结合三代测序与二代测序技术揭示蜜蜂球囊菌孢子转录组的复杂性

【目的】蜜蜂球囊菌(Ascosphaera apis)是一种专性侵染蜜蜂幼虫的致死性真菌病原。本研究旨在利用PacBio单分子实时(singlemoleculereal-time,SMRT)测序技术对蜜蜂球囊菌孢子(AaS)中基因的可变剪切(alternative splicing,AS)和可变多聚腺苷酸化(alternative polyadenylation,APA)以及长链非编码RNA (long non-coding RNA,lncRNA)进行鉴定和分析,进而揭示蜜蜂球囊菌孢子中转录组的复杂性。【方法】采用Suppa软件对蜜蜂球囊菌孢子中基因的AS事件进行鉴定。通过RT-PCR对不同类型的AS事件进行验证。采用TAPIS pipeline对蜜蜂球囊菌孢子基因的APA位点进行鉴定。利用MEME软件分析孢子全长转录本的poly(A)剪接位点上游50bp的序列特征并鉴定motif。联用CPC和CNCI软件和比对Swiss-prot数据库的方法预测lncRNA,取三者的交集作为高可信度的lncRNA集合。进一步比较lncRNA和mRNA的转录本长度,外显子数量与长度,内含子长度,GC含...     

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.     

LncRNA ZEB1‐AS1 down‐regulation suppresses the proliferation and invasion by inhibiting ZEB1 expression in oesophageal squamous cell carcinoma

Multiple studies have unveiled that long non‐coding RNAs (lncRNAs) play a pivotal role in tumour progression and metastasis. However, the biological role of lncRNA ZEB1‐AS1 in oesophageal squamous cell carcinoma (ESCC) remains under investigation, and thus, the current study was to investigate the functions of ZEB1‐AS1 in proliferation and invasion of ESCC. Here, we discovered that ZEB1‐AS1 and ZEB1 were markedly up‐regulated in ESCC tissues and cells relative to their corresponding normal control. ZEB1‐AS1 and ZEB1 overexpressions were both related to TNM staging and lymph node metastasis as well as poor prognosis in ESCC. The hypomethylation of ZEB1‐AS1 promoter triggered ZEB1‐AS1 overexpression in ESCC tissues and cells. In addition, ZEB1‐AS1 knockdown mediated by siRNA markedly suppressed the proliferation and invasion in vitro in EC9706 and TE1 cells, which was similar with ZEB1 siRNA treatment, coupled with EMT alterations including the up‐regulation of E‐cadherin level as well as the down‐regulation of N‐cadherin and vimentin levels. Notably, ZEB1‐AS1 depletion dramatically down‐regulated ZEB1 expression in EC9706 and TE1 cells, and ZEB1 overexpression obviously reversed the inhibitory effects of proliferation and invasion triggered by ZEB1‐AS1 siRNA. ZEB1‐AS1 shRNA evidently inhibited tumour growth and weight, whereas ZEB1 elevation partly recovered the tumour growth in ESCC EC9706 and TE1 xenografted nude mice. In conclusion, ZEB1‐AS1 overexpression is tightly involved in the development and progression of ESCC, and it exerts the antitumour efficacy by regulating ZEB1 level in ESCC.     

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.     

Comprehensive bioinformatics analysis identifies lncRNA HCG22 as a migration inhibitor in esophageal squamous cell carcinoma

