Dual-luciferase Reporter Gene Experiment Research Articles

Exosome-mediated transfer of lncRNA RP3-340B19.3 promotes the progression of breast cancer by sponging miR-4510/MORC4 axis

BackgroundThis study aims to explore the molecular mechanism of lncRNA RP3-340B19.3 on breast cancer cell proliferation and metastasis and clinical significance of lncRNA RP3-340B19.3 for breast cancer.MethodsThe subcellular localization of lncRNA RP3-340B19.3 was identified using RNA fluorescence in situ hybridization (FISH). The expression of lncRNA RP3-340B19.3 in breast cancer cells, breast cancer tissues, as well as the serum and serum exosomes of breast cancer patients, was measured through quantitative RT-PCR. In the in vitro setting, we conducted experiments to observe the effects of RP3-340B19.3 on both cell migration and proliferation. This was achieved through the utilization of transwell migration assays as well as clone formation assays. Meanwhile, transwell migration assays and clone formation assays were used to observe the effects of MDA-MB-231-exosomes enriched in RP3-340B19.3 on breast cancer microenvironment cells MCF7 and BMMSCs. Additionally, western blotting techniques were used to assess the expression levels of proteins associated with essential cellular processes such as proliferation, apoptosis, and metastasis. In vivo, the impact of RP3-340B19.3 knockdown on tumour weight and volume was observed within a nude mice model. We aimed to delve into the intricate molecular mechanisms involving RP3-340B19.3 by using bioinformatics analysis, dual luciferase reporter gene experiments and western blotting. Moreover, the potential correlations between RP3-340B19.3 expression and various clinical pathological characteristics were analyzed.ResultsOur investigation revealed that RP3-340B19.3 was expressed in both the cytoplasm and nucleus, with a noteworthy increase in breast cancer cells. Notably, we found that RP3-340B19.3 exerted a promoting influence on the proliferation and migration of breast cancer cells, both in vitro and in vivo. MDA-MB-231-exosomes enriched in RP3-340B19.3 promoted the proliferation and migration of MCF7 and BMMSCs in vitro. Mechanistically, RP3-340B19.3 demonstrated the capability to modulate the expression of MORC4 by forming a complex with miR-4510. This interaction subsequently triggered the activation of the NF-κB and Wnt-β-catenin signaling pathways. Furthermore, our study highlighted the potential diagnostic utility of RP3-340B19.3. We discovered its presence in the serum and exosomes of breast cancer patients, showing promising efficacy as a diagnostic marker. Notably, the diagnostic potential of RP3-340B19.3 was particularly significant in relation to distinguishing between different pathological types of breast cancer and correlating with tumour diameter.ConclusionOur findings establish that RP3-340B19.3 plays a pivotal role in driving the proliferation and metastasis of breast cancer. Additionally, exosomes enriched in RP3-340B19.3 could influence MCF7 and BMMSCs in tumour microenvironment, promoting the progression of breast cancer. This discovery positions RP3-340B19.3 as a prospective novel candidate for a tumour marker, offering substantial potential in the realms of breast cancer diagnosis and treatment strategies.

Mechanistic study on lncRNA XIST/miR-124-3p/ITGB1 axis in renal fibrosis in obstructive nephropathy

ObjectiveThe purpose of this study was to investigate the role and possible mechanism of lncRNA XIST in renal fibrosis and to provide potential endogenous targets for renal fibrosis in obstructive nephropathy (ON). MethodsThe study included 50 cases of ON with renal fibrosis (samples taken from patients undergoing nephrectomy due to ON) and 50 cases of normal renal tissue (samples taken from patients undergoing total or partial nephrectomy due to accidental injury, congenital malformations, and benign tumors). Treatment of human proximal renal tubular epithelium (HK-2) cells with TGF-β1 simulated renal fibrosis in vitro. Cell viability and proliferation were measured by CCK-8 and EdU, and cell migration was measured by transwell. XIST, miR-124-3p, ITGB1, and epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, α-SMA, and fibronectin) were detected by PCR and immunoblot. The targeting relationship between miR-124-3p and XIST or ITGB1 was verified by starBase and dual luciferase reporter gene experiments. In addition, The left ureter was ligated in mice as a model of unilateral ureteral obstruction (UUO), and the renal histopathology was observed by HE staining and Masson staining. ResultsON patients with renal fibrosis had elevated XIST and ITGB1 levels and reduced miR-124-3p levels. The administration of TGF-β1 exhibited a dose-dependent promotion of HK-2 cell viability, proliferation, migration, and EMT. Conversely, depleting XIST or enhancing miR-124-3p hindered HK-2 cell viability, proliferation, migration, and EMT in TGF-β1-damaged HK-2 cells HK-2 cells. XIST functioned as a miR-124-3p sponge. Additionally, miR-124-3p negatively regulated ITGB1 expression. Elevating ITGB1 weakened the impact of XIST depletion on TGF-β1-damaged HK-2 cells. Down-regulating XIST improved renal fibrosis in UUO mice. ConclusionXIST promotes renal fibrosis in ON by elevating miR-124-3p and reducing ITGB1 expressions.

