Dual-luciferase Reporter Experiment Research Articles

The Role of miR-29a and miR-143 on the Anti-apoptotic MCL-1/cIAP-2 Genes Expression in EGFR Mutated Non-small Cell Lung Carcinoma Patients.

The survival rate of lung cancer is low due to the high frequency of drug resistance in patients with mutations in the driver genes. Overexpression of anti-apoptotic genes is one of the most prominent features of tumor drug resistance. EGFR signaling induces the expression of anti-apoptotic genes. Also, microRNAs (miRNAs) have a critical role in regulating biological functions such as apoptosis; a process mostly eluded in cancer progression. The mutation screening was performed on one thousand non-small cell lung carcinoma patients to enroll clinical samples in this study. Bioinformatics analysis predicted that miRNAs (miR-29a, miR-143) might regulate MCL-1 and cIAP-2 expression. We investigated the expression of MCL-1, cIAP-2, miR-29a, and miR-143 encoding genes in adenocarcinoma patients with or without EGFR mutations before treatment. The potential role of miR-29a and miR-143 on gene expression was evaluated by overexpression and luciferase assays in HEK-293T cells. EGFR mutations were found in 262 patients (26.2%) with a greater incidence in females (36.23% vs. 20.37%, P = 0.001). The expression levels of MCL-1 and cIAP-2 genes in patients with mutated EGFR were higher than those of wild-type EGFR. In contrast, compared to those of patients with wild-type EGFR, the expression levels of miR-29a and miR-143 were lower in the patients carrying EGFR mutations. In cell culture, overexpression of miR-29a and miR-143 significantly downregulated the expression of MCL-1 and cIAP-2. Dual-luciferase reporter experiments confirmed that miR-29a and miR-143 target MCL-1 and cIAP-2 mRNAs, respectively. Our results suggest that upregulation of EGFR signaling in lung cancer cells may increase anti-apoptotic MCL-1 and cIAP-2 gene expression, possibly through downregulation of miR-29a-3p and miR-143-3p.

Acidity-induced ITGB6 promote migration and invasion of lung cancer cells by epithelial-mesenchymal transition and focal adhesion

Non-small cell lung cancer (NSCLC) is a prevalent tumor and acidic tumor microenvironment provides an energy source driving tumor progression. We previously demonstrated significantly upregulated Integrin β6 (ITGB6) in NSCLC cells. This study was designed to investigate the role of ITGB6 in NSCLC metastasis and explore the potential mechanisms. The expression of ITGB6 was evaluated in patients with NSCLC. Migration and invasion assays were utilized to investigate the role of ITGB6, and ChIP-qPCR and dual-luciferase reporter experiments preliminarily analyzed the relationship between ETS proto-oncogene 1 (ETS1) and ITGB6. Bioinformatics analysis and rescue models were performed to explore the underlying mechanisms. The results demonstrated that ITGB6 was upregulated in NSCLC patients and the difference was even more pronounced in patients with poor prognosis. Functionally, acidity-induced ITGB6 promoted migration and invasion of NSCLC cells in vitro, and epithelial-mesenchymal transition (EMT) and focal adhesion were the important mechanisms responsible for ITGB6-involved metastasis. Mechanistically, we revealed ETS1 enriched in the ITGB6 promoter region and promoted transcription to triggered the activation of subsequent signaling pathways. Moreover, ChIP-qPCR and dual-luciferase reporter experiments demonstrated that ETS1 played an important role in directly mediating ITGB6 expression. Furthermore, we found ITGB6 was responsible for the acidic microenvironment-mediated migration and invasion processes in NSCLC by performing rescue experiments with ITGB6 knockdown. Our findings indicated acidic microenvironment directly induced ETS1 to regulate the expression of ITGB6, and then the highly expressed ITGB6 further mediate EMT and activates the downstream focal adhesion pathways, eventually promotes the invasion and migration in NSCLC progression and metastasis.

Suppression of MALAT1 promotes human synovial mesenchymal stem cells enhance chondrogenic differentiation and prevent osteoarthritis of the knee in a rat model via regulating miR-212-5p/MyD88 axis.

