associated With High mRNA Maintenance in Duckling Liver via m6A Modification.

Host translation is generally modulated by viral infection, including duck hepatitis A virus (DHAV) infection. Previously, we reported that cellular protein synthesis in a cell model of duck embryo fibroblasts is significantly inhibited by DHAV infection but not viral proteins, suggesting that an important viral-host interaction occurs at the translational level. In this study, we aim to further understand the impact of DHAV virulence on cellular N6-methyladenosine (m6A) modification, which is essential to a wide variety of RNA biological processes, such as mRNA stabilization and translation. Using m6A antibody-based immunoprecipitation, m6A-seq, and LC-MS/MS, we observed that m6A-modified mRNA exists in both virulent and attenuated DHAV-infected duckling livers. Importantly, m6A levels in mRNA were much higher in attenuated DHAV-infected livers compared with virulent DHAV-infected livers, suggesting virulence-dependent regulation of m6A modification. Analysis of modification motifs indicated that GAAGAAG is the most enriched motif. Combined m6A-seq and RNA-seq data analysis indicated a generally positive correlation between m6A and mRNA expression levels in DHAV-infected duckling livers. GO analysis of genes with decreased or increased m6A levels showed that these genes were enriched in various terms, including oxidation-reduction processes and antiviral immune responses. Collectively, our work reveals DHAV virulence-dependent coordination between m6A modification and mRNA expression in duckling livers.

TCTP participates in hepatic metabolism by regulating gene expression involved in insulin resistance.

Translationally controlled tumor protein (TCTP) has various cellular functions and molecular interactions, many related to its growth-promoting and antiapoptotic properties. Recently, TCTP expression was reported to increases in insulin-resistant mice fed with high-fat diet. TCTP is a multifunctional protein, but its role in liver metabolism is unclear. Here, we investigated the function and mechanism of TCTP in HepG2 cells. Knock-down of TCTP led to 287 differentially expressed genes (DEGs) that were highly associated with cellular apoptosis and signal response, TNF and NF-κB signaling pathways, glycolysis/gluconeogenesis, insulin resistance, FoxO and insulin signaling pathways, adipocytokine and AMPK signaling pathways. shTCTP downregulated the expression of the key gluconeogenesis enzyme phosphoenolpyruvate carboxykinase (PCK1). Furthermore, TCTP regulated the alternative splicing of genes enriched in the phospholipid biosynthetic process and glycerophospholipid metabolism. We further showed that shTCTP down-regulated the intracellular levels of triglyceride and total cholesterol. Our results showed that TCTP regulates the liver cell transcriptome at both the transcriptional and alternative splicing levels. The TCTP regulatory network predicts the biological functions of TCTP in glucose and lipid metabolism, and also insulin resistance, which may be associated with liver metabolism and diseases such as nonalcoholic fatty liver disease.

DDX41 regulates the expression and alternative splicing of genes involved in tumorigenesis and immune response.

DEAD­box helicase 41 (DDX41) is an RNA helicase and accumulating evidence has suggested that DDX41 is involved in pre­mRNA splicing during tumor development. However, the role of DDX41 in tumorigenesis remains unclear. In order to determine the function of DDX41, the human DDX41 gene was cloned and overexpressed in HeLa cells. The present study demonstrated that DDX41 overexpression inhibited proliferation and promoted apoptosis in HeLa cells. RNA­sequencing analysis of the transcriptomes in overexpressed and normal control samples. DDX41 regulated 959 differentially expressed genes compared with control cells. Expression levels of certain oncogenes were also regulated by DDX41. DDX41 selectively regulated the alternative splicing of genes in cancer­associated pathways including the EGFR and FGFR signaling pathways. DDX41 selectively upregulated the expression levels of five antigen processing and presentation genes (HSPA1A, HSPA1B, HSPA6, HLA­DMB and HLA­G) and downregulated other immune­response genes in HeLa cells. Additionally, DDX41­regulated oncogenes and antigen processing and presentation genes were associated with patient survival rates. Moreover, DDX41 expression was associated with immune infiltration in cervical and endocervical squamous cancer. The present findings showed that DDX41 regulated the cancer cell transcriptome at both the transcriptional and alternative splicing levels. The DDX41 regulatory network predicted the biological function of DDX41 in suppressing tumor cell growth and regulating cancer immunity, which may be important for developing anticancer therapeutics.