METTL3-mediated pre-miR-665/DLX3 m6A methylation facilitates the committed differentiation of stem cells from apical papilla

Methyltransferase-like 3 (METTL3) is a crucial element of N6-methyladenosine (m6A) modifications and has been extensively studied for its involvement in diverse biological and pathological processes. In this study, we explored how METTL3 affects the differentiation of stem cells from the apical papilla (SCAPs) into odonto/osteoblastic lineages through gain- and loss-of-function experiments. The m6A modification levels were assessed using m6A dot blot and activity quantification experiments. In addition, we employed Me-RIP microarray experiments to identify specific targets modified by METTL3. Furthermore, we elucidated the molecular mechanism underlying METTL3 function through dual-luciferase reporter gene experiments and rescue experiments. Our findings indicated that METTL3+/− mice exhibited significant root dysplasia and increased bone loss. The m6A level and odonto/osteoblastic differentiation capacity were affected by the overexpression or inhibition of METTL3. This effect was attributed to the acceleration of pre-miR-665 degradation by METTL3-mediated m6A methylation in cooperation with the “reader” protein YTHDF2. Additionally, the targeting of distal-less homeobox 3 (DLX3) by miR-665 and the potential direct regulation of DLX3 expression by METTL3, mediated by the “reader” protein YTHDF1, were demonstrated. Overall, the METTL3/pre-miR-665/DLX3 pathway might provide a new target for SCAP-based tooth root/maxillofacial bone tissue regeneration.

HucMSCs Delay Muscle Atrophy After Peripheral Nerve Injury Through Exosomes by Repressing Muscle-Specific Ubiquitin Ligases.

Cell therapy based on mesenchymal stem cells (MSCs) alleviate muscle atrophy caused by diabetes and aging; however, the impact of human umbilical cord mesenchymal stem cells on muscle atrophy following nerve injury and the underlying mechanisms remain unclear. In this study, we evaluated the therapeutic efficacy of human umbilical cord MSCs (hucMSCs) and hucMSC-derived exosomes (hucMSC-EXOs) for muscle atrophy following nerve injury and identified the underlying molecular mechanisms. Sciatic nerve crush injury in rats and the induction of myotubes in L6 cells were used to determine the ameliorating effect of hucMSCs and hucMSC-EXOs on muscle atrophy. Q-PCR and Western blot analyses were used to measure the expression of muscle-specific ubiquitin ligases Fbxo32 (Atrogin1, MAFbx) and Trim63 (MuRF-1). Dual-luciferase reporter gene experiments were conducted to validate the direct binding of miRNAs to their target genes. Local injection of hucMSCs and hucMSC-EXOs mitigated atrophy in the rat gastrocnemius muscle following sciatic nerve crush injury. In vitro, hucMSC-EXOs alleviated atrophy in L6 myotubes. Mechanistic analysis indicated the upregulation of miR-23b-3p levels in L6 myotubes following hucMSC-EXOs treatment. MiR-23b-3p significantly inhibited the expression of its target genes, Fbxo32 and Trim63, and suppressed myotube atrophy. Notably, an miR-23b-3p inhibitor reversed the inhibitory effect of miR-23b-3p on myotube atrophy in vitro. These results suggest that hucMSCs and their exosomes alleviate muscle atrophy following nerve injury. MiR-23b-3p in exosomes secreted by hucMSCs contributes to this mechanism by inhibiting the muscle-specific ubiquitination ligases Fbxo32 and Trim63.

The role of HMGA2 in activating the IGFBP2 expression to promote angiogenesis and LUAD metastasis via the PI3K/AKT/VEGFA signaling pathway.

