LncRNA MALAT1: A potential therapeutic target in DSSinduced ulcerative colitis progression in vitro

Purpose: Ulcerative colitis is a severe disease affecting human health worldwide. Studies have shown that lncRNA MALAT1 has a significant correlation with breast, pancreatic, colon and liver cancers, but its effects on colitis is yet to be discovered. In this study, the potential role of lncRNA MALAT1 and the underlying molecular mechanism in DSS-induced colitis were investigated in vitro.Methods: Colorectal mucosal cell line FHC was induced with dextran sulphate sodium (DSS) to form an in vitro colitis model. Transfection procedure was employed to up- or down-regulate the expressions of lncRNA MALAT1 or miR-30c-5p in FHC cells. Cell viabilities were detected by CCK-8 assay. RT-qPCR was applied for evaluating gene expressions in normal FHC and DSS-induced FHC cell lines, while protein expression levels of target genes were examined by Western blot analysis. Starbase was used to predict the molecular interaction between MALAT1 and miR-30c-5p, while luciferase reporter assay was utilized to verify the binding sites between the two genes.Results: Expression of MALAT1 in the DSS-induced FHC cells was high with low cell viabilities, compared to the normal FHC cells. In the DSS-induced colitis-like FHC cells, overexpression of MALAT1 inhibited cell viabilities, while its downregulation promoted it. MiR-30c-5p directly targets MALAT1 and inhibited its expression in DSS-treated FHC cells. Upregulation of miR-30c-5p increased cell viabilities. Bcl-xL expression was inhibited by the up-regulation of MALAT1, while that of Bax was enhanced and the mimics of miR-30c-5p reversed these observations, suggesting that the enhancement of apoptosis promoted by oe-MALAT1 could be inhibited by miR-30c-5p. The interaction between MALAT1 and miR-30c-5p regulated NF-κB/TGF-β/Wnt-β-catenin signaling pathway.Conclusion: Overexpression of MALAT1 led to inhibition of cell viability, while apoptosis and inflammation were promoted by targeting miR-30c-5p via NF-κB/TGF-β/Wnt-β-catenin signaling pathway. These findings suggest MALAT1 as a therapeutic target for treating colitis. Keywords: Colitis, MALAT1, miR-30c-5p, NF-κB/TGF-β/Wnt-β-catenin

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LncRNA MALAT1 Regulates iPSC-derived Β-cell Differentiation by Targeting the miR-15b-5p/Ihh Axis

10.21203/rs.3.rs-89983/v1 ◽

2020 ◽

Author(s):

Yao Wang ◽

Xinxing Lin ◽

Jin Lv ◽

Jiachen Zhu ◽

Haowen Fan ◽

...

Keyword(s):

Cell Differentiation ◽

Cell Therapy ◽

Target Genes ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Β Cells ◽

Pancreatic Β Cells ◽

Lncrna Malat1

Abstract Background: iPSCs-derived β-like cell differentiation provides a novel strategy for type 1 diabetes treatment. Clarifying the regulatory mechanisms of lncRNAs in β-like cells derived from induced pluripotent stem cells (iPSCs) is not only significant for understanding the development of pancreas or pancreatic β cells, but also helpful for improving the quality of β-like cells for stem cell therapy.Methods: β-like cells derived from iPSCs followed a three-step protocol. RNA-sequencing was carried out to screen the differentially expressed lncRNAs which was probably involved in the differentiation of pancreatic β cells. Bioinformatics was performed to analyze the putative target genes of significantly differentially expressed lncRNAs. LncRNA Malat1 was chosen for further research. Lentivirus victor, siRNA victor, antagomir victor and mimic victor were constructed for overexpression of lncRNA Malat1, knockdown of lncRNA Malat1, knockdown of miR-15b-5p and overexpression of miR-15b-5p respectively. Quantitative Real-Time PCR (qRT-PCR), Western Blot and Immunofluorescence (IF) staining were carried out to detect the functions of pancreatic β cells at mRNA and protein level separately. Cytoplasmic and nuclear RNA fractionation and Fluorescence in situ hybridization (FISH) were to ventilate the subcellar location of lncRNA Malat1 in β-like cells. Flow cytometry and ELISA were performed to examine differentiation efficiency and function of insulin secretion from β-like cells after being stimulated with different concentrations of glucose. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p and Ihh were detected by Dual luciferase reporter assay (LRA).Results: We found that expression of lncRNA Malat1 was on the decline during the differentiation and overexpression of this lncRNA obviously impaired the differentiation and maturation of β-like cells derived from iPSCs in vitro and in vivo. Localized to the cytoplasm, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according the bioinformatic prediction, mechanistic analysis and downstream experiments.Conclusion: This study built an unreported regulatory network of lncRNA Malat1 and miR-15b-5p/Ihh axis during differentiation of iPSCs into β-like cells. Except for acting as a proverbial oncogene promoting tumorigenesis, lncRNA Malat1 may provide effective and novel molecule for diabetes cell therapy in the future.

