Direct Binding Relationship Research Articles

Long non‑coding RNA SNHG1 promotes autophagy in vascular smooth muscle cells induced by facilitating CLEC7A.

Long non‑coding RNAs serve a crucial role in autophagy of vascular smooth muscle cells (VSMCs). The present study aimed to investigate the effect of small nucleolar RNA host gene 1 (SNHG1) on autophagy in VSMCs and the associated underlying mechanisms. Rapamycin was used to induce autophagy in VSMCs and the effects of SNHG1 on the proliferation and migration of VSMCs and the change in phenotype were tested following overexpression and silencing of SNHG1. The target gene of SNHG1 was predicted and validated. SNHG1‑regulated autophagy of VSMCs via C‑type lectin domain family 7 member A (CLEC7A) was determined by combined silencing of SNHG1 and overexpression of CLEC7A. Rapamycin‑induced autophagy in VSMCs changed the cell phenotype from contractile to synthetic, with decreased expression of α‑smooth muscle actin and smooth muscle protein 22a and increased expression of osteopontin. Overexpression of SNHG1 caused the same change in phenotype while the opposite change was observed following SNHG1 silencing. Overexpression of SNHG1 promoted the proliferation and migration of VSMCs. CLEC7A was identified as a target gene of SNHG1 and a direct binding relationship between them was confirmed by RNA immunoprecipitation and RNA pull‑down assays. Overexpression of SNHG1 increased the expression of CLEC7A. The expression of both SNHG1 and CLEC7A was increased during autophagy of VSMCs. Overexpression of SNHG1 promoted autophagy of VSMCs and silencing of CLEC7A reduced this effect of SNHG1. In conclusion, SNHG1 and CLEC7A were increased in VSMCs following autophagy. SNHG1 promotes the conversion of VSMCs from a contractile phenotype to a synthetic phenotype by facilitating CLEC7A expression.

IL-18 serves as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway

ABSTRACT Background: N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA. However, its role in non-small cell lung cancer (NSCLC) has not been completely elucidated. Objective: To explore whether methyltransferase like 3 (METTL3) in cancer associated fibroblasts (CAFs) affects the secretion of IL-18, which drives NSCLC cells to regulate PD-L1-mediated immunosuppression via the nuclear factor kappa B (NF-κB) pathway. Methods: Histopathological features of NSCLC tissues were identified by H&E and IHC staining. The levels of m6A writers (METTL3), IL-18 and NF-κB pathway related genes were assessed. The quantity of CD8+ T cells was evaluated by flow cytometry (FCM). The direct binding relationship between METTL3 and IL-18 mRNA was detected by RIP assay and RNA pulldown and confirmed by dual – luciferase reporter assay. The level of RNA m6A was detected by RNA m6A dot blot and meRIP assays. A heterotopic implantation model of NSCLC was established in NOD-SCID mice for further explore the effect of CAF derived METTL3 on immunosuppression of NSCLC in vivo. Results: Our results illustrated that METTL3 was down-regulated in CAFs, and CAF derived METTL3 alleviated PD-L1-mediated immunosuppression of NSCLC through IL-18. Subsequently, we found that IL-18 was main effector of CAF-derived METTL3 against immunosuppression of NSCLC, and IL-18 accelerated immunosuppression of NSCLC by driving NF-κB pathway. In vivo, METTL3 knockdown-derived CAFs accelerated immunosuppression of NSCLC. Conclusion: IL-18 served as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway.

LncRNA MEG3 Promotes Osteogenic Differentiation of Tendon Stem Cells Via the miR-129-5p/TCF4/β-Catenin Axis and thus Contributes to Trauma-Induced Heterotopic Ossification.

Heterotopic ossification (HO) is one of the most intractable conditions following injury to the musculoskeletal system. In recent years, much attention has been paid to the role of lncRNA in musculoskeletal disorders, but its role in HO was still unclear. Therefore, this study attempted to determine the role of lncRNA MEG3 in the formation of post-traumatic HO and further explore the underlying mechanisms. On the basis of high-throughput sequencing and qPCR validation, elevated expression of the lncRNA MEG3 was shown during traumatic HO formation. Accordingly, in vitro experiments demonstrated that lncRNA MEG3 promoted aberrant osteogenic differentiation of tendon-derived stem cells (TDSCs). Mechanical exploration through RNA pulldown, luciferase reporter gene assay and RNA immunoprecipitation assay identified the direct binding relationship between miR-129-5p and MEG3, or miR-129-5p and TCF4. Further rescue experiments confirmed the miR-129-5p/TCF4/β-catenin axis to be downstream molecular cascade responsible for the osteogenic-motivating effects of MEG3 on the TDSCs. Finally, experiments in a mouse burn/tenotomy model corroborated the promoting effects of MEG3 on the formation of HO through the miR-129-5p/TCF4/β-catenin axis. Our study demonstrated that the lncRNA MEG3 promoted osteogenic differentiation of TDSCs and thus the formation of heterotopic ossification, which could be a potential therapeutic target.