Osteoarthritis (OA) is one of the most common diseases of the skeleton. Long non-coding RNAs (lncRNAs) are emerging as key players in OA pathogenesis. This work sets out to determine the function of lncRNA MALAT1 in OA and the mechanisms by which it does so. Mesenchymal stem cells isolated from the human synovial membrane are called hSMSCs. The hSMSCs' surface markers were studied using flow cytometry. To determine whether or not hSMSC might differentiate, researchers used a number of different culture settings and labeling techniques. The expression levels of associated genes and proteins were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), and immunostaining. A dual luciferase reporter experiment and RNA immunoprecipitation (RIP) test demonstrated the direct association between miR-212-5p and MALAT1 or MyD88. MALAT1 was downregulated during the chondrogenic differentiation of hSMSCs, and underexpression of MALAT1 promotes chondrogenesis in hSMSCs. Using dual luciferase reporter and RIP assays facilitated the identification of MALAT1 as a competitive endogenous RNA (ceRNA) that sequesters miR-212-5p. Additionally, the expression of MYD88 was regulated by MALAT1 through direct binding with miR-212-5p. Significantly, the effects of MALAT1 on the chondrogenic differentiation of hSMSCs were counteracted by miR-212-5p/MYD88. Furthermore, our in vivo investigation revealed that the inhibition of MALAT1 mitigated osteoarthritis progression in rat models. In conclusion, the promotion of chondrogenic differentiation in hSMSCs and the protective effect on cartilage tissue in OA can be achieved by suppressing MALAT1, which regulates the miR-212-5p/MyD88 axis.

Up-regulation of lncRNA WT1-AS ameliorates Aβ-stimulated neuronal injury through modulation of miR-186-5p/CCND2 axis in Alzheimer's disease.

As a common neurodegenerative disorder, Alzheimer's disease (AD) seriously threatens human life. Long non-coding RNAs (lncRNAs) exhibit essential functions in AD development. Nevertheless, the detailed effects and possible mechanisms of lncRNA Wilms tumor 1 Antisense RNA (WT1-AS) in AD are largely unknown. In our studies, a total of 30 serum samples from AD patients were collected, and WT1-AS expressions were detected through qRT-PCR analysis. Additionally, an in vitro AD model was constructed by treating Aβ1-42 in human neuroblastoma cells. Functional assays were implemented to assess the impacts of WT1-AS on Aβ1-42-stimulated human neuroblastoma cell proliferation together with apoptosis. Moreover, relationship of WT1-AS, microRNA (miR)-186-5p as well as cyclin D2 (CCND2) could be predicted through bioinformatics tools as well as proved via dual-luciferase reporter experiments. Our results showed that WT1-AS together with CCND2 were low-expressed, while miR-186-5p presented high expression in AD serum samples together with Aβ1-42-stimulated human neuroblastoma cells. WT1-AS over-expression or miR-186-5p depletion notably promoted the proliferation, reduced the apoptosis, and decreased the p-Tau protein expressions of human neuroblastoma cells induced with Aβ1-42. Moreover, miR-186-5p combined with WT1-AS, and CCND2 was modulated by miR-186-5p. Furthermore, CCND2 elevation partially offsets the impacts of miR-186-5p elevation on Aβ1-42-stimulated cell proliferation as well as apoptosis mediated with WT1-AS up-regulation. Our results indicated that up-regulation of lncRNA WT1-AS ameliorated Aβ-stimulated neuronal damage through modulating miR-186-5p/CCND2 axis, offering a novel direction for AD therapy.

Silencing lncRNA-DARS-AS1 suppresses nonsmall cell lung cancer progression by stimulating miR-302a-3p to inhibit ACAT1 expression.

Long noncoding RNAs (LncRNAs) have been gaining attention as potential therapeutic targets for lung cancer. In this study, we investigated the expression and biological behavior of lncRNA DARS-AS1, its predicted interacting partner miR-302a-3p, and ACAT1 in nonsmall cell lung cancer (NSCLC). The transcript level of DARS-AS1, miR-302a-3p, and ACAT1 was analyzed using qRT-PCR. Endogenous expression of ACAT1 and the expression of-and changes in-AKT/ERK pathway-related proteins were determined using western blotting. MTS, Transwell, and apoptosis experiments were used to investigate the behavior of cells. The subcellular localization of DARS-AS1 was verified using FISH, and its binding site was verified using dual-luciferase reporter experiments. The binding of DARS-AS1 to miR-302a-3p was verified using RNA co-immunoprecipitation. In vivo experiments were performed using a xenograft model to determine the effect of DARS-AS1 knockout on ACAT1 and NSCLC. lncRNA DARS-AS1 was upregulated in NSCLC cell lines and tissues and the expression of lncRNA DARS-AS1 was negatively correlated with survival of patients with NSCLC. Knockdown of DARS-AS1 inhibited the malignant behaviors of NSCLC via upregulating miR-302a-3p. miR-302a-3p induced suppression of malignancy through regulating oncogene ACAT1. This study demonstrates that the DARS-AS1-miR-302a-3p-ACAT1 pathway plays a key role in NSCLC.

CircFN1 promotes acute myeloid leukemia cell proliferation and invasion but refrains apoptosis via miR-1294/ARHGEF10L axis.

