Retracted: microRNA-129-5p involved in the neuroprotective effect of dexmedetomidine on hypoxic-ischemic brain injury by targeting COL3A1 through the Wnt/β-catenin signaling pathway in neonatal rats

Our study aims to elucidate the mechanisms how microRNA-129-5p (miR-129-5p) involved in the neuroprotective effect of dexmedetomidine (DEX) on hypoxic-ischemic brain injury (HIBI) by targeting the type III procollagen gene (COL3A1) through the Wnt/β-catenin signaling pathway in neonatal rats. A total of 120 rats were obtained, among which 15 rats were selected as sham group and rest rats as model, DEX, DEX + negative control (DEX + NC), DEX + miR-129-5p mimics, DEX + miR-129-5p inhibitors, DEX + XAV-939, and DEX + miR-129-5p inhibitors + XAV-939 groups. A dual-luciferase reporter assay was performed for the target relationship between miR-129-5p and COL3A1. Weight rate and water content of cerebral hemisphere were detected. Quantitative real-time polymerase chain reaction and Western blot analysis were conducted to detect miR-129-5p expression and expressions of COL3A1, E-cadherin, T-cell factor (TCF)− 4, and β-catenin. The DEX, DEX + miR-129-5p mimics, DEX + XAV-939 groups had increased weight rate of the cerebral hemisphere, but decreased water content of left cerebral hemisphere, levels of COL3A1, β-catenin, TCF-4, and E-cadherin in the hippocampus compared with the model and DEX + miR-129-5p inhibitors groups. COL3A1 was verified as the target gene of the miR-129-5p. Compared with the DEX + NC and DEX + miR-129-5p inhibitors + XAV-939 groups, the DEX + XAV-939 and DEX + miR-129-5p mimics groups had elevated weight rate of the cerebral hemisphere, but reduced water content of left cerebral hemisphere, levels of COL3A1, β-catenin, TCF-4, and E-cadherin in the hippocampus. Our findings demonstrate that miR-129-5p improves the neuroprotective role of DEX in HIBI by targeting COL3A1 through the Wnt/β-catenin signaling pathway in neonatal rats.     

MicroRNA‐140‐5p elevates cerebral protection of dexmedetomidine against hypoxic–ischaemic brain damage via the Wnt/β‐catenin signalling pathway

Hypoxia–ischaemia (HI) remains a major cause of foetal brain damage presented a scarcity of effective therapeutic approaches. Dexmedetomidine (DEX) and microRNA‐140‐5p (miR‐140‐5p) have been highlighted due to its potentially significant role in the treatment of cerebral ischaemia. This study was to investigate the role by which miR‐140‐5p provides cerebral protection using DEX to treat hypoxic–ischaemic brain damage (HIBD) in neonatal rats via the Wnt/β‐catenin signalling pathway. The HIBD rat models were established and allocated into various groups with different treatment plans, and eight SD rats into sham group. The learning and memory ability of the rats was assessed. Apoptosis and pathological changes in the hippocampus CA1 region and expressions of the related genes of the Wnt/β‐catenin signalling pathway as well as the genes responsible of apoptosis were detected. Compared with the sham group, the parameters of weight, length growth, weight ratio between hemispheres, the rate of reaching standard, as well as Bcl‐2 expressions, were all increased. Furthermore, observations of increased levels of cerebral infarction volume, total mortality rate, response times, total response duration, expressions of Wnt1, β‐catenin, TCF‐4, E‐cadherin, apoptosis rate of neurons, and Bax expression were elevated. Following DEX treatment, the symptoms exhibited by HIBD rats were ameliorated. miR‐140‐5p and si‐Wnt1 were noted to attenuate the progression of HIBD. Our study demonstrates that miR‐140‐5p promotes the cerebral protective effects of DEX against HIBD in neonatal rats by targeting the Wnt1 gene through via the negative regulation of the Wnt/β‐catenin signalling pathway.     