Esophageal cancer is one of the most lethal malignancies worldwide, and esophageal squamous cell carcinoma (ESCC) is the dominant histological type. However, the long noncoding RNA (lncRNA) alterations in ESCC have not been elucidated to date. In this study, reliable databases from Gene Expression Omnibus (GEO), which analyzed lncRNA expression in ESCC tumor tissues and adjacent normal tissues were searched, and common differentially expressed lncRNAs and genes were analyzed. Next, cis- trans analysis was performed to predict the underlying relationships between altered lncRNAs and mRNAs, and the lncRNA-mRNA regulatory network was established. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of altered lncRNA-related genes were performed. The promising lncRNA HCG22 was validated by quantitative polymerase chain reaction (qPCR), and clinicopathological data were collected to identify the relationship between lncRNA HCG22 expression level and clinical features. Finally, Transwell assays were performed to explore the biological functions of lncRNA HCG22 in ESCC cells. Two hundred forty-one lncRNAs and 835 mRNAs were observed to be remarkably altered between ESCC tumor tissues and adjacent normal tissues. The lncRNA-mRNA regulatory network showed the coexpression association between lncRNA HCG22 and SPINK7 and ADAMTS12. GO and KEGG analyses showed that HCG22 and ADAMTS12 had potential biological functions in the cell migration of ESCC. The downregulation of lncRNA HCG22 in ESCC tumor tissues was validated by qPCR, and the clinicopathological data showed a noticeable correlation between lncRNA HCG22 expression level and the ESCC differentiational degree and clinical TNM stage. Kaplan-Meier analysis showed that patients with ESCC having low lncRNA HCG22 expression in ESCC tissues had considerably shorter overall survival compared with patients with ESCC having high lncRNA HCG22 expression. Following Transwell assays confirmed the migratory role of lncRNA HCG22 in ESCC cells. In conclusion, lncRNA HCG22 was downregulated in ESCC tissues and can be a migration inhibitor of ESCC cells, and SPINK7 and ADAMTS12 are promising to be the regulatory targets of lncRNA HCG22.     

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.     

The Molecular Mechanism of Long Non-Coding RNA MALAT1-Mediated Regulation of Chondrocyte Pyroptosis in Ankylosing Spondylitis

Long non-coding RNAs (lncRNAs) may be important regulators in the progression of ankylosing spondylitis (AS). The competing endogenous RNA (ceRNA) activity of lncRNAs plays crucial roles in osteogenesis. We identified the mechanism of the differentially expressed lncRNA MALAT1 in AS using bioinformatic analysis and its ceRNA mechanism. The interaction of MALAT1, microRNA-558, and GSDMD was identified using integrated bioinformatics analysis and validated. Loss- and gain-of-function assays evaluated their effects on the viability, apoptosis, pyroptosis and inflammation of chondrocytes in AS. We found elevated MALAT1 and GSDMD but reduced miR-558 in AS cartilage tissues and chondrocytes. MALAT1 contributed to the suppression of cell viability and facilitated apoptosis and pyroptosis in AS chondrocytes. GSDMD was a potential target gene of miR-558. Depletion of MALAT1 expression elevated miR-558 by inhibiting GSDMD to enhance cell viability and inhibit inflammation, apoptosis and pyroptosis of chondrocytes in AS. In summary, our key findings demonstrated that knockdown of MALAT1 served as a potential suppressor of AS by upregulating miR-558 via the downregulation of GSDMD expression.     

LncRNA DGCR5 represses the development of hepatocellular carcinoma by targeting the miR-346/KLF14 axis

Long non-coding RNAs (lncRNAs) are a class of regulatory noncoding RNAs. Emerging evidence highlights the critical roles of lncRNAs in the progression of hepatocellular carcinoma (HCC). Although many lncRNAs have been identified in the development of HCC, the association between DiGeorge syndrome critical region gene 5 (DGCR5) and HCC remains unclear. In the current study, we focused on the biological role of DGCR5 in HCC. We observed that DGCR5 was decreased in HCC cells, including SMCC7721, Hep3B, HepG2, MHCC-97L, MHCC-97H, and SNU449 hepatocellular carcinoma cells, compared with the normal human liver cell line THLE-3 normal human liver cells. In addition, DGCR5 overexpression could repress HCC cell growth, migration, and invasion considerably. Increasing studies have indicated the interactions between lncRNAs and microRNAs. MicroRNAs are endogenous small noncoding RNAs and they can play important roles in tumorigenesis. MicroRNA 346 (miR-346) has been demonstrated in various human cancer types, including HCC. MiR-346 was found to be increased in HCC cells and DGCR5 can act as a sponge of miR-346 to modulate the progression of HCC. The binding correlation between DGCR5 and miR-346 was validated in our research. Subsequently, Krüppel-like factor 14 (KLF14) was predicted as a downstream target of miR-346 and miR-346 can induce the development of HCC by inhibiting KLF14. Finally, we proved that DGCR5 can rescue the inhibited levels of KLF14 repressed by miR-346 mimics in MHCC-97H and Hep3B cells. Taken together, it was indicated in our study that DGCR5 can restrain the progression of HCC through sponging miR-346 and modulating KLF14 in vitro.     