This study investigates the molecular mechanism of HMGA2-mediated regulation of IGFBP2 expression in the PI3K/AKT/VEGFA signaling pathway, which is involved in angiogenesis and LUAD metastasis. Target genes with prognostic implications for LUAD patients were selected using bioinformatics, and previously published literature was referenced to predict the molecular regulatory mechanisms. A549 cells were used for in vitro validation. Cell proliferation and viability were assessed using CCK-8 and EdU assays, while cell migration ability was evaluated using Transwell and wound healing assays. Changes in angiogenesis were examined using an angiogenesis assay. The targeted binding of HMGA2 with the IGFBP2 promoter was confirmed through dual luciferase reporter gene experiments and ChIP assays. In vivo validation was performed using a xenograft mouse model, and changes in angiogenesis and tumor metastasis were observed using western blot, immunofluorescence, and H&E staining. Bioinformatics analysis revealed that HMGA2 was one of the AAGs that differed between normal individuals and LUAD patients and could serve as a critical mRNA for predicting LUAD prognosis. Results from in vitro experiments demonstrated that the expression of the HMGA2 gene was significantly upregulated in LUAD cell lines. Through mediating the expression of IGFBP2, the HMGA2 gene activated the PI3K/AKT/VEGFA signaling pathway, promoting the proliferation, migration, and angiogenesis of A549 cells. In vivo, animal experiments further confirmed that HMGA2 facilitated angiogenesis and the development and metastasis of LUAD through mediating IGFBP2 expression and activating the PI3K/AKT/VEGFA signaling pathway. HMGA2 promotes angiogenesis and healthy growth and metastasis of LUAD by activating the PI3K/AKT/VEGFA signaling pathway by mediating IGFBP2 expression.

Mechanism of non-small cell lung cancer cell-derived exosome miR-196b-5p promoting pyroptosis of tumor T cells and tumor cell proliferation by downregulating ING5.

In the world, lung cancer is one of the most common malignant cancers and has become the leading cause of death of cancers in China, among which non-small cell lung cancer (NSCLC) accounts for a relatively high proportion, but there is a lack of effective treatment at present. An animal model of NSCLC was established, and BEAS-2b, H1299, Lewis, and T cells were used for subsequent experimental verification. The level of miR-196b-5p was detected by quantitative real-time polymerase chain reaction. Growth inhibitor 5 (ING5), CD9, CD63, HSP70, Caspase-1, NLRP3, and GSDMD-NT were detected by western blot. The level of ING5 was confirmed by immunohistochemistry, the location of miR-196b-5p was analyzed by fluorescence in situ hybridization (FISH), cell viability was investigated by Cell Counting Kit-8 kit, and interleukin (IL)-1β and IL-18 were confirmed by enzyme-linked immunosorbent assay. Cell apoptosis was detected by flow cytometry. In addition, the binding site was verified by dual-luciferase reporter gene experiments. Tumor volume was measured. TUNEL staining was used to detect apoptosis. Flow cytometry was used to measure the levels of CD8 T, CD4 T, and Treg cells in tumors. miR-196-5p was highly expressed in exosomes secreted by tumor cells. miR-196-5p negatively targeted ING5 to promote the growth of tumor cells. Cancer-derived exosomes promote pyroptosis of T cells to further aggravate the development of cancer. Exosome-derived miR-196b-5p promoted pyroptosis of T cells. Exosome-derived miR-196b-5p inhibited the level of ING5 to promote tumor growth and accelerate the process of NSCLC.

The role and mechanism of miR-425–3p regulating neuronal pyroptosis -mediated inorganic arsenic-induced generalized anxiety disorder

Pyroptosis plays a critical role in the pathogenesis of mental disorders. However, its specific role and mechanism in arsenic (As)-induced generalized anxiety disorder (GAD) remain elusive. We utilized the data from CtdBbase, Phenopedia and DisGeNet to analyze genes that interact with arsenic poisoning and GAD. Subsequently KEGG and GO enrichment analysis were conducted to preliminatively predict the mechanism of inorganic arsenic-induced GAD. Male Wistar rats were administered water containing NaAsO2 (50, 100 μg/L) to evaluate GAD-like behavior through open field test and elevated plus maze. The expression of differential miRNAs including miR-425–3p, and pyroptosis in the prefrontal cortex of rats were detected. Furthermore, SKNSH cells were stimulated with NaAsO2 to examine the molecular changes, and then miR-425–3p mimic was transfected into SKNSH cells to detect pyroptosis in order to verify the function of miR-425–3p. Inorganic arsenic was confirmed to induce GAD-like behavior in rats, characterized by decreased locomotor activity and exploratory activities. Rats with inorganic arsenic-induced GAD exhibited reduced miR-425–3p expression levels in the prefrontal cortex and increased expression of pyroptosis-related proteins, including NF-κB, NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18. Treating with different concentrations of NaAsO2 showed that inorganic arsenic exposure downregulates miR-425–3p expression in SKNSH cells and upregulates the expression levels of pyroptosis-related proteins. Dual-luciferase reporter gene experiments demonstrated that miR-425–3p targets the NFKB1. Overexpressing miR-425–3p reversed the inorganic arsenic-induced pyroptosis in SKNSH cells by inhibiting the expression of NF-κB, NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18. Our findings suggest that inorganic arsenic exposure may induce GAD-like behavior in rats by downregulating miR-425–3p in prefrontal cortex, which targets NF-κB and regulates pyroptosis in neuronal cells.

