Antisense RNA1 Research Articles

Effects of antisense lncRNA PCBP1-AS1 on biological behaviors of vulvar squamous carcinoma cells by regulating TRAF5 and NF-κB expression

Background: Given antisense long noncoding RNAs (lncRNAs) poly C binding protein-1 antisense RNA1 (PCBP1-AS1) was significantly co-expressed with tumor necrosis factor receptor associated factor 5 (TRAF5) mRNA in vulvar squamous cell carcinoma (VSCC). TRAF5 could mediate nuclear factor kappa B (NF-κB) activation and regulate biological behaviors of tumor cells. Herein, we determined whether PCBP1-AS1 could affect the tumor behaviors of VSCC by regulating TRAF5 and NF-κB expression. Methods: The expression of PCBP1-AS1 and TRAF5 mRNA in VSCC tissues and cells was detected via quantitative real-time polymerase chain reaction, while the protein expression levels of TRAF5, p65, p-p65, c-Rel, and IκBα were examined by immunoblotting. After the overexpression of PCBP1-AS1 and RNA interference knockdown of TRAF5 in SW954 cells, NF-κB activation-nuclear translocation fluorescence, the CCK-8 assay, Annexin V-FITC & PI fluorescent staining, the wound healing assay, and the Transwell invasion assay were performed to evaluate the activity of NF-κB, cellular proliferation, apoptosis, migration, and invasion. Results: PCBP1-AS1 and TRAF5 mRNA expression were downregulated in VSCC tissues and were correlated with patient clinicopathological characteristics. The protein expression of TRAF5, p65, p-p65, c-Rel, and IκBα protein in VSCC tissues was significantly decreased. We demonstrated that PCBP1-AS1 positively regulated the expression of TRAF5. PCBP1-AS1 overexpression or knockdown of TRAF5 regulated the activity of the NF-κB pathway. Increase of PCBP1-AS1 significantly inhibited proliferation and enhanced apoptosis via TRAF5-mediated regulation of the NF-κB signaling pathway. Additionally, PCBP1-AS1 can suppress migration and invasion in an NF-κB-independent manner. Conclusions: As a tumor suppressor gene, PCBP1-AS1 regulates the proliferation and apoptosis of VSCC via TRAF5-mediated expression of the NF-κB.

Effects of antisense lncRNA PCBP1-AS1 on biological behaviors of vulvar squamous carcinoma cells by regulating TRAF5 and NF-κB expression.

BackgroundGiven antisense long noncoding RNAs (lncRNAs) poly C binding protein-1 antisense RNA1 (PCBP1-AS1) was significantly co-expressed with tumor necrosis factor receptor associated factor 5 (TRAF5) mRNA in vulvar squamous cell carcinoma (VSCC). TRAF5 could mediate nuclear factor kappa B (NF-κB) activation and regulate biological behaviors of tumor cells. Herein, we determined whether PCBP1-AS1 could affect the tumor behaviors of VSCC by regulating TRAF5 and NF-κB expression.MethodsThe expression of PCBP1-AS1 and TRAF5 mRNA in VSCC tissues and cells was detected via quantitative real-time polymerase chain reaction, while the protein expression levels of TRAF5, p65, p-p65, c-Rel, and IκBα were examined by immunoblotting. After the overexpression of PCBP1-AS1 and RNA interference knockdown of TRAF5 in SW954 cells, NF-κB activation-nuclear translocation fluorescence, the CCK-8 assay, Annexin V-FITC & PI fluorescent staining, the wound healing assay, and the Transwell invasion assay were performed to evaluate the activity of NF-κB, cellular proliferation, apoptosis, migration, and invasion.ResultsPCBP1-AS1 and TRAF5 mRNA expression were downregulated in VSCC tissues and were correlated with patient clinicopathological characteristics. The protein expression of TRAF5, p65, p-p65, c-Rel, and IκBα protein in VSCC tissues was significantly decreased. We demonstrated that PCBP1-AS1 positively regulated the expression of TRAF5. PCBP1-AS1 overexpression or knockdown of TRAF5 regulated the activity of the NF-κB pathway. Increase of PCBP1-AS1 significantly inhibited proliferation and enhanced apoptosis via TRAF5-mediated regulation of the NF-κB signaling pathway. Additionally, PCBP1-AS1 can suppress migration and invasion in an NF-κB-independent manner.ConclusionsAs a tumor suppressor gene, PCBP1-AS1 regulates the proliferation and apoptosis of VSCC via TRAF5-mediated expression of the NF-κB.