Abstract 3771: PiR-hsa-4447944 promotes the progression of prostate cancer through NEFH and induces androgen-independent growth

Abstract Introduction and Objective: Non-coding RNAs (ncRNAs) represent a diverse family of regulatory transcripts that drive tumorigenesis of prostate cancer (PCa) and various other types of cancers by their hyperactivity or diminished function. PIWI-interacting RNAs (piRNAs) are a class of small ncRNAs mechanistically similar to but much less studied than microRNAs. As the most diversified small ncRNAs, piRNAs are widely involved in the pathogenesis of different types of cancers. Our study aimed to investigate the role of piRNAs in the development of castration-resistant prostate cancer (CRPC). Methods: Small RNA sequencing was used to explore the CRPC associated piRNAs on a basis of 10 benign prostate tissues, 10 paired hormone-sensitive PCa tissues and CRPC tissues from the same patients. The top 12 upregulated piRNAs were validated through qRT-PCR in a bigger cohort. piR-hsa-4447944 was selected and investigated its biological function by gain of function in vitro. To study its loss of function role, an androgen-independent PCa cell (LNCaP-AI) was established. For mechanistic investigation, high-throughput RNA sequencing was conducted to identify dysregulated genes in PCa cells with control or piR-hsa-4447944 overexpression. Then, potential downstream targets were further predicted by miRanda program and validated by qRT-PCR, western-blot and dual-luciferase reporter assay. Results: piR-hsa-4447944 demonstrated a relatively higher expression level in both CRPC tissues and CRPC cell lines when compared with PCa tissues and cells. Functionally, overexpression of piR-hsa-4447944 could confer resistance to androgen deprivation and anti-androgen in PCa cells in vitro, whereas anti-sense RNA against the piR-hsa-4447944 could potentiate the sensitivity to androgen deprivation in LNCaP-AI cell. In addition, piR-hsa-4447944 showed the ability in promoting migration and invasion of PCa cells. Mechanistically, mRNA sequencing results identified a panel of downstream target-genes. Among them, neurofilament heavy chain (NEFH) was predicted and validated to have a direct binding relationship with piR-hsa-4447944, and was successfully verified to be negatively regulated by piR-hsa-4447944 in PCa cells. Conclusions: Our study showed, for the first time, that piR-hsa-4447944 can contribute to androgen insensitivity in CRPC. This sheds a new insight into the regulation of CRPC by a small ncRNA piRNA, which may form the basis of a novel target in the treatment of CRPC. Citation Format: Qiang Peng, Jingkai Sun, Peter Ka-Fung Chiu, Tingting Xie, Jeremy Yuen-Chun Teoh, Chi-Fai Ng. PiR-hsa-4447944 promotes the progression of prostate cancer through NEFH and induces androgen-independent growth. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3771.

MP20-12 PiR-hsa-24683 IS ESSENTIAL FOR PROSTATE TUMOR-PROPAGATING CELLS AND PROMOTE THE DEVELOPMENT OF CASTRATION RESISTANCE

