Pulsed Electromagnetic Fields Combined With Adipose-Derived Stem Cells Protect Ischemic Myocardium by Regulating miR-20a-5p/E2F1/p73 Signaling.

Myocardial infarction (MI) is a serious threat to human health. Although monotherapy with pulsed electromagnetic fields (PEMFs) or adipose-derived stem cells (ADSCs) has been reported to have positive effect on the treatment of MI, a satisfactory outcome has not yet been achieved. In recent years, combination therapy has attracted widespread interest. Herein, we explored the synergistic therapeutic effect of combination therapy with PEMFs and ADSCs on MI and found that the combination of PEMFs and ADSCs effectively reduced infarct size, inhibited cardiomyocyte apoptosis and protected the cardiac function in mice with MI. In addition, bioinformatics analysis and RT-qPCR showed that the combination therapy could affect apoptosis by regulating the expression of miR-20a-5p. A dual-luciferase reporter gene assay also confirmed that the miR-20a-5p could target E2F transcription factor 1 (E2F1) and inhibit cardiomyocyte apoptosis by regulating the E2F1/p73 signaling pathway. Therefore, our study systematically demonstrated the effectiveness of combination therapy on the inhibition of cardiomyocyte apoptosis by regulating the miR-20a-5p/E2F1/p73 signaling pathway in mice with MI. Thus, our study underscored the effectiveness of the combination of PEMFs and ADSCs and identified miR-20a-5p as a promising therapeutic target for the treatment of MI in the future.

miR-20a: a key regulator of orthodontic tooth movement via BMP2 signaling pathway modulation.

AIM: In this study, we aimed to establish a rat tooth movement model to assess miR-20's ability in enhancing the BMP2 signaling pathway and facilitate alveolar bone remodeling. METHOD: 60 male SD rats had nickel titanium spring devices placed between their left upper first molars and incisors, with the right side serving as the control. Forces were applied at varying durations (18h, 24h, 30h, 36h, 42h, 1d, 3d, 5d, 7d, 14d), and their bilateral maxillary molars and surrounding alveolar bones were retrieved for analysis. Fluorescent quantitative PCR was conducted to assess miR-20a, BMP2, Runx2, Bambi and Smad6 gene expression in alveolar bone, and western blot was performed to determine the protein levels of BMP2, Runx2, Bambi, and Smad6 after mechanical loading. RESULT: We successfully established an orthodontic tooth movement model in SD rats and revealed upregulated miR-20a expression and significantly increased BMP2 and Runx2 gene expression and protein synthesis in alveolar bone during molar tooth movement. Although Bambi and Smad6 gene expression did not significantly increase, their protein synthesis was found to decrease significantly. CONCLUSION: MiR-20a was found to be involved in rat tooth movement model alveolar bone remodeling, wherein it promoted remodeling by reducing Bambi and Smad6 protein synthesis through the BMP2 signaling pathway.

Analysis of MiR-20b, MIR-197 markers for differentiation between forensic body fluids encountered in sexual assault cases.

Identifying body fluids can be a critical clue that aids in reconstructing the crime scene. Semen and vaginal fluid identification is crucial, especially in cases of sexual assault. The majority of forensic studies focused on identifying normal body fluids and neglected the expression variation of semen in pathology. To differentiate between vaginal fluids, fertile and infertile semen samples (oligospermia and azoospermia) using miR 20b and miR197. A total of 48 body fluid samples, divided as 16 vaginal fluids, 16 fertile semen, and 16 infertile semen samples (8 with oligospermia and 8 with azoospermia), were collected, and the expression levels of miR-20b and miR-197 were detected by the SYBR Green real-time quantitative PCR technique. Our results showed significant different expression of these miRNAs in normal semen compared to vaginal and infertile semen. Moreover, we designed a model based on Fisher's discriminant function to forecast the group affiliations of unidentified samples. With three novel equations, we were able to accurately distinguish between semen and vaginal fluid, fertile and infertile semen, and oligospermia and azoospermia semen samples with validation accuracy of 81.3%, 100%, and 100%, respectively. MiR-20b and miR-197 expression levels are efficient and appropriate markers to distinguish semen from vaginal fluid and to differentiate between fertile and infertile semen samples. However, the present study is a preliminary study based on clinical samples, and the potential role of these markers in differentiating real crime scene samples is still unknown, so we recommend further research to investigate these markers expression while using forensic samples.