HDAC2-miRNA183 Epigenetic Circuit Contributes to the Growth of Philadelphia-Positive Acute Lymphoblastic Leukemia Via BCR-ABL/PTEN/p-AKT and c-MYC Signaling Pathways

Purpose: To characterize the expression and function of leukemia related/specific microRNAs (miRNAs) in Philadelphia-positive Acute Lymphoblastic Leukemia (Ph-positive ALL), and to identify the mechanistic signaling pathways in Ph-positive ALL. Method: Differentially expressed miRNAs in Ph-positive ALL patient samples and cell line were identified using RNA-seq and qPCR. Cell proliferation and apoptosis were detected with CCK-8 and flow cytometry assays, respectively. Bioinformatics software predicted the upstream and downstream regulatory pathway of miRNA183; the dual luciferase reporter gene analysis verified the regulatory effect of miRNA183 on the target gene BCR-ABL; ChIP experiment detected the recruitment of HDACs in the miRNA183 promoter region, and western blot detected the expression of related protein in the miRNA183 related pathway. Results: We identified thirty-one differentially expressed miRNAs in three Ph-positive ALL samples vs. normal karyotype B-ALL samples. Among these alternately expressed miRNAs, we found miRNA183 was significantly downregulated in Ph-positive ALL. qPCR verified the down-expression of miRNA183 in Ph-positive ALLcasesamples and Ph-positive ALL cell line SUP-B15. Forced over-expression of miRNA183 inhibited proliferation of SUP-B15 cells, but had no effect on NALM-6 cells (normal karyotype B-ALL). Furthermore, either forced over-expression of miRNA183 or silence down of BCR-ABL1 promoted SUP-B15 cell apoptosis.TargetScan software prediction found that miRNA183 and BCR-ABL (e1a2) had potential binding sites, dual luciferase reporter gene experiments further proved that miRNA183 could direct bind to BCR-ABL gene; qPCR and western blot further showed that miRNA183 inhibited the expression of P190. Our results showed that BCR-ABL (e1a2) is the target gene of miRNA183. In addition, forced over-expression of miRNA183 and silence down of BCR-ABL1 regulated the downstream proteins including PTEN, PARP, p-AKT and c-MYC with the same change trend. In complementary loss-of-function studies, inhibition of miRNA183 restored the expression of these proteins.Treatment of SUP-B15 cells with decitabine (DAC) and histone deacetylase inhibitor (HDACi), respectively, resulted in decrease of cell viability in a dose-dependent manner. Surprisingly, miRNA183 levels were significantly increased in HDACi exposure but not in DAC exposure. Furthermore, miRNA183 inhibitor reduced HDACi-induced upregulation of miRNA183 expression and attenuated HDACi-mediated apoptosis in SUP-B15 cells. Biology software prediction showed the presence of a transcription start site (TSS) 5 kb upstream of miRNA183. ChIP assay further demonstrated that HDAC2 but not HDAC1 was specifically recruited to the miRNA183 promoter region. Additionally, after HDACi treatment, HDACi-induced H3K27ac was recruited and accumulated at the miRNA183 promoter region. Western blotassay further proved the changes of the related proteins. Conclusion :Our results indicate that miRNA183 is a potential biomarker for Ph-positive ALL and suggest that a novel “HDAC2-miRNA183 Epigenetic Circuit” may be involved in the growth and progression of Ph-positive ALL by PTEN/p-AKT and c-MYC signaling pathways. Taken together, this study may provide novel approaches to the design of targeted drugs for Ph-positive ALL.

β-elemene enhances cisplatin sensitivity of non-small cell lung cancer cells via the miR-17-5p/STAT3 axis.