An integrated analysis of lncRNA and mRNA expression profiles in the kidneys of mice with lupus nephritis.

Long noncoding RNAs (lncRNAs) are persistently expressed and have been described as potential biomarkers and therapeutic targets in various diseases. However, there is limited information regarding lncRNA expression in the tissue of kidney exhibiting lupus nephritis (LN)a serious complication of systemic lupus erythematosus (SLE). In this study, RNA sequencing (RNA-seq) was performed to characterize the lncRNA and mRNA expression in kidney tissues from LN (MRL/lpr) and control mice. We identified 12,979 novel lncRNAs in mouse. The expression profiles of both mRNAs and lncRNAs were differed significantly between LN and control mice. In particular, there were more upregulated lncRNAs and mRNAs than downregulated ones in the kidney tissues of LN mice. However, GO analysis showed that more downregulated genes were enriched in immune and inflammatory response-associated pathways. KEGG analysis showed that both downregulated and upregulated genes were enriched in a number of pathways, including the SLE pathway, and approximately half of these SLE-associated genes encoded inflammatory factors. Moreover, we observed that 2,181 DElncRNAs may have targeted and regulated the expression of 778 mRNAs in LN kidney tissues. The results of this study showed that 11 DElncRNAs targeted and were co-expressed with six immune and SLE-associated genes. qPCR analysis confirmed that lncRNA Gm20513 positively regulated the expression of the SLE-associated gene H2-Aa. In conclusion, the results of our study demonstrates that lncRNAs influence the progression of LN and provide some cues for further study of lncRNAs in LN. These results regarding the lncRNA-mRNAregulatory network may have important value in LN diagnosis and therapy.

The m6A Reader IGF2BP2 Regulates Macrophage Phenotypic Activation and Inflammatory Diseases by Stabilizing TSC1 and PPARγ.

Phenotypic polarization of macrophages is regulated by a milieu of cues in the local tissue microenvironment. Currently, little is known about how the intrinsic regulators modulate proinflammatory (M1) versus prohealing (M2) macrophages activation. Here, it is observed that insulin-like growth factor 2 messenger RNA (mRNA)-binding protein 2 (IGF2BP2)-deleted macrophages exhibit enhanced M1 phenotype and promote dextran sulfate sodium induced colitis development. However, the IGF2BP2-/- macrophages are refractory to interleukin-4 (IL-4) induced activation and alleviate cockroach extract induced pulmonary allergic inflammation. Molecular studies indicate that IGF2BP2 switches M1 macrophages to M2 activation by targeting tuberous sclerosis 1 via an N6-methyladenosine (m6A)-dependent manner. Additionally, it is also shown a signal transducer and activators of transcription 6 (STAT6)-high mobility group AT-hook 2-IGF2BP2-peroxisome proliferator activated receptor-γ axis involves in M2 macrophages differentiation. These findings highlight a key role of IGF2BP2 in regulation of macrophages activation and imply a potential therapeutic target of macrophages in the inflammatory diseases.

Erratum to: TfR1 Extensively Regulates the Expression of Genes Associated with Ion Transport and Immunity.

It is hereby certified that there is no shared co-first authorship in this paper. Dr. Nan HUANG is the only first author of this article.

TfR1 Extensively Regulates the Expression of Genes Associated with Ion Transport and Immunity.

