特异性蛋白1(SP1)上调FOXP4-AS1促进结直肠癌细胞增殖

大量的证据表明,长链非编码RNAs (long non-coding RNAs)在结直肠癌发生发展中发挥重要作用。有迹象表明,lncRNA FOXP4-AS1推动结直肠癌的进程。本文通过数据库信息分析发现,FOXP4-AS1在结直肠癌中高表达且与患者较差的预后正相关。实时荧光定量PCR方法也证实,FOXP4-AS1在结直肠癌细胞和组织中的表达量均高于正常细胞和组织。其中,FOXP4-AS1在结肠癌细胞LOVO中表达量最高,是正常结直肠细胞的13倍。生物信息学预测结合双荧光素酶报告基因实验和染色质沉淀研究结果表明,特异性蛋白1（specificity protein 1,SP1）可直接结合在FOXP4-AS1启动子上,上调其活性。过表达FOXP4-AS1,可下调p16和p18表达,同时上调CDK4、CDK6和细胞周期蛋白 D1表达,最终促进结直肠癌细胞增殖。相反,敲低FOXP4-AS1将上调p16和p18表达,抑制CDK4、CDK6和细胞周期蛋白 D1,获得相反的结果。总之,特异性蛋白1(SP1)上调FOXP4-AS1,促进结直肠癌细胞增殖。     

Association between SNP rs527616 in lncRNA AQP4-AS1 and susceptibility to breast cancer in a southern Brazilian population

Breast cancer (BC) is the leading cause of death by this disease in women worldwide. Among the factors involved in tumorigenesis, long non-coding RNAs (lncRNAs) and their differential expression have been associated. Differences in gene expression may be triggered by variations in DNA sequence, including single nucleotide polymorphisms (SNPs). In the present study, we analyzed the rs527616 (C>G), located in the lncRNA AQP4-AS1, using PCR-SSP in 306 BC patients and 312 controls, from a Brazilian population. In the BC group, the frequency found for CG heterozygotes was above the expected and the overdominant model is the best one to explain our results (OR: 1.70, IC 95%: 1.23-2.34, P<0.001). Furthermore, the SNP were associated with age at BC diagnosis and the risk genotype more frequent in the older age group. According to TCGA data, AQP4-AS1 is down-regulated in BC tissue, and the overexpression is associated with better prognoses, including Luminal A, HER2-, stage 1 of disease and smaller tumor. In conclusion, the CG genotype is associated with increased susceptibility in the southern Brazilian population. This SNP is mapped in the lncRNA AQP4-AS1, showing differential expression in BC samples. Based on these results, we emphasize the potential of the role of AQP4-AS1 in cancer.     

Comprehensive characterization of the alternative splicing landscape in ovarian cancer reveals novel events associated with tumor-immune microenvironment

Background: Ovarian cancer (OV) is a serious threat to women’s health. Immunotherapy is a new approach. Alternative splicing (AS) of messenger RNA (mRNA) and its regulation are highly relevant for understanding every cancer hallmark and may offer a broadened target space.Methods: We downloaded the clinical information and mRNA expression profiles of 587 tumor tissues from The Cancer Genome Atlas (TCGA) database. We constructed a risk score model to predict the prognosis of OV patients. The association between AS-based clusters and tumor-immune microenvironment features was further explored. The ESTIMATE algorithm was also carried out on each OV sample depending on the risk score groups. A total of three immune checkpoint genes that have a significant correlation with risk scores were screened.Results: The AS-events were a reliable and stable independent risk predictor in the OV cohort. Patients in the high-risk score group had a poor prognosis (P<0.001). Mast cells activated, NK cells resting, and Neutrophils positively correlated with the risk score. The number of Macrophages M1 was also more numerous in the low-risk score group (P<0.05). Checkpoint genes CD274, CTLA-4, and PDCD1LG2, showed a negative correlation with the risk score of AS in OV.Conclusions: The proposed AS signature is a promising biomarker for estimating overall survival (OS) in OV. The AS-events signature combined with tumor-immune microenvironment enabled a deeper understanding of the immune status of OV patients, and also provided new insights for exploring novel prognostic predictors and precise therapy methods.     

RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides –9 to –3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides –8 to –3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an ~7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix–loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix–loop groove, which may also play a role in RNA binding.     

LncRNA OIP5-AS1/cyrano sponges RNA-binding protein HuR

The function of the vast majority of mammalian long noncoding (lnc) RNAs remains unknown. Here, analysis of a highly abundant mammalian lncRNA, OIP5-AS1, known as cyrano in zebrafish, revealed that OIP5-AS1 reduces cell proliferation. In human cervical carcinoma HeLa cells, the RNA-binding protein HuR, which enhances cell proliferation, associated with OIP5-AS1 and stabilized it. Tagging OIP5-AS1 with MS2 hairpins to identify associated microRNAs revealed that miR-424 interacted with OIP5-AS1 and competed with HuR for binding to OIP5-AS1. We further identified a ‘sponge’ function for OIP5-AS1, as high levels of OIP5-AS1 increased HuR-OIP5-AS1 complexes and prevented HuR interaction with target mRNAs, including those that encoded proliferative proteins, while conversely, lowering OIP5-AS1 increased the abundance of HuR complexes with target mRNAs. We propose that OIP5-AS1 serves as a sponge or a competing endogenous (ce)RNA for HuR, restricting its availability to HuR target mRNAs and thereby repressing HuR-elicited proliferative phenotypes.     

Circular RNA circABCC4 as the ceRNA of miR‐1182 facilitates prostate cancer progression by promoting FOXP4 expression

In recent years, circular RNAs (circRNAs) have been identified to be essential regulators of various human cancers. However, knowledge of the functions of circRNAs in prostate cancer remains very limited. The correlation between circABCC4 and human cancer is largely unknown. This study aims to investigate the biological functions of circABCC4 in prostate cancer progression and illustrate the underlying mechanism. We found that circABCC4 was remarkably up‐regulated in prostate cancer tissues and cell lines and promoted FOXP4 expression by sponging miR‐1182 in prostate cancer cells. CircABCC4 knockdown markedly suppressed prostate cancer cell proliferation, cell‐cycle progression, migration and invasion in vitro. Furthermore, silencing of the circRNA also delayed tumor growth in vivo. Taken together, our findings indicated that circABCC4 facilitates the malignant behaviour of prostate cancer by promoting FOXP4 expression through sponging of miR‐1182. The circABCC4–miR‐1182‐FOXP4 regulatory loop may be a promising therapeutic target for prostate cancer intervention.     

Synovial explant inflammatory mediator production corresponds to rheumatoid arthritis imaging hallmarks: a cross-sectional study

Introduction

Despite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study was to compare site-specific release of inflammatory mediators and evaluate the corresponding anatomical sites by examining colour Doppler ultrasound (CDUS) and MRI scans.

Methods

RA patients were evaluated on the basis of CDUS and 3-T MRI scans and subsequently underwent synovectomy using a needle arthroscopic procedure of the hand joints. The synovial tissue specimens were incubated for 72 hours, and spontaneous release of monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), macrophage inflammatory protein 1β (MIP-1β) and IL-8 was measured by performing multiplex immunoassays. Bone marrow oedema (BME), synovitis and erosion scores were estimated on the basis of the rheumatoid arthritis magnetic resonance imaging score (RAMRIS). Mixed models were used for the statistical analyses. Parsimony was achieved by omitting covariates with P > 0.1 from the statistical model.