You have accessJournal of UrologyCME1 Apr 2023MP20-12 PiR-hsa-24683 IS ESSENTIAL FOR PROSTATE TUMOR-PROPAGATING CELLS AND PROMOTE THE DEVELOPMENT OF CASTRATION RESISTANCE Qiang Peng, Peter Ka-Fung Chiu, Tingting Xie, Jeremy Yuen-Chun Teoh, and Chi-Fai Ng Qiang PengQiang Peng More articles by this author , Peter Ka-Fung ChiuPeter Ka-Fung Chiu More articles by this author , Tingting XieTingting Xie More articles by this author , Jeremy Yuen-Chun TeohJeremy Yuen-Chun Teoh More articles by this author , and Chi-Fai NgChi-Fai Ng More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs (sncRNAs) mechanistically similar to but much less studied than microRNAs. piRNAs represent the most diversified sncRNAs that are widely involved in the pathogenesis of different types of cancers including prostate cancer (PCa). Our study aimed to investigate the role of piRNAs in the development of castration-resistant prostate cancer (CRPC). METHODS: Small RNA sequencing was used to explore the CRPC associated piRNAs on a basis of 10 benign prostate tissues, 10 paired hormone-sensitive PCa tissues and CRPC tissues from the same patients. The top 8 upregulated piRNAs were validated through qRT-PCR in a bigger cohort. piR-hsa-24683 was selected and investigated its biological function by gain of function in vitro. To study its loss of function role, an androgen-independent PCa cell (LNCaP-AI) was established. For mechanistic investigation, the mRNA sequencing analysis of differentially expressed genes (DEGs) was conducted in PCa cells transfected with control/piR-hsa-24683 mimics. Then, potential downstream targets were further predicted by miRanda program and validated by qRT-PCR. RESULTS: piR-hsa-24683 demonstrated a relatively higher expression level in both CRPC tissues and CRPC cell lines when compared with PCa tissues and cells. Functionally, overexpression of piR-hsa-24683 could confer resistance to androgen deprivation and anti-androgen in PCa cells in vitro, whereas anti-sense RNA against the piR-hsa-24683 could potentiate the sensitivity to androgen deprivation in LNCaP-AI cell. In addition, piR-hsa-24683 showed the ability in promoting sphere formation, migration and invasion of PCa cells. Mechanistically, mRNA sequencing results identified a panel of downstream target-genes. Among them, serine/threonine kinase 3 (STK3) was predicted to have a direct binding relationship with piR-hsa-24683, and was successfully verified to be negatively regulated by piR-hsa-24683. CONCLUSIONS: Our study showed, for the first time, that piR-hsa-24683 can contribute to androgen insensitivity in CRPC. This sheds a new insight into the regulation of CRPC by a sncRNA piRNA, which may form the basis of a novel target in the treatment of CRPC. Source of Funding: UGC Research Matching Fund 8601404 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e279 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qiang Peng More articles by this author Peter Ka-Fung Chiu More articles by this author Tingting Xie More articles by this author Jeremy Yuen-Chun Teoh More articles by this author Chi-Fai Ng More articles by this author Expand All Advertisement PDF downloadLoading ...

S-18-5: ENDOTHELIAL GATA5 POSITIVELY REGULATES ANGIOGENESIS VIA CATHEPSIN S-MEDIATED ANGPT2/FLK1 AND MMP2/9 SIGNALING PATHWAYS

Objective: Although GATA5 is vital in maintaining the function of endothelial cells, the relationship between GATA5 and angiogenesis, however, remains unclear. Our study aims to determine how endothelial GATA5 mediates angiogenesis. Design and method: 1. C57BL/6 mice were injected with endothelial cell-specific GATA5-overexpressing adeno-associated virus. One week after virus infection, the hindlimb ischemia model was established by ligating the femoral artery of mice, and the blood perfusion ratio of the operated limb/healthy limb was detected by a blood perfusion imager. CD31 staining of mouse gastrocnemius muscle showed the number of new capillaries per unit area. Western Blot and qPCR were used to detect the expression of GATA5 and related angiogenesis factors. 2. On human umbilical vein endothelial cells, transfected lentivirus to knock down the expression of GATA5 and/or administered vascular endothelial growth factor-165 intervention, through CCK-8 experiments were used to detect cell proliferation, scratch experiments to detect cell migration, and endothelial tube formation experiments to detect the ability of cells to form tubes. Western blot and qPCR assays were used to detect the protein and mRNA levels of GATA5, cathepsin S, Angpt2 and Flk1, and MMP2/MMP9. The direct binding of GATA5 to cathepsin S was verified by co-immunoprecipitation experiments. 3. GATA5 knockdown human umbilical vein endothelial cells were transfected with plasmids overexpressing GATA5 or cathepsin S to increase the expression levels of GATA5 or cathepsin S. Cell function and expression of related factors were detected. Results: 1. The blood perfusion ratio and CD31 immunofluorescence expression of the mice in the endothelium-specific overexpression GATA5 group were significantly higher than those of the negative control mice, and the mRNA and protein levels of angiogenesis factors Angpt2 and Flk1 were increased as well. 2. Cell experiments showed that the knockdown of GATA5 could inhibit cell proliferation, migration and endothelial tube formation stimulated by VEGF-165, and at the same time, reducing the expression of cathepsin S, Angpt2, Flk1 and MMP2/MMP2. Co-immunoprecipitation experiments showed that GATA5 had a direct binding relationship with cathepsin S. 3. Overexpression of GATA5 or cathepsin S reversed the decline in cell migration and tube formation caused by GATA5 knockdown, and up-regulated the expressions of Angpt2/Flk1 and MMP2/MMP9. Conclusion: Endothelial GATA5 regulates angiogenesis by regulating endothelial cell proliferation, migration and tube formation through the cathepsin S-Angpt2/Flk1 and MMP2/MMP9 signaling pathways.

CircNFIX stimulates the proliferation, invasion, and stemness properties of ovarian cancer cells by enhancing SH3RF3 mRNA stability via binding LIN28B.