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LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

10.21203/rs.3.rs-89983/v2 ◽

2021 ◽

Author(s):

Yao Wang ◽

Xinxing Lin ◽

Jin Lv ◽

Jiachen Zhu ◽

Haowen Fan ◽

...

Keyword(s):

Stem Cell ◽

Target Genes ◽

Induced Pluripotent Stem Cell ◽

Luciferase Reporter ◽

Β Cells ◽

Pancreatic Β Cells ◽

Protein Levels ◽

Lncrna Malat1

Abstract Background: Differentiation of induced pluripotent stem cell (iPSC)-derived β-like cells is a novel strategy for treatment of type 1 diabetes. Elucidation of the regulatory mechanisms of long noncoding RNAs (lncRNAs) in β-like cells derived from iPSCs is important for understanding the development of the pancreas and pancreatic β-cells and may improve the quality of β-like cells for stem cell therapy.Methods: β-like cells were derived from iPSCs in a three-step protocol. RNA sequencing and bioinformatics analysis were carried out to screen the differentially expressed lncRNAs and identify the putative target genes separately. LncRNA Malat1 was chosen for further research. Series of loss and gain of functions experiments were performed to study the biological function of this lncRNA. Quantitative real-time PCR (qRT-PCR), Western blot analysis and immunofluorescence (IF) staining were carried out to separately detect the functions of pancreatic β-cells at the mRNA and protein levels. Cytoplasmic and nuclear RNA fractionation and fluorescence in situ hybridization (FISH) were used to determine the subcellar location of lncRNA Malat1 in β-like cells. Flow cytometry and enzyme-linked immunosorbent assays (ELISAs) were performed to examine the differentiation and insulin secretion of β-like cells after stimulation with different glucose concentrations. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p and Ihh were detected by dual luciferase reporter assays (LRAs).Results: We found that the expression of lncRNA Malat1 declined during differentiation, and overexpression of this lncRNA notably impaired the differentiation and maturation of β-like cells derived from iPSCs in vitro and in vivo. Localized to the cytoplasm, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according to bioinformatics prediction, mechanistic analysis and downstream experiments.Conclusion: This study established an unreported regulatory network of lncRNA Malat1 and the miR-15b-5p/Ihh axis during the differentiation of iPSCs into β-like cells. In addition to acting as an oncogene promoting tumorigenesis, lncRNA Malat1 may be an effective and novel target for treatment of diabetes in the future.

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Circular RNA circSDHC serves as a sponge for miR-127-3p to promote the proliferation and metastasis of renal cell carcinoma via the CDKN3/E2F1 axis

Molecular Cancer ◽

10.1186/s12943-021-01314-w ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Junjie Cen ◽

