Analysis of association between MALAT1 haplotype and the severity of normal-tension glaucoma (NTG)

MALAT1, which is disorderly expressed in the growth, invasion, migration and cancer cell apoptosis, was shown to be associated with normal-tension glaucoma (NTG), a type of optic neuropathy. The haplotype in MALAT1 affects its expression and is correlated with human diseases like normal-tension glaucoma (NTG). However, the underlying detailed mechanism remains unclear. In this study, we aimed to analyse the association between MALAT1 haplotype and the severity of NTG in a molecular level. Quantitative real-time PCR, ELISA and luciferase assays were performed to establish the underlying signalling pathways. RNFL thickness, RA and C/D ratio were calculated for NTG patients. Accordingly, GGGT haplotype was demonstrated to be associated with a decreased risk of NTG. The MALAT1 level in serum of NTG patients carrying GGGT haplotype was significantly decreased compared with NTG patients carrying other haplotypes, along with elevated miR-1 expression and diminished IL-6 expression. NTG patients carrying GGGT haplotype had thicker RNFL and RA, but a smaller C/D ratio. Sequence analysis found potential target sites of miR-1 on MALAT1 and IL-6, and luciferase assay confirmed the inhibitory effect of miR-1 on MALAT1 and IL-6 expression. Meanwhile, MALAT1 also down-regulated miR-1 expression and consequently up-regulated IL-6 expression. This study presented evidence for a regulatory network containing MALAT1, miR-1 and IL-6, and further demonstrated the effect of MALAT1 haplotype on the risk and severity of NTG.     

Identification and development of long non‐coding RNA‐associated regulatory network in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of cancer‐associated death globally. Long non‐coding RNAs (lncRNAs) have been identified as micro RNA (miRNA) sponges in a competing endogenous RNA (ceRNA) network and are involved in the regulation of mRNA expression. This study aims to construct a lncRNA‐associated ceRNA network and investigate the prognostic biomarkers in CRC. A total of 38 differentially expressed (DE) lncRNAs, 23 DEmiRNAs and 27 DEmRNAs were identified by analysing the expression profiles of CRC obtained from The Cancer Genome Atlas (TCGA). These RNAs were chosen to develop a ceRNA regulatory network of CRC, which comprised 125 edges. Survival analysis showed that four lncRNAs, six miRNAs and five mRNAs were significantly associated with overall survival. A potential regulatory axis of ADAMTS9‐AS2/miR‐32/PHLPP2 was identified from the network. Experimental validation was performed using clinical samples by quantitative real‐time PCR (qRT‐PCR), which showed that expression of the genes in the axis was associated with clinicopathological features and the correlation among them perfectly conformed to the ‘ceRNA theory’. Overexpression of ADAMTS9‐AS2 in colon cancer cell lines significantly inhibited the miR‐32 expression and promoted PHLPP2 expression, while ADAMTS9‐AS2 knockdown had the opposite effects. The constructed novel ceRNA network may provide a comprehensive understanding of the mechanisms of CRC carcinogenesis. The ADAMTS9‐AS2/miR‐32/PHLPP2 regulatory axis may serve as a potential therapeutic target for CRC.     

The functional pathway analysis and clinical significance of miR-20a and its related lncRNAs in breast cancer

BackgroundmiR-20a is a critical molecule in various biological processes and cancer progression procedures. However, its relationships with lncRNAs and their functional pathway analysis in breast tumorigenesis are less intensively studied.MethodsThe expression data from TCGA database and multiple bioinformatics resources were used to check the expression levels, survival curves, interactions and functional illustrations of miR-20a and its related lncRNAs (XIST, H19 and MALAT1) in breast cancer patients. The luciferase reporter assays and Pearson's correlation analyses were utilized to verify the direct regulatory relationship between miR-20a and three lncRNAs (XIST, H19 and MALAT1). In vitro cell proliferation, migration and invasion assays, were performed to check the biological effects of miR-20a and XIST in different breast cancer cell lines. The receiver operating characteristic curve (ROC) analyses were done for evaluating diagnostic values of serum miR-20a and XIST in breast cancer patients.ResultsThe miR-20a expression was significantly up-regulated in both breast cancer samples and serum samples, and correlated with poor survival rate in breast cancer patients. LncRNAs (XIST, H19 and MALAT1) directly bound to hsa-miR-20a and were negatively correlated with hsa-miR-20a expression in breast cancer patient samples. For functional illustrations and downstream signaling pathways analysis, XIST, H19 and MALAT1 mainly shared their regulatory functions in cell motility and interleukin signaling in breast cancer progression. Additionally, over-expression of miR-20a and inhibition of XIST promoted breast cancer cell growth, migration and invasion in vitro, and serum miR-20a and XIST served as potential diagnostic biomarkers for breast cancer with the area under ROC curve (AUC) of 0.87 (95% CI = 0.78 to 0.97), and 0.78 (95% CI = 0.67 to 0.89) respectively.ConclusionsTaken together, these findings provide us novel insights and avenues for utilizing miR-20a and its related lncRNAs as potential diagnostic biomarkers and promising therapeutic targets for breast cancer treatment.     