We aimed to study the regulatory roles and mechanism of circular nuclear factor IX (circNFIX) in cancer growth and stemness properties of ovarian cancer (OC). CircNFIX and SH3RF3 levels in OC tissues and cells were tested by quantitative real-time PCR. RNase R treatment quantified circNFIX RNA stability. Molecular interaction among circNFIX, LIN28B, and SH3RF3 was predicted by bioinformatics software and validated through RNA immunoprecipitation (RIP) assay. The gain- or loss-experiments of circNFIX on capabilities of metastasis and stemness in vitro were assessed using Cell Counting Kit-8, Transwell, western blot, and sphere-formation assays. CircNFIX and SH3RF3 were markedly elevated in OC tissues and OC cells. Knocking down circNFIX repressed the proliferation, migration, invasion, and stemness properties of A2780 and SKOV3 cells. The RIP assay verified the direct binding relationship between LIN28B, circNFIX, and SH3RF3. Additionally, overexpression of circNFIX elevated the SH3RF3 expression, while this effect was reversed by LIN28B silence. Rescue experiments demonstrated that the overexpression of SH3RF3 reversed the knockdown of circNFIX on OC cells' proliferation, metastasis, and stemness properties. CircNFIX improved the mRNA stability and translation of SH3RF3 via recruiting LIN28B, thus promoting the proliferation, invasion, and stemness properties of OC cells in vitro.

Emodin inhibiting epithelial-mesenchymal transition in pulmonary fibrosis through the c-MYC/miR-182-5p/ZEB2 axis.

Emodin is a natural anthraquinone compound, which is the main component found in the traditional Chinese herb Polygonum cuspidatum. The anti-fibrosis effects of Emodin have been reported. This study aimed to explore the specific mechanism of Emodin in the epithelial-mesenchymal transition (EMT) of pulmonary fibrosis. The pulmonary fibrosis mice models were constructed with bleomycin, the EMT models of alveolar epithelial cells were stimulated by TGF-β1, and Emodin was used for intervention. c-MYC and miR-182-5p were overexpressed or silenced by cell transfection. Our results demonstrated that Emodin attenuated pulmonary fibrosis induced by bleomycin in mice, and inhibited EMT, meanwhile downregulated c-MYC, upregulated miR-182-5p, and downregulated ZEB2 in vitro and vivo. Next, overexpression of c-MYC promoted EMT, while silencing c-MYC and overexpressing miR-182-5p inhibited EMT. Then, c-MYC negatively regulated the expression of miR-182-5p with a direct binding relationship. And miR-182-5p inhibited ZEB2 expression in a targeted manner. Finally, Emodin inhibited EMT that had been promoted by overexpression of c-MYC. In conclusion, Emodin could attenuate pulmonary fibrosis and EMT by regulating the c-MYC/miR-182-5p/ZEB2 axis, which might provide evidence for the application of Emodin in the treatment of pulmonary fibrosis.

Human Bone Marrow Mesenchymal Stem Cell (hBMSCs)-Derived miR-29a-3p-Containing Exosomes Impede Laryngocarcinoma Cell Malignant Phenotypes by Inhibiting PTEN.

Although microRNA-29a-3p was reported to inhibit laryngocarcinoma progression, the potential mechanisms have not been explored clearly. Laryngocarcinoma tissues were collected for analyzing the levels of miR-29a-3p and phosphatase and tensin homolog (PTEN). The miR mimics or inhibitor was transfected into laryngocarcinoma cell lines M4E and Hep2 for the investigation of the biological functions (proliferative, invasion, migratory rates, and apoptotic rates) of this miRNA. The exosomes (Exo) from human bone marrow mesenchymal stem cells (hBMSCs) after the transfection of miR mimics/inhibitor/si-PTEN were isolated and used to stimulate M4E and Hep2 cells. The in vivo mouse model was constructed to verify our findings. The miR-29a-3p level was decreased, and PTEN level was elevated in laryngocarcinoma tissues and the cancer cell lines. MiR mimics could inhibit proliferative, invasive migratory rates while promoting apoptotic rates of M4E and Hep2 cells. MiR-29a-3p was enriched in hBMSC-derived Exo, and the Exo from miR-29a-3p mimics transfected hBMSCs could inhibit laryngocarcinoma cell malignant phenotypes in vitro and prevent tumor progression in vivo. In addition, the direct binding relationship between miR-29a-3p and PTEN in this disease was determined. In conclusion, hBMSC-derived Exo with upregulated miR-29a-3p inhibited laryngocarcinoma progression via regulating PTEN, providing a potential diagnostic and therapeutic target in this disease.

Circ_0058063 contributes to oral squamous cell carcinoma development by sponging miR-145-5p and upregulating SERPINE1.