Yanping Liang ◽

Yong Huang ◽

Yihui Pan ◽

Guannan Shu ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Renal Cell ◽

Target Genes ◽

Expression Patterns ◽

Luciferase Reporter ◽

Circular Rnas ◽

Proliferation And Metastasis

Abstract Background There is increasing evidence that circular RNAs (circRNAs) have significant regulatory roles in cancer development and progression; however, the expression patterns and biological functions of circRNAs in renal cell carcinoma (RCC) remain largely elusive. Method Bioinformatics methods were applied to screen for circRNAs differentially expressed in RCC. Analysis of online circRNAs microarray datasets and our own patient cohort indicated that circSDHC (hsa_circ_0015004) had a potential oncogenic role in RCC. Subsequently, circSDHC expression was measured in RCC tissues and cell lines by qPCR assay, and the prognostic value of circSDHC evaluated. Further, a series of functional in vitro and in vivo experiments were conducted to assess the effects of circSDHC on RCC proliferation and metastasis. RNA pull-down assay, luciferase reporter and fluorescent in situ hybridization assays were used to confirm the interactions between circSDHC, miR-127-3p and its target genes. Results Clinically, high circSDHC expression was correlated with advanced TNM stage and poor survival in patients with RCC. Further, circSDHC promoted tumor cell proliferation and invasion, both in vivo and in vitro. Analysis of the mechanism underlying the effects of circSDHC in RCC demonstrated that it binds competitively to miR-127-3p and prevents its suppression of a downstream gene, CDKN3, and the E2F1 pathway, thereby leading to RCC malignant progression. Furthermore, knockdown of circSDHC caused decreased CDKN3 expression and E2F1 pathway inhibition, which could be rescued by treatment with an miR-127-3p inhibitor. Conclusion Our data indicates, for the first time, an essential role for the circSDHC/miR-127-3p/CDKN3/E2F1 axis in RCC progression. Thus, circSDHC has potential to be a new therapeutic target in patients with RCC.

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MiR-30a-5p Downregulation Induces Neuronal Autophagy by Activating AMPK After 2856MHz Microwave Radiation

10.21203/rs.3.rs-1063942/v1 ◽

2021 ◽

Author(s):

Yanhui Hao ◽

Wenchao Li ◽

Hui Wang ◽

Jing Zhang ◽

Haoyu Wang ◽

...

Keyword(s):

Microwave Radiation ◽

Hippocampal Neurons ◽

Target Genes ◽

Neuronal Damage ◽

Luciferase Reporter ◽

Potential Health ◽

Downstream Target ◽

Neuronal Autophagy

Abstract Background With the development of science and technology, microwaves are being widely used. More and more attention has been paid to the potential health hazards of microwave exposure. The regulation of miR-30a-5p (miR-30a) on autophagy is involved in the pathophysiological process of many diseases. Our previous study found that 30 mW/cm2 microwave radiation could reduce miR-30a expression and activate neuronal autophagy in rat hippocampus. However, the roles played by miR-30a in microwave-induced neuronal autophagy and related mechanisms remain largely unexplored. Results In the present study, we established neuronal damage models by exposing rat hippocampal neurons and rat adrenal pheochromocytoma (PC12) cell-derived neuron-like cells to 30 mW/cm2 microwave, which resulted in miR-30a downregulation and autophagy activation in vivo and in vitro. Bioinformatics analysis was conducted, and Beclin1, Prkaa2, Irs1, Pik3r2, Rras2, Ddit4, Gabarapl2 and autophagy-related gene 12 (Atg12) were identified as potential downstream target genes of miR-30a involved in regulating autophagy. Based on our previous findings that microwave radiation can cause a neuronal energy metabolism disorder, Prkaa2, encoding adenosine 5’-monophosphate-activated protein kinase α2 (AMPKα2, an important catalytic subunit of energy sensor AMPK), was selected for further analysis. Dual-luciferase reporter assay results showed that Prkaa2 is a downstream target gene of miR-30a. Microwave radiation increased the expression and phosphorylation (Thr172) of AMPKα both in vivo and in vitro. Moreover, the transduction of cells with miR-30a mimics suppressed AMPKα2 expression, inhibited AMPKα (Thr172) phosphorylation and reduced autophagy flux in neuron-like cells. Importantly, miR-30a mimics abolished microwave-activated autophagy and inhibited microwave-induced AMPKα (Thr172) phosphorylation. Conclusions AMPKα2 was a newly founded downstream gene of miR-30a involved in autophagy regulation, and miR-30a downregulation after microwave radiation could promote neuronal autophagy by increasing AMPKα2 expression and activating AMPK signaling.