Correlation of gene expression and clinical parameters identifies a set of genes reflecting LV systolic dysfunction and morphological alterations.

To gain new insights into the complex pathophysiology of dilated cardiomyopathy (DCM) we performed a quantitative approach to identify genes with expression patterns that linearly correlate with parameters of cardiac morphology (left ventricular end-diastolic diameter indexed by body surface are (LVEDDI), systolic function [LV ejection fraction (LVEF)], and serum levels of cardiac peptide hormone NH2-terminal probrain natriuretic peptide (NT-proBNP) in human endomyocardial biopsies of 47 DCM patients and eight individuals with normal LVEF. A set of genes was identified as common heart failure markers characterized by correlation of their expression with cardiac morphology, systolic function, and NT-proBNP. Among them are already known genes encoding e.g., the natriuretic peptide hormones NPPA and NPPB and its converting enzyme corin, but also potential new heart failure markers like EP300 antisense RNA1 and dimethylarginine dimethylaminohydrolase 1 (DDAH1) along with other genes with so far unknown relation to heart function. In contrast, the expression of other genes including the Ca2+ flux regulating genes phospholamban (PLN), sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA), and extracellular matrix proteins showed significant correlation with LVEF and LVEDDI only. Those genes seem to reflect more specifically pathological alterations of systolic function and morphology in DCM hearts.

LncRNA HNF1A-AS1 modulates non-small cell lung cancer progression by targeting miR-149-5p/Cdk6.

Growing evidence have shown the important regulation of lncRNAs (long noncoding RNAs) in non-small cell lung cancer (NSCLC). lncRNAhepatocyte nuclear factor 1 homeobox A (HNF1A)-antisense RNA 1 (AS1), an "oncogene", was reported to regulate human tumors progression. However, the molecular mechanism of HNF1A-AS1 involved in the development of NSCLC is still under investigation. In the current study, we found that HNF1A-AS1 was relatively upregulated in both NSCLC patient tissues and cell lines. Functional studies established that overexpression of HNF1A-AS1 promoted cell proliferation, cell cycle, invasion, and migration of NSCLC cells in vitro. The promotion abilities of HNF1A-AS1 on NSCLC cell progression were suppressed via knockdown of HNF1A-AS1. miR-149-5p was then proved to be a novel target of HNF1A-AS1, whose expression was negatively correlated with HNF1A-AS1 in NSCLC patient tissues and cell lines. HNF1A-AS1 increased the expression of cyclin-dependent kinase 6 (Cdk6) via sponging with miR-149-5p. Gain- and loss-of-functional studies indicated that HNF1A-AS1 promoted NSCLC progression partially through inhibition of miR-363-3p and induction of Cdk6. Subcutaneous xenotransplanted tumor model confirmed that interference of HNF1A-AS1 suppressed the tumorigenic ability of NSCLC via upregulation of miR-149-5p and downregulation of Cdk6 in vivo. In conclusion, our findings clarified the biologic significance of the HNF1A-AS1/miR-149-5p/Cdk6 axis in NSCLC progression and provided novel evidence that HNF1A-AS1 may be a new potential therapeutic target for the treatment of NSCLC.

Decreased expression of SPINT1-AS1 and SPINT1 mRNA might be independent unfavorable prognostic indicators in esophageal squamous cell carcinoma.

Purpose: The serine peptidase inhibitor, Kunitz type 1 antisense RNA1 (SPINT1-AS1), a long non-coding RNA , has been linked to cancer progression. In this study, we aimed to explore the SPINT1-AS1 expression in matched esophageal squamous cell carcinoma (ESCC) and normal tissues, and analyze the potential correlations of SPINT1-AS1 expression with clinicopathological characteristics, in order to evaluate its prognosis and therapeutic value.Methods: SPINT1-AS1 expression was detected in 99 cases of matched ESCC and normal tissues samples using the quantitative real-time polymerase chain reaction method.Results: The expression level (△Ct) of SPINT1-AS1 and SPINT1 mRNA was significantly downregulated in ESCC tissues compared with matched normal tissues (P=0.0005; P=0.0002, respectively), and there was an obvious positive correlation between SPINT1-AS1 and SPINT1 mRNA expression. Clinicopathological characteristics indicated that SPINT1-AS1 expression was correlated with age and tumor size, while SPINT1 mRNA expression was correlated with age and gender. The receiver operating characteristic (ROC) curve analysis of the expression level of SPINT1-AS1 and SPINT1 mRNA yielded an area under the ROC curve value of 0.638 and 0.625, respectively. The overall survival is shorter in patients with low SPINT1-AS1 expressed than those with high levels of SPINT1-AS1 (P=0.044), and SPINT1 mRNA expression level is associated with the OS (P=0.001). Univariate and multivariate analysis suggested that SPINT1-AS1 was an independent prognostic indicator in ESCC.Conclusions: We found that the expression of SPINT1-AS1 and SPINT1 mRNA is downregulated in ESCC tissues, which could contribute to tumor progression. SPINT1-AS1 and SPINT1 mRNA may be therapeutic targets and prognosis markers for ESCC.

OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.

Antisense long non-coding RNAs (AS lncRNAs) have increasingly been recognized as important regulators of gene expression and they have been found to play key roles in several diseases. However, very little is known about the role of AS lncRNAs in fibrotic diseases such as systemic sclerosis (SSc). Our recent screening experiments by RNA sequencing showed that ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) and its sense gene OTUD6B were significantly downregulated in SSc skin biopsies. Therefore, we aimed to identify key regulators of OTUD6B-AS1 and to analyze the functional relevance of OTUD6B-AS1 in SSc. OTUD6B-AS1 and OTUD6B expression in SSc and healthy control (HC) dermal fibroblasts (Fb) after stimulation with transforming growth factor-β (TGFβ), Interleukin (IL)-4, IL-13, and platelet-derived growth factor (PDGF) was analyzed by qPCR. To identify the functional role of OTUD6B-AS1, dermal Fb or human pulmonary artery smooth muscle cells (HPASMC) were transfected with a locked nucleic acid antisense oligonucleotide (ASO) targeting OTUD6B-AS1. Proliferation was measured by BrdU and real-time proliferation assay. Apoptosis was measured by Caspase 3/7 assay and Western blot for cleaved caspase 3. While no difference was recorded at the basal level between HC and SSc dermal Fb, the expression of OTUD6B-AS1 and OTUD6B was significantly downregulated in both SSc and HC dermal Fb after PDGF stimulation in a time-dependent manner. Only mild and inconsistent effects were observed with TGFβ, IL-4, and IL-13. OTUD6B-AS1 knockdown in Fb and HPASMC did not affect extracellular matrix or pro-fibrotic/proinflammatory cytokine production. However, OTUD6B-AS1 knockdown significantly increased Cyclin D1 expression at the mRNA and protein level. Moreover, silencing of OTUD6B-AS1 significantly reduced proliferation and suppressed apoptosis in both dermal Fb and HPASMC. OTUD6B-AS1 knockdown did not affect OTUD6B expression at the mRNA level and protein level. Our data suggest that OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner. This is the first report investigating the function of OTUD6B-AS1. Our data shed light on a novel apoptosis resistance mechanism in Fb and vascular smooth muscle cells that might be relevant for pathogenesis of SSc.

Expression of long non-coding RNA hypoxia-inducible factor-1α antisense RNA-2 in hepatocellular carcinoma and its effect on proliferation and invasion

Objective To detect the expression of long non-coding RNA hypoxia-inducible factor-1α antisense RNA-2 (lncRNA-HIF1A-AS2) in hepatocellular carcinoma (HCC) and cell lines and its effect on proliferation and invasion of hepatoma cells. Methods Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of HIF1A-AS2 in 49 HCC tissues and adjacent normal tissues, four HCC cell lines(Huh7, HepG2, SMMC-7721, Bel-7402) and one normal liver cell line L02, then analyze the correlation between HIF1A-AS2 expression and clinicopathological features; Two high-expression HIF1A-AS2 HCC cells were transfected into small interfering RNA (si-HIF1A-AS2) and negative control (si-NC); Cell counting kit 8 (CCK-8) assay and Colony-forming assay were conducted to evaluate the cell proliferation; Transwell assay was conducted to evaluate the cell invasion. Results The expression of HIF1A-AS2 in HCC tissues was higher than that in adjacent tissues (-12.060±0.657 vs. -12.450±0.683, P<0.01); The expression of HIF1A-AS2 was correlated with age, tumor size and tumor differentiation (χ2=4.573, 4.871, 8.353, P<0.05); The expression of HIF1A-AS2 in HCC cell line was also significantly higher than that in normal liver cell line, and the expression was highest in Huh7 and HepG2 (Huh7: 5.349±0.992, HepG2: 5.660±0.726, P<0.01); CCK8 assay and colony-forming assay showed that the cell proliferation ability after transfection of si-HIF1A-AS2 was significantly lower than that of the control group in Huh7 and HepG2 (Huh7: 0.668±0.067 vs. 0.558±0.055, 0.931±0.881 vs. 0.670±0.072, 1.769±0.079 vs. 1.036±0.074; HepG2: 1.063±0.0604 vs. 0.803±0.097, 1.767±0.091 vs. 1.083±0.080, P<0.01) (Huh7: 542.70±25.50 vs. 378.00±19.31, HepG2: 530.00±14.53 vs. 217.30±18.15, P<0.01); Transwell assay showed that the number of cell invasion was significantly lower after transfection of si-HIF1A-AS2 [Huh7: (116.300±6.506) cells vs. (520.000±9.539) cells, HepG2: (88.330±5.686) cells vs. (55.000±5.245) cells, P<0.01]. Conclusion HIF1A-AS2 is highly expressed in HCC and cell lines, and down-regulation of HIF1A-AS2 expression can inhibit the proliferation and invasion ability of HCC cells. Key words: Hepatocellular carcinoma; Long non-coding RNA hypoxia-inducible factor-1α antisense RNA-2; Proliferation; Invasion