miR-129-5p inhibits proliferation,migration, and invasion in rectal adenocarcinoma cells through targeting E2F7

microRNAs (miRNAs), a kind of small noncoding RNAs, are considered able to regulate expression of genes and mediate RNA silencing. miR-129-5p was shown to be a cancer-related miRNA. However, the influence of miR-129-5p in rectal adenocarcinoma (READ) development remains to be determined. Based on the TCGA data, downregulation of miR-129-5p in READ samples was observed. Manual restoration of the miR-129-5p in SW1463 and SW480 cell lines significantly inhibited invasion, migration, and proliferation of READ cell lines, while the apoptosis ability was enhanced. Meanwhile, we found E2F7 acted as a potential target of miR-129-5p and was upregulated in READ samples. E2F7 upregulation reversed the repression of miR-129-5p on READ development. Finally, in vivo experiments showed that inhibition of tumor growth in nude mice was achieved through upregulating miR-129-5p. Overall, our findings suggest increasing of miR-129-5p leads to the suppression of READ progression through regulating the expression of E2F7, which may provide novel insights into the treatment of READ.     

Down-Regulation of miR-129-5p Inhibits Growth and Induces Apoptosis in Laryngeal Squamous Cell Carcinoma by Targeting APC

miRNAs regulate gene expression and are key mediators of tumourigenesis. miR-129 has diverse effects in tumours, but its role in laryngeal squamous cell carcinoma (LSCC) remains unknown. This article focuses on the role of miR-129-5p in LSCC. We show miR-129-5p is upregulated in primary LSCC tumours and correlated with advanced disease. Down-regulating miR-129-5p suppressed cell proliferation and migration, and caused cell cycle arrest in Hep-2 cell lines. Downregulation of miR-129-5p alone is sufficient to induce apoptosis both in vivo and in vitro. Moreover, the growth of LSCC xenograft exposed to miR-129-5p antisense oligonucleotides (ASO) in BALB/c mice was markedly inhibited. In addition, we found that miR-129-5p targeted adenomatous polyposis coli (APC) to release inhibition of Wnt signalling causing cell growth and tumourigenesis. Our results suggest miR-129-5p functions as an oncogene in LSCC by repressing APC and is a potential therapeutic target for LSCC.     

A2AR敲除对新生小鼠缺氧缺血脑区cyt C表达的影响

腺苷（adenosine）A2A受体（A2A receptor,A2AR）作为腺苷四种受体亚型之一,对新生鼠脑缺氧缺血的作用尚存在争议,探讨其作用机制将有助于新生儿缺氧缺血性脑病（hypoxic-ischemic encephalopathy,HIE）的临床治疗。为此,该实验观察了新生鼠脑缺氧缺血后A2AR敲除对其神经行为学的影响;采用TUNEL技术结合HE染色检测神经细胞凋亡;采用免疫组织化学法检测活化天冬氨酸特异性半胱氨酸蛋白酶3（caspase 3）及胞浆中细胞色素C（cytochrome C,cyt C）的表达。结果发现,A2AR敲除损伤了新生鼠神经行为功能,使神经细胞的凋亡和胞浆中cyt C的表达增加、caspase3活化增强。其中,在脑缺氧缺血后的1,3,7 d,神经细胞凋亡和caspase 3活化较野生型显著增加（P〈0.01）,而胞浆中cyt C的表达仅在脑缺氧缺血后的1,3 d显著增加（P〈0.01）,且其表达与神经细胞凋亡、caspase 3的活化均呈显著正相关。这提示,A2AR敲除后可能通过促使cyt C由线粒体释放出来增加新生鼠脑缺氧缺血后神经细胞的凋亡。     

Inhibition of lncRNA MEG3 protects renal tubular from hypoxia-induced kidney injury in acute renal allografts by regulating miR-181b/TNF-α signaling pathway