Regulatory mechanism of miR-20a-5p in neuronal damage and inflammation in lipopolysaccharide-induced BV2 cells and MPTP-HCl-induced Parkinson's disease mice.

BACKGROUND: Parkinson's disease (PD) is a prevailing neurodegenerative disorder increasingly affecting the elderly population. The involvement of microRNAs (miRNAs) in PD has been confirmed. We sought to explore the molecular mechanism of miR-20a-5p in PD. METHODS: Lipopolysaccharide (LPS)-induced BV2 cell model and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP-HCl)-induced PD mouse model were established. miR-20a-5p, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1, and IL-10 expression in BV2 cells was examined by reverse transcription - quantitative polymerase chain reaction. Cell viability was assessed by MTT assay. The apoptotic rate and levels of Bcl-2, Bax, cleaved caspase-3, and signal transducer and activator of transmission (STAT)3 were examined by flow cytometry and Western blot. Bioinformatics software predicted the potential binding sites of miR-20a-5p and STAT3. Dual-luciferase experiment verified the binding relationship. Iba1-positive and tyrosine hydroxylase (TH)-positive cell numbers in substantia nigra pars compacta were detected by immunohistochemistry. The effect of miR-20a-5p on motor function in MPTP-induced PD mice was detected by Rota-rod test, Pole test, Traction test and Beam-crossing task. RESULTS: miR-20a-5p was under-expressed in LPS-induced BV2 cells. Overexpression of miR-20a-5p increased the viability of LPS-induced BV2 cells and reduced apoptosis rates. Moreover, overexpression of miR-20a-5p reduced cleaved caspase-3, Bax, iNOS, IL-6, and TNF-α and increased Bcl-2 and TGF-ß1, and IL-10. miR-20a-5p targeted STAT3. STAT3 overexpression partially reversed miR-20a-5p overexpression-mediated effects on LPS-induced BV2 cell viability, apoptosis, and inflammatory responses. miR-20a-5p overexpression inhibited MPTP-induced STAT3 and α-synuclein levels, microglia activation, and inflammatory response, and reduced the loss of TH-positive cells in mice. miR-20a-5p overexpression ameliorated MPTP-induced dyskinesia in PD model mice. CONCLUSION: miR-20a-5p alleviates neuronal damage and suppresses inflammation by targeting STAT3 in PD.

MiR-20a-5p alleviates kidney ischemia/reperfusion injury by targeting ACSL4-dependent ferroptosis.

Ischemia/reperfusion injury (IRI) is prone to occur after kidney transplantation, leading to delayed graft function (DGF). MicroRNAs play a crucial role in the pathogenesis of ischemia/reperfusion-induced acute kidney injury, and miR-20a-5p was found to be the most significantly upregulated gene in a DGF patient cohort. However, the roles of microRNAs in transplanted kidneys remain largely unknown. In this study, we found that miR-20a-5p was upregulated in the kidneys of acute kidney injury mice and in patients with DGF. We identified early growth response-1 as a critical upstream target and verified the binding of early growth response-1 to a predicted sequence in the promoter region of the miR-20a-5p gene. Functionally, the miR-20a-5p mimic attenuated IRI and postischemic renal fibrosis, whereas the miR-20a-5p inhibitor delivery aggravated IRI and fibrosis. Importantly, delivery of the miR-20a-5p mimic or inhibitor in the donor kidneys attenuated or aggravated renal loss and structural damage in cold storage transplantation injury. Furthermore, our study identified miR-20a-5p as a negative regulator of acyl-CoA synthetase long-chain family member 4 (ACSL4) by targeting the 3' untranslated region of ACSL4 mRNA, thereby inhibiting ACSL4-dependent ferroptosis. Our results suggest a potential therapeutic application of miR-20a-5p in kidney transplantation through the inhibition of ACSL4-dependent ferroptosis.

Non-coding RNAs are correlated to TGF-ß receptor type 2 in patients with colorectal cancer.