Previous studies have proved circFN1 is highly expressed in acute myeloid leukemia (AML) patients and AML cell lines. This study aims to investigate the impact of circFN1 on AML and its mechanism. Via real-time quantitative PCR to detect circFN1, miR-1294, ARHGEF10L expressions in clinical plasma samples and AML cell lines, AML cells were cultured in vitro and transfected with si-circFN1, pcDNA3.1-circFN1, and si-ARHGEF10L, respectively, or co-transfected pcDNA3.1-circFN1 + miR-1294 mimic and pcDNA3.1-circFN1 + si-ARHGEF10L. Using dual luciferase reporter experiment to detect the relationship between circFN1 and miR-1294, as well as miR-1294 and ARHGEF10L. CCK-8 was used to detect cell proliferation, Transwell to cell invasion, TUNEL staining and flow cytometry to detect cell apoptosis, RT-qPCR to circFN1 RNA, miR-1294, and ARHGEF10L expression levels in HL-60 cells, and western blot to ARHGEF10L protein expression level in HL-60 cells. We found highly expressed circFN1 and ARHGEF10L, as well as low-expressed miR-1294 in AML patients and AML cell lines. In contrast to si-NC group, si-circFN1 group could signally inhibit HL-60 cell proliferation and migration, but promote cell apoptosis; compared with mimic NC group, miR-1294 mimic group could visually inhibit HL-60 cell proliferation and migration, but promote cell apoptosis. miR-1294 was the target of circFN1, and ARHGEF10L was the target of miR-1294. Over-expressing miR-1294 or silencing ARHGEF10L could signally inhibit circFN1 promoting HL-60 cell proliferation and migration and repressing cell apoptosis. circFN1 promotes proliferation and invasion of AML cell and represses cell apoptosis via regulating miR-1294/ARHGEF10L axis, which provides new insight for molecular targeted-treatment for AML.

LncRNA PRKCA-AS1 promotes LUAD progression and function as a ceRNA to regulate S100A16 by sponging miR-508-5p.

Objective: This study aimed to elucidate the underlying mechanism of LncRNA PRKCA-AS1 in lung adenocarcinoma (LUAD). Methods: The expression of LncRNA PRKCA-AS1, miR-508-5p and S100A16, in LUAD tissues or cell lines (NCI-H520 and H1299) was analyzed with qRT-PCR. The clinical diagnostic value of LncRNA PRKCAAS1, miR-508-5p and S100A16 in LUAD were analyzed by receptor operating characteristic (ROC) curve. Then we knockdown or overexpression of PRKCAAS1 in NCI-H520 and H1299 cells, and the cell function test was applied to detect the activity and metastasis level of cells in different transfection groups. Then Pearson correlation analysis was used for the correlation between miR-508-5p and PRKCA-AS1. The dual-luciferase reporter experiment and CHIRP analysis was conducted to verify the target binding relationship of PRKCA-AS1, miR-508-5p or S100A16. FISH assay analyzed the colocalization of PRKCA-AS1 and miR-508-5p in NCI-H520 and H1299 cells. Rescue experiment and tumorigenesis experiment in nude mice further explore the regulatory mechanisms of LncRNA PRKCA-AS1, miR-508-5p and S100A16 on LUAD progression in vitro and in vivo. Results: From the results, PRKCA-AS1 and S100A16 were up-regulated in LUAD tissues, while miR-508-5p was downregulated compared with the adjacent tissues. And gain-of-function revealed that PRKCA-AS1 knock-down apparently suppressed the cell proliferation and metastasis, whereas miR-508-5p inhibitors or S100A16 overexpression showed a opposite effect. In addition, there is evidence that PRKCA-AS1, miR-508-5p and S100A16 have a targeted regulatory relationship. Moreover, rescue experiment and tumorigenesis experiment in nude mice further confirmed that LncRNA PRKCA-AS1 regulates S100A16 through sponging miR-508-5p to regulate LUAD progression in vitro and in vivo. Conclusion: These results demonstrated that LncRNA PRKCA-AS1 might regulate LUAD by acting as a ceRNA via sponging miR-508-5p and regulating S100A16 expression, indicating that manipulation of PRKCA-AS1 might be a potential therapeutic strategy in LUAD.