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Abstract WMP80: Micro RNA-Therapy in Murine Thromboembolic Stroke

Stroke ◽

10.1161/str.48.suppl_1.wmp80 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Kumar Vaibhav ◽

Shannon Williams ◽

Sumbul Fatima ◽

Babak Baban ◽

Krishnan M Dhandapani ◽

...

Keyword(s):

Target Genes ◽

Infarct Volume ◽

Protein Translation ◽

Micro Rna ◽

Thromboembolic Stroke ◽

Gene Expressions ◽

Mirna Array ◽

Micro Rnas ◽

Pai 1

Background: Micro RNAs (miRNAs) could target multiple mRNAs, repressing the protein translation. We report acute changes in humoral miRNAome in a murine thromboembolic stroke model (eMCAo), and demonstrate the benefits of miRNA therapy in improving cerebral blood flow (CBF). Methods: Non-biased micro RNA (miRNA) array and bioinformatics analysis was performed in plasma collected at 4h post-eMCAo from male mice (C57/B6, 16-weeks). Individual PCR for miRNAs was also performed in brain tissues at 24h post-eMCAo. Moreover, frozen human plasma samples collected at ~4.5h post-stroke were also used for miRNA analysis. Finally, the miRNA mimic that was predicted to target genes of our interest was also tested in vivo and in vitro . Results: Principal component analysis (PCA) of the miRNA-array showed ~68% variance in the humoral miRNAome 4h after eMCAo in mice, and a significant change in Stroke vs. Sham groups (Cut off value >2 fold; p<0.05). Of interest, the hairpin precursor of miR-449b was downregulated (~2.35 fold, p<0.05) at 4h post-eMCAo, while the mature miR-449b was also significantly reduced at 24h post-eMCAo. Mature miR-449b was significantly reduced in human stroke plasma, too. In human brain endothelial cells, miR-449b mimic downregulated gene expressions of both plasminogen activator inhibitor (PAI-1) and alpha 2- antiplasmin (α-AP) only in hypoxia but not during normoxia. Therefore, we finally tested the cholesterol-conjugated miR-449b mimic in the murine eMCAo model. Pre-treatment with miR-449b mimic (8 mg/kg bwt) increased the absolute CBF and reduced edema (as determined by MRI), and also improved the neurological outcomes and reduced % infarct volume (p<0.05). Results: The miR-449b mimic could be a possible therapy to suppress aberrant gene expressions of PAI-1 and α-AP, which will allow more spontaneous reperfusion and benefits from low dose tPA.

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LukS-PV-Regulated MicroRNA-125a-3p Promotes THP-1 Macrophages Differentiation and Apoptosis by Down-Regulating NF1 and Bcl-2

Cellular Physiology and Biochemistry ◽

10.1159/000485415 ◽

2017 ◽

Vol 44 (3) ◽

pp. 1093-1105 ◽

Cited By ~ 8

Author(s):

Xiao-Xi Sun ◽

Shan-Shan Zhang ◽

Chun-Yang Dai ◽

Jing Peng ◽

Qing Pan ◽

...

Keyword(s):