LncRNA NR2F2‐AS1 promotes tumourigenesis through modulating BMI1 expression by targeting miR‐320b in non‐small cell lung cancer

Recently, long noncoding RNAs (lncRNAs) are attracting wide attention in the field of cancer research because of its important role in cancer diagnosis and prognosis. But studies on the biological effects and relevant mechanisms of lncRNAs in non‐small cell lung cancer (NSCLC) remain few and need to be enriched. Our study discussed the expression and biological effects of LncRNA NR2F2‐AS1, and further explored its possible molecular mechanisms. As a result, elevated expression of NR2F2‐AS1 was detected in NSCLC tissues and cells and was remarkably associated with the tumor, node, metastasis (TNM) stage and the status of lymphatic metastasis of patients. Down‐regulated NR2F2‐AS1 contributed to the promotion of cell apoptosis and the inhibition of cell proliferation and invasion in A549 and SPC‐A‐1 cells in vivo and vitro. Through bioinformatics analysis, NR2F2‐AS1 functions as a ceRNA directly binding to miR‐320b, BMI1 was a direct target of miR‐320b. Combined with the following cellular experiments, the data showed that NR2F2‐AS1 may influence the NSCLC cell proliferation, invasion and apoptosis through regulating miR‐320b targeting BMI1.     

HOTAIR promotes osteosarcoma development by sponging miR-217 and targeting ZEB1

Long noncoding RNAs (lncRNAs) have drawn increasing attention because of the role which they play in various diseases, including osteosarcoma. So far, the function and mechanism of HOTAIR in osteosarcoma are unclear. In our study, we observed that HOTAIR was elevated accompanied with a decrease of miR-217 and an increase of ZEB1 in human osteosarcoma cells including U2OS, MG63, Saos-2, and SW1353 compared with human osteoblast cell line hFOB. In addition, the subsequent functional assay exhibited that silencing HOTAIR could significantly repress osteosarcoma cell growth, migration, invasion, and induce cell apoptosis capacity, which indicated that HOTAIR exerted an oncogenic role in osteosarcoma. Moreover, it was revealed by using bioinformatics analysis that HOTAIR can be targeted by microRNA-217 (miR-217). miR-217 has been recognized as a crucial tumor suppressive gene in cancers. We verified that mimics of miR-217 were able to suppress the osteosarcoma development. Furthermore, real-time quantitative PCR showed that HOTAIR siRNA increased miR-217 expression. Besides these, ZEB1 was identified as a downstream gene of miR-217 and we found that HOTAIR can mediate osteosarcoma progress by upregulating ZEB1 expression via acting as a competitive endogenous RNA (ceRNA) via miR-217. Taken these together, our findings in this study indicated that HOTAIR/miR-217/ZEB1 axis, as a novel research point can provide new insights into molecular mechanism of osteosarcoma development.     

Overexpression of MALAT1 contributes to cervical cancer progression by acting as a sponge of miR-429

Cervical cancer remains a malignant type of tumor and is the fourth leading cause of cancer-related death among females. MALAT1 has been identified as a tumor oncogene in various cancers. Our present study aimed to explore the biological role of MALAT1 in cervical cancer. We observed that MALAT1 was significantly upregulated in human cervical cancer cell lines compared with the ectocervical epithelial cells. MALAT1 was repressed by transfection with LV-shMALAT1, whereas increased by LV-MALAT1 in HeLa and Caski cells. Silencing of MALAT1 obviously reduced cervical cell viability, induced cell apoptosis, and repressed cell invasion capacity. Conversely, overexpression of MALAT1 exhibited an opposite phenomenon. Furthermore, miR-429 was predicted as a direct target of MALAT1, and it was dramatically decreased in cervical cancer cells. It has been shown that miR-429 plays a crucial role in cervical cancer progression. In our current study, the targeting correlation between MALAT1 and miR-429 was confirmed by luciferase reporter assays and RIP experiments. Finally, in vivo animal models were established, and we indicated that MALAT1 inhibited cervical cancer progression via targeting miR-429. These findings revealed that MALAT1 can sponge miR-429 and regulate cervical cancer pathogenesis in vivo and in vitro. In conclusion, we indicated that the MALAT1/miR-429 axis was involved in cervical cancer development.     

