Histone lysine demethylase KDM5B facilitates proliferation and suppresses apoptosis in human acute myeloid leukemia cells through the miR-140-3p/BCL2 axis.

The histone lysine demethylase KDM5B is frequently up-regulated in various human cancer cells. However, its expression and functional role in human acute myeloid leukemia (AML) cells remain unclear. Here, we found that the expression level of KDM5B is high in primary human AML cells. We have demonstrated that knocking down KDM5B leads to apoptosis and impairs proliferation in primary human AML and some human AML cell lines. We further identified miR-140-3p as a downstream target gene of KDM5B. KDM5B expression was inversely correlated with the miR-140-3p level in primary human AML cells. Molecular studies showed that silencing KDM5B enhanced H3K4 trimethylation (H3K4me3) at the promoter of miR-140-3p, leading to high expression of miR-140-3p, which in turn inhibited B-cell CLL/lymphoma 2 (BCL2) expression. Finally, we demonstrate that the defective proliferation induced by KDM5B knockdown (KD) can be rescued with the miR-140-3p inhibitor or enhanced by combining KDM5B KD with a BCL2 inhibitor. Altogether, our data support the conclusion that KDM5B promotes tumorigenesis in human AML cells through the miR-140-3p/BCL2 axis. Targeting the KDM5B/miR-140-3p/BCL2 pathway may hold therapeutic promise for treating human AML.

Reversing the miRNA -5p/-3p stoichiometry reveals physiological roles and targets of miR-140 miRNAs.

The chondrocyte-specific miR-140 miRNAs are necessary for normal endochondral bone growth in mice. miR-140 deficiency causes dwarfism and craniofacial deformity. However, the physiologically important targets of miR-140 miRNAs are still unclear. The miR-140 gene (Mir140) encodes three chondrocyte-specific microRNAs, miR-140-5p, derived from the 5' strand of primary miR-140, and miR140-3p.1 and -3p.2, derived from the 3' strand of primary miR-140. miR-140-3p miRNAs are 10 times more abundant than miR-140-5p likely due to the nonpreferential loading of miR-140-5p to Argonaute proteins. To differentiate the role of miR-140-5p and -3p miRNAs in endochondral bone development, two distinct mouse models, miR140-C > T, in which the first nucleotide of miR-140-5p was altered from cytosine to uridine, and miR140-CG, where the first two nucleotides of miR-140-3p were changed to cytosine and guanine, were created. These changes are expected to alter Argonaute protein loading preference of -5p and -3p to increase -5p loading and decrease -3p loading without changing the function of miR140-5p. These models presented a mild delay in epiphyseal development with delayed chondrocyte maturation. Using RNA-sequencing analysis of the two models, direct targets of miR140-5p, including Wnt11, were identified. Disruption of the predicted miR140-5p binding site in the 3' untranslated region of Wnt11 was shown to increase Wnt11 mRNA expression and caused a modest acceleration of epiphyseal development. These results show that the relative abundance of miRNA-5p and -3p can be altered by changing the first nucleotide of miRNAs in vivo, and this method can be useful to identify physiologically important miRNA targets.

Circ-AGTPBP1 promotes white matter injury through miR-140-3p/Pcdh17 axis role of Circ-AGTPBP1 in white matter injury.