Transferrin receptor 1 (TfR1), encoded by the TFRC gene, is the gatekeeper of cellular iron uptake for cells. A variety of molecular mechanisms are at work to tightly regulate TfR1 expression, and abnormal TfR1 expression has been associated with various diseases. In the current study, to determine the regulation pattern of TfR1, we cloned and overexpressed the human TFRC gene in HeLa cells. RNA-sequencing (RNA-seq) was used to analyze the global transcript levels in overexpressed (OE) and normal control (NC) samples. A total of 1669 differentially expressed genes (DEGs) were identified between OE and NC. Gene ontology (GO) analysis was carried out to explore the functions of the DEGs. It was found that multiple DEGs were associated with ion transport and immunity. Moreover, the regulatory network was constructed on basis of DEGs associated with ion transport and immunity, highlighting that TFRC was the node gene of the network. These results together suggested that precisely controlled TfR1 expression might be not only essential for iron homeostasis, but also globally important for cell physiology, including ion transport and immunity.

Tristetraprolin-RNA interaction map reveals a novel TTP-RelB regulatory network for innate immunity gene expression.

Tristetraprolin (TTP) regulates inflammatory and immune responses by destabilizing target mRNAs via binding to their 3'-UTR AREs. We have recently reported that TTP preferentially up-regulates the expression level of innate immunity genes involved in the type I interferon-mediated signaling pathway and viral response in cancer cells. To elucidate the role of TTP-RNA interaction in TTP-mediated upregulation of gene expression, we performed iRIP-seq experiments to obtain the RNA interaction map consisting of direct and indirect binding sites of TTP in HeLa cells. We found substantial TTP binding signals in mRNA regions and the introns. ARE-motif AUUUA is over-represented in TTP binding peaks. Strikingly, AUUUA frequency is high both in 3'UTR and intronic regions, and the intronic peaks were more associated with TTP-regulated genes. Analysis of the over-represented motifs in TTP peaks revealed the high frequencies of UAGG and GUGUG motifs reported for hnRNPA2/B1 and CELF1 respectively in the 3'UTR and introns, and also the UGGAC motif overlapping with the m6A motif GGACU in the CDS regions. We further demonstrated that TTP binds to multiple intronic and exonic sites in the pre-mRNA/mRNA of the transcription factor RelB, correlating with the TTP-upregulated expression of RelB. TTP-up-regulated genes without a TTP binding site, but not those with, are highly enriched in innate immunity pathways and show higher tendency of harboring RelB binding sites in their promoter regions. These findings support a model in which TTP binding of RelB pre-mRNA/mRNA coordinates the RelB upregulation and activation of the innate immunity for antiviral response.

Poly(A)-seq: A method for direct sequencing and analysis of the transcriptomic poly(A)-tails.

Poly(A) tails at the 3' end of eukaryotic messenger RNAs control mRNA stability and translation efficiency. Facilitated by various NGS methods, alternative polyadenylation sites determining the 3'-UTR length of gene transcripts have been extensively studied. However, poly(A) lengths demonstrating dynamic and developmental regulation remain largely unexplored. The recently developed NGS-based methods for genome-wide poly(A) profiling have promoted the study of genom-wide poly(A) dynamics. Here we present a straight forward NGS-method for poly(A) profiling, which applies a direct 3'-end adaptor ligation and the template switching for 5'-end adaptor ligation for cDNA library construction. Poly(A) lengths are directly calculated from base call data using a self-developed pipeline pA-finder. The libraries were directly sequenced from the 3'-UTR regions into the followed poly(A) tails, firstly on NextSeq 500 to produce single-end 300-nt reads, demonstrating the method feasibility and that optimization of the fragmented RNA size for cDNA library construction could detecting longer poly (A) tails. We next applied Poly(A)-seq cDNA libraries containing 40-nt and 120-nt poly(A) tail spike-in RNAs on HiSeq X-ten and NovaSeq 6000 to obtain 150-nt and 250-nt pair-end reads. The sequencing profiles of the spike-in RNAs demonstrated both high accuracy and high quality score in reading poly(A) tails. The poly(A) signal bleeding into the 3' adaptor sequence and a sharp decreased quality score at the junction were observed, allowing the modification of pA-finder to remove homopolymeric signal bleeding. We hope that wide applications of Poly(A)-seq help facilitate the study of the development- and disease-related poly(A) dynamics and regulation, and of the recent emerging mixed tailing regulation.

hnRNPDL extensively regulates transcription and alternative splicing.