Early damage to transplanted organs initiates excess inflammation that deteriorates existing injury, which is a leading cause of graft loss. Long noncoding RNAs (lncRNAs) are recently thought to play a significant role in cellular homeostasis during pathological process of kidney diseases. The aim of this study was to assess the function and mechanism of lncRNA, maternally expressed gene 3 (MEG3), on early renal allografts pathogenesis. Real-time polymerase chain reaction (RT-PCR) analysis found that the levels of MEG3 and miR-181b-5p were increased and decreased respectively in grafted kidney. The Western blot assay showed that TNF-alpha was upregulated in the kidney and in HK-2 cells. Administering MEG3-specific small interfering RNA (siRNA) in mice silenced MEG3 expression and protected kidney renal allograft from injury. Bioinformatical analysis and luciferase assay indicated that MEG3 is a target of miR-181b-5p. MEG3 inhibition and overexpression promoted and suppressed miR-181b-5p levels respectively. In addition, Western blot and immunohistochemical staining suggested that decreased TNF-alpha expression was observed in the kidney. In contrary to MEG3, miR181b overexpression attenuated hypoxia-induced HK-2 cell apoptosis, as well as suppressed hypoxia-induced TNF-alpha upregulation. In luciferase reporter assay, we confirmed that miR-181b directly bound to the 3′-untranslated region (3′-UTR) of TNF-alpha, thereby negatively regulating the TNF-alpha expression. Our data suggested that MEG3 functions as a competing endogenous RNA for miR-181b to regulate the TNF-alpha expression in hypoxia-induced kidney injury in acute renal allografts.     

Upregulation of miR-582-5p regulates cell proliferation and apoptosis by targeting AKT3 in human endometrial carcinoma

The human endometrial carcinoma is one of the most common female malignancies, and there is an urgent requirement to explore new therapeutic strategies. There is accumulating evidence that microRNAs (miRNAs) can serve as potential diagnostic and prognostic biomarkers for various types of cancer, but the significance of miR-582-5p still remains largely unknown in the endometrial carcinoma. The aims of this study were to understand and identify the influence of miR-582-5p on the proliferation and apoptosis of human endometrial carcinoma and its relevant mechanism. First, quantitative real-time PCR (qRT-PCR) was used to detect miR-582-5p and AKT3 expression in human tissue samples and cells. Then, CyQuant assay and 2D colony assay were employed to evaluate cell proliferation. Western blotting was used to determine protein expression. Subsequently, the luciferase reporter assay was used to identify the target of miR-582-5p. Finally, Annexin V assay was used to detect cell apoptosis. We found that miR-582-5p expression was significantly decreased in human endometrial carcinoma tissues, and miR-582-5p upregulation in human endometrial carcinoma cells inhibit cell proliferation and promote apoptosis. Moreover, AKT3 was validated as a target of miR-582-5p and AKT3 expression was inversely correlated with miR-582-5p expression. Besides, AKT3 upregulation efficiently abrogates the effect of miR-582-5p on the cells. These results demonstrated that miR-582-5p regulates cell proliferation and apoptosis in human endometrial carcinoma via AKT3. Thus, miR-582-5p represents a potential therapeutic target in human endometrial carcinoma meriting further investigation.     

LncRNA MEG3 negatively modified osteosarcoma development through regulation of miR-361-5p and FoxM1

This study was aimed to figure out whether long noncoding RNA MEG3/miR-361-5p/FoxM1 signaling would contribute to improved proliferation and metastasis of osteosarcoma cells. We altogether collected 204 pairs of osteosarcoma tissues and adjacent normal tissues, and obtained four human osteosarcoma cell lines. Then pcDNA3.1-MEG3, si-MEG3, miR-361-5p mimic, miR-361-5p inhibitor, pcDNA3.1-FoxM1, si-FoxM1, and negative control (NC) were, respectively, transfected into the osteosarcoma cells. Furthermore, real time polymerase chain reaction was utilized to determine the mRNA expressions of maternally expressed gene 3 (MEG3) and miR-361-5p, and western blot analysis was applied for determining the FoxM1 expression. Besides, dual luciferase reporter gene assay was adopted to verify if MEG3 can be directly targeted by miR-361-5p. Finally, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, colony formation assay, flow cytometry, wound healing assay, and transwell assay were conducted to investigate the influence of MEG3, miR-361-5p, and FoxM1 expressions on the viability, proliferation, apoptosis, migration, and invasion of osteosarcoma cells. MEG3 and miR-361-5p were observed to be significantly downregulated within both osteosarcoma tissues and cell lines, whereas FoxM1 was upregulated in osteosarcoma tissues and cell lines (p < 0.05). MEG3 directly bound to miR-361-5p, and significantly upgraded its expression (p < 0.05). The upregulated MEG3 and miR-361-5p or the downregulated FoxM1 appeared to substantially inhibit proliferation, migration, and invasion of osteosarcoma cells (p < 0.05). Finally, the proliferation, migration, invasion, and motility of osteosarcoma cells within the miR-NC + pcDNA3.1-FoxM1 group and pcDNA + pcDNA-FoxM1 group were markedly promoted when compared with the miR-361-5p mimic group and pcDNA3.1-MEG3 group (p < 0.05). The MEG3/miR-361-5p/FoxM1 axis could potentially serve as therapeutic targets or diagnostic biomarkers for osteosarcoma.     