We aimed to demonstrate the effects circ_0058063 exerted on oral squamous cell carcinoma (OSCC) and its downstream mechanism associated with miR-145-5p and SERPINE1. The relevant contents of miR-145-5p, circ 0058063, and SERPINE1 mRNAs in OSCC were evaluated using quantitative reverse transcription polymerase chain reaction. Functional experiments including CCK-8, Transwell, Western blot, and in vivo experiment were implemented to investigate the biological impacts on OSCC cells. Using dual-luciferase reporter, RIP, and RNA pull-down assays, the direct binding relationship between miR-145-5p, circ 0058063, and SERPINE1, SMAD3, CYR61, and IGF1R mRNAs was verified. In OSCC, Circ 0058063 was significantly overexpressed. Knockdown of circ_0058063 suppressed OSCC cell migration and proliferation, but enhanced cell apoptosis. Functionally and mechanistically, circ_0058063 could specifically bind with miR-145-5p and thus upregulated expression of downstream target SERPINE1, which together contributed to the progression of OSCC. Circ_0058063 could promote the malignant behavior of OSCC by upregulating SERPINE1 through sponging miR-145-5p.

CircEXOC5 promotes ferroptosis by enhancing ACSL4 mRNA stability via binding to PTBP1 in sepsis-induced acute lung injury

BackgroundSepsis causes severe acute lung injury (ALI). Circular RNA is involved in the regulation of sepsis-related ALI progression. The regulation mechanism of circEXOC5 in sepsis-induced ALI is still unclear. Whether circEXOC5 is involved in the regulation of ferroptosis remains to be explored. MethodsWe constructed a mouse model of sepsis through cecal ligation and puncture (CLP). LPS induced mouse lung microvascular endothelial cells (MPVECs) to construct a sepsis cell model. The expression of circEXOC5 in the sepsis model was detected by qPCR. The extent of lung injury in mice was analyzed by HE staining. The contents of GSH/GSSG, iron, MDA and 4HNE in mice lung tissues and cells were detected by the kit. And further the ROS content was detected in the cells. Finally, the binding relationship between circEXOC5 and PTBP1 was detected by RIP and RNA pulldown. ResultsOur results showed that the circEXOC5 expression was significantly increased in the in vivo and in vitro models of sepsis. And after inhibiting circEXOC5, it improved the lung injury of septic mice. It was confirmed in cell models that ROS levels and ferroptosis in cells were reduced after knocking down circEXOC5. In addition, the expressions of ACSL4 and Gpx4 proteins were regulated by the level of circEXOC5. Finally, we also found that circEXOC5 had a direct binding relationship with PTBP1. ConclusionOur study found that the expression of cell ferroptosis and circEXOC5 increased in ALI induced by sepsis, and circEXOC5 aggravated ferroptosis in septic cells by regulating the PTBP1/ACSL4 axis.

TRPM2-AS Promotes Bladder Cancer by Targeting miR-22-3p and Regulating GINS2 mRNA Expression.

BackgroundBladder cancer (BLCA) refers to the malignancy growth that spreads from the bladder linings to the bladder muscles. However, the impact of miR-22-3p and lncRNA TRPM2-AS on this tumor has generated divergent views in the literature. This research aimed to study the effects of lncRNA TRPM2-AS on BLCA and its interaction with miR-22-3p and GINS2 mRNA.MethodsqRT-PCR was employed to measure the expression of TRPM2-AS, miR-22-3p and GINS2 mRNA in bladder tissues and cells. The subcellular localization of TRPM2-AS in T24 and 5637 cell lines was identified using a cell fractionation system. Luciferase assay, RIP assay and RNA pull-down assay were later performed to validate the direct binding relationship between TRPM2-AS, miR-22-3p and GINS2 mRNA. Several experiments were conducted to determine the viability, proliferation, colony formation and apoptosis of the cell lines.ResultsFindings indicated that TRPM2-AS was significantly upregulated in BLCA tissues and cell lines. Apart from that, it was observed that TRPM2-AS knockdown significantly inhibited the viability, proliferation and colony formation of BCLA cells, but it promoted the apoptosis of the BCLA cells. A significant downstream target of TRPM2-AS, miR-22-3p was found to show a lower expression level in BLCA tissues and cell lines. However, the inhibition of miR-22-3p considerably enhanced BLCA cell phenotypes. As well as discovering that GINS2 mRNA was a downstream target of miR-22-3p and was significantly upregulated in BLCA, experimental results also indicated that the knockdown of GINS2 suppressed BLCA cell phenotypes.ConclusionThis research confirmed that TRPM2-AS could promote BCLA by binding to miR-22-3p to increase GINS2 expression. This novel interactome in BLCA cell lines might provide more insights into BLCA therapy.