BACKGROUND: Multiple molecular expression alterations, particularly in non-coding RNAs, play fundamental roles in the regulations of cellular processes and may relate to the occurrence and progression of colorectal cancer (CRC). In the present study, we investigated the associations between TGFBR2, miR20a-5p and long non-coding RNA (lncRNA) LAMTOR5-AS1 in CRC patients. METHODS: Colorectal cancer and adjacent normal tissue samples (n = 34) were prepared from CRC patients. The associations between TGFBR2, miR20a-5p and lncRNA LAMTOR5-AS1 were predicted using bioinformatics tools. The expression levels of TGFBR2, miR20a-5p and lncRNA LAMTOR5-AS1 were measured using a quantitative real-time polymerase chain reaction technique. The TGFBR2 protein values were measured by western blotting. The clinical importance of lncRNA LAMTOR5-AS1 was assessed using receiver operating characteristic curve. RESULTS: The up-regulated levels of TGFBR2 (p = 0.02), TGFBR2 protein (p = 0.008) and lncRNA LAMTOR5-AS1 (p = 0.02) were significantly observed in CRC tissues compared to the adjacent normal tissues. The miR20a-5p expression level (p = 0.009) was downregulated in CRC tissues. In addition, the miR20a-5p expression level was inversely correlated to the TGFBR2 gene (r2  = 0.88, p < 0.0001), protein (r2  = 0.95, p < 0.0001) and lncRNA LAMTOR5-AS1 gene (r2  = 0.93, p < 0.0001) expression levels. Based on the area under curve, the increase of lncRNA LAMTOR5-AS1 expression level with a sensitivity of 64.52% and specificity of 65.52% was considered in CRC patients. CONCLUSIONS: We propose that miR20a-5p is inversely related to long non-coding RNA (lncRNA) LAMTOR5-AS, such that it may be involved in the regulation of TGFBR2 expression level in CRC patients.

UCMSCs-derived exosomal circHIPK3 promotes ulcer wound angiogenesis of diabetes mellitus via miR-20b-5p/Nrf2/VEGFA axis.

AIMS: Experiments confirmed that circular RNAs contributed to the pathogenesis of diabetic foot ulcers (DFUs). CircHIPK3 was upregulated in type 2 diabetes mellitus (T2DM), but its role in DFU remained unknown. Our study aimed to investigate the regulatory functions of exosomal circHIPK3 and its potential mechanisms in DFU. METHODS: Exosomal size and distribution, marker proteins, and circHIPK3 levels were evaluated by transmission electron microscope, ExoView R200, western blot, and qRT-PCR. Flow cytometry, MTT, Wound healing assays, and tube formation assays were used to assess the roles of exosomal circHIPK3 in high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs). The relationships between Nrf2/VEGFA/circHIPK3 and miR-20b-5p, and between Nrf2 and VEGFA were determined by luciferase reporter assay and RNA immunoprecipitation. We used cell and mice models to investigate the mechanisms of exosomal circHIPK3 under diabetic conditions. RESULTS: CircHIPK3 was significantly upregulated in exo-circHIPK3 rather than exo-vector. Exo-circHIPK3 remarkably inhibited cell apoptosis but promoted cell proliferation, migration, and tube formation in HG-treated HUVECs. Luciferase reporter and RIP assays showed that miR-20b-5p targeted and inhibited Nrf2 and VEGFA, and circHIPK3 acted as a ceRNA of miR-20b-5p to inhibit the binding to its downstream genes Nrf2 and VEGFA. Mechanistically, circHIPK3 promoted cell proliferation, migration, and angiogenesis via downregulating miR-20b-5p to upregulate Nrf2 and VEGFA. However, the overexpressed miR-20b-5p could abolish the promoting effects of circHIPK3 overexpression on cell proliferation, migration, and tube formation under HG conditions. CONCLUSION: UCMSCs-derived exosomal circHIPK3 protected HG-treated HUVECs via miR-20b-5p/Nrf2/VEGFA axis. The exosomal circHIPK3 might be a therapeutic candidate to treat DFU.

The dual role of microRNA (miR)-20b in cancers: Friend or foe?

MicroRNAs, as non-coding transcripts, modulate gene expression through RNA silencing under normal physiological conditions. Their aberrant expression has strongly associated with tumorigenesis and cancer development. MiR-20b is one of the crucial miRNAs that regulate essential biological processes such as cell proliferation, apoptosis, autophagy, and migration. Deregulated levels of miR-20b contribute to the early- and advanced stages of cancer. On the other hand, investigations emphasize the tumor suppressor ability of miR-20b. High-throughput strategies are developed to identify miR-20b potential targets, providing the proper insight into its molecular mechanism of action. Moreover, accumulated results suggest that miR-20b exerts its effects through diverse signaling pathways, including PI3K/AKT/mTOR and ERK axes. Restoration of the altered expression levels of miR-20b induces cell apoptosis and reduces invasion and migration. Further, miR-20b can be used as a biomarker in cancer. The current comprehensive review could lead to a better understanding of the miR-20b in either tumorigenesis or tumor regression that may open new avenues for cancer treatment. Video Abstract.