CircRNA amyloid precursor protein by competitive adsorption of microRNA-6838-5p mediates CDV3 expression to enhance malignant behavior and Warburg effect in Gastrointestinal Stromal Tumor

Dysregulated circular RNA (circRNA) expression profiles and their carcinogenic contributions have been noted in nearly all human cancers. This study aimed to unearth the role of circRNA Amyloid Precursor Protein (circAPP), an abnormally highly expressed circRNA in Gastrointestinal Stromal Tumors (GIST). As the results found, circAPP was upregulated in GIST tissues and cells. FISH experiment, dual-luciferase reporter experiment, and RIP experiment confirmed that circAPP promoted CDV3 expression by absorption of miR-6838-5p in GIST. Cell experiments confirmed that silencing circAPP inhibited GIST cell proliferation, migration, invasion, glucose consumption, lactate production, ATP level, expression of HK2 and PKM2, decreased ATP/ADP, and increased NAD+/NADH, but promoted apoptosis, whereas overexpression of circAPP did the exact opposite. Furthermore, miR-6838-5p depletion and CDV3 overexpression abolished the influences of downregulating circAPP and overexpressing circAPP on GIST cells, respectively. Animal experiments displayed that circAPP knockdown inhibited GIST tumor growth and liver metastasis. All in all, circAPP promotes GIST cell proliferation and the Warburg effect by miR-6838-5p/CDV3 axis and circAPP may be a potential future therapeutic target for GIST.

MiR-497-5p Expression and Biological Activity in Gastric Cancer.

Background: This research aims to investigate the expression and biological roles of miR-497-5p in gastric cancer (GC), and its possible mechanisms. Methods: Real Time Quantitative PCR (RT-qPCR) was performed to detect miR-497-5p in GC and normal tissues, as well as GC cell lines versus normal gastric mucosal cells (GES-1). The effects of miR-497-5p overexpression on proliferation were measured by the cell counting kit-8 (CCK8) assay and ethidium bromide (EdU) assay. Flow cytometry was used to assess the cell cycle. The migration and invasion were evaluated by scratch assay and Transwell assay, respectively. Gene targets of miR-497-5p were predicted using "multiMiR" R package combined with mirTarPathway database. And then luciferase reporter experiment was used to evaluate the activity of ERBB2 by miR-497-5p mimics in GC cell line. Besides, functional experiments were performed to verify the impact of miR-497-5p /ERBB2 on phenotypes of GC cells. Results: Compared with the normal tissues and mucosal cells, miR-497-5p was reduced in GC tissues and GC cell lines. miR-497-5p significantly decreased proliferation, migration, and invasion capacity, with an elevated apoptosis ratio of gastric cancer cells. Bioinformatics indicated that ERBB2 might be the potential target of miR-497-5p Dual-luciferase reporter experiments showed it adversely regulated ERBB2 3'UTR luciferase activity. The expression of ERBB2 in GC tissues and cells is significantly higher compared to normal tissues and cells. Over-expression of ERBB2 in gastric cancer cells significantly reduced miR-497-5p's inhibitory effect on the malignant behavior of GC cells. Conclusion: miR-497-5p was significantly down-regulated in GC tissues and cells, which inhibited the malignant features of GC cells by targeting ERBB2.

Volatile oil of Angelica sinensis Radix improves cognitive function by inhibiting miR-301a-3p targeting Ppp2ca in cerebral ischemia mice

Ethnopharmacological relevanceAngelica Sinensis Radix (ASR) is a commonly used Chinese medicine known for its effects on tonifying blood, promoting blood circulation, and alleviating pain associated with menstrual regulation. Additionally, it has been used in the treatment of vascular cognitive impairment (VCI). The primary pharmacodynamic agent within ASR is volatile oil of Angelica Sinensis Radix (VOASR), which has demonstrated efficacy in combating cognitive impairment, although its mechanism remains unclear. ObjectiveThis study aimed to elucidate the potential molecular mechanisms underlying VOASR's improvement of cognitive function in cerebral ischemic mice. MethodsA model of cerebral ischemic mice was established through unilateral common carotid artery occlusion (UCCAO) surgery, followed by intervention with VOASR. Cognitive function was assessed using the Morris water maze (MWM) test, while RT-qPCR was utilized to measure the differential expression of miR-301a-3p in the hippocampus. To evaluate cognitive function and hippocampal protein differences, wild-type mice and miR-301a-3p knockout mice were subjected to the MWM test and iTRAQ protein profiling. The relationship between miR-301a-3p and potential target genes was validated through a Dual-Luciferase Reporter experiment. RT-qPCR and Western blot were employed to determine the differential expression of Ppp2ca and synaptic plasticity-related proteins in the mouse hippocampus. ResultsIntervention with VOASR significantly improved cognitive impairment in cerebral ischemic mice and reduced the expression of miR-301a-3p in the hippocampus. Our findings suggest that miR-301a-3p may regulate cognitive function by targeting Ppp2ca. Furthermore, VOASR intervention led to an increase in the expression of Ppp2ca and synaptic plasticity-related proteins. ConclusionOur study indicates that VOASR may be involved in regulating cognitive function by inhibiting miR-301a-3p, consequently increasing the expression of Ppp2ca and synaptic plasticity proteins. These results provide a new target and direction for the treatment of cognitive dysfunction.

Tetralogy of Fallot: variants of MYH6 gene promoter and cellular functional analyses.