In China, β-elemene, a sesquiterpene compound derived from Curcuma wenyujin, is clinically used to treat many human malignancies, including non-small cell lung cancer (NSCLC). Nonetheless, the role of β-elemene in regulating cisplatin sensitivity of NSCLC cells and the related mechanisms are not clear. This study was conducted to investigate the role of β-elemene in sensitizing NSCLC cells to cisplatin. In this work, cisplatin-resistant NSCLC cell lines were constructed. CCK-8, colony formation, and flow cytometry assays were executed to examine cell viability, growth, and apoptosis. MiR-17-5p and STAT3 expression levels in cells were detected by qRT-PCR. Western blot was executed to determine the expression levels of STAT3 and apoptosis-related proteins (Bax and Bcl-2) in the cells. Dual-luciferase reporter gene experiments were performed to verify the targeting relationship between miR-17-5p and STAT3. Herein, we report that, β-elemene inhibits the viability, and induces the apoptosis of cisplatin-resistant NSCLC cells. Additionally, β-elemene induces the upregulation miR-17-5p and downregulation of STAT3. STAT3 is validated to be a target of miR-17-5p in NSCLC cells. Additionally, the role of β-elemene to repress the viability of cisplatin-resistant NSCLC cells is partially counteracted by miR-17-5p inhibitor or STAT3 overexpression. In summary, our study suggests that β-elemene enhances cisplatin sensitivity of NSCLC cells by modulating miR-17-5p/STAT3 axis, and it may be a choice for the complementary treatment of NSCLC patients.

MiR-3195 suppresses the malignant progression of osteosarcoma cells via targeting SOX4

BackgroundOsteosarcoma (OS) is a highly invasive primary malignancy of the bone that is common in children and adolescents. MicroRNAs (miRNAs) are novel diagnostic and predictive biomarkers for cancers. The miRNA miR-3195 is aberrantly expressed in multiple types of tumors. However, the expression levels and biological functions of miR-3195 in OS remain unclear.MethodsTwo Gene Expression Omnibus (GEO) datasets (GSE69470 and GSE16088) were used to analyze differentially expressed miRNAs and mRNAs in osteosarcoma cell lines and OS tissues. Quantitative RT-PCR was used to detect the expression levels of miR-3195 and the SRY-box transcription factor 4 (SOX4) mRNA in OS tissues and cell lines. The relationship between miR-3195 and the 3’-upstream region (3’-UTR) in the SOX4 mRNA (predicted through bioinformatics) was analyzed using Pearson's correlation analysis and confirmed by a dual-luciferase reporter gene experiment. Cell counting kit-8 assays, colony formation assays, flow cytometry, wound healing assays, transwell assays, and western blotting were performed to explore the effects of miR-3195 levels on SOX4 affected OS cell biological behavior.ResultsOur results revealed that miR-3195 was the most down-regulated miRNA and SOX4 was the most up-regulated mRNA by Bioinformatic analysis. It was further confirmed miR-3195 had low expression, and SOX4 had high expression levels in clinical OS tissue samples; the expression levels of both genes were negatively correlated with each other in OS tissues. Overexpression of miR-3195 in OS cell lines significantly inhibited cell proliferation, migration, and invasiveness, while promoting apoptosis; all these effects were reversed by increasing SOX4 expression levels. We also found that miR-3195 could directly bind with the SOX4 gene and down-regulate SOX4 expression.ConclusionsmiR-3195 can modulate proliferation, migration, invasiveness, and apoptosis in OS cells by regulating the SOX4 gene. Thus, the miR-3195/SOX4 signaling may be a novel therapeutic target in OS treatment.

Exosomal miR-320e through wnt2targeted inhibition of the Wnt/β-catenin pathway allevisate cerebral small vessel disease and cognitive impairment.