Circular RNA ITCH increases sorafenib-sensitivity in hepatocellular carcinoma via sequestering miR-20b-5p and modulating the downstream PTEN-PI3K/Akt pathway.

BACKGROUNDS: Sorafenib-resistance leads to poor prognosis and high mortality in advanced hepatocellular carcinoma (HCC), and this study aims to investigate the functional role of a circular RNA ITCH (circITCH) in regulating the sorafenib-resistance of HCC and its underlying mechanisms. METHODS: The expression of circITCH in HCC tissues and cell lines were detected by performing quantitative real-time polymerase chain reaction. Sorafenib-resistant HCC cells were transfected with PLCDH-circITCH to upregulate circITCH and intervened with sorafenib, and MTT assay, flow cytometry and transwell assay were used to test the cell viability, apoptosis and migration ability, respectively. The downstream target of circITCH were explored by using bioinformatic analysis, dual luciferase reporter system and Western blot. RESULTS: CircITCH was significantly down-regulated in HCC tissues and cell lines, compared with their normal counterparts. Especially, in contrast with the sorafenib-sensitive HCC cells, continuous sorafenib treatment decreased the expression levels of circITCH in the sorafenib-resistant HCC cells. Overexpression of circITCH increased sorafenib-sensitivity, promoted cell apoptosis and reduced cell migration abilities in the sorafenib-resistant HCC cells. Mechanically, circITCH elevated PTEN expression to inactivate the PI3K/Akt signals through negatively regulating miR-20b-5p in HCC, and upregulating miR-20b-5p or inhibiting PTEN abolished the enhancing effect of circITCH overexpression on sorafenib-induced cytotoxicity in sorafenib-resistant HCC cells. CONCLUSION: Taken together, this study proves that circITCH enhances sorafenib-sensitivity in sorafenib-resistant HCC cells via regulating the miR-20b-5p/PTEN/PI3K/Akt signaling cascade, which highlights the potential value of circITCH as a target for enhancing the sorafenib-sensitivity in HCC.

Dynamic effects of miR-20a-5p on hippocampal ripple energy after status epilepticus in rats.

To determine the dynamic effects of miR-20a-5p on hippocampal ripple energy in rats after status epilepticus (SE). A lithium pilocarpine (LiCl-PILO)-induced rat model of status epilepticus (SE) was established, and the rats were divided into the normal control (Control, CTL), epileptic control (PILO), valproic acid (VPA + PILO), miR-20a-5p overexpression lentivirus vector (miR + PILO), sponges blocking lentivirus vector (Sponges + PILO), and scramble sequence negative control (Scramble + PILO) groups (n = 6). Electroencephalograms (EEGs) were used to analyze changes in hippocampal ripple energy before and after SE. Quantitative polymerase chain reaction (q-PCR) analysis showed that miR-20a-5p levels gradually increased after miR-20a-5p overexpression lentivirus vector injection into the lateral ventricle, and the miR-20a-5p levels were significantly higher than that in CTL group on days 7 and 36 (P < 0.001). The miR-20a-5p levels decreased significantly on days 7 and 36 after blocking by sponges lentivirus vector injected into the lateral ventricle (P < 0.001). After injection of PILO, the average ripple energy expression in each group gradually increased, and reached the peak before chloral hydrate injection (compared with 1 day before SE, P < 0.05). The ripple energy in the VPA + PILO and Sponges + PILO groups was significantly lower than that in the PILO group at 60 min and 70 min after PILO injection and before chloral hydrate injection (P < 0.05), and maintained lower until 2 h after chloral hydrate injection in VPA + PILO (P < 0.05). Compared with the VPA + PILO group, the mean ripple energy of the Sponges + PILO group had no difference at all time points (P ≥ 0.05). After SE, ripple distribution of space and energy is closely related to the occurrence of epilepsy. Inhibition of miR20a-5p expression can downregulate ripple oscillation energy during seizure.

miR-20b attenuates airway inflammation by regulating TXNIP and NLRP3 inflammasome in ovalbumin-induced asthmatic mice.