LncRNA MEG3 regulates breast cancer proliferation and apoptosis through miR-141-3p/RBMS3 axis

Maternally expressed 3 (MEG3) and RNA binding motif single stranded interacting protein 3 (RBMS3) are abnormally expressed in breast cancer susceptibility genes (BRCA), but the mechanism of the two in breast cancer (BC) is unclear. By performing in vivo and in vitro experiments, we found that MEG3 and RBMS3 were low-expressed, negatively correlated with high-expressed miR-141-3p, were positively correlated with each other in BC. MEG3 targeted miR-141-3p, and miR-141-3p targeted RBMS3. MEG3, which was mainly distributed in BC cytoplasm, could down-regulate miR-141-3p and up-regulate RBMS3, and reverse effect of miR-141-3p on related gene expressions and on promoting cancer development. Overexpressed MEG3 inhibited growth of xenografts, promoted cell apoptosis via regulating apoptosis related factors, and up-regulated RBMS3 expression but down-regulated miR-141-3p. The findings of this study showed that MEG3 inhibited proliferation and promoted apoptosis of BC cells through the miR-141-3p/RBMS3 axis, and MEG3 inhibited growth of xenografts through miR-141-3p.     

Inhibition of microRNA-129-5p expression ameliorates ultraviolet ray-induced corneal epithelial cell injury via upregulation of EGFR

Existing evidence has highlighted the effect of ultraviolet light radiation leading to corneal epithelium impairment. During this study, we aim to investigate the effect of microRNA-129-5p (miR-129-5p) on the wound healing process of corneal epithelial cells (CECs) induced by ultraviolet rays in mice by targeting epidermal growth factor receptor (EGFR). First, mouse models of ultraviolet ray-induced CEC injury were established and intrastromally injected with different mimic, inhibitor, and short interfering RNA (siRNA) to detect the effect of miR-129-5p on CEC injury. Subsequently, the corneal tissues were obtained to detect the antioxidant ability and EGFR-positive expression rate. The dual-luciferase reporter gene assay was used to test whether EGFR could directly target miR-129-5p. To further investigate the specific mechanism of miR-129-5p and EGFR in CEC injury, CECs were cultured and transfected with miR-129-5p mimic, miR-129-5p inhibitor, siRNA-EGFR, and miR-129-5p inhibitor + siRNA-EGFR. miR-129-5p has been proven to directly target EGFR. Inhibition of miR-129-5p is able to increase the antioxidant capacity, EGFR-positive rate and the expressions of EGFR, B-cell lymphoma-2, zonula occluden-1, occludin, and keratinocyte growth factor-2, but decrease the expression of vascular endothelial growth factor, BCL2-associated X protein, interleukin (IL)-1β, and IL-4. Inhibition of miR-129-5p arrests cells at the S and G2 phases and decreases apoptosis. Our study provides evidence stating that inhibiting miR-129-5p and upregulating EGFR could aid in the repair of mice CEC injury induced by ultraviolet radiation. Therefore, inhibition of miR-129-5p might provide a basic theory in the repair of CEC injury caused by ultraviolet rays.     