Nuclear lncRNA HOXD-AS1 suppresses colorectal carcinoma growth and metastasis via inhibiting HOXD3-induced integrin \u03b23 transcriptional activating and MAPK/AKT signalling

BackgroundLong noncoding RNAs (lncRNAs) have been indicated to play critical roles in cancer development and progression. LncRNA HOXD cluster antisense RNA1 (HOXD-AS1) has recently been found to be dysregulated in several cancers. However, the expression levels, cellular localization, precise function and mechanism of HOXD-AS1 in colorectal carcinoma (CRC) are largely unknown.MethodsReal-time PCR and in situ hybridization were used to detect the expression of HOXD-AS1 in CRC tissue samples and cell lines. Gain- and loss-of-function experiments were performed to investigate the biological roles of HOXD-AS1 in CRC cell line. RNA pull down, RNA immunoprecipitation and chromatin immunoprecipitation assays were conducted to investigate the mechanisms underlying the functions of HOXD-AS1 in CRC.ResultsWe observed that HOXD-AS1 was located in the nucleus of CRC cells and that nuclear HOXD-AS1 was downregulated in most CRC specimens and cell lines. Lower levels of nuclear HOXD-AS1 expression were associated with poor outcomes of CRC patients. HOXD-AS1 downregulation enhanced proliferation and migration of CRC cells in vitro and facilitated CRC tumourigenesis and metastasis in vivo. Mechanistic investigations revealed that HOXD-AS1 could suppress HOXD3 transcription by recruiting PRC2 to induce the accumulation of the repressive marker H3K27me3 at the HOXD3 promoter. Subsequently, HOXD3, as a transcriptional activator, promoted Integrin β3 transcription, thereby activating the MAPK/AKT signalling pathways.ConclusionOur results reveal a previously unrecognized HOXD-AS1-HOXD3-Integrin β3 regulatory axis involving in epigenetic and transcriptional regulation constitutes to CRC carcinogenesis and progression.

Upregulation and hypomethylation of lncRNA AFAP1‑AS1 predicts a poor prognosis and promotes the migration and invasion of cervical cancer

Although lncRNA AFAP1 antisense RNA1 (AFAP1‑AS1) is considered an oncogenic lncRNA, little is known about the role of AFAP1‑AS1 in cervical cancer. In the present study, we found that AFAP1‑AS1 was elevated and hypomethylated in cervical cancer and was associated with a poor prognosis of patients with cervical cancer by analyzing the Cancer RNA‑Seq Nexus (CRN), MethCH and UCSC XENA databases. Subsequently, we knocked AFAP1‑AS1 expression down using siRNAs in cervical cancer cells. Wound healing experiments and matrigel invasion experiments revealed that the downregulation of AFAP1‑AS1 suppressed the migration and invasion of cervical cancer cells. Furthermore, western blot analysis demonstrated that the antitumor effects induced by the silencing of AFAP1‑AS1 were mainly mediated through the regulation of the Rho/Rac signaling pathway and epithelial‑mesenchymal transition (EMT)‑related genes. Taken together, the findings of the present study indicate that the expression level of AFAP1‑AS1 may be involved in the development of cervical cancer. Thus, AFAP1‑AS1 may be a novel prognostic biomarker and a potential therapeutic target for patients with cervical cancer.