OBJECTIVES: Asthma is a chronic inflammatory disorder of the airway and is associated with pyroptosis. microRNAs (miRNAs) underlie pathogenic mechanism in asthma. This study is expected to evaluate the role of miR-20b in asthma-induced airway inflammation via regulating thioredoxin-interacting protein (TXNIP) and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. METHODS: The asthmatic mouse model was established via ovalbumin (OVA) induction. Expressions of miR-20b, TXNIP, and NLRP3 in lung tissues were determined. Bronchial hyperresponsiveness was appraised, cells in bronchoalveolar lavage fluid were counted and categorized, and histopathological damage was observed. Levels of inflammatory and pyroptotic cytokines were measured. The binding relationship of miR-20b and TXNIP was testified. Co-location and interaction between TXNIP and NLRP3 were detected. Mice were infected with the lentivirus packaged with pcDNA3.1-TXNIP or pcDNA3.1-NLRP3 for joint experiments to observe the pathological changes of mice. RESULTS: miR-20b was poorly expressed, while TXNIP and NLRP3 were highly expressed in OVA-induced mice. miR-20b overexpression attenuated airway inflammation and pyroptosis, manifested by alleviation of histopathological damage, declined numbers of total cells and inflammatory cells, lowered bronchial hyperresponsiveness, decreased levels of pro-inflammatory and pyroptotic cytokines, and increased anti-inflammatory cytokines. miR-20b targeted TXNIP and inhibited TXNIP expression, and TXNIP can bind to NLRP3 and upregulated NLRP3 expression. Upregulation of TXNIP or NLRP3 could reverse the protecting role of miR-20b overexpression in OVA-induced mice. CONCLUSION: miR-20b inhibited TXNIP expression to reduce the binding of TXNIP and NLRP3, thus restricting pyroptosis and airway inflammation of asthmatic mice.

Downregulation of miR-20b-5p Contributes to the Progression of Liver Fibrosis via the STAT3 Signaling Pathway In Vivo and In Vitro.

BACKGROUND: Activated hepatic stellate cells (HSCs) are primarily involved in liver fibrosis and portal hypertension (PHT). We aimed to investigate the effect of miR-20b-5p on HSCs, liver fibrosis, and PHT. METHODS: MiR-20b-5p expression in HSCs and in mouse liver fibrosis was determined by qPCR. Further, the effects of miR-20b-5p mimic on HSCs migration, proliferation, and apoptosis were investigated in vitro. A dual-luciferase reporter assay was performed to confirm the direct interaction between miR-20b-5p and STAT3. In vivo, mouse liver fibrosis was established by common bile duct ligation and intervened by agomiR-20b-5p. Sirius red staining and hydroxyproline content were used to evaluate collagen deposition. The α-SMA expression in the liver was detected by IHC and Western blotting. The STAT3 signaling pathway and its downregulated cytokines as well as portal pressure and angiogenesis were explored. RESULTS: MiR-20b-5p was significantly downregulated during HSCs activation and in mouse liver fibrosis. The functional analyses demonstrated that miR-20b-5p inhibited cell proliferation, activation, and promoted apoptosis in HSCs in vitro. Moreover, miR-20b-5p regulated STAT3 expression by binding to the 3'UTR of its miRNA directly. Overexpression of miR-20b-5p facilitated HSC activation and proliferation by inhibiting the STAT3 signaling pathway. MiR-20b-5p overexpression suppressed the STAT3 and its downstream cytokines and ameliorated liver fibrosis in mice. The intra- and inter-hepatic angiogenesis were also effectively inhibited. The inhibition of liver fibrosis and neoangiogenesis contributed to the decrease of portal pressure. CONCLUSIONS: MiR-20b-5p plays an important role in the fibrosis and angiogenesis of liver fibrosis by targeting the STAT3 signaling pathway.

Identifying the potential role of serum miR-20a as a biomarker for olfactory dysfunction in patients with Parkinson's disease.