White matter injury (WMI) resulting from intracerebral hemorrhage (ICH) is closely associated with adverse prognoses in ICH patients. Although Circ-AGTPBP1 has been reported to exhibit high expression in the serum of premature infants with WMI, its effects and mechanisms in ICH-induced WMI remain unclear. This study aimed to investigate the role of circ-AGTPBP1 in white matter injury after intracerebral hemorrhage. An intracerebral hemorrhage rat model was established by injecting autologous blood into rat left ventricles and circ-AGTPBP1 was knocked down at the ICH site using recombinant adeno-associated virus, AAV2/9. Magnetic resonance imaging (MRI) and gait analysis were conducted to assess long-term neurobehavioral effects. Primary oligodendrocyte progenitor cells (OPCs) were isolated from rats and overexpressed with circ-AGTPBP1. Downstream targets of circ-AGTPBP1 in OPCs were investigated using CircInteractome, qPCR, FISH analysis, and miRDB network. Luciferase gene assay was utilized to explore the relationship between miR-140-3p and Pcdh17 in OPCs and HEK-293T cells. Finally, CCK-8 assay, EdU staining, and flow cytometry were employed to evaluate the effects of mi-RNA-140-3p inhibitor or silencing of sh-pcd17 on the viability, proliferation, and apoptosis of OPCs. Low expression of circ-AGTPBP1 alleviates white matter injury and improves neurological functions in rats after intracerebral hemorrhage. Conversely, overexpression of circ-AGTPBP1 reduces the proliferative and migrative potential of oligodendrocyte progenitor cells and promotes apoptosis. CircInteractome web tool and qPCR confirmed that circ-AGTPBP1 binds with miR-140-3p in OPCs. Additionally, miRDB network predicted Pcdh17 as a downstream target of miR-140-3p. Moreover, pcdh17 expression was increased in the brain tissue of rats with intracerebral-induced white matter injury. Furthermore, inhibiting miR-140-3p suppressed the proliferation and migration of OPCs and facilitated apoptosis through Pcdh17. Circ-AGTPBP1 promotes white matter injury through modulating the miR-140-3p/Pcdh17 axis. The study provides a new direction for developing therapeutic strategies for white matter injury.

CircACTR2 attenuated the effects of tetramethylpyrazine on human kidney cell injury.

Tetramethylpyrazine (TMP) is one of the active ingredients of Chuan Xiong that has been reported to have effects on numerous diseases, including diabetic nephropathy (DN). Whereas, related molecular mechanisms are not fully elucidated. We aimed to explore circACTR2's role in TMP-mediated protective effects on DN. In vitro DN condition was established in human kidney cells (HK-2) by treating high glucose (HG). CCK-8 assay and flow cytometry assay were used to observe cell viability and survival. Oxidative stress was determined by the associated markers using kits. The release of inflammatory factors was detected using ELISA kits. Quantitative real-time PCR (qPCR) and western blot were utilized for expression analysis of cricACTR2, miR-140-5p, and GLI pathogenesis-related 2 (GLIPR2). The binding between miR-140-5p and circACTR2 or GLIPR2 was confirmed by dual-luciferase, RIP, and pull-down studies. HG largely induced HK-2 cell apoptosis, oxidative stress, and inflammation, which were alleviated by TMP. CircACTR2's expression was enhanced in HG-treated HK-2 cells but attenuated in HG + TMP-treated HK-2 cells. CircACTR2 overexpression attenuated the functional effects of TMP and thus restored HG-induced cell apoptosis, oxidative stress, and inflammation. CircACTR2 bound to miR-140-5p to enhance the expression of GLIPR2. MiR-140-5p restoration or GLIPR2 inhibition reversed the role of circACTR2 overexpression. CircACTR2 attenuated the protective effects of TMP on HG-induced HK-2 cell damages by regulating the miR-140-5p/GLIPR2 network, indicating that circACTR2 was involved in the functional network of TMP in DN.

UBE2C, targeted by miR-140-3p, promotes the progression of osteosarcoma via PI3K/AKT signaling pathway.

BACKGROUND: UBE2C was reported to play carcinogenic effects in diverse cancers. However, the role of UBE2C in osteosarcoma was poorly understood, and its functional mechanisms were not fully clarified. METHODS: RT-qPCR was used to assess the expression of UBE2C mRNA and miR-140-3p, and western blot technique was used to examine the UBE2C protein and PI3K/AKT pathway-associated proteins. CCK-8 test was applied to measure cell proliferation, and wound healing assay were used to measure migration. Using animal studies, the function of UBE2C in vivo was evaluated. Dual-luciferase reporter assay was used to confirm the potential interaction among UBE2C and miR-140-3p. RESULTS: In osteosarcoma cells as well as tumor samples, UBE2C was strongly expressed. Osteosarcoma cell proliferation as well as cell migration were inhibited by UBE2C knockdown, and PI3K/AKT signaling activity was diminished. In addition, UBE2C knockdown impeded tumor growth in animal models. UBE2C expression was lessened by miR-140-3p as miR-140-3p targets it. UBE2C is overexpressed which promote osteosarcoma proliferation as well as migration, and strengthened the PI3K/AKT signaling activity, while forced miR-140-3p expression partially abolished these effects. CONCLUSION: UBE2C, targeted by miR-140-3p, drove carcinogenic effects in osteosarcoma through activating the PI3K/AKT pathway.