Tetralogy of Fallot (TOF) is a common form of congenital heart disease. The MYH6 gene has important effects on cardiovascular growth and development. In 608 subjects, including 315 TOF patients, we investigated the MYH6 gene promoter variants and verified the effect on gene expression by using cellular functional experiments with three cell lines (HEK-293, HL-1, and H9C2 cells) and bioinformatics analysis. In the MYH6 gene promoter, 12 variants were identified from 608 subjects. Five variants were found only in patients with TOF and two of them (g.3384G>T and g.4518T>C) were novel. Electrophoretic mobility shift assay with three cell lines (HEK-293, HL-1, and H9C2) showed significant changes in the transcription factors bound by the promoter variants compared to the wild-type. Dual luciferase reporter showed that four of the five variants reduced the transcriptional activity of the MYH6 gene promoter (p < 0.05). This study is the first to test the cellular function of variants in the promoter region of the MYH6 gene in patients with TOF, which provides new insights into the genetic basis of TOF and provides a basis for further study of the mechanism of TOF formation. DNA from 608 human subjects was sequenced for MYH6 gene promoter region variants with five variants found only in TOF patients and two were novel. EMSA and dual luciferase reporter experiments in three cell lines found these variants pathological. Prediction by JASPAR database indicated that these variants alter the transcription factor binding sites. The study, for the first time, confirmed that there are variants at the MYH6 gene promoter region and these variants alter the cellular function. The variants found in this study suggest the possible pathological role in the formation of TOF.

Deregulation of circRNA hsa_circ_0009109 promotes tumor growth and initiates autophagy by sponging miR-544a-3p in gastric cancer.

Autophagy death of cancer cells is detrimental to apoptosis induced by therapeutic drugs, which promotes tumor progression to a certain extent. Increasing reports have demonstrated the regulatory role of circular RNAs (circRNAs) in autophagy. Here, we aimed to determine the role of hsa_circ_0009109 in autophagy in gastric cancer (GC). The effects of hsa_circ_0009109 on autophagy were examined using quantitative real-time polymerase chain reaction (qPCR), transmission electron microscopy, Western blot, and immunofluorescence. The mechanism of hsa_circ_0009109 regulating the miR-544a-3p/bcl-2 axis was analysed using fluorescence in situ hybridization, dual-luciferase reporter, and rescue experiments. Functional testing indicated that hsa_circ_0009109 was significantly down-expressed in GC tissues and cell lines. A reduction in cytoplasmic-derived hsa_circ_0009109 could promote GC progression by accelerating cell proliferation, enhancing migration and invasion, inhibiting apoptosis, and accelerating the cell cycle progression. Besides, hsa_circ_0009109 was found to exert the effect of an autophagy inhibitor such as 3-Methyladenine (3-MA), which was manifested by the weakening of the immunofluorescence of LC3B and the reduction in autophagy-related proteins after overexpression of hsa_circ_0009109, while increased autophagosomes were observed after interference with hsa_circ_0009109. Subsequently, the crosstalk between hsa_circ_0009109 and miR-544a-3p/bcl-2 was verified using dual-luciferase reporter assay. The autophagy status was altered under the regulation of the hsa_circ_0009109-targeted miR-544a-3p/bcl-2 axis. The hsa_circ_0009109 mediated a novel autophagy regulatory network through targeting the miR-544a-3p/bcl-2 axis, which may shed new light on the exploration of therapeutic targets for the clinical treatment of GC.

Alternative Polyadenylation of Malic Enzyme 1 Is Essential for Accelerated Adipogenesis.

Understanding the mechanism of adipogenesis is an important basis for improving meat quality traits of livestock. Alternative polyadenylation (APA) is a vital mechanism to regulate the expression of eukaryotic genes. However, how the individual APA functions in adipogenesis remains elusive. This study was intended to investigate the effect of malic enzyme 1 (ME1) APA on adipogenesis. Here, intracellular lipid droplets were stained using Oil red O. 3' RACE was used to verify APA events of the ME1 gene. Interactions between ME1 3' untranslated region (3' UTR)-APA isoforms and miRNAs, as well as differential expression of isoforms, were examined using dual-luciferase reporter and molecular experiments. The mechanism of ME1 APA on adipogenesis was explored by gain and loss of function assays. In this study, two ME1 isoforms with different 3' UTR lengths were detected during adipogenesis. Moreover, the ME1 isoform with a short 3' UTR was significantly upregulated compared with the one with a long 3' UTR. Mechanistically, only the long ME1 isoform was targeted by miR-153-3p to attenuate adipogenesis, while the short one escaped the regulation of miR-153-3p to accelerate adipogenesis. Our results reveal a novel mechanism of ME1 APA in regulating adipogenesis.

Altered HCAR3 expression may underlying the blunted niacin responses of the psychiatric disorders and the risk of schizophrenia.