Exosomal miRNAs play crucial roles in many central nervous system diseases. Cerebral small vessel disease (CVSD) is a small vessel disease that is affected by various factors. This study aimed to investigate the role of exosomal miR-320e in the Wnt/β-catenin pathway stimulated by oxidative stress and assess its clinical correlation with psychiatric symptoms in patients with CVSD. To explore whether exosomal miR-320e could suppress the Wnt/β-catenin pathway and play a protective role in CVSD progression, as well as examine its potential correlation with cognitive impairment and depression in patients with CVSD. Differentially expressed exosomal miRNAs were filtered by sequencing plasma exosomes from patients with CVSD and healthy controls. Bioinformatics and dual luciferase analyses were used to confirm the binding of miR-320e to Wnt2, and the mRNA and protein levels of downstream components in the Wnt/β-catenin pathway were evaluated when overexpressed or with knockdown of miR-320e under H2O2-induced oxidative stress. In addition, Wnt2-targeting siRNA was used to confirm the role of miR-320e in the Wnt2-mediated inhibition of the Wnt/β-catenin pathway. A retrospective analysis was conducted among patients with CVSD to confirm the correlation between miR-320e expression and the severity of cognitive impairment and depression, which were quantified using the Montreal Cognitive Assessment (MoCA)/Executive Function Assessment (EFA), and the Hamilton Depression Scale (HAMD)/Beck Depression Inventory (BDI), respectively. High-throughput sequencing revealed that exosomal miR-320e was downregulated in patients with CVSD. Bioinformatics analysis and dual-luciferase reporter gene experiments showed that exosomal miR-320e inhibited the Wnt/β-catenin pathway in response to oxidative stress by targeting the 3' noncoding region of Wnt2. Uptake of exosomes carrying miR-320e into endothelial cells could also target Wnt2 and inhibit the Wnt2/β-catenin pathway. Elevated miR-320e expression may protect patients with CVSD from relatively severe cognitive impairment and depression, as it was found to have a positive correlation with the MoCA/EFA and HAMD/BDI scores. Our results suggest that exosomal miR-320e suppresses the Wnt/β-catenin pathway and may play a protective role in CVSD progression.

CircRNA_0088196 Regulates Trophoblast Proliferation and Apoptosis in Preeclampsia Through the miR-379-5p/HSPA5 Axis.

Existing research has confirmed the dysregulation of circular RNA (circRNA) in a wide variety of human diseases. Thus, in this study, we explored the potential mechanism of circRNA_0088196 in preeclampsia (PE). We performed quantitative real-time PCR to examine circRNA_0088196 expression and verified the function of circRNA_0088196 in vitro using CCK-8, TUNEL, flow cytometry, and Western blotting analyses. Additionally, we studied the mechanism using dual-luciferase reporter gene experiments. The results of our research revealed the up-regulation of circRNA_0088196 in PE patients' placentas and Heat Shock 70 kDa Protein 5 (HSPA5)-stimulated trophoblast (HTR-8/SVneo) cells. An investigation of the mechanism also showed that there was a binding between miR-379-5p and circRNA_0088196. Additionally, circRNA_0088196 inhibited HTR-8/SVneo cell proliferation and promoted cell apoptosis via the miR-337-3p/HSPA5 axis, thereby facilitating PE. In vivo experiments indicated that circRNA_0088196 regulated HTR-8/SVneo cell production through miR-379-5p. Overall, the findings of this study illustrate that circRNA_0088196 interference promotes cell apoptosis and inhibits HTR-8/SVneo proliferation via the miR-379-5p/HSPA5 axis, thereby accelerating the development of PE.

Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells

To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05). Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Abstract 496: Inhibition Of Tirna-gly-gcc Ameliorates Neointimal Formation Via Cbx3-mediated Vascular Smooth Muscle Cells Phenotypic Switching

Introduction: Vascular smooth muscle cells (VSMCs) are the main component of blood vessels and synthetic VSMCs play an essential role in vascular diseases. tRNA-derived fragments (tRFs) are a new class of non-coding RNAs involved in a variety of pathological processes, but their biological functions and mechanisms in VSMCs phenotype transition and vascular intimal hyperplasia are unclear. Hypothesis: We assessed the hypothesis that tRFs play important role in VSMCs phenotype transition, regulation of tRFs could ameliorates neointimal formation. Methods: Differential expression profiles of tRFs from different phenotype of VSMCs was acquired using small-RNA sequence. The expression of tiRNA-Gly-GCC was confirmed in VSMCs, vascular tissues and plasma from healthy and atherosclerosis using quantitative real-time PCR and RNA fluorescence in situ hybridization. Gain-and loss- of function were performed using mimics and inhibitors to investigate the role of tiRNA-Gly-GCC in VSMCs phenotypic transition and neointimal formation after vascular injury. Dual-luciferase reporter gene and rescue experiments were performed to demonstrate the relationship between tiRNA-Gly-GCC and CBX3. Results: tiRNA-Gly-GCC is upregulated in synthetic VSMCs, atherosclerotic arteries, plasma and the balloon injured carotid artery of rats. Functionally, the inhibition of tiRNA-Gly-GCC represses VSMCs proliferation, migration, and reversed dedifferentiation, whereas the overexpression of tiRNA-Gly- GCC have contrary effects. Mechanistically, tiRNA-Gly-GCC performs these functions on VSMCs via downregulating the expression of CBX3. Finally, the inhibition of tiRNA-Gly-GCC could ameliorate neointimal formation after vascular injury in vivo . Conclusions: In conclusion, tiRNA-Gly-GCC is a mediator of VSMCs phenotypic switching by targeting CBX3, inhibition of tiRNA-Gly-GCC suppresses neointimal formation.