Mechanistic study on ursolic acid inhibiting the growth of colorectal cancer cells through the downregulation of TGF-ß3 by miR-140-5p.

Colorectal cancer (CRC) is a common digestive tract tumor with a high incidence and a poor prognosis. Traditional chemotherapy drugs are usually accompanied by unpleasant side effects, highlighting the importance of exploring new adjunctive drugs. In this study, we aimed to explore the role of ursolic acid (UA) in CRC cells. Specifically, HT-29 cells were treated with UA at different concentrations (10, 20, 30, and 40 µM), and the expression of miR-140-5p, tumor growth factor-ß3 (TGF-ß3), ß-catenin, and cyclin D1 was determined by real-time quantitative PCR. The cell cycle and apoptosis were checked by flow cytometry, and cell proliferation was detected by Cell Counting Kit-8 assay. The HT-29 cell model was established through overexpression (miR-140-5p mimics) and interference (miR-140-5p inhibitor) of miR-140-5p. Western blot was used to detect the protein expression of TGF-ß3. We found that UA could inhibit the proliferation of HT-29 cells, block cells in the G1 phase, and promote cell apoptosis. After UA treatment, the expression of miR-140-5p increased and TGF-ß3 decreased. Notably, miR-140-5p downregulated the expression of TGF-ß3, while the overexpression of miR-140-5p exerted a similar function to UA in HT-29 cells. Additionally, the messenger RNA expression of TGF-ß3, ß-catenin, and cyclin D1 was decreased in HT-29 cells after UA treatment. In conclusion, UA inhibited CRC cell proliferation and cell cycle and promoted apoptosis by regulating the miR-140-5p/TGF-ß3 axis, which may be related to the inhibition of Wnt/ß-catenin signaling pathway.

Circ_0004674 regulation of glycolysis and proliferation mechanism of osteosarcoma through miR-140-3p/TCF4 pathway.

As a subclass of noncoding RNAs, circular RNA play an important role in tumour development. The aim of this study was to investigate the role of circ_0004674 in osteosarcoma glycolysis and the molecular mechanism of its regulation. We examined the expression of circ_0004674, miR-140-3p, TCF4 and glycolysis-related proteins (including HK2, PKM2, GLUT1 and LDHA) in osteosarcoma cells and tissues by quantitative reverse transcription-polymerase chain reaction and immunoblotting (Western blot analysis). The role of circ_0004674, miR-140-3p and TCF4 in the proliferation, apoptosis, migration and invasion of OS cells was examined using CCK8 assay, Apoptosis assay, Wound healing assay, Transwell migration and Matrigel invasion assay. The interaction of circ_0004674/miR-140-3p and miR-1543/TCF4 was also analysed using a dual luciferase reporter assay. Finally, the glycolytic process was assessed by glucose uptake assays and lactate production measurements. The results showed that the expression of circ_0004674 and TCF4 was significantly higher in MG63 and U2OS cells compared to hFOB1.19 cells, while the expression of miR-140-3p was downregulated. Silencing of circ_0004674 gene significantly inhibited the proliferation, migration and invasion of cancer cells and promoted apoptosis of cancer cells. Experiments such as dual luciferase reporter analysis showed that circ_0004674 regulates the expression of glycolysis-related proteins through the miR-140-3p/TCF4 pathway, and inhibition of this gene attenuated the depletion of glucose content and the production of lactate in cancer cells. Furthermore, inhibition of miR-140-3p or overexpression of TCF could reverse the phenotypic changes in cancer cells induced by circ_0004674 silencing. In summary, this study elucidated the specific function and potential mechanisms of circ_0004674 in osteosarcoma glycolysis. The findings demonstrate that miR-140-3p and TCF4 function respectively as a tumor suppressor gene and an oncogene in osteosarcoma. Notably, they influence glycolysis and associated pathways, regulating osteosarcoma proliferation. Therefore, circ_0004674 promotes osteosarcoma glycolysis and proliferation through the miR-140-3p/TCF4 pathway, enhancing the malignant behaviour of tumours, and it is expected to be a potential molecular target for osteosarcoma treatment.

Reciprocal negative feedback between Prrx1 and miR-140-3p regulates rapid chondrogenesis in the regenerating antler.