Blunted niacin response (BNR) was an endophenotype of schizophrenia, but the underlying mechanism remains unclarified. The objective of this study was to verify whether genes associated with BNR pathway constitute the genetic basis and the pathological mechanism of BNR phenotypic psychiatric patients. Two independent sample sets consisting of 971 subjects were enrolled in this study. A total of 62 variants were genotyped in the discovery set, then the related variants were verified in the verification set. The published PGC GWAS data were used to validate the associations between the variants and psychiatry disorders. RT-PCR analysis, eQTL data, and Dual-Luciferase Reporter experiment were used to investigate the potential molecular mechanisms of the variants underlying BNR. The results showed that two SNPs, rs56959712 in HCAR2 and rs2454721 in HCAR3 were significantly associated with niacin response. The risk allele T of rs2454721 could affect the niacin responses of psychiatric patients through elevated HCAR3 gene expression. These two genes, especially HCAR3, were significantly associated with the risk of schizophrenia, as identified in this study and verified using the published GWAS data. HCAR3 is a novel schizophrenia susceptibility gene which is significantly associated with blunted niacin response in schizophrenia. In-depth investigation of HCAR3 is of great significance for uncovering the pathogenesis and propose new therapeutic targets for psychiatric disorders, especially for the BNR subgroup patients.

The linear ANRIL transcript P14AS regulates the NF-κB signaling to promote colon cancer progression

BackgroundThe linear long non-coding RNA P14AS has previously been reported to be dysregulated in colon cancer, but the mechanistic role that P14AS plays in colon cancer progression has yet to be clarified. Accordingly, this study was developed to explore the regulatory functions of ANRIL linear transcript-P14AS in cancer.MethodsThe expression of P14AS, ANRIL, miR-23a-5p and their target genes were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell supernatants of IL6 and IL8 were measured by Enzyme linked immunosorbent (ELISA) assay. Dual-luciferase reporter assays, RNA immunoprecipitation, or pull-down assays were used to confirm the target association between miR-23a-5p and P14AS or UBE2D3. Cell proliferation and chemosensitivity of NF-κB inhibitor BAY 11-7085 were evaluated by cell counting kit 8 (CCK8).ResultsWhen P14AS was overexpressed in colon cancer cell lines, enhanced TNF-NF-κB signaling pathway activity was observed together with increases in IL6 and IL8 expression. The Pita, miRanda, and RNA hybrid databases revealed the ability of miR-23a-5p to interact with P14AS, while UBE2D3 was further identified as a miR-23a-5p target gene. The results of dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation experiments confirmed these direct interactions among P14AS/miR-23a-5p/UBE2D3. The degradation of IκBa mediated by UBE2D3 may contribute to enhanced NF-κB signaling in these cells. Moreover, the beneficial impact of P14AS on colon cancer cell growth was eliminated when cells were treated with miR-23a-5p inhibitors or UBE2D3 was silenced. As such, these findings strongly supported a role for the UBE2D3/IκBa/NF-κB signaling axis as a mediator of the ability of P14AS to promote colon cancer progression.ConclusionsThese data suggested a mechanism through which the linear ANRIL transcript P14AS can promote inflammation and colon cancer progression through the sequestration of miR-23a-5p and the modulation of NF-κB signaling activity, thus highlighting P14AS as a promising target for therapeutic intervention efforts.

MiR-26 inhibits proliferation and promotes apoptosis of multiple myeloma cells by targeting BNIP3.

This study was carried out to investigate the molecular mechanism of microRNA-26 (miR-26) targeting BNIP3 to mediate proliferation and apoptosis of multiple myeloma (MM) cells. The expression of miR-26 and BNIP3 in MM and normal tissues was detected by qRT-PCR and Western blot. According to the average expression of miR-26 and BNIP3, the patients were divided into 12 cases with high miR-26 expression group, 18 cases with low miR-26 expression group, 20 cases with BNIP3 high expression group, and 10 cases with BNIP3 low expression group. The correlation between the expression of miR-26 and BNIP3 and the clinicopathological characteristics of MM patients was compared and analyzed. The effect of up-regulation of miR-26 expression and BNIP3 overexpression on the proliferation of multiple myeloma cells RPMI8226 was examined by MTT assay. Flow cytometry was used to detect the effect of miR-26 expression and BNIP3 overexpression on the apoptosis of RPMI8226 cells. The dual luciferase reporter assay validated the targeted regulation of miR-26 on BNIP3. The expression level of miR-26 in MM tissues was lower than that in normal tissues (P<0.05), and the expression level of BNIP3 in MM tissues was higher than that in normal tissues (P<0.05). miR-26 was closely related to clinical stage, M protein type and light chain type (P<0.05), while BNIP3 was closely related to M protein type and light chain type (P<0.05). After up-regulating miR-26 expression, cell viability was significantly decreased (P<0.05), apoptosis rate was significantly increased (P<0.05) Dual luciferase reporter experiments confirmed that miR-26 could target BNIP3 and negatively regulate the expression of BNIP3 (P<0.05). Overexpression of BNIP3 reversed the effect of up-regulation of miR-26 expression on proliferation and apoptosis of RPMI8226 cells. Up-regulation of miR-26 expression inhibits MM cell proliferation and promotes apoptosis by targeting BNIP3.