Long non-coding RNA ZFAS1 promotes proliferation and metastasis of clear cell renal cell carcinoma via targeting miR-10a/SKA1 pathway

BackgroundLncRNA ZFAS1 (ZNFX1 antisense RNA1) plays key roles in the occurrence and progression of various cancers, including colorectal cancer, glioma, lung cancer, gastric cancer, and so on. To date, relatively little is known about its potential role in development and/or progression of clear cell renal cell carcinoma (ccRCC). MethodsExpression level of ZFAS1 and miR-10a in 60 ccRCC and 20 adjacent non-tumor tissues were determined by using qRT-PCR. The effect of knockdown of ZFAS1 on cell proliferation, migration and invasion were measured by CCK-8 assay, transwell migration and invasion assay, respectively. The correlation of ZFAS1 and miR-10a was analyzed by bioinformatics DIANA TOOLS. Protein and mRNA expression of spindle and kinetochore-associated protein 1(SKA1) were determined by western blot and qRT-PCR analysis, respectively. Interactions between ZFAS1 and miR-10a were verified by luciferase reporter assay and RNA immunoprecipitation (RIP) assay, and interactions between miR-10a and SKA1 was verified by a luciferase reporter assay. ResultsWe observed that high-level expression of ZFAS1 is positively correlated with poor prognosis and shorter overall survival (OS) in patients with ccRCC. Knockdown of ZFAS1 significantly suppressed proliferation, migration and invasion of ccRCC cells. Furthermore, miR-10a was identified as a target of ZFAS1. Silencing miR-10a could attenuate the ability of ZFAS1 in promoting proliferation and metastasis of ccRCC. Subsequently, our studies validated that SKA1, as a key downstream target protein for miR-10a, is responsible for the biological role of ZFAS1. ZFAS1 positively regulated SKA1 expression via sponging miR-10a. ConclusionsTaken together, our findings suggest that ZFAS1 promotes growth and metastasis of ccRCC via targeting miR-10a/SKA1 pathway, which may represent a novel therapeutic target or biomarker for ccRCC.

Long non-coding RNA UASR1 promotes proliferation and migration of breast cancer cells through the AKT/mTOR pathway.

Long non-coding RNAs (lncRNAs) are non-coding RNAs longer than 200 nucleotides that function as regulatory factors in many human diseases, including cancer. However, majority of lncRNAs remain to be characterized. In this study, we characterized a novel lncRNA transcript, named UNC5B antisense RNA1 (UASR1). UASR1 is 647bp in length consisting of two exons. This lncRNA is an antisense of intron 1 of unc-5 netrin receptor B (UNC5B) gene. In breast cancer tissues, UASR1 was upregulated. Ectopic expression of UASR1 promoted proliferation and clonogenic growth of breast cancer cells MCF7 and MDA-MB-231. The migration of these cells also increased as demonstrated by wound healing and transwell assays. In contrast, silencing of UASR1 suppressed cell proliferation and migration. Further studies showed that UASR1 activated AKT and AKT-mediated mTOR signaling pathway to stimulate cell proliferation and growth. In these cells, active pAKT, pTSC2, p4EBP1 and pp70S6K were increased. Taken together, our data suggest that UASR1 plays an oncogenic role in breast cancer cells through activation of the AKT/mTOR signaling pathway, being a novel RNA oncogene.

&lt;p&gt;The inhibitive effect of sh-HIF1A-AS2 on the proliferation, invasion, and pathological damage of breast cancer via targeting miR-548c-3p through regulating HIF-1&amp;alpha;/VEGF pathway in vitro and vivo&lt;/p&gt;

BackgroundBreast cancer (BC) has been the commonest malignant tumor with a low survival rate among woman. Long non-coding RNA hypoxia-inducible factor-1 alpha antisense RNA-2 (HIF1A-AS2) was correlated with various cancers.PurposeThe study aimed to investigate the roles and related underlying molecular mechanisms of HIF1A-AS2 in BC.Material and methodsTarget relationships were speculated by Targetscan 7.0 and confirmed by dual luciferase reporter assay. Proteins levels were monitored by RT-qPCR, Western blot and immunohistochemistry assays. CCK-8 assay, SA-β-gal staining and transwell assay were used to detect proliferation, senescence and invasion, respectively. Xenograft nude mice were put into use to evaluate the tumor growth and motility.ResultsThe present study exhibited that HIF1A-AS2 and hypoxia-inducible factor-1 alpha (HIF-1α) were upregulated while miR-548c-3p was downregulated in MDA-MB-231, MCF-7, ZR-75-1, and BT-549 BC cell lines. Bioinformatics analysis showed HIF1A-AS2 and HIF-1α were two targets of miR-548c-3p, and the target relationship was further confirmed by dual luciferase reporter assay. Moreover, knockdown of HIF1A-AS2 by shRNA (sh-HIF1A-AS2) markedly elevated miR-548c-3p level, and the enhanced miR-548c-3p noticeably suppressed cell proliferation, invasion, and epithelial–mesenchymal transition, and promoted senescence in vitro. In addition, overexpression of HIF-1α promoted MCF-7 cell invasion. Intriguingly, low expression of HIF1A-AS2 reduced HIF-1α level by upregulating the expression of miR-548c-3p. Furthermore, experiment in xenograft nude mice has indicated that sh-HIF1A-AS2 inhibited tumor growth and motility by targeting miR-548c-3p through regulating HIF-1α/vascular endothelial growth factor (VEGF) pathway in vivo.ConclusionThe inhibitive effect of HIF-1α/VEGF pathway by sh-HIF1A-AS2 through targeting miR-548c-3p plays crucial regulatory roles in BC. Therefore, designing targeted drugs against HIF1A-AS2 provides a new direction for the treatment of BC.