During growth phase, antlers exhibit a very rapid rate of chondrogenesis. The antler is formed from its growth center reserve mesenchyme (RM) cells, which have been found to be the derivatives of paired related homeobox 1 (Prrx1)-positive periosteal cells. However, the underlying mechanism that drives rapid chondrogenesis is not known. Herein, the miRNA expression profiles and chromatin states of three tissue layers (RM, precartilage, and cartilage) at different stages of differentiation within the antler growth center were analyzed by RNA-sequencing and ATAC-sequencing. We found that miR-140-3p was the miRNA that exhibited the greatest degree of upregulation in the rapidly growing antler, increasing from the RM to the cartilage layer. We also showed that Prrx1 was a key upstream regulator of miR-140-3p, which firmly confirmed by Prrx1 CUT&Tag sequencing of RM cells. Through multiple approaches (three-dimensional chondrogenic culture and xenogeneic antler model), we demonstrated that Prrx1 and miR-140-3p functioned as reciprocal negative feedback in the antler growth center, and downregulating PRRX1/upregulating miR-140-3p promoted rapid chondrogenesis of RM cells and xenogeneic antler. Thus, we conclude that the reciprocal negative feedback between Prrx1 and miR-140-3p is essential for balancing mesenchymal proliferation and chondrogenic differentiation in the regenerating antler. We further propose that the mechanism underlying chondrogenesis in the regenerating antler would provide a reference for helping understand the regulation of human cartilage regeneration and repair.

Circ_0059457 Promotes Proliferation, Metastasis, Sphere Formation and Glycolysis in Breast Cancer Cells by Sponging miR-140-3p to Regulate UBE2C.

Circular RNA (circRNA) has been confirmed to regulate breast cancer (BC) progression. However, the role of circ_0059457 in BC progression is still unclear.The expression of circ_0059457, taspase 1 (TASP1), microRNA (miR)-140-3p and ubiquitin-binding enzyme E2C (UBE2C) was detected by quantitative real-time PCR. Cell proliferation, migration, invasion and sphere formation ability were assessed by cell counting kit-8 assay, EdU assay, wound healing assay, transwell assay and sphere formation assay. Cell glycolysis was assessed by detecting glucose uptake, lactate levels and ATP/ADP ratio. Dual-luciferase reporter assay, RIP assay, RNA pull-down assay were used to validate RNA interaction. Xenograft tumor model to assess the effect of circ_0059457 on BC tumor growth in vivo. Circ_0059457 had elevated expression in BC tissues and cells. Circ_0059457 knockdown inhibited BC cell proliferation, metastasis, sphere formation ability, and glycolysis. In terms of mechanism, circ_0059457 sponged miR-140-3p, and miR-140-3p targeted UBE2C. MiR-140-3p inhibition reversed the effect of circ_0059457 knockdown on BC cell malignant behaviors. Besides, miR-140-3p overexpression inhibited BC cell proliferation, metastasis, sphere formation ability and glycolysis, and these effects were abrogated by UBE2C enhancement. Furthermore, circ_0059457 regulated UBE2C expression through sponging miR-140-3p. Additionally, circ_0059457 knockdown obviously inhibited BC tumor growth in vivo. Circ_0059457 promoted BC progression via miR-140-3p/UBE2C axis, which provided potential target for the treatment of BC.

MicroRNA-140-3p inhibits proliferation and promotes apoptosis in non-small cell lung cancer by targeting MDIG.

BACKGROUND: MicroRNAs (miRNAs) are associated with cancer progression. MiR-140-3p is a tumor suppressor. Nevertheless, its function in non-small cell lung cancer (NSCLC) is unclear. METHODS: MiR-140-3p expression in NSCLC clinical specimens was examined using the TCGA database and real-time PCR. NSCLC cell proliferation and apoptosis were investigated after the miRNA overexpression. Then, mineral dust-induced gene (MDIG) levels in NSCLC clinical specimens were monitored by real-time PCR and western blotting. Bioinformatics predicated the binding of miR-140-3p to MDIG, and their relationship was validated by luciferase reporter assay. The miR-140-3p/MDIG axis was further validated through rescue experiments. The involvement of STAT3 signaling in the actions of miR-140-3p/MDIG axis was investigated. RESULTS: MiR-140-3p was decreased in NSCLC tissues and negatively correlated with MDIG expression. Additionally, it was also lower in high-grade specimens than in low-grade ones. MiR-140-3p restrained cell proliferation, facilitated apoptosis, and inhibited STAT3 signaling in NSCLC. Interestingly, MDIG was a target of this miRNA. Furthermore, MDIG upregulation abolished miR-140-3p's effect on cell proliferation, apoptosis, and STAT3 pathway in NSCLC cells. CONCLUSION: MiR-140-3p restrained NSCLC development through the regulation of the STAT3 pathway by targeting MDIG. This axis may be a promising target for NSCLC treatment.