MiR-95-3p/EPM2A/MMP2 contributes to the pathogenesis of severe preeclampsia through the regulation of trophoblast biological behaviour

ObjectivePreeclampsia (PE) is a maternal multisystem disease with an unclear mechanism. Data showed that MiR-95-3p promoted cell migration, invasion and proliferation, leading to the occurrence and development of many cancers, and placental trophoblasts and tumor cells had similar migration, invasion and proliferation abilities. Meanwhile we found that MiR-95-3p was differentially expressed in PE and normal placenta. Therefore, this article aimed to explore the biological function and mechanism of miR-95-3p in PE. MethodsThe expression of miR-95-3p in PE and normal placental tissue was explored by high-throughput sequencing and qRT–PCR. The effects of miR-95-3p on trophoblast migration, invasion, proliferation, angiogenesis and apoptosis were investigated by Transwell migration and invasion assays, cell viability assay, tube formation assay and flow cytometry in two trophoblast cell lines (HTR-8/SVneo and JAR). The miR-95-3p target gene EPM2A was identified and verified by unique identifier mRNA next-generation sequencing and dual-luciferase reporter gene experiments. Rescue experiments were conducted to investigate whether miR-95-3p regulated EPM2A to participate in trophoblast migration and invasion. Finally, the effects of miR-95-3p and EPM2A on the expression of angiogenic factors and inflammation-related factors were investigated by ELISA. ResultsWe found that miR-95-3p was expressed at low levels in the placental tissue of patients with PE and was negatively correlated with EPM2A expression. In vitro upregulation of miR-95-3p and downregulation of EPM2A promote trophoblast migration, invasion and proliferation. Furthermore, EPM2A was confirmed as a target mRNA of miR-95-3p. Upregulation of EPM2A mitigated miR-95-3p-mediated promotion of trophoblast migration and invasion and vice versa. Finally, both miR-95-3p and EPM2A regulate the expression of trophoblast angiogenesis-related factors and inflammation-related factors. ConclusionOur findings demonstrated that miR-95-3p promoted the migration and invasion of trophoblast cells by targeting EPM2A to inhibit the occurrence and development of PE.

The effect of miR-155-5p on M1 polarization of Kupffer cells and immune response during liver transplantation through regulating the expression of KDM5D.

To explore the effect and its specific mechanism of miR-155-5p on M1 polarization of Kupffer cells (KCs) and immune response in liver transplantation (LT) through KDM5D. Primary KCs were isolated from Wistar rats and identified by cell culture, ink-swallowing test and flow cytometry. The cells identified as KCs were induced into LT acute rejection (AR) model cells by LPS/IFN-γ, flow cytometry was used for cell sorting and apoptosis detection. Enzyme-linked immunosorbent assay (ELISA) kit was used to detect the levels of inflammatory factors, macrophages and liver function markers. RT-qPCR detected the expression of miR-155-5p and KDM5D mRNA. The protein expression of KDM5D was detected by Western blot. Dual luciferase reporter gene experiment verified the targeting relationship between miR-155-5p and KDM5D. The separated KCs adhered after being cultured for 24h, had pseudopodia and phagocytosis, and the proportion of F4/80 positive cells was more than 90%. The expression of miR-155-5p was increased in LPS/IFN-γ-induced KCs. And knockdown of miR-155-5p inhibited H3K4me3 and H3K27me3 of TNF-α promoter, M1 polarization of KCs and the immune response of AR model cells by upregulating KDM5D. In animal experiments, knockdown of miR-155-5p was found to inhibit liver damage and immune response in rats with allogeneic orthotopic LT. These results confirmed that miR-155-5p inhibited M1 polarization of KCs induced by LPS/IFN-γ, thereby alleviating AR and liver function impairment after LT by upregulating KDM5D.

MicroRNA-22-3p Regulates the Expression of Kruppel-like Factor 6 to Affect the Cardiomyocyte-like Differentiation of Bone Marrow Mesenchymal Stem Cell

Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.

A novel TBK1/IKKϵ is involved in immune response and interacts with MyD88 and MAVS in the scallop Chlamys farreri.