Bone Marrow Mesenchymal Stem Cell Exosomal miR-345-3p Ameliorates Cerebral Ischemia-reperfusion Injury by Targeting TRAF6.

The purpose of this study was to investigate the effects of bone marrow mesenchymal stem cells (BMSCs) exosomal miR-345-3p and tumor necrosis factor receptorassociated factor 6 (TRAF6) on cerebral ischemia reperfusion (CIR) injury. Exosomes (Exos) derived from BMSCs were isolated and identified. PC12 (rat pheochromocytoma) cells were used to establish an oxygen and glucose deprivation/reoxygenation (OGD/R) model. Cell counting kit-8, TUNEL staining, lactate dehydrogenase staining, RT-qPCR, and western blotting were utilized for analyzing the functions of miR-345-3p about PC12 cells. Dualluciferase reporter experiment was then to confirm the link between miR-345-3p and TRAF6. Finally, using male SD rats, the middle cerebral artery occlusion (MCAO) model was constructed. Regulation of I/R damage in MCAO rats of miR-345-3p and TRAF6 were further explored in the changes of modified neurological severity score, cerebral infarction pictures, relative infarct volume, and histopathological changes. After OGD/R treatment, neuronal apoptosis was dramatically increased. After treatment with exosomal miR-345-3p, OGD/R-induced neuroapoptosis was dramatically inhibited. Exosomal miR-345-3p inhibited OGD/R-induced neuroapoptosis by downregulating the expression of TRAF6. However, the miR-345-3p inhibitor aggravated the changes caused by OGD/R. The corresponding regulations of miR-345-3p were reversed with TRAF6 overexpression. The animal experiments in vivo further verified that miR-345-3p ameliorated brain I/R injury in MCAO rats by targeting TRAF6. This study found that BMSCs-exosomal miR-345-3p protected against CIR injury by decreasing TRAF6.

LINC01343 targets miR-526b-5p to facilitate the development of hepatocellular carcinoma by upregulating ROBO1

Long noncoding RNAs (lncRNAs) contribute to hepatocellular carcinoma (HCC) progression and development. However, the function and molecular mechanisms of action of LINC01343 in HCC remain unclear. qRT-PCR and western blotting were performed to assess miR-526b-5p, LINC01343, and ROBO1 levels in HCC cell lines and tissue samples. Flow cytometry, transwell, and cell counting kit-8 assays were conducted in vitro to assess how LINC01343 influences the apoptosis, migration, and proliferation of HCC cells. In addition, the role of LINC01343 in the growth of tumors was verified using an in vivo xenograft tumor assay. Specific binding of miR-526b-5p to LINC01343/ROBO1 was validated using RNA immunoprecipitation and dual-luciferase reporter experiments. LINC01343 was upregulated in HCC cells and tissues. In vitro, LINC01343-knockdown Hep3B and Huh-7 cells exhibited enhanced apoptosis and suppressed proliferation and migration. An in vivo study further validated that LINC01343-knockdown repressed tumor growth. In terms of mechanisms, LINC01343 directly sponged miR-526b-5p, negatively modulating its expression. Moreover, further experiments revealed that inhibiting miR-526b-5p could counteract the tumor-suppressive effects of LINC01343-knockdown in Hep3B and Huh-7 cells. ROBO1 was identified as a direct target of miR-526b-5p. ROBO1 knockdown weakens the migratory and proliferative abilities of Hep3B and Huh-7 cells. Nonetheless, the inhibition of miR-526b-5p mitigated this effect. These findings revealed that LINC01343 serves as a vital oncogene in HCC. Moreover, the LINC01343/miR-526b-5p/ROBO1 axis may be a prospective target for HCC treatment.