Downregulation of circ-STK39 suppresses pancreatic cancer progression by sponging mir-140-3p and regulating TRAM2-mediated epithelial-mesenchymal transition.

BACKGROUND: Pancreatic cancer (PC) is amongst the most lethal gastrointestinal tumors, which is the seventh leading reason of cancer-related mortality worldwide. Previous studies have indicated that circular RNAs (circRNAs), which is a new type of endogenous noncoding RNA (ncRNA), can mediate tumor progression in diverse tumor types including PC. Whereas precise roles regarding circRNAs and their underlying regulatory mechanisms in PC remain unknown. METHODS: In the current study, we employed next generation sequencing (NGS) to characterize abnormally expressed circRNAs among PC tissues. Next, we assessed expression levels of one identified circRNA, circ-STK39, in PC cell lines and tissues. Then, using bioinformatics analysis, luciferase reporter, Transwell migration, EdU and CCK-8 assays, we examined the regulatory mechanisms and targets of circ-STK39. Finally, our group explored the circ-STK39 role in PC tumor growth and metastasis in vivo. RESULTS: Our team discovered that circ-STK39 expression increased in PC tissues and cells, suggesting that circ-STK39 may have a role in PC progression. Downregulation of circ-STK39 inhibited PC proliferation and migration. Bioinformatics and luciferase reporter outcomes demonstrated that TRAM2 and miR-140-3p were circ-STK39 downstream targets. TRAM2 overexpression reversed the miR-140-3p overexpression effects upon migration, proliferation and the epithelial-mesenchymal transition (EMT). CONCLUSION: In this regard, we showed that circ-STK39 downregulation led to decreased migration, proliferation and the EMT of PC via the miR-140-3p/TRAM2 axis.

miR-140-5p attenuates hepatic fibrosis by directly targeting TGFßR1.

OBJECTIVE: To explore the protective effect and related mechanism of miR-140-5p on liver fibrosis by interfering with TGF-ß/Smad signaling pathway. METHODS: Liver fibrosis mice models were established by intraperitoneal injection of CCL4. Hematoxylin and eosin (HE) staining was used to detect the structural and morphological changes of the liver. Masson staining was used to detect collagen deposition. Human hepatic stellate cells (HSCs, LX-2) were transfected with miR-140-5p mimic or inhibitor then treated with TGF-ß1. The qRT-PCR and Western blotting was used to detect the expression of related molecules. The luciferase reporter assay was used to identify the target of miR-140-5p. RESULTS: Our results indicated that miR-140-5p expression was downregulated in fibrotic liver tissues of model mice and LX-2 cells treated with TGF-ß1. The overexpression of miR-140-5p decreased the expression of collagen1(COL1) and α-smooth muscle actin(α-SMA), inhibited the phosphorylation of Smad-2/3 (pSmad-2/3) in LX-2 cells. Conversely, the knockdown of miR-140-5p upregulated COL1 and α-SMA expression, increased Smad-2/3 phosphorylation. A dual-luciferase reporter assay showed that TGFßR1 was a target gene of miR-140-5p. The overexpression of miR-140-5p suppressed TGFßR1 expression in LX-2 cells. Additionally, knockdown of TGFßR1 decreased the expression of COL1 and α-SMA. Conversely, the overexpression of TGFßR1 reversed the inhibitory effect of miR-140-5p upregulation on expression of COL1 and α-SMA. CONCLUSION: miR-140-5p bound to TGFßR1 mRNA 3'-untranslated region(3'UTR) and inhibited the expression of TGFßR1, pSmad-2/3, COL1 and α-SMA, thereby exerting a potential therapeutic effect on hepatic fibrosis.