Results

Tissue samples from 58 synovial sites were obtained from 25 patients. MCP-1 was associated with CDUS activity (P = 0.009, approximate Spearman’s ρ = 0.41), RAMRIS BME score (P = 0.01, approximate Spearman’s ρ = 0.42) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.31). IL-6 was associated with RAMRIS synovitis score (P = 0.04, approximate Spearman’s ρ = 0.50), BME score (P = 0.04, approximate Spearman’s ρ = 0.31) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.35). MIP-1β was associated with CDUS activity (P = 0.02, approximate Spearman’s ρ = 0.38) and RAMRIS synovitis scores (P = 0.02, approximate Spearman’s ρ = 0.63). IL-8 associations with imaging outcome measures did not reach statistical significance.

Conclusions

The association between imaging activity and synovial inflammatory mediators underscores the high sensitivity of CDUS and MRI in the evaluation of RA disease activity. The associations found in our present study have different implications for synovial mediator releases and corresponding imaging signs. For example, MCP-1 and IL-6 were associated with both general inflammation and bone destruction, in contrast to MIP-1β, which was involved solely in general synovitis. The lack of association of IL-8 with synovitis was likely underestimated because of a large proportion of samples above assay detection limits among the patients with the highest synovitis scores.     

Long noncoding RNA complementarity and target transcripts abundance

Eukaryotic mRNA metabolism regulates its stability, localization, and translation using complementarity with counter-part RNAs. To modulate their stability, small and long noncoding RNAs can establish complementarity with their target mRNAs. Although complementarity of small interfering RNAs and microRNAs with target mRNAs has been studied thoroughly, partial complementarity of long noncoding RNAs (lncRNAs) with their target mRNAs has not been investigated clearly. To address that research gap, our lab investigated whether the sequence complementarity of two lncRNAs, lincRNA-p21 and OIP5-AS1, influenced the quantity of target RNA expression. We predicted a positive correlation between lncRNA complementarity and target mRNA quantity. We confirmed this prediction using RNA affinity pull down, microarray, and RNA-sequencing analysis. In addition, we utilized the information from this analysis to compare the quantity of target mRNAs when two lncRNAs, lincRNA-p21 and OIP5-AS1, are depleted by siRNAs. We observed that human and mouse lincRNA-p21 regulated target mRNA abundance in complementarity-dependent and independent manners. In contrast, affinity pull down of OIP5-AS1 revealed that changes in OIP5-AS1 expression influenced the amount of some OIP5-AS1 target mRNAs and miRNAs, as we predicted from our sequence complementarity assay. Altogether, the current study demonstrates that partial complementarity of lncRNAs and mRNAs (even miRNAs) assist in determining target RNA expression and quantity.     

SNP-mediated lncRNA-ENTPD3-AS1 upregulation suppresses renal cell carcinoma via miR-155/HIF-1α signaling

Over the last decade, more than 10 independent SNPs have been discovered to be associated with the risk of renal cell carcinoma among different populations. However, the biological functions of them remain poorly understood. In this study, we performed eQTL analysis, ChIP-PCR, luciferase reporter assay, and Cox regression analysis to identify the functional role and underlying mechanism of rs67311347 in RCC. The ENCORI database, which contains the lncRNA–miRNA–mRNA interactions, was used to explore the possible target miRNA of ENTPD3-AS1. The results showed that the G > A mutation of rs67311347 created a binding motif of ZNF8 and subsequently upregulated ENTPD3-AS1 expression by acting as an enhancer. The TCGA-KIRC and our cohorts both confirmed the downregulation of ENTPD3-AS1 in RCC tissues and demonstrated that increased ENTPD3-AS1 expression was associated with good OS and PFS. Furthermore, ENTPD3-AS1 interacted with miR-155-5p and activated the expression of HIF-1α, which was an important tumor suppressor gene in the development of RCC. The functional experiments revealed that overexpression of ENTPD3-AS1 inhibited cell proliferation in RCC cell lines and the effect could be rescued by knocking down HIF-1α. Our findings reveal that SNP-mediated lncRNA-ENTPD3-AS1 upregulation suppresses renal cell carcinoma via miR-155/HIF-1α signaling.Subject terms: Cancer epigenetics, Renal cell carcinoma