MicroRNA-30a inhibits cell proliferation in a sepsis-induced acute kidney injury model by targeting the YAP-TEAD complex

BackgroundAcute kidney injury (AKI) is a primary feature of renal complications in patients with sepsis. MicroRNA (miRNA/miR)-30a is an essential regulator of cardiovascular diseases, tumors, phagocytosis, and other physical processes, but whether it participates in sepsis-induced AKI (sepsis-AKI) is unknown. We aimed to elucidate the functions and molecular mechanism underlying miR-30a activity in sepsis-AKI. MethodsThe classical cecal ligation and puncture (CLP) method and lipopolysaccharide (LPS)-induced Human Kidney 2 (HK-2) cells were used to establish in vivo and in vitro sepsis-AKI models. Specific pathogen-free and mature male Sprague-Dawley (SD) rats, aged 6–8 weeks (weight 200–250 g), were randomly divided into five-time phase subgroups. Fluid resuscitation with 30 mL/kg 37 °C saline was administered after the operation, without antibiotics. Formalin-fixed, paraffin-embedded kidney sections were stained with hematoxylin and eosin. SD rat kidney tissue samples were collected for analysis by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. HK-2 cells were transfected with hsa-miR-30a-3p mimics or inhibitors, and compared with untreated normal controls. RNA, protein, and cell viability were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot, and cell counting kit-8 methods. A Dual-Luciferase Assay Kit (Promega) was used to measure luciferase activity 48 h after transfection with miR-30a-3p mimics. ResultsExpression levels of miR-30a-3p and miR-30a-5p in renal tissues of the sepsis group were significantly reduced at 12 h and 24 h (P <0.05). Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly increased in renal tissue 3 h after the operation in rats (P <0.05), and gradually decreased 6 h, 12 h, and 24 h after CLP. Levels of miR-30a-5p and miR-30a-3p were significantly down-regulated at 3 h after LPS treatment (P <0.05), and gradually decreased in HK-2 cells. One hour after LPS (10 µg/mL) treatment, TNF-α and IL-1β levels in HK-2 cells were significantly up-regulated (P < 0.05), and they were markedly down-regulated after 3 h (P <0.05). IL-6 expression levels began to rise after LPS treatment of cells, peaked at 6 h (P <0.05), and then decreased to the initial level within a few hours. Stimulation with 10 µg/mL LPS promoted HK-2 cells proliferation, which was inhibited after miR-30a-3p-mimic transfection. Bioinformatics prediction identified 37 potential miR-30a-3p target genes, including transcriptional enhanced associate domain 1 (TEAD1). After transfection of HK-2 cells with miR-30a-3p mimics and miR-30a-3p inhibitor, TEAD1 transcript was significantly up- and down-regulated, respectively (both P <0.05). After LPS treatment (24 h), expression of TEAD1 in the inhibitors group was significantly increased (P <0.01), while that in the mimics group was significantly suppressed (P <0.01). In the dual luciferase reporter experiment, miR-30a-3p overexpression decreased fluorescence intensity (P <0.01) from TEAD1-wt-containing plasmids, but did not influence fluorescence intensity from TEAD1-muta-containing plasmids. LPS may promote HK-2 cells proliferation through the miR-30a-3p/TEAD1 pathway. ConclusionIn a background of expression of inflammatory factors, including TNF-α, IL-1β, and IL-6, which were transiently increased in the sepsis-AKI model, miR-30a was down-regulated. Down-regulated miR-30a-3p may promote cell proliferation by targeting TEAD1 in LPS-induced HK-2 cells, demonstrating its potential as a biomarker for early sepsis-AKI diagnosis.

Circ_SPEF2 Regulates the Balance of Treg Cells by Regulating miR-16-5p/BACH2 in Lymphoma and Participates in the Immune Response.

This study aims to explore the potential mechanism of action of the newly discovered hsa_circ_0128899 (circSPEF2) in diffuse large B-cell lymphoma (DLBCL). circSPEF2, miR-16-5p and BTB and CNC homologue 2 (BACH2) expression patterns in DLBCL patients and cell lines were studied by RT-qPCR. The biological function of circSPEF2 in vitro and in vivo was investigated by function acquisition experiments. The proliferation activity of lymphoma cells was detected by MTT. Bax, Caspase-3, and Bcl-2 were determined by Western Blot. Apoptosis and the ratio of CD4 to Treg of immune cells in the co-culture system were analyzed by flow cytometry. The mechanism of action of circSPEF2 in DLBCL progression was further investigated by RIP and dual luciferase reporter experiments. circSPEF2 was a circRNA with abnormally down-regulated expression in DLBCL. Increasing circSPEF2 expression inhibited the proliferative activity and induced apoptosis of lymphoma cells in vitro and in vivo, as well as increased CD4+T cells and decreased Treg cell proportion of immune cells in the tumor microenvironment. Mechanically, circSPEF2 was bound to miR-16-5p expression, while BACH2 was targeted by miR-16-5p. circSPEF2 overexpression-mediated effects on lymphoma progression were reversible by upregulating miR-16-5p or downregulating BACH2. circSPEF2 can influence DLBCL progression by managing cellular proliferation and apoptosis and the proportion of immune cells Treg and CD4 through the miR-16-5p/BACH2 axis.