Silencing circ_0080425 alleviates high-glucose-induced endothelial cell dysfunction in diabetic nephropathy by targeting miR-140-3p/FN1 axis.

BACKGROUND: Hsa_circ_0080425 (circ_0080425) is newly identified to correlate with the progression of diabetic nephropathy (DN). However, its role and mechanism in DN process is not very clear. METHODS: Cell counting kit-8 assay, flow cytometry, scratch wound assay, and western blotting were performed to measure endothelial cell dysfunction. Expression of circ_0080425, microRNA (miR)-140-3p and fibronectin 1 (FN1) were determined by quantitative real-time PCR and western blotting. The direct interaction was confirmed by dual-luciferase reporter assay. RESULTS: High-glucose (HG) treatment could induce inhibition of cell proliferation, cell cycle entrance and wound healing rate in human umbilical vein endothelial cells (HRGEC), and enhancement of apoptosis rate. Circ_0080425 expression was upregulated by HG, and exhausting circ_0080425 could attenuate HG-induced above effects in HRGEC. MiR-140-3p was sponged by circ_0080425, and its inhibitor reversed the regulation of circ_0080425 knockdown on HG-induced HRGEC injury. FN1 was targeted by miR-140-3p, and its overexpression also restored the inhibitory effect of miR-140-3p on HC-induced HRGEC injury. CONCLUSION: Circ_0080425 expression might contribute to HG-induced endothelial cell injury, and circ_0080425/miR-140-3p/FN1 axis was a potential therapeutic approach to interfere DN process.

Apigenin inhibits angiogenesis in retinal microvascular endothelial cells through regulating of the miR-140-5p/HDAC3-mediated PTEN/PI3K/AKT pathway.

BACKGROUND: Diabetic retinopathy (DR) is a common cause of visual impairment. Apigenin has been shown to have antiangiogenic effects in various diseases. Our study aimed to investigate the role of apigenin in DR and elucidate the underlying mechanism. METHODS: Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to establish a DR model. HRMECs were treated with apigenin. Then we knocked down or overexpressed miR-140-5p and HDAC3, and added PI3K/AKT inhibitor LY294002. The expression levels of miR-140-5p, HDAC3, and PTEN were measured using qRT-PCR. Western blot analysis was performed to assess the expression of HDAC3, PTEN, and PI3K/AKT pathway-related proteins. Finally, cell proliferation and migration were evaluated using MTT, wound-healing assay, and transwell assay, while angiogenesis was examined using the tube formation assay. RESULTS: HG treatment resulted in reduced miR-140-5p expression and overexpression of miR-140-5p suppressed proliferation, migration, and angiogenesis of the HG-induced HRMECs. Apigenin treatment significantly restored the decreased level of miR-140-5p caused by HG treatment and inhibited proliferation, migration, and angiogenesis of the HG-induced HRMECs by upregulating miR-140-5p. Moreover, miR-140-5p targeted HDAC3, and overexpression of miR-140-5p reversed the HG-inducted upregulation of HDAC3 expression. HDAC3 was found to bind to the promoter region of PTEN, inhibiting its expression. Knockdown of HDAC3 suppressed the PI3K/AKT pathway by elevating PTEN expression. Furthermore, apigenin inhibited angiogenesis in DR cell models through the regulating of the miR-140-5p/HDAC3-mediated PTEN/PI3K/AKT pathway. CONCLUSIONS: Apigenin effectively suppressed angiogenesis in HG-induced HRMECs by modulating the miR-140-5p/HDAC3-mediated PTEN/PI3K/AKT pathway. Our study may contribute to the development of novel therapeutic approaches and identification of potential targets for the treatment of DR.

Critical microRNAs and regulatory motifs in cleft palate identified by a conserved miRNA-TF-gene network approach in humans and mice.