Silencing lncRNA-DGCR5 increased trophoblast cell migration,invasion and tube formation,and inhibited cell apoptosis via targeting miR-454-3p/GADD45A axis

Long noncoding RNA (lncRNA)-DGCR5 has been recognized as a potential tumor progression regulator, while its expression and specific functions in preeclampsia (PE) development remain unveiled. The expressions of miR-454-3p, lncRNA-DiGeorge syndrome critical region gene 5 (DGCR5) and growth arrest and DNA damage protein-inducible 45A (GADD45A) in placental tissues from PE patients or HTR-8/SVneo cells were assessed by Western blot or qRT-PCR. Dual-luciferase reporter assay determined the binding relations between miR-454-3p and GADD45A and between miR-454-3p and lncRNA-DGCR5. The viability, apoptosis, migration, invasiveness and tube formation of HTR-8/SVneo cell were evaluated using cell counting kit (CCK)-8, Annexin-V/Propidium iodide staining, wound healing, transwell and tube formation assays, respectively. miR-454-3p was low-expressed in PE tissue, and upregulation of miR-454-3p increased viability and promoted migration, invasion and tube formation in HTR-8/SVneo cells while inhibiting apoptosis. Then, miR-454-3p was found to directly target GADD45A which was high-expressed in PE tissues. Overexpressing GADD45A decreased the viability and inhibited the migration, invasion and tube formation of HTR-8/SVneo cells while enhancing apoptosis, and it neutralized the effect of miR-454-3p upregulation. In turn, miR-454-3p upregulation reversed the effect of GADD45A overexpression. Meanwhile, miR-454-3p could also target lncRNA-DGCR5. Silencing lncRNA-DGCR5 increased miR-454-3p expression and cell viability and promoted migration, invasion and tube formation in HTR-8/SVneo cells while inhibiting apoptosis, and it counteracted the effect of miR-454-3p downregulation. As usual, miR-454-3p downregulation reversed the effect of lncRNA-DGCR5 silencing. To conclude, silencing lncRNA-DGCR5 increased viability, promoted migration, invasion and tube formation, and inhibited apoptosis in HTR-8/SVneo cells by rescuing the inhibition of GADD45A expression caused by miR-454-3p.

Graphical abstract

     

Long non-coding RNA MEG3 promotes autophagy and apoptosis of nasopharyngeal carcinoma cells via PTEN up-regulation by binding to microRNA-21

Long non-coding RNAs (lncRNAs) have been highlighted as attractive markers for diagnosis and prognosis as well as new therapeutic targets in multiple cancers, including nasopharyngeal carcinoma (NPC). Here, we attempted to investigate the underlying regulatory role of the lncRNA maternally expressed gene 3 (MEG3) in NPC development. As determined by RT-qPCR, MEG3 expression was down-regulated in NPC cells. Online RNA crosstalk analysis predicted the binding of miR-21 to MEG3 and PTEN, respectively. MEG3 was validated to bind to miR-21 while PTEN was identified as a target of miR-21 by dual-luciferase reporter gene assay. Exogenous transfection was done to change the levels of MEG3, miR-21 and PTEN in HK-1 cells to investigate their effects on the autophagy and apoptosis of NPC cells. The results suggested that MEG3 overexpression in HK-1 cells up-regulated PTEN and down-regulated miR-21, by which MEG3 further inhibited autophagy and apoptosis ability of NPC cells. The tumour formation ability was tested after injecting the HK-1 cells into nude, mice and tumour growth was monitored. Consistently, MEG3 overexpression inhibited the tumour formation in vivo. Collectively, MEG3 promotes the autophagy and apoptosis of NPC cells via enhancing PTEN expression by binding to miR-21.     

LncRNA MALAT1 targeting miR-124-3p regulates DAPK1 expression contributes to cell apoptosis in Parkinson's Disease