Cell Differentiation ◽

Cellular Differentiation ◽

Target Genes ◽

B Cell Lymphoma ◽

Neurofibromatosis Type ◽

Luciferase Reporter ◽

Loss Of Function ◽

Leukaemia Cell Line ◽

Reporter Assays

Background/Aims: LukS-PV is a component of Panton-Valentine leukocidin (PVL). We have previously demonstrated that LukS-PV potently promoted differentiation and induced apoptosis in THP-1 cells. However, the precise mechanisms of these actions remain unknown. MicroRNAs (miRs) play important roles in cellular differentiation and apoptosis. This study aimed to investigate the role of miR-125a-3p in LukS-PV-regulated differentiation and apoptosis and its underlying mechanism in THP-1 cells. Methods: MicroRNA profiling analyses were conducted to determine differential miRNA expression levels in THP-1 cells treated with LukS-PV. Cell differentiation and apoptosis were measured in THP-1 cells by gain-of-function and loss-of-function experiments. Bioinformatics analysis and luciferase reporter assays were used to confirm the targets of miR-125a-3p. The effects of the miR-125a-3p targets on cellular differentiation were determined by knocking them down. Results: MiR-125a-3p was up-regulated after treating the human monocytic leukaemia cell line THP-1 with LukS-PV. In addition, miR-125a-3p positively regulated apoptosis and differentiation in THP-1 cells treated with LukS-PV. Concordantly, luciferase reporter assays confirmed that neurofibromatosis type 1 (NF1) and B-cell lymphoma 2 (Bcl-2) were direct target genes of miR-125a-3p. Moreover, NF1 knockdown in THP-1 cells significantly promoted differentiation in vitro. Finally, the extracellular signal-regulated kinase (ERK) pathway, a downstream target of NF1, was activated after NF1 knockdown. Conclusions: These findings confirm that miR-125a-3p is involved in LukS-PV-mediated cell differentiation and apoptosis in THP-1 cells.

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Decreased miR-4512 Levels in Monocytes and Macrophages of Individuals With Systemic Lupus Erythematosus Contribute to Innate Immune Activation and Neutrsophil NETosis by Targeting TLR4 and CXCL2

Frontiers in Immunology ◽

10.3389/fimmu.2021.756825 ◽

2021 ◽

Vol 12 ◽

Author(s):

Binbin Yang ◽

Xinwei Huang ◽

Shuangyan Xu ◽

Li Li ◽

Wei Wu ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Lupus Erythematosus ◽

Target Genes ◽

Mononuclear Cells ◽

Therapeutic Potential ◽

Luciferase Reporter ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear ◽

Monocytes And Macrophages

ObjectiveSystemic lupus erythematosus (SLE) is an autoimmune disease with complex etiology that is not yet entirely understood. We aimed to elucidate the mechanisms and therapeutic potential of microRNAs (miRNAs) in SLE in a Tibetan population.MethodsPeripheral blood mononuclear cells from SLE patients (n = 5) and healthy controls (n = 5) were used for miRNA–mRNA co-sequencing to detect miRNAs related to immune abnormalities associated with SLE. Luciferase reporter assay was used to identify potential targets of candidate miRNA. The target genes were verified in miRNA-agomir/antagomir transfection assays with multiple cells lines and by expression analysis. The effects of candidate miRNA on monocyte and macrophage activation were evaluated by multiple cytokine profiling. Neutrophil extracellular traps (NETs) formation was analyzed in vitro by cell stimulation with supernatants of monocytes and macrophages transfected with candidate miRNA. The rodent MRL/lpr lupus model was used to evaluate the therapeutic effect of CXCL2Ab on SLE and the regulation effect of immune disorders.ResultsIntegrated miRNA and mRNA expression profiling identified miRNA-4512 as a candidate miRNA involved in the regulation of neutrophil activation and chemokine-related pathways. MiR-4512 expression was significantly reduced in monocytes and macrophages from SLE patients. MiR-4512 suppressed the TLR4 pathway by targeting TLR4 and CXCL2. Decreased monocyte and macrophage miR-4512 levels led to the expression of multiple proinflammatory cytokines in vitro. Supernatants of miR-4512 antagomir-transfected monocytes and macrophages significantly promoted NETs formation (P < 0.05). Blocking of CXCL2 alleviated various pathogenic manifestations in MRL/lpr mice, including kidney damage and expression of immunological markers of SLE.ConclusionsWe here demonstrated the role of miR-4512 in innate immunity regulation in SLE. The effect of miR-4512 involves the regulation of monocytes, macrophages, and NETs formation by direct targeting of TLR4 and CXCL2, indicating the miR-4512-TLR4-CXCL2 axis as a potential novel therapeutic target in SLE.