Cleft palate (CP) is the second most common congenital birth defect. The etiology of CP is complicated, with involvement of various genetic and environmental factors. To investigate the gene regulatory mechanisms, we designed a powerful regulatory analytical approach to identify the conserved regulatory networks in humans and mice, from which we identified critical microRNAs (miRNAs), target genes and regulatory motifs (miRNA-TF-gene) related to CP. Using our manually curated genes and miRNAs with evidence in CP in humans and mice, we constructed miRNA and transcription factor (TF) co-regulation networks for both humans and mice. A consensus regulatory loop (miR17/miR20a-FOXE1-PDGFRA) and eight miRNAs (miR-140, miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-451a and miR-92a) were discovered in both humans and mice. The role of miR-140, which had the strongest association with CP, was investigated in both human and mouse palate cells. The overexpression of miR-140-5p, but not miR-140-3p, significantly inhibited cell proliferation. We further examined whether miR-140 overexpression could suppress the expression of its predicted target genes (BMP2, FGF9, PAX9 and PDGFRA). Our results indicated that miR-140-5p overexpression suppressed the expression of BMP2 and FGF9 in cultured human palate cells and Fgf9 and Pdgfra in cultured mouse palate cells. In summary, our conserved miRNA-TF-gene regulatory network approach is effective in detecting consensus miRNAs, motifs, and regulatory mechanisms in human and mouse CP.

CircZNF644 aggravates lipopolysaccharide-induced HK-2 cell impairment via the miR-140-5p/MLKL axis.

Circular RNAs (circRNAs) play vital roles in human diseases, including acute kidney injury (AKI). In this paper, we focused on the effect of circRNA zinc finger protein 644 (circZNF644) on AKI cell model progression. qRT-PCR was conducted for the levels of circZNF644, ZNF644, miR-140-5p and mixed lineage kinase domain like pseudokinase (MLKL). RNase R assay, actinomycin D assay and subcellular fraction analysis were conducted to analyze the features of circZNF644. CCK-8 assay and EdU assay were used to explore cell proliferation. Flow cytometry analysis was conducted to analyze cell cycle and cell apoptosis. Western blot assay was executed for protein levels. ELISA was performed for the levels of inflammatory cytokines. The relationships among circZNF644, miR-140-5p and MLKL were analyzed by dual-luciferase reporter assay and RIP assay. CircZNF644 was upregulated in LPS-stimulated HK-2 cells. LPS-mediated inhibitory effects on cell proliferation and cell cycle and promotional effects on apoptosis and inflammation were reversed by circZNF644 knockdown. CircZNF644 directly interacted with miR-140-5p and MLKL was the target gene of miR-140-5p. The impact of circZNF644 knockdown on HK-2 cell injury was relieved by miR-140-5p inhibition. Moreover, miR-140-5p enhancement alleviated LPS-triggered HK-2 cell damage, while MLKL elevation reversed the effect. CircZNF644 knockdown protected HK-2 cells from LPS-induced injury by altering miR-140-5p/MLKL pathway, suggesting that circZNF644 may be a hopeful therapeutic target for AKI.

Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes.

OBJECTIVE: MicroRNA 140 (miR-140) is a chondrocyte-specific endogenous gene regulator implicated in osteoarthritis (OA). As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes. METHOD: Primary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140, in combination with Cas9 endonuclease. Combinations of sgRNAs and single/double transfections were tested. Gene editing was measured by T7 endonuclease 1 (T7E1) assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140-/- mice. RESULTS: High efficiency gene editing of miR-140 (90-98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P < 0.01), we validated three previously identified ones (septin 2, bone morphogenetic protein 2 and fibroblast growth factor 2). Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium. CONCLUSION: We describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive miR-140 targets of disease relevance.

Fluid shear stress-induced down-regulation of microRNA-140-5p promotes osteoblast proliferation by targeting VEGFA via the ERK5 pathway.

PURPOSE: Fluid shear stress (FSS) plays a critical role in osteoblast proliferation. However, the role of miRNA in osteoblast proliferation induced by FSS and the possible molecular mechanisms remain to be defined. The aim of the present study was to investigate whether miR-140-5p regulates osteoblast proliferation under FSS and its molecular mechanism. MATERIALS AND METHODS: miR-140-5p expression was measured by qRT-PCR. Western blot was used to measure the expressions of P-ERK1/2, ERK1/2, P-ERK5 and ERK5. The levels of VEGFA, PCNA, CDK4 and Cyclin D1 were identified through qRT-PCR and western blot, respectively. Cell proliferation was detected by CCK-8 assay and EdU labeling assay. Dual-luciferase reporter assay was used to validate the target of miR-140-5p. RESULTS: miR-140-5p was significantly down-regulated when MC3T3-E1 cells were exposed to FSS. We then confirmed that up-regulation of miR-140-5p inhibited and down-regulation of miR-140-5p promoted osteoblast proliferation. In addition, FSS promotes osteoblast proliferation via down-regulating miR-140-5p. Luciferase reporter assay demonstrated that VEGFA is a direct target of miR-140-5p. Furthermore, transfection of mimic-140-5p inhibited the up-regulation of VEGFA protein level induced by FSS, suggesting that FSS regulates VEGFA protein expression via miR-140-5p. Further investigations demonstrated that VEGFA could promote osteoblast proliferation. Lastly, we demonstrated that miR-140-5p regulates osteoblast proliferation and ERK5 activation through VEGFA. CONCLUSIONS: Our study demonstrates that FSS-induced the down-regulation of miR-140-5p promotes osteoblast proliferation through activing VEGFA/ERK5 signaling pathway. These findings may provide a novel mechanism of FSS-induced osteoblast proliferation and offer a new avenue to further investigate osteogenesis induced by mechanical loading.