Death associated protein kinase 1 (DAPK1) was initially discovered in the progress of gamma-interferon induced programmed cell death, it is a key factor in the central nervous system, including Parkinson's disease (PD). However, the underlying mechanisms of DAPK1 in PD remain unclear and this research work aims to explore the potential mechanisms of DAPK1 in PD. In the study, we exposed SH-SY5Y cells to MPP⁺ and treated mice with MPTP to investigate the roles of DAPK1 in PD and the underlying mechanisms. The results indicated that the expression of DAPK1 is significantly upregulated and negatively correlated with miR-124-3p levels in SH-SY5Y cells treated by MPP⁺, and miR-124-3p mimics could effectively inhibit DAPK1 expressions and alleviate MPP⁺-induced cell apoptosis. In addition, knockdown MALAT1 reduces the levels of DAPK1 and the ratio of SH-SY5Y cell apoptosis, which is reversed via miR-124-3p inhibitor in vitro. Similarly, knockdown MALAT1 could improve behavioral changes and reduce apoptosis by miR-124-3p upregulation and DAPK1 downregulation in MPTP induced PD mice. Taken together, our data showed that lncRNA MALAT1 positively regulates DAPK1 expression by targeting miR-124-3p, and mediates cell apoptosis and motor disorders in PD. In summary, these results suggest that MALAT1/miR-124-3p /DAPK1 signaling cascade mediates cell apoptosis in vitro and in vivo, which may provide experimental evidence of developing potential therapeutic strategies for PD.     

MicroRNA-424通过增加转录因子的表达减轻小鼠缺血性脑损伤

目的研究microRNA-424（miR-424）对小鼠脑缺血后神经细胞凋亡及转录因子表达的影响。方法将制备的慢病毒Lenti-miR-424（10’U／mL,8斗L）通过脑室注射,7d后采用大脑中动脉线栓闭塞（MCAO）的方法建立小鼠脑缺血模型,动物分4组：假手术组,假手术＋miR-424慢病毒,MCAO模型组,MCAO＋miR-424慢病毒处理组（n=6）。缺血8h后取脑组织,石蜡切片进行TUNEL染色,观察神经细胞凋亡的情况;Westernblot检测缺血脑组织中转录因子Pu．1、低氧诱导因子-la（hypoxiainduciblefactor-1a,HIF-1a）、凋亡相关蛋白p53的表达。结果TUNEL免疫荧光观察结果显示,miR-424可以减轻小鼠脑缺血后8h的神经细胞凋亡;Westernblot结果显示,在缺血前和缺血8h后,miR-424对正常小鼠或MCAO模型脑组织中转录因子的调节趋势是相同的,均增加转录因子PU．1蛋白、HIF．1a蛋白、以及凋亡相关蛋白p53的表达。结论miR-424可能通过增加小鼠脑组织转录因子PU．1和HIF-la,以及凋亡相关蛋白p53的表达,从而减轻脑缺血后神经细胞的凋亡。     

Aagab acts as a novel regulator of NEDD4-1-mediated Pten nuclear translocation to promote neurological recovery following hypoxic-ischemic brain damage

Hypoxic-ischemic encephalopathy (HIE) is a main cause of mortality and severe neurologic impairment in the perinatal and neonatal period. However, few satisfactory therapeutic strategies are available. Here, we reported that a rapid nuclear translocation of phosphatase and tensin homolog deleted on chromosome TEN (PTEN) is an essential step in hypoxic-ischemic brain damage (HIBD)- and oxygen-glucose deprivation (OGD)-induced neuronal injures both in vivo and in vitro. In addition, we found that OGD-induced nuclear translocation of PTEN is dependent on PTEN mono-ubiquitination at the lysine 13 residue (K13) that is mediated by neural precursor cell expressed developmentally downregulated protein 4-1 (NEDD4-1). Importantly, we for the first time identified α- and γ-adaptin binding protein (Aagab) as a novel NEDD4-1 regulator to regulate the level of NEDD4-1, subsequently mediating Pten nuclear translocation. Finally, we demonstrated that genetic upregulation of Aagab or application of Tat-K13 peptide (a short interference peptide that flanks K13 residue of PTEN) not only reduced Pten nuclear translocation, but also significantly alleviated the deficits of myodynamia, motor and spatial learning and memory in HIBD model rats. These results suggest that Aagab may serve as a regulator of NEDD4-1-mediated Pten nuclear translocation to promote functional recovery following HIBD in neonatal rats, and provide a new potential therapeutic target to guide the clinical treatment for HIE.Subject terms: Cognitive ageing, Neurological disorders