LncRNA FEZF1-AS1 Is Associated With Prognosis in Lung Adenocarcinoma and Promotes Cell Proliferation, Migration, and Invasion

Long noncoding RNAs (lncRNAs) have been reported to play important roles in tumorigenesis. In the present study, we demonstrated that lncRNA forebrain embryonic zinc finger protein 1 (FEZF1) antisense RNA1 (FEZF1-AS1) is markedly upregulated in human lung adenocarcinoma (LAD) tissues and cell lines and is associated with poor prognosis. Loss of function revealed that deletion of FEZF1-AS1 expression significantly inhibited the LAD cell proliferation, invasion, and migration. Further studies revealed that downregulation of FEZF1-AS1 reduced mRNA and protein expression of its sense-cognate gene FEZF1 in LAD cells, and vice versa. Correlation analysis indicated that there was a positive correlation between FEZF1-AS1 and FEZF1 expression in LAD tissues. Additionally, rescue assay confirmed that the function of FEZF1-AS1 in LAD was mediated by FEZF1. Our findings suggested that dysregulation of FEZF1-AS1 contributed to the progression of LAD, which might be a potential target for LAD therapy.

Long non-coding RNA AFAP1-AS1 promoting epithelial-mesenchymal transition of endometriosis is correlated with transcription factor ZEB1.

The role of the long non-coding RNA (lncRNA) actin filament associated protein 1 antisense RNA1 (AFAP1-AS1) in the etiology of endometriosis is unknown. Expression of epithelial-mesenchymal transition (EMT) markers was quantified using qRT-PCR, immunohistochemistry, and Western blotting. The proliferation, migration, and invasion of ectopic endometrial epithelial cells and Ishikawa cells were evaluated by MTT, EdU, wound healing, and transwell assays. Inflammatory cytokine levels were detected by ELISA. Luciferase assays were used to measure activity of the ZEB1 promoter site pGL3-P886. AFAP1-AS1 levels were much higher in ectopic endometrial tissues than that in eutopic tissues. Expression of ZEB1, E-cadherin, and keratin was obviously higher in eutopic tissues than those in ectopic tissues. In contrast, expression of vimentin and N-cadherin was significantly lower in eutopic tissue than those in ectopic tissues. After knockdown of AFAP1-AS1, the morphology of endometrial epithelial cells varied from spindle fiber shaped to polygon epithelioid and proliferation, migration, and invasion were each inhibited. The knockdown of AFAP1-AS1 significantly inhibited expression from promoter site pGL3-P886 of the EMT-related transcription factor ZEB1. The size of subcutaneous tumours in nude mice was significantly reduced after down-regulation of AFAP1-AS1 expression. Higher expression of AFAP1-AS1 positively correlated with greater EMT in ectopic endometrium of patients with endometriosis. Knockdown of AFAP1-AS1 inhibited E2-induced activity of promoter site pGL3-P886 of transcription factor ZTB1, suggesting that AFAP1-AS1 knockdown inhibited growth of endometrial epithelial cells and that pathogenesis may be correlated with EMT.