INTRODUCTION: Olfactory dysfunction (OD), one of the most common non-motor symptoms in Parkinson's disease (PD), is a cardinal prodromal symptom that can appear years before the onset of motor symptoms. Ongoing studies have demonstrated that microRNAs (miRNAs) are suitable biomarkers for PD, while there is a lack of robust miRNAs that can serve as markers for OD in PD. METHODS: The concordantly differentially expressed miRNAs (DE miRNAs) in the damaged olfactory system were first identified in 2 OD-related Gene Expression Omnibus (GEO) datasets. Then, they were verified in another PD-related GEO dataset and only one miRNA (miR-20a) was found to be significantly altered. Serum levels of miR-20a were further measured by qPCR in 79 PD patients with OD (PD-OD), 52 PD patients without OD (PD-NOD), and 52 healthy controls (HC). Objective measure of OD was defined by 16-item Sniffin' Sticks odor identification test. All the participants underwent a demographic and comprehensive PD-related clinical assessment. RESULTS: Our results proved that miR-20a was significantly downregulated in PD-OD compared with PD-NOD and the area under curve (AUC) for OD detection by miR-20a was 0.803 (95% confidence interval, 0.724-0.883). In addition, PD-OD had higher scores of Movement Disorder Society-Unified Parkinson's Disease Rating Scale (UPDRS) II, Hoehn and Yahr stage (H-Y), Non-Motor Symptoms Scale (NMSS) 3, NMSS 5, NMSS 9, Hamilton Rating Scale for Depression (HAMD), Hamilton Anxiety Scale (HAMA), Activity of Daily Living (ADL), and lower scores of Mini-Mental State Examination (MMSE) and 39-item PD Quality of Life Questionnaire (PDQ-39) than PD-NOD. Binary regression model further presented that lower expressions of miR-20a and poorer cognitive function acted as promoting factors in the development of OD. CONCLUSION: Our results suggest that miR-20a could be a novel biomarker for OD in PD and PD-OD patients tend to have higher disease stage, poorer motor aspects of experiences of daily living, worse cognitive scores, and inferior quality of life, and were more likely to have mental disorders. Cognitive function, in particular, is strongly associated with OD in PD patients.

The miR-20a/miR-92b Profile Is Associated with Circulating γδ T-Cell Perturbations in Mild Psoriasis.

Psoriasis vulgaris (PV) is an autoinflammatory dermatosis of unknown etiology. Current evidence suggests a pathogenic role of γδT cells, but the growing complexity of this population has made the offending subset difficult to pinpoint. The work on γδTCRint and γδTCRhi subsets, which express intermediate and high levels of γδTCR at their surface, respectively, is particularly scarce, leaving their inner workings in PV essentially unresolved. We have shown here that the γδTCRint/γδTCRhi cell composition and their transcriptom are related to the differential miRNA expression by performing a targeted miRNA and mRNA quantification (RT-qPCR) in multiplexed, flow-sorted γδ blood T cells from healthy controls (n = 14) and patients with PV (n = 13). A significant loss of miR-20a in bulk γδT cells (~fourfold decrease, PV vs. controls) largely mirrored increasing Vδ1-Vδ2- and γδintVδ1-Vδ2- cell densities in the bloodstream, culminating in a relative excess of γδintVδ1-Vδ2- cells for PV. Transcripts encoding DNA-binding factors (ZBTB16), cytokine receptors (IL18R1), and cell adhesion molecules (SELPLG) were depleted in the process, closely tracking miR-20a availability in bulk γδ T-cell RNA. Compared to controls, PV was also associated with enhanced miR-92b expression (~13-fold) in bulk γδT cells that lacked association with the γδT cell composition. The miR-29a and let-7c expressions remained unaltered in case-control comparisons. Overall, our data expand the current landscape of the peripheral γδT cell composition, underlining changes in its mRNA/miRNA transcriptional circuits that may inform PV pathogenesis.

Plasmatic MicroRNAs and Treatment Outcomes of Patients with Metastatic Castration-Resistant Prostate Cancer: A Hospital-Based Cohort Study and In Silico Analysis.

Prostate cancer (PCa) is one of the most common malignancies among men worldwide. Inevitably, all advanced PCa patients develop metastatic castration-resistant prostate cancer (mCRPC), an aggressive phase of the disease. Treating mCRPC is challenging, and prognostic tools are needed for disease management. MicroRNA (miRNA) deregulation has been reported in PCa, constituting potential non-invasive prognostic biomarkers. As such, this study aimed to evaluate the prognostic potential of nine miRNAs in the liquid biopsies (plasma) of mCRPC patients treated with second-generation androgen receptor axis-targeted (ARAT) agents, abiraterone acetate (AbA) and enzalutamide (ENZ). Low expression levels of miR-16-5p and miR-145-5p in mCRPC patients treated with AbA were significantly associated with lower progression-free survival (PFS). The two miRNAs were the only predictors of the risk of disease progression in AbA-stratified analyses. Low miR-20a-5p levels in mCRPC patients with Gleason scores of <8 were associated with worse overall survival (OS). The transcript seems to predict the risk of death regardless of the ARAT agent. According to the in silico analyses, miR-16-5p, miR-145-5p, and miR-20a-5p seem to be implicated in several processes, namely, cell cycle, proliferation, migration, survival, metabolism, and angiogenesis, suggesting an epigenetic mechanism related to treatment outcome. These miRNAs may represent attractive prognostic tools to be used in mCRPC management, as well as a step further in the identification of new potential therapeutic targets, to use in combination with ARAT for an improved treatment outcome. Despite the promising results, real-world validation is necessary.