Long non-coding RNA ZNF667-AS1 retards the development of esophageal squamous cell carcinoma via modulation of microRNA-1290-mediated PRUNE2

Abnormal long non-coding RNAs (lncRNAs) have been detected in esophageal squamous cell carcinoma (ESCC). Here, we focused on lncRNA ZNF667-AS1 and its downstream mechanism in ESCC progression. Differentially expressed lncRNAs in ESCC were predicted by bioinformatics analysis. ZNF667-AS1, microRNA-1290 (miR-1290), and prune homolog 2 with BCH domain (PRUNE2) expression was determined with their relationship in cell biological processes analyzed also by means of gain- and loss-of-function assays. Xenograft mouse models were performed to re-produce the in vitro findings. We found a decline in ZNF667-AS1 expression in ESCC tissues and cell lines. ZNF667-AS1 overexpression indicated a favorable prognosis of ESCC sufferers. ZNF667-AS1 overexpression suppressed ESCC cell malignant potentials. ZNF667-AS1 reduced miR-1290 to result in upregulation of the miR-1290 target gene PRUNE2. The inhibiting property of ZNF667-AS1 on the malignant characteristics of ESCC cells was achieved by disrupting the miR-1290-mediated downregulation of PRUNE2. ZNF667-AS1 suppressed the tumorigenesis of ESCC in vivo. Collectively, our study demonstrates that ZNF667-AS1 can function as a tumor suppressor in ESCC by upregulating PRUNE2 and downregulating miR-1290.     

lncRNA NR2F1‐AS1 promotes breast cancer angiogenesis through activating IGF‐1/IGF‐1R/ERK pathway

Long non‐coding RNAs (lncRNAs) take various effects in cancer mostly through sponging with microRNAs (miRNAs). lncRNA NR2F1‐AS1 is found to promote tumour progression in hepatocellular carcinoma, endometrial cancer and thyroid cancer. However, the role of lncRNA NR2F1‐AS1 in breast cancer angiogenesis remains unknown. In this study, we found lncRNA NR2F1‐AS1 was positively related with CD31 and CD34 in breast cancer through Pearson's correlation analysis, while lncRNA NR2F1‐AS1 transfection promoted human umbilical vascular endothelial cell (HUVEC) tube formation. In breast cancer cells, lncRNA NR2F1‐AS1 enhanced the HUVEC proliferation, tube formation and migration ability through tumour‐conditioned medium (TCM). In zebrafish model, lncRNA NR2F1‐AS1 increased the breast cancer cell‐related neo‐vasculature and subsequently promoted the breast cancer cell metastasis. In mouse model, lncRNA NR2F1‐AS1 promoted the tumour vessel formation, increased the micro vessel density (MVD) and then induced the growth of primary tumour. Mechanically, lncRNA NR2F1‐AS1 increased insulin‐like growth factor‐1 (IGF‐1) expression through sponging miRNA‐338‐3p in breast cancer cells and then activated the receptor of IGF‐1 (IGF‐1R) and extracellular signal‐regulated kinase (ERK) pathway in HUVECs. These results indicated that lncRNA NR2F1‐AS1 could promote breast cancer angiogenesis through IGF‐1/IGF‐1R/ERK pathway.     

Knockdown of DGCR5 enhances the radiosensitivity of human laryngeal carcinoma cells via inducing miR-195