CircPAN3 ameliorates myocardial ischaemia/reperfusion injury by targeting miR-421/Pink1 axis-mediated autophagy suppression

AbstractCardiovascular diseases are considered the leading cause of death worldwide. Myocardial ischaemia/reperfusion (I/R) injury is recognized as a critical risk factor for cardiovascular diseases. Although increasing advances have been made recently in understanding the mechanisms of I/R injury, they remain largely unknown. In this study, we found that the expression of circPAN3 (circular RNA PAN3) was decreased in a mouse model of myocardial I/R. Overexpression of circPAN3 significantly inhibited autophagy and alleviated cell apoptosis of cardiomyocytes, which was further verified in vivo by decreased autophagic vacuoles and reduced myocardial infarct sizes. Moreover, miR-421 (microRNA-421) was identified as a downstream target involved in circPAN3-mediated myocardial I/R injury. Additionally, miR-421 could negatively regulate Pink1 (phosphatase and tensin homologue-induced putative kinase 1) via a direct binding relationship. Furthermore, the mitigating effects of circPAN3 overexpression on myocardial I/R injury by suppressing autophagy and apoptosis were abolished by knockdown of Pink1. Our findings reveal a novel role for circPAN3 in modulating autophagy and apoptosis in myocardial I/R injury and the circPAN3–miR-421–Pink1 axis as a regulatory network, which might provide potential therapeutic targets for cardiovascular diseases.Cardiovascular diseases are the leading cause of death worldwide. Myocardial ischaemia/reperfusion (I/R) injury is a major risk for cardiovascular disease. Herein, the authors demonstrate that circPAN3 ameliorates myocardial I/R injury by absorbing miR-421 to regulate Pink1-mediated autophagy, which may provide potential therapeutic targets in I/R injury.

LncRNA MSTRG.29039.1 Promotes Proliferation by Sponging hsa-miR-12119 via JAK2/STAT3 Pathway in Multiple Myeloma.

Noncoding RNA (ncRNA) is involved in the occurrence, development, metastasis, and drug resistance of tumors and involves a variety of biological functions. In addition, miRNA can regulate proliferation and migration and even regulate epigenetics to promote the development of multiple myeloma (MM). However, the mechanism of ncRNA involved in MM is still unclear, and there are many unknown ncRNAs to be explored. This research is aimed at discovering the unknown lncRNA in MM through high-throughput sequencing and to study the mechanism and role of competitive endogenous RNA (ceRNA) involved in the pathogenesis of MM for the development of novel molecular markers and potential new targeted drugs. We screened out 262 new lncRNAs with statistical differences by RNA sequencing and selected the lncRNA MSTRG.29039.1 according to the expression and function of lncRNAs and their target genes in MM. We verified that MSTRG.29039.1 and its target gene OSMR were highly expressed in MM. After knockdown of MSTRG.29039.1 in MM cell lines, the expression of OSMR was decreased, and the expression of hsa-miR-12119 was upregulated which can also promote cell apoptosis and inhibit proliferation. Then, we knocked down hsa-miR-12119 and MSTRG.29039.1, we found that apoptosis of MM cells was reduced, and cell proliferation was increased compared with just knocking down hsa-miR-12119. We further verified the direct binding relationship between MSTRG.29039.1 and OSMR by the dual-luciferase reporter assay system. Thus, MSTRG.29039.1 can competitively bind with miRNA to counteract the inhibitory effect of miRNA on OSMR, which regulates cell proliferation and apoptosis through the JAK2/STAT3 pathway. In a conclusion, lncRNA MSTRG.29039.1 could promote proliferation by sponging hsa-miR-12119 via the JAK2/STAT3 pathway in multiple myeloma. This may be a molecular marker and a potential therapeutic target for MM.

LncRNA HOXA-AS2 accelerates the proliferation and migration and inhibits the apoptosis of vascular smooth muscle cells by absorbing miRNA-877-3p.

The aim of this study was to clarify the role of long non-coding RNA (lncRNA) HOXA-AS2 in influencing the proliferative, migratory and apoptotic abilities of human aortic vascular smooth muscle cells (HA-VSMCs) by absorbing microRNA-877-3p (miRNA-877-3p). HOXA-AS2 level in HA-VSMCs treated with different doses of oxidized low-density lipoprotein (ox-LDL) and for different time points was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). After transfection of si-HOXA-AS2 in HA-VSMCs undergoing ox-LDL treatment, the viability, apoptotic rate and migration of cells were detected, respectively. Meanwhile, the subcellular distribution of HOXA-AS2 was analyzed. The Dual-Luciferase reporter gene assay was applied to verify the binding relationship between HOXA-AS2 and miRNA-877-3p. MiRNA-877-3p level in HA-VSMCs treated with different doses of ox-LDL was determined as well. Furthermore, the regulatory effects of HOXA-AS2/miRNA-877-3p axis on cellular behaviors of HA-VSMCs were determined. HOXA-AS2 expression was upregulated by ox-LDL treatment in a time- and dose-dependent manner. After being treated with 100 mg/L ox-LDL for 48 h, the proliferative and migratory abilities of HA-VSMCs were significantly enhanced, while apoptosis was inhibited. Conversely, these changes were reversed by transfection of si-HOXA-AS2. HOXA-AS2 was mainly distributed in the nuclear fraction. Dual-Luciferase reporter gene assay confirmed the direct binding relationship between HOXA-AS2 and miRNA-877-3p. Moreover, miRNA-877-3p was markedly downregulated after transfection of si-HOXA-AS2. MiRNA-877-3p expression decreased gradually with an increased dose of ox-LDL. In addition, knockdown of miRNA-877-3p could reverse the regulatory effects of HOXA-AS2 on proliferative, migratory and apoptotic abilities of HA-VSMCs. HOXA-AS2 is upregulated after HA-VSMCs injury, which accelerates the proliferative and migratory abilities, and inhibits the apoptosis of vascular smooth muscle cells by absorbing miRNA-877-3p.