Inhibitor of κB kinase (IKK) family proteins are key signaling molecules in the animal innate immune system and are considered master regulators of inflammation and innate immunity that act by controlling the activation of transcription factors such as NF-κB. However, few functional studies on IKK in invertebrates have been conducted, especially in marine mollusks. In this study, we cloned the IKK gene in the Zhikong scallop Chlamys farreri and named it CfIKK3. CfIKK3 encodes a 773-amino acid-long protein, and phylogenetic analysis showed that CfIKK3 belongs to the invertebrate TBK1/IKKϵ protein family. Quantitative real-time PCR analysis showed that CfIKK3 mRNA is ubiquitously expressed in all tested scallop tissues. The expression of CfIKK3 transcripts was significantly induced after challenge with lipopolysaccharide, peptidoglycan, or poly(I:C). Co-immunoprecipitation (co-IP) assays confirmed the direct interaction of CfIKK3 with MyD88 (the key adaptor in the TLR pathway) and MAVS (the key adaptor in the RLR pathway), suggesting that this IKK protein plays a crucial role in scallop innate immune signal transduction. In addition, the CfIKK3 protein formed homodimers and bound to CfIKK2, which may be a key step in the activation of its own and downstream transcription factors. Finally, in HEK293T cells, dual-luciferase reporter gene experiments showed that overexpression of CfIKK3 protein activated the NF-κB reporter gene in a dose-dependent manner. In conclusion, our experimental results confirmed that CfIKK3 could respond to PAMPs challenge and participate in scallop TLR and RLR pathway signaling, ultimately activating NF-κB. Therefore, as a key signaling molecule and modulator of immune activity, CfIKK3 plays an important role in the innate immune system of scallops.

LncRNA MEG3 promotes cisplatin sensitivity of cervical cancer cells by regulating the miR-21/PTEN axis

BackgroundCervical cancer (CC) is a common gynecological malignancy worldwide. Some patients perform serious resistance after chemotherapy, and long-stranded non-coding RNA MEG3 is reported to be involved in the regulation of chemoresistance in many solid tumors. However, its involvement in cervical adenocarcinoma has not been reported.MethodsHela cell lines, cisplatin-resistant cell lines (Hela-CR) and nude mice were used in this study. After MEG3 was overexpressed or knocked down in cells by the lentivirus vector, cell growth was detected by the CCK-8 assay, and cell migration was evaluated using Transwell assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression of MEG3, miR-21 and PTEN mRNA. Apoptosis was detected by flow cytometry. The targeting relationship between mRNAs was predicted and verified using dual-luciferase reporter gene experiments. Western blot was executed to examine Bax, cleaved-caspase 3, Bcl-2, PTEN and GAPDH expression. Cells were injected into the mice to form xenograft tumors to compare tumorigenesis capacity.ResultsWe demonstrated that MEG3 was down-regulated in cervical cancer by analyzing the TCGA database. Moreover, knockdown of MEG3 promoted CC cell proliferation, migration and inhibited the apoptosis. These changes of CC cells were more pronounced under cisplatin treatment. Further studies showed that the MEG3/miR-21/PTEN axis affected cisplatin sensitivity in cervical cancer cells, and these results of recue assay were used to confirm this conclusion.ConclusionsMEG3 performing as ceRNA promotes cisplatin sensitivity in CC cells through the miR-21/PTEN axis.

LINC00511 promotes cervical cancer progression by regulating the miR-497-5p/MAPK1 axis

BackgroundLong non-coding RNA (lncRNA) exhibits a crucial role in multiple human malignancies. The expression of lncRNA LINC00511, reportedly, is aberrantly up-regulated in several types of tumors. Our research was aimed at deciphering the role and mechanism of LINC00511 in the progression of cervical cancer (CC).MethodQuantitative real-time polymerase chain reaction (qRT-PCR) was performed to quantify the expression levels of LINC00511, miR-497-5p and MAPK1 mRNA in CC tissues and cell lines. Cell counting kit-8 (CCK-8), 5-bromo-2’-deoxyuridine (BrdU) and Transwell assays were conducted for detecting the proliferation, migration and invasion of CC cells. Dual-luciferase reporter gene experiments were performed to verify the targeting relationships amongst LINC00511, miR-497-5p and MAPK1. Besides, MAPK1 expression in CC cells was detected via Western blot after LINC00511 and miR-497-5p were selectively regulated.ResultsUp-regulation of LINC00511 expression in CC tissues and cell lines was observed, which was in association with tumor size, clinical stage and lymph node metastasis of the patients. LINC00511 overexpression facilitated the proliferation, migration and invasion of CC cells, while opposite effects were observed after knockdown of LINC00511. Mechanistically, LINC00511 was capable of targeting miR-497-5p and up-regulating MAPK1 expression.ConclusionLINC00511/miR-497-5p/MAPK1 axis regulates CC progression.