Long noncoding RNAs (lncRNAs) exert critical roles in the development of various cancers, including human laryngeal cancer. Radioresistance contributes to the predominant causes of laryngeal cancer recurrence after radiotherapy. The aim of our study was to investigate the association of dysregulated lncRNA and radiation resistance in human larynx squamous carcinoma. Here, we investigated the biological roles of lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) in radioresistance of human laryngeal cancer. Two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R), with different radiosensitivities in vitro were used in the present study. We observed that DGCR5 was significantly upregulated in Hep-2R cells. Inhibition of DGCR5 by LV-shDGCR5 transfection restrained Hep-2R cell proliferation and sensitized cells to radiation. Reversely, overexpression of DGCR5 exhibited an opposite phenomenon in vitro. In addition, microRNA (miR)-195 was predicted as a direct downstream target of DGCR5. Dual-luciferase reporter and RNA immunoprecipitation assays verified the direct interaction between them. Meanwhile, miR-195 was observed to be reduced in Hep-2R cells and miR-195 mimics repressed Hep-2 cell growth. Moreover, radiosensitivity of Hep-2R cells was greatly enhanced by overexpression of miR-195, which could be reversed by upregulation of DGCR5. Finally, in vivo experiments were used to validate that knockdown of DGCR5 suppressed laryngeal carcinoma via targeting miR-195. In conclusion, we indicated that DGCR5 could contribute to the radioresistance of human laryngeal carcinoma cells via sponging miR-195.     

Systematic analysis of survival-associated alternative splicing signatures in clear cell renal cell carcinoma

Alternative splicing (AS) constitutes a major reason for messenger RNA (mRNA) and protein diversity. Increasing studies have shown a link to splicing dysfunction associated with malignant neoplasia. Systematic analysis of AS events in kidney cancer remains poorly reported. Therefore, we generated AS profiles in 533 kidney renal clear cell carcinoma (KIRC) patients in The Cancer Genome Atlas (TCGA) database using RNA-seq data. Then, prognostic models were developed in a primary cohort (N = 351) and validated in a validation cohort (N = 182). In addition, splicing networks were built by integrating bioinformatics analyses. A total of 11 268 and 8083 AS variants were significantly associated with patient overall survival time in the primary and validation KIRC cohorts, respectively, including STAT1, DAZAP1, IDS, NUDT7, and KLHDC4. The AS events in the primary KIRC cohorts served as candidate AS events to screen the independent risk factors associated with survival in the primary cohort and to develop prognostic models. The area under the curve of the receiver-operator characteristic curve for prognostic prediction in the primary and validation KIRC cohorts was 0.84 and 0.82 at 2500 days of overall survival, respectively. In addition, splicing correlation networks revealed key splicing factors (SFs) in KIRC, such as HNRNPH1, HNRNPU, KHDBS1, KHDBS3, SRSF9, RBMX, SFQ, SRP54, HNRNPA0, and SRSF6. In this study, we analyzed the AS landscape in the TCGA KIRC cohort and detected predictors (prognostic) based on AS variants with high performance for risk stratification of the KIRC cohort and revealed key SFs in splicing networks, which could act as underlying mechanisms.     

Protein phosphatase 1 activation and alternative splicing of Bcl-X and Mcl-1 by EGCG + ibuprofen

Epigallocatechin-3-gallate (EGCG) and ibuprofen synergistically act to suppress proliferation and enhance apoptosis of prostate cancer cell lines, PC-3 and LNCaP. The purpose of this study was to investigate the mechanism of underlying this synergism. Most interestingly, EGCG + ibuprofen treatment in PC-3 cells resulted in altering the ratio of the splice variants of Bcl-X and Mcl-1, downregulating the mRNA levels of anti-apoptotic Bcl-X(L) and Mcl-1(L) with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-X(s) and Mcl-1(s). However, there were no apparent changes in splicing variants in either ibuprofen or EGCG treated cells. Induction of alternative splicing was correlated with increased activity of protein phosphatase 1 (PP1) in EGCG + ibuprofen-treated cells, since pretreatment with calyculin A and tautomycin blocked EGCG + ibuprofen-induced alternative splicing in PC-3 cells in contrast to pretreatment with okadaic acid. On the other hand, EGCG + ibuprofen treatment in LNCaP cells did not alter splicing variants of Bcl-X and Mcl-1, despite the increase in protein phosphatase activity. In both cell lines, EGCG + ibuprofen inhibited cell proliferation synergistically. Taken together, this study demonstrate for the first time that EGCG + ibuprofen upregulated PP1 activity, which in turn induced alternative splicing of Bcl-X and Mcl-1 in a cell-type specific manner. Our study also demonstrates that the activation of PP1 contributes to the alternative splicing of Mcl-1.