RETRACTED: linc01088 promotes cell proliferation by scaffolding EZH2 and repressing p21 in human non-small cell lung cancer

Non-small cell lung cancer (NSCLC), characterized by extensive metastasis and poor prognosis, is the most common type of lung cancer. Dysregulation of certain lncRNAs is known to be linked to the tumorigenesis of NSCLC. However, the specific roles in NSCLC for many other lncRNAs, such as linc01088, remain largely unknown. The expression patterns of linc01088, p21 and EZH2 were examined both in NSCLC tissues and cell lines using RT-qPCR assay. CCK-8, colony formation, immunofluorescence staining, and flow cytometry assays were employed to evaluate the effects of linc01088 on NSCLC cell proliferation properties. RNA immunoprecipitation (RIP) assay was performed to determine the direct binding relationship between linc01088 and zeste homolog 2 (EZH2). Western blot and RT-qPCR analysis were performed to assess p21 level within knockdown of either linc01088 or EZH2. Nude mouse subcutaneous NSCLC models were constructed for further validating the effects and mechanisms of linc01088 in vivo. linc01088 and EZH2 were highly expressed both in NSCLC tissues and cell lines. Knockdown of linc01088 suppressed the proliferation of NSCLC cells, and prolonged the G1 phase while shortened S and G2-M phases. RIP assay revealed the direct binding relationship between linc01088 and EZH2. Knockdown of either linc01088 or EZH2 induced up-regulation of p21 expression, which subsequently inhibited the tumor growth. We demonstrated that linc01088 could promote cell proliferation via binding with EZH2 to repress p21, which aggravates the tumorigenesis of NSCLC. Therefore, linc01088 might be a potential oncogene and target for novel anti-tumor therapies.

LncRNA MALAT1 inhibits hypoxia/reoxygenation-induced human umbilical vein endothelial cell injury via targeting the microRNA-320a/RAC1 axis.

Angiogenesis is believed to protect against hypoxia/reoxygenation (H/R)-induced cell injury. MALAT1 and microRNA-320a (miR-320a) are involved in cancer angiogenesis. To investigate the function of the MALAT1/miR-320a axis in H/R-induced cell injury, human umbilical vein endothelial cell (HUVEC) angiogenesis was detected using the Cell Counting Kit-8 (CCK-8), Transwell migration, cell adhesion and tube formation assays. The expression of MALAT1 and miR-320a was revealed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The direct binding relationship between miR-320a and MALAT1 was detected by RNA immunoprecipitation (RIP) and dual luciferase reporter assays. The data indicated that H/R induces angiogenesis injury and that the expression of MALAT1 was augmented in H/R-stimulated HUVECs. Overexpression of MALAT1 alleviated H/R-stimulated HUVEC dysfunction, whereas silencing of MALAT1 exerted the opposite effects. MALAT1 also reduced miR-320a levels in HUVECs. Overexpression of miR-320a repressed the function of MALAT1 on H/R-stimulated HUVECs, whereas inhibition of miR-320a exerted the opposite effect. Additionally, miR-320a inhibition alleviated H/R-stimulated HUVEC injury via RAC1. Taken together, this investigation concluded that MALAT1 represses H/R-stimulated HUVEC injury by targeting the miR-320a/RAC1 axis.

MiR-221 Promotes Hepatocellular Carcinoma Cells Migration via Targeting PHF2.