TCF7L2 activated HOXA-AS2 decreased the glucocorticoid sensitivity in acute lymphoblastic leukemia through regulating HOXA3/EGFR/Ras/Raf/MEK/ERK pathway

Acute lymphoblastic leukemia (ALL) is characterized by abnormal lymphoblasts accumulation in the bone marrow and blood. Despite great efforts have been made in exploring novel therapeutic method, the prognosis of children with ALL is still unsatisfied. Glucocorticoid (GC) resistance is a great obstacle for the clinical treatment of ALL. Therefore, it is essential to investigate the molecular mechanism underlying the GC resistance. According to previous reports, long noncoding RNAs (lncRNAs) are involved in drug resistance of various human cancers. LncRNA HOXA cluster antisense RNA2 (HOXA-AS2) has been reported in several human malignancies due to its oncogenic property. However, the molecular mechanism of HOXA-AS2 involved in the GC resistance of ALL still needs to be further clarified. At first, we found that lncRNA HOXA-AS2 was highly expressed both in prednisone insensitive ALL cell lines and patient samples. Gain or loss-of-function assays revealed that HOXA-AS2 enhanced GC resistance via promoting cell proliferation and inhibiting cell apoptosis. Furthermore, we validated that HOXA-AS2 upregulated HOXA3, thereby activating EGFR/Ras/Raf/MEK/ERK signaling pathway. Our findings showed that HOXA-AS2 may be a potential therapeutic target for ALL patients with poor GC resistance.

Lowly expressed EGFR-AS1 promotes the progression of preeclampsia by inhibiting the EGFR-JAK/STAT signaling pathway.

To detect the differentially expressed long non-coding RNA (lncRNA) EGFR-AS1 in the placenta tissues of preeclampsia (PE) patients and normal pregnancies. We also investigated the underlying mechanism of EGFR-AS1 in regulating PE development. 21 PE patients and 21 normal pregnancies admitted in our hospital were selected. The expression of EGFR Antisense RNA1 (EGFR-AS1) in the placenta tissues of PE patients and normal pregnancies was detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The regulatory effect of EGFR-AS1 on proliferation of HTR-8 cells was observed by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. Cell cycle and apoptosis of HTR-8 cells after overexpression or knockdown of EGFR-AS1 were detected by flow cytometry. The expression changes of EGFR and proteins related to JAK/STAT signaling pathway after knockdown of EGFR-AS1 in HTR-8 cells were detected by Western blot. Rescue experiments were carried out after upregulating EGFR expression in HTR-8 cells. QRT-PCR results showed that the mRNA expression of EGFR-AS1 in PE patients was lower than that in normal pregnancies. CCK-8 assay and colony formation assay showed that knockdown of EGFR-AS1 in HTR-8 cells inhibited cell proliferation. Flow cytometry results showed that knockdown of EGFR-AS1 blocked the cell cycle of HTR-8 cells. Overexpression of EGFR-AS1 obtained opposite results. Western blot results showed that the protein expressions of EGFR, p-JAK and p-STAT were decreased after knockdown of EGFR-AS1. Alterations in protein expressions of p-JAK and p-STAT, and proliferative ability of HTR-8 cells induced by EGFR-AS1 knockdown could be rescued after upregulation of EGFR. The expression of lncRNA EGFR-AS1 is decreased in PE patients, which can promote the progression of preeclampsia by inhibiting EGFR-JAK/STAT signaling pathway.

Down-regulation of long non-coding RNA ITGB2-AS1 inhibits osteosarcoma proliferation and metastasis by repressing Wnt/β-catenin signalling and predicts favourable prognosis

Osteosarcoma is the most common and highly aggressive bone neoplasm often occurs among adolescents and young people. In despite of the major advances in the treatment, the overall survival of osteosarcoma patients remains poor. Long non-coding RNAs (lncRNAs) have been identified as key regulators involved in tumorigenesis and progression of osteosarcoma. However, the exact roles and mechanisms of lncRNAs in osteosarcoma remain unclear. In this study, the functions and underlying molecular mechanisms of a novel lncRNA, ITGB2 antisense RNA1 (ITGB2-AS1) were investigated in osteosarcoma. The expression levels of ITGB2-AS1 were up-regulated in osteosarcoma tissue samples and cell lines determined by qRT-PCR assay. Osteosarcoma patients with high ITGB2-AS1 expression had a significantly poor prognosis. In addition, knockdown of ITGB2-AS1 inhibited the proliferation and induced apoptosis of osteosarcoma cells using MTT assay and flow cytometry detection. Furthermore, wound healing and transwell invasion assay revealed that depletion of ITGB2-AS1 suppressed the migration and invasion abilities of osteosarcoma cells. Molecular mechanism study indicated that ITGB2-AS1 inhibition impaired Wnt/β-catenin signalling pathway in osteosarcoma cells. Taken together, our study suggested that ITGB2-AS1 played critical roles in the development and progression of osteosarcoma via Wnt/β-catenin signalling, indicating that ITGB2-AS1 might be a valuable diagnostic biomarker and potential anticancer therapeutic target for osteosarcoma.