MEF2C-AS1 regulates its nearby gene MEF2C to mediate cervical cancer cell malignant phenotypes in vitro.

Cervical cancer (CC) is the second most common malignancy among women. GEPIA demonstrated that MEF2C-AS1 and its nearby gene MEF2C present downregulation in CC tissues. We attempted to clarify molecular mechanism between MEF2C-AS1 and MEF2C underlying CC progression. RT-qPCR was used to measure expression levels and subcellular distribution of MEF2C-AS1 and MEF2C in CC cell lines. Gain-of-function assays were conducted to reveal roles of MEF2C-AS1 and MEF2C in CC cell behaviors. Bioinformatics, RNA pull down, and RIP assays were performed to assess association of MEF2C-AS1 or MEF2C with miR-20 b-5p in CC cells. Rescue assays were done to assess regulatory function of the MEF2C-AS1-miR-20 b-5p-MEF2C axis in CC cellular processes. MEF2C-AS1 and its nearby gene MEF2C showed downregulation and had a positive expression correlation in CC tissues. MEF2C-AS1 and MEF2C presented downregulation in CC cells, and they majorly distributed in CC cell cytoplasm. MEF2C-AS1 and MEF2C upregulation repressed CC cell proliferative, migratory, and angiogenic abilities. MEF2C-AS1 competitively bound with miR-20 b-5p to upregulate MEF2C in CC cells. The impacts of MEF2C-AS1 elevation on CC cell proliferative, migratory, and angiogenic capabilities were countervailed by miR-20 b-5p overexpression. The impacts of miR-20 b-5p inhibitor on CC cell proliferative, migratory and angiogenic capabilities were countervailed by MEF2C depletion. To sum up, MEF2C-AS1 and its nearby gene MEF2C present downregulation and serve as tumor suppressors in CC cells. MEF2C-AS1 suppresses CC cell malignancy in vitro through sponging miR-20 b-5p to upregulate MEF2C, which may provide a potential new direction for seeking therapeutic plans of CC.

LncRNA CALML3-AS1 suppresses papillary thyroid cancer progression via sponging miR-20a-5p/RBM38 axis.

BACKGROUND: The incidence and mortality of thyroid cancer (TC) has been steadily rising in the past decades. It is imperative to have a better understanding of the molecular mechanisms underlying TC development and identify novel therapeutic targets. This study characterized the role of lncRNA CALML3-AS1 (CALML3-AS1) in the development of papillary thyroid cancer (PTC). METHOD: Related mRNAs expression were validated in the tumor and adjacent normal tissues from 52 PTC patients and PTC cell lines by qRT-PCR. Expression of RBM38 was detected by Western blot. We have also conducted CCK-8 and colony formation assays were used to detect the effect of CALML3-AS1 on cell proliferation, Transwell assay was utilized to evaluate cell migration and invasion, apoptosis detected by flow cytometry assay, RNA pull-down and luciferase assays were performed to validate gene predictions. RESULTS: The results indicated that the expression of both CALML3A-S1 and RBM38 were significantly downregulated in PTC tissues (p < 0.01), while the expression of miR-20a-5p was increased in PTC (p < 0.01). Functionally, CALML3-AS1 overexpression inhibited PTC cell proliferation in vitro and in vivo. Mechanistically, CALML 3-AS1 sponged miR-20a-5p, which in turn leads to the suppression of RBM38 expression and PTC progression. CONCLUSIONS: CALML3-AS1 functions as a ceRNA for miR-20a-5p in the regulation of the expression of RBM38 in PTC. Higher level of CALML3-AS1 serves as a good prognostic indicator of survival in PTC patients. Targeting CALML3-AS1/ miR-20a-5p/RBM38 axis may represent a novel therapeutic strategy in the treatment of PTC.

Knockdown of lncRNA H19 alleviates ox-LDL-induced HCAECs inflammation and injury by mediating miR-20a-5p/HDAC4 axis.