MicroRNAs (MiRNAs), which regulate the gene expression leading to translational inhibition or mRNA degradation, are involved in carcinogenesis and tumor progression. Previous studies have demonstrated that miR-221 was one of the most consistent overexpressed miRNAs in several types of cancer. However, the role of miR-221 in human liver cancer progression is not yet fully elucidated. Levels of miR-221 and plant homeodomain finger 2 (PHF2) expressions in human hepatocellular carcinoma (HCC) tissues and cell lines were detected using western blotting and quantitative real-time PCR (qRT-PCR). Cell migration was studied using the transwell assays. A dual-luciferase reporter system was used to validate the target gene of miR-221. The results indicated that miR-221 promoted HCC cell migration. By performing subsequent systematic bioinformatic analyses, we found PHF2 was the target gene of miR-221 and the direct binding relationship was further validated by dual-luciferase reporter assay. In addition, lower expression of PHF2 promoted HCC cell migration and linked to worse overall survival in HCC patients. Finally, the negative correlation between miR-221 and PHF2 expression levels in HCC specimens was further confirmed. Taken together, our findings implied that miR-221 could be a potential candidate for the therapeutics of HCC metastasis.

Akirin2 is modulated by miR-490-3p and facilitates angiogenesis in cholangiocarcinoma through the IL-6/STAT3/VEGFA signaling pathway

Akirin2 is a key regulator of embryonic development and the innate immunity response. However, this regulator’s role in tumorigenesis especially in cholangiocarcinoma (CCA) development has not been thoroughly elucidated to date. In the current work, we used RT-qPCR, western blot analysis, and immunohistochemistry (IHC) to explore the expression level of Akirin2, and the relationship between Akirin2 levels and clinicopathological characteristics was evaluated. The biological functions of Akirin2 were examined in vitro and in vivo by using a lentiviral vector system. Luciferase reporter assays were applied to detect the direct binding relationship between the 3′-UTR of Akirin2 mRNA and miR-490-3p. The results showed that Akirin2 was overexpressed in CCA and this upregulation was associated with a shorter overall survival. Silencing or overexpressing Akirin2 by lentiviral approaches significantly influenced CCA cell proliferation, migration, invasion, and angiogenesis. An in vivo tumor model further validated the oncogenic effect of Akirin2 on CCA cell growth, metastasis, and angiogenesis. Mechanistic studies demonstrated that Akirin2 induced angiogenesis by increasing the expression of VEGFA by activating the IL-6/STAT3 signaling pathway. Akirin2 promoted cell migratory and invasive potential by affecting the epithelial–mesenchymal transition (EMT) process. In addition, Akirin2 expression was negatively controlled by miR-490-3p in CCA cells, and miR-490-3p attenuated cell migration and angiogenesis in CCA cells by silencing Akirin2. Taken together, the data indicated that Akirin2 could be regulated by miR-490-3p at the posttranscriptional level and facilitate CCA cell progression via the IL-6/STAT3/VEGFA signaling pathway. The present study may expedite the development of novel therapeutic strategies for CCA.

Identification of a novel microRNA-mRNA regulatory biomodule in human prostate cancer

Our recent study identified a list of differentially expressed microRNAs (miRNAs) in human prostate cancer (PCa) tissues compared to adjacent benign prostate tissues. In the current study, to identify the crucial miRNA–mRNA regulatory biomodule involved into prostate carcinogenesis based on the previous miRNA expression profile in PCa, we proposed an integrated systematic approach which combined miRNA-mediated gene expression regulatory network analysis, experimental validations in vitro and in vivo, as well as clinical significance evaluation. As a result, the CCND1-RNASEL-CDKN1A-TP73-MDM2-UBE2I axis was identified as a bottleneck in the miRNA-mediated gene expression regulatory network of PCa according to network topological analysis. The direct binding relationship between TP73 and PCa downregulated miR-193a-5p, and the direct binding relationship between UBE2I and PCa upregulated miR-188-5p were both experimentally validated. In addition, miR-193a-5p had a more significant regulatory effect on the tumor promoter isoform of TP73-deltaNp73 than on the tumor suppressive isoform of TP73-TAp73. Importantly, the deregulation of either the miR-193a-5p-TP73 or miR-188-5p-UBE2I axes was significantly associated with aggressive progression and poor prognosis in PCa patients. Gain- and loss-of-function experiments showed that miR-193a-5p efficiently inhibited in vitro PCa cell proliferation, migration, and invasion, and in vivo tumor growth, and markedly induced PCa cell apoptosis via regulating TP73 with a corresponding suppression of the CCND1-RNASEL-CDKN1A-MDM2 axis. In contrast, miR-188-5p exerted its tumor promoter roles through targeting UBE2I with a subsequent activation of the CCND1-RNASEL-CDKN1A-MDM2 axis. Taken together, this integrated analysis revealed the potential roles of the miR-193a-5p/TP73 and miR-188-5p/UBE2i negative regulation pairs in PCa. In addition to the significant clinical relevance, miR-193a-5p- and miR-188-5p-regulated CCND1-RNASEL-CDKN1A-TP73-MDM2-UBE2I signaling may be a novel regulatory biomodule in prostate carcinogenesis.