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Interaction of c-Myb with p300 Is Required for the Induction of Acute Myeloid Leukemia by Human AML Oncogenes, and Represents a Potential Therapeutic Target.

Blood ◽

10.1182/blood.v120.21.2402.2402 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2402-2402

Author(s):

Diwakar R Pattabiraman ◽

Konstantin Shakhbazov ◽

Valerie Barbier ◽

Crystal McGirr ◽

Keerthana Krishnan ◽

...

Keyword(s):

Therapeutic Target ◽

Therapeutic Strategy ◽

Target Genes ◽

Conflicts Of Interest ◽

Therapeutic Index ◽

Therapeutic Window ◽

Small Peptides ◽

Transforming Activity ◽

Haemopoietic Cells

Abstract Abstract 2402 The MYB oncogene is widely expressed in acute leukaemias and is important for the continued proliferation of leukemia cells, raising the possibility that MYB may be a therapeutic target. However realization of this potential requires (i) a significant therapeutic window for MYB inhibition, given its essential role in normal haemopoiesis; and (ii) an approach for developing an effective therapeutic. We previously showed that the interaction of Myb with the coactivator CBP/p300 is essential for Myb's intrinsic transforming activity (1). Here we use haemopoietic cells from the Booreana mouse strain, which carries a mutation in Myb that prevents interaction with CBP/p300 (see Fig. 1 and ref. 2), to examine the requirement for this interaction in myeloid transformation and leukaemogenesis. Using this strain and a strain (plt6) carrying a “complementary” mutation in p300 (3), we show that the Myb-p300 interaction is essential for in vitro transformation by the myeloid leukaemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type (WT) mice, Booreana cells fail to induce leukaemia upon transplantation into irradiated recipients following transduction with an AML1-ETO9a retrovirus (Fig. 2). These data, as well as reinforcing the notion that MYB is an essential cofactor for myeloid leukaemogenesis, highlight disruption of the Myb-p300 interaction as a potential therapeutic strategy for AML. Furthermore, our results suggest that such a strategy would have a useable therapeutic index since Booreana mice, unlike Myb null mice, are viable. We have started to explore the use of small peptides to provide proof-of-principle for this approach. We have also begun to explore the molecular basis of the requirement for MYB, and the Myb-p300 interaction, in AML using gene expression profiling. This has highlighted several Myb target genes – identified by our previous ChIP-Seq analysis (4) - that have been previously implicated in myeloid leukaemogenesis, as being differentially expressed between WT and Booreana cells transduced with AML1-ETO9a. One striking example is the apparent Myb-p300 dependent activation of Ets2 and Gfi1, accompanied by repression of Gata2 and Gfi1b; this pattern has previously been reported in MLL-AF9 and MOZ-TIF-induced murine AML (5). Disclosures: No relevant conflicts of interest to declare.

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MicroRNA-34a Attenuates Paclitaxel Resistance in Prostate Cancer Cells via Direct Suppression of JAG1/Notch1 Axis

Cellular Physiology and Biochemistry ◽

10.1159/000494004 ◽

2018 ◽

Vol 50 (1) ◽

pp. 261-276 ◽

Cited By ~ 13

Author(s):

Xiaobing Liu ◽

Xing Luo ◽

Yuqi Wu ◽

Ding Xia ◽

Wei Chen ◽

...