LncRNA CCAT1 Upregulates ATG5 to Enhance Autophagy and Promote Gastric Cancer Development by Absorbing miR-140-3p.

BACKGROUND: Long noncoding RNA colon cancer-associated transcript 1 (LncRNA CCAT1) is highly expressed in gastric cancer tissues and plays a role in autophagy. However, the underlying mechanism still needs to be further clarified. OBJECTIVE: To study the role of LncRNA CCAT1 in regulating autophagy of gastric cancer cells, analyze its downstream targets, and elucidate the mechanism. METHODS: qPCR detected the expression of LncRNA CCAT1 in gastric cancer cells. The proliferation, migration, and invasion ability of LncRNA CCAT1 and the expression level of autophagy-related proteins in gastric cancer cells were detected. Bioinformatics method predicted the downstream targets of LncRNA CCAT1, and they were verified by dual-luciferase assay. The relationship between LncRNA CCAT1, miR-140, and ATG5 was verified by co-transfection, and the expression levels of ATG5 and ATG5-ATG12 complex proteins were detected. Finally, the role of LncRNA CCAT1 in vivo was confirmed by gastric cancer transplantation model. RESULTS: LncRNA CCAT1 was highly expressed in gastric cancer cells. LncRNA CCAT1 can promote the proliferation, migration, invasion, and autophagy activity of gastric cancer cells. LncRNA CCAT1 can bind to miR-140-3p and regulate its expression, while miR-140-3p further regulates the expression of ATG5. Overexpression of LncRNA CCAT1 can promote tumor growth in nude mice. After LncRNA CCAT1 silencing, the positive expression rate of ATG5 in nude mice was low. CONCLUSION: LncRNA CCAT1 may inhibit the expression of miR-140-3p by sponge adsorption, thus weakening its inhibitory effect on ATG5. Eventually, gastric cancer cells were more prone to autophagy under the pressure of stress.

Exosomal miR-140-5p inhibits osteogenesis by targeting IGF1R and regulating the mTOR pathway in ossification of the posterior longitudinal ligament.

BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) is a disabling disease whose pathogenesis is still unclear, and there are no effective cures or prevention methods. Exosomal miRNA plays an important role in the osteogenesis of ectopic bone. Therefore, we focused on the downregulation of miR-140-5p in OPLL cell-derived exosomes to explore the mechanism by which exosomal miR-140-5p inhibits osteogenesis in OPLL. RESULTS: Exosomes were isolated by differential centrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis, and exosomal markers. Exosomal RNA was extracted to perform miRNA sequencing and disclose the differentially expressed miRNAs, among which miR-140-5p was significantly downregulated. Confocal microscopy was used to trace the exosomal miR-140-5p delivered from OPLL cells to human mesenchymal stem cells (hMSCs). In vitro, we verified that exosomal miR-140-5p inhibited the osteoblast differentiation of hMSCs by targeting IGF1R and suppressing the phosphorylation of the IRS1/PI3K/Akt/mTOR pathway. In vivo, we verified that exosomal miR-140-5p inhibited ectopic bone formation in mice as assessed by micro-CT and immunohistochemistry. CONCLUSIONS: We found that exosomal miR-140-5p could inhibit the osteogenic differentiation of hMSCs by targeting IGF1R and regulating the mTOR pathway, prompting a further potential means of drug treatment and a possible target for molecular therapy of OPLL.