RNA binding proteins (RBPs) are key players of genome regulation. Here we report the transcriptome study of HnRNP D-Like protein, which belongs to the hnRNP family. We used RNA-seq to analyze the global transcript level and alternative splicing on hnRNPDL shRNA-treated cells and control. Sh-hnRNPDL extensively increased in the expression of genes involved in female pregnancy, cell apoptosis, cell proliferation and cell migration. HnRNPDL regulated alternative splicing of hundreds of genes enriched in transcription regulation and signaling pathways including NOD-like receptor signaling, Notch signaling, and TNF signaling. This study provides the first transcriptome-wide analysis of hnRNPDL regulation of gene expression, which adds to the understanding of critical hnRNPDL functions.

Grape seed proanthocyanidins inhibit proliferation of pancreatic cancer cells by modulating microRNA expression.

MicroRNAs (miRNAs) are small non-coding RNAs of 18-25 nucleotides that modulate gene expression at the post-transcriptional level. Grape seed proanthocyanidins (GSPs), which are biologically active components in grape seeds, have been demonstrated to exhibit anticancer effects. The current study investigated whether GSPs can regulate miRNA expression and the possible anticancer molecular mechanisms of GSPs. Pancreatic cancer (PC) cell samples, SS3, SS12 and SS24, were treated with 20 µg/ml GSPs for 3, 12 and 24 h, respectively. Control samples, SC3, SC12 and SC24, were also prepared. Using miRNA-seq, transcriptome analysis identified 24, 83 and 83 differentially expressed (DE) miRNAs in SS3 vs. SC3, SS12 vs. SC12 and SS24 vs. SC24, respectively. This indicated that treatment with GSPs could modulate the expression of miRNAs. Subsequently, 74, 598 and 1,204 target genes for the three sets of DE miRNAs were predicted. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed that multiple target genes were associated with the proliferation and apoptosis of PC cells. In addition, a network was constructed of the DE miRNAs and the target genes associated with PC. The associations identified suggested that treatment with GSPs may inhibit the proliferation of PC cells through the modulation of miRNA expression.

Transcriptome profiling reveals transcriptional and alternative splicing regulation in the early embryonic development of hair follicles in the cashmere goat.

The undercoat fiber of the cashmere goat, from the secondary hair follicle (HF), possesses commercial value. However, very few studies have focused on the molecular details of primary and secondary HF initiation and development in goat embryos. In this study, skin samples at embryonic day 45, 55, and 65 (E45, E55, and E65) were collected and prepared for RNA sequencing (RNA-seq). We found that the HF probably initiated from E55 to E65 by analyzing the functional pathways of differentially expressed genes (DEGs). Most key genes in canonical signaling pathways, including WNT, TGF-ß, FGF, Hedgehog, NOTCH, and other factors showed clear expression changes from E55 to E65. We, for the first time, explored alternative splicing (AS) alterations, which showed distinct patterns among these three stages. Functional pathways of AS-regulated genes showed connections to HF development. By comparing the published RNA-seq samples from the E60, E120, and newborn (NB) stages, we found the majority of WNT/ß-catenin signaling genes were important in the initiation of HF development, while other factors including FOXN1, GATA3, and DLX3 may have a consistent influence on HF development. Our investigation supported the time points of embryonic HF initiation and identified genes that have potential functions of embryonic HF initiation and development. We further explored the potential regulatory roles of AS in HF initiation, which extended our knowledge about the molecular mechanisms of HF development.