SP1-induced lncRNA-ZFAS1 contributes to colorectal cancer progression via the miR-150-5p/VEGFA axis

Increasing long non-coding RNAs (lncRNAs) have been reported to play key roles in the development and progression of various malignancies. ZNFX1 antisense RNA1 (ZFAS1) has been reported to be aberrant expression and suggested as a tumor suppressor or oncogene in many cancers. However, the biological role and underlying molecular mechanism of ZFAS1, especially the miRNA sponge role of which in CRC remain largely unknown. We found that ZFAS1 expression was higher in CRC tissues, where it was associated with poor overall survival (OS), we also showed that ZFAS1 upregulation was induced by nuclear transcription factor SP1. Moreover, ZFAS1 and VEGFA are both targets of miR-150-5p, while ZFAS1 binds to miR-150-5p in an AGO2-dependent manner. Additionally, ZFAS1 upregulation markedly promoted as well as ZFAS1 knockdown significantly suppressed CRC cell proliferation, migration, invasion and angiogenesis, and the inhibitory effect caused by ZFAS1 knockdown could be reversed by antagomiR-150-5p. Lastly, we demonstrated that ZFAS1 knockdown inhibited EMT process and inactivated VEGFA/VEGFR2 and downstream Akt/mTOR signaling pathway in CRC. Our data demonstrated that SP1-induced ZFAS1 contributed to CRC progression by upregulating VEGFA via competitively binding to miR-150-5p, which acts as a tumor suppressor by targeting VEGFA in CRC.

Long non-coding RNA MYU promotes prostate cancer proliferation by mediating the miR-184/c-Myc axis.

Long non-coding RNAs (lncRNAs) play critical roles in tumorigenesis and cancer progression. The c-Myc upregulated lncRNA MYU (VPS9D1 antisense RNA1, annotated as VPS9D1-AS1) has been reported in several common types of human cancers, which has revealed that lncRNA MYU could function as either an oncogene or a tumor-suppressor gene in different cancer types. However, the function of lncRNA MYU in prostate cancer remains unknown. In the present study, we demonstrated that lncRNA MYU is significantly upregulated in prostate cancer tissues. MYU knockdown impaired prostate cancer cell growth and migration as shown from cell viability, colony formation, Transwell and wound healing assays. In contrast, MYU overexpression displayed opposite effects. No correlation was noted between MYU and its cognate VPS9D1 expression level. Moreover, lncRNA MYU did not regulate the expression of VPS9D1 either at the mRNA or protein level as detected using qRT-PCR and western blotting assays. Furthermore, lncRNA MYU was able to be transported into the extracellular milieu by means of exosomes, and then promoted adjacent cell proliferation and migration. Mechanistically, lncRNA MYU upregulated c-Myc by competitively binding miR-184 and then induced the proliferation of prostate cancer. Thus, this study demonstrated that lncRNA MYU functions as an oncogene in prostate cancer at least in part through the miR-184/c-Myc axis, and may serve as a potential diagnostic biomarker and therapeutic target.

Long non-coding RNA SBF2-AS1 promotes hepatocellular carcinoma progression through regulation of miR-140-5p-TGFBR1 pathway

A growing number of studies has suggested that long non-coding RNAs (lncRNAs) exert essential roles in the development and progression of hepatocellular carcinoma (HCC). However, the roles of lncRNA and its molecular mechanism in HCC are largely unknown. In the present study, the functions and molecular mechanisms of a novel lncRNA, SET-binding factor 2 (SBF2) antisense RNA1 (SBF2-AS1), were investigated in HCC tissues and cell lines. We found that the expression levels of SBF2-AS1 were significantly up-regulated in HCC tissues and correlated with poor prognosis. SBF2-AS1 knockdown could inhibit the proliferation of HCC cells and attenuate the development of HCC tumor in vivo. Moreover, wound healing and Transwell assays revealed that down-regulation of SBF2-AS1 suppressed the migration and invasion of HCC cells by modulating epithelial-mesenchymal transition (EMT) ability. Mechanistically, we observed that SBF2-AS1 served as a competing endogenous RNA (ceRNA) of miR-140–5p. Subsequently, transforming growth factor beta receptor 1 (TGFBR1) was certified as a direct target of miR-140–5p and enforcing SBF2-AS1 expression elevated TGFBR1 expression in HCC. Taken together, our study suggested that SBF2-AS1 modulated TGFBR1 through sponging miR-140–5p in HCC development and progression indicating that SBF2-AS1 might be further chosen as a potential anticancer therapeutic target and a promising prognostic biomarker for HCC.