Keyword(s):

Prostate Cancer ◽

Tumor Growth ◽

Western Blot ◽

Target Genes ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Qrt Pcr

Background/Aims: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically centered on paclitaxel-based chemotherapy. In this study, we aimed to evaluate whether miR-34a attenuates chemoresistance to paclitaxel by regulating target genes associated with drug resistance. Methods: We used data from The Cancer Genome Atlas to compare miR-34a expression levels in prostate cancer (PC) tissues with normal prostate tissues. The effects of miR-34a inhibition and overexpression on PC proliferation were evaluated in vitro via Cell Counting Kit-8 (CCK-8) proliferation, colony formation, apoptosis, and cell-cycle assays. A luciferase reporter assay was employed to identify the interactions between miR-34a and specific target genes. To determine the effects of up-regulation of miR-34a on tumor growth and chemo-resistance in vivo, we injected PC cells overexpressing miR-34a into nude mice subcutaneously and evaluated the rate of tumor growth during paclitaxel treatment. We examined changes in the expression levels of miR-34a target genes JAG1 and Notch1 and their downstream genes via miR-34a transfection by quantitative reverse transcription PCR (qRT-PCR) and western blot assay. Results: miR-34a served as an independent predictor of reduced patient survival. MiR-34a was down-regulated in PC-3PR cells compared with PC-3 cells. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to paclitaxel while miR-34a down-regulation resulted in chemoresistance to paclitaxel in vitro. A study of gain and loss in a series of functional assays revealed that PC cells expressing miR-34a were chemosensitive. Furthermore, the overexpression of miR-34a increased the sensitivity of PC-3PR cells to chemotherapy in vivo. The luciferase reporter assay confirmed that JAG1 and Notch1 were directly targeted by miR-34a. Interestingly, western blot analysis and qRT-PCR confirmed that miR-34a inhibited the Notch1 signaling pathway. We found that miR-34a increased the chemosensitivity of PC-3PR cells by directly repressing the TCF1/ LEF1 axis. Conclusion: Our results showed that miR-34a is involved in the development of chemosensitivity to paclitaxel. By regulating the JAG1/Notch1 axis, miR-34a or its target genes JAG1 or Notch1 might serve as potential predictive biomarkers of response to paclitaxel-based chemotherapy and/or therapeutic targets that will help to overcome chemoresistance at the mCRPC stage.

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Long Noncoding RNA TUG1 Promotes the Function in ox-LDL-Treated HA-VSMCs via miR-141-3p/ROR2 Axis

Cardiovascular Therapeutics ◽

10.1155/2020/6758934 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Yu Tang ◽

Jing Hu ◽

Zhiying Zhong ◽

Yanfeng Liu ◽

Yunxia Wang

Keyword(s):

Binding Sites ◽

Noncoding Rna ◽

Severe Disease ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Orphan Receptor ◽

Luciferase Reporter ◽

Ki 67 ◽

Positive Effects

Background. Atherosclerosis (AS) is a common severe disease around the world. The merging paper reported that long noncoding RNAs (lncRNAs) took part in diversified pathological processes of AS, although the mechanism remains unknown. This study is aimed at uncovering the profile of lncRNA taurine-upregulated gene 1 (TUG1), which has biological function, and potential mechanism in AS progression in vitro. Methods. Oxidized low-density lipoprotein (ox-LDL) was used for AS model construction in vitro. Levels of lncRNA TUG1, miR-141-3p, and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in AS tissues or in ox-LDL-treated vascular smooth muscle cells (HA-VSMCs). The biofunctional effects were examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and transwell assays. The expression of proliferation-related proteins (CyclinD1, Ki-67) and metastasis-associated proteins (β-catenin, Vimentin) and ROR2 in cells was determined by western blot analysis. The potential binding sites were predicted by starBase software online and confirmed by dual-luciferase reporter analysis. Results. The expression of TUG1 and ROR2 was promoted in AS tissues and ox-LDL-treated HA-VSMCs. While the low expression of miR-141-3p negatively correlated with that of TUG1 or ROR2 in AS tissues. Silencing of TUG1 inhibited the proliferation, migration, invasion, and metastasis in ox-LDL-treated HA-VSMCs. Moreover, the putative binding sites between miR-141-3p and TUG1 or ROR2 were predicted by starBase software online. Also, miR-141-3p deletion reversed the positive effects of TUG1 knockdown on cells. Besides, downregulation of miR-141-3p disrupted the biofunctional results from ROR2 silencing. Conclusion. TUG1 enhanced the progression of AS in vitro by regulating the miR-141-3p/ROR2 axis.

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