Upregulation of long noncoding TNFSF10 contributes to osteoarthritis progression through the miR-376-3p/FGFR1 axis

Osteoarthritis (OA) is a common joint disease with high morbidity, but there is still no definitive treatment for it. Long noncoding RNAs (lncRNAs) have been confirmed to play key roles in OA progression. This work was done to investigate the roles and action mechanism of lncRNA TNFSF10 in OA. The messenger RNA levels of TNFSF10 in articular cartilage samples from patients or chondrocytes were detected by Quantitative real-time PCR assay (qRT-PCR). The effects of TNFSF10 on chondrocytes were evaluated on the basis of cell growth, apoptosis, and inflammation. Then, the interaction between TNFSF10 and miR-376-3p was explored by dual-luciferase reporter test, RNA-binding protein immunoprecipitation, and RNA pull-down assay. Finally, various cell experiments, Western blot analysis, and qRT-PCR were performed to study the interaction among TNFSF10, miR-376-3p, and fibroblast growth factor receptor 1 (FGFR1). It was found that TNFSF10 was upregulated in OA cartilages and stimulated cell proliferation, antiapoptosis, and inflammation for chondrocytes. In addition, TNFSF10 acted as a competing endogenous RNA to downregulate miR-376-3p, and the influence of TNFSF10 on chondrocytes was partly reversed by miR-376-3p. Moreover, FGFR1, as a target of miR-376-3p, had reversal functions on the outcomes mediated by miR-376-3p. The further analysis displayed that there was a negative relationship between TNFSF10 and miR-376-3p as well as miR-376-3p and FGFR1, while FGFR1 was positively related with TNFSF10. Altogether, TNFSF10 overexpression probably stimulated proliferation and inflammation, and inhibited apoptosis by regulating the miR-376-3p/FGFR1 axis, implying that its increase contributed to OA progression. Our study provided a new potential biomarker or therapeutic target-TNFSF10, which was helpful to develop an efficient approach to cure OA.     

Transcriptome wide analysis of long non-coding RNA-associated ceRNA regulatory circuits in psoriasis

Long non-coding RNAs (lncRNAs) play critical roles in regulating immune-associated diseases and chronic inflammatory disorders. Here, we found that lncRNAs involve in the pathogenesis of psoriasis through integrative analysis of RNA-seq data sets from a psoriasis cohort. Then, lncRNA-protein-coding genes (PCGs) co-expression network analysis demonstrated that lncRNAs extensively interact with IFN-γ signalling pathway-associated genes. Further, we validated 3 lncRNAs associate with IFN-γ signalling pathway activation upon IFN-γ stimulated in HaCaT cells, and loss of function experiments indicate their functional roles in the activation of inflammatory cytokine genes. Additionally, microRNA target screening analysis showed that lncRNAs may regulate JAK/STAT pathway activity through complete endogenous RNA (ceRNA) mechanism. Further experimental validation of PRKCQ-AS1/STAT1/miR-545-5p regulatory circuitry showed that lncRNAs regulate the expression of JAK/STAT signalling pathway genes through competing for miR-545-5p. In summary, our results demonstrated that dysregulation of lncRNA-JAK/STAT pathway axis promotes the inflammation level in psoriasis and thus provide potential therapeutic targets for psoriasis treatments.     

Construction of a paclitaxel-related competitive endogenous RNA network and identification of a potential regulatory axis in pancreatic cancer

BackgroundIncreasing numbers of studies have elucidated the role of competitive endogenous RNA (ceRNA) networks in carcinogenesis. However, the potential role of the paclitaxel-related ceRNA network in the innate mechanism and prognosis of pancreatic cancer has not been identified.MethodsComprehensive bioinformatics analyses were performed to identify drug-related miRNAs (DRmiRNAs), drug-related mRNAs (DRmRNAs) and drug-related lncRNAs (DRlncRNAs) and construct a ceRNA network. The ssGSEA and CIBERSORT algorithms were utilized for immune cell infiltration analysis. Additionally, we validated our paclitaxel-related ceRNA regulatory axis at the gene expression level; functional experiments were conducted to explore the biological functions of the key genes.ResultsA total of 182 mRNAs, 13 miRNAs, and 53 lncRNAs were confirmed in the paclitaxel-related ceRNA network. In total, 6 mRNAs, 4 miRNAs, and 6 lncRNAs were identified to establish a risk signature and exhibited optimal prognostic effects. The mRNA signature can predict the abundance of immune cell infiltration and the sensitivity of different chemotherapeutic drugs and may also have a guiding effect in immune checkpoint therapy. A potential PART1/hsa-mir-21/SCRN1 axis was confirmed according to the ceRNA theory and was verified by qPCR. The results indicated that PART1 knockdown markedly increased hsa-mir-21 expression but inhibited SCRN1 expression, weakening the proliferation and migration abilities.ConclusionsWe hypothesized that the paclitaxel-related ceRNA network strongly influences the innate mechanism, prognosis, and immune infiltration of pancreatic cancer. Our risk signatures can accurately predict survival outcomes and provide a clinical basis.     

Caspase-7 mediates caspase-1-induced apoptosis independently of Bid

Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.