BACKGROUND: Coronary artery disease (CAD) seriously disturbs the life of people. LncRNA H19 is reported to promote the progression of CAD; Nevertheless, the detailed mechanism by which H19 modulates CAD development is unclear. METHODS: Clinical samples of CAD patients were collected, meanwhile we established in vitro and in vivo models of CAD by treating HCAECs with ox-LDL and feeding ApoE-/- mice with high fat diets (HFD). MTT assay was adopted to assess the cell viability. Transwell detection was applied to test the migration, and apoptosis was tested by flow cytometry. The levels of inflammatory cytokines were examined by ELISA. The relation among H19, miR-20a-5p and HDAC4 was explored by dual luciferase reporter and RIP assay. RESULTS: H19 and HDAC4 levels were elevated, while miR-20a-5p was reduced in plasma of CAD patients and ox-LDL-treated HCAECs. ox-LDL increased H19 level and induced apoptosis and inflammation in HCAECs, while silencing of H19 rescued this phenomenon. In addition, the level of H19 was negatively correlated with miR-20a-5p, and miR-20a-5p inhibitor restored the effect of H19 silencing on HCAECs function. HDAC4 was the downstream mRNA of miR-20a-5p, and miR-20a-5p upregulation reversed ox-LDL-induced HCAECs injury through targeting HDAC4. Furthermore, H19 silencing significantly alleviated the coronary atherosclerotic plaques and inhibited the inflammatory responses in vivo. CONCLUSIONS: We proved that knockdown of H19 alleviated ox-LDL-induced HCAECs injury via miR-20a-5p/HDAC4 axis, which might provide a new tactics against CAD.

Identification of the circ_PRKDC/miR-20a-3p/RASA1 axis in regulating HaCaT keratinocyte migration.

Migration of keratinocytes plays a crucial role in the re-epithelialization phase during wound healing. Circular RNA (circRNA) protein kinase, DNA-activated, catalytic subunit (circ_PRKDC, hsa_circ_0084443) has been identified as a regulator of keratinocyte migration. However, the molecular basis governing it remains unclear. The levels of circ_PRKDC, microRNA (miR)-20a-3p, and RAS p21 protein activator 1 (RASA1) were assessed by quantitative real-time PCR (qRT-PCR) or western blot. Subcellular localization, Actinomycin D, and Ribonuclease (RNase) R assays were performed to characterise circ_PRKDC. Cell migration was gauged by transwell and wound-healing assays. A direct relationship between miR-20a-3p and circ_PRKDC or RASA1 was verified by dual-luciferase reporter and RNA pull-down assays. Circ_PRKDC expression was reduced in wound skin during wound healing. Circ_PRKDC modulated migration of HaCaT keratinocytes. Mechanistically, circ_PRKDC directly targeted miR-20a-3p. The regulation of circ_PRKDC on HaCaT keratinocyte migration was mediated by miR-20a-3p. RASA1 was identified as a direct and functional target of miR-20a-3p, and miR-20a-3p-mediated inhibition of RASA1 impacted HaCaT keratinocyte migration. Circ_PRKDC acted as a post-transcriptional modulator of RASA1 expression through miR-20a-3p. Moreover, circ_PRKDC modulated migration of HaCaT keratinocytes by RASA1. Our findings demonstrated a novel molecular basis, the miR-20a-3p/RASA1 axis, for the regulation of circ_PRKDC on HaCaT keratinocyte migration.

Combined high-throughput library screening and next generation RNA sequencing uncover microRNAs controlling human cardiac fibroblast biology.

BACKGROUND: Myocardial fibrosis is a hallmark of the failing heart, contributing to the most common causes of deaths worldwide. Several microRNAs (miRNAs, miRs) controlling cardiac fibrosis were identified in recent years; however, a more global approach to identify miRNAs involved in fibrosis is missing. METHODS AND RESULTS: Functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated miRNAs inducing proliferation. In parallel, miRNA deep sequencing was performed after subjecting HCFs to proliferating and resting stimuli, additionally enabling discovery of novel miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p and miR-132-3p. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated by synthetic siRNA. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by miR-20a-5p and miR-132-3p explain some of their pro-fibrotic nature on a mechanistic level. CONCLUSION: We here identified both miR-20a-5p and miR-132-3p as crucial regulators of fibrotic pathways in an in vitro model of human cardiac fibroblast biology.