scholarly journals Investigation of the role of the miR17-92 cluster in BMP9-induced osteoblast lineage commitment

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Yunyuan Zhang ◽  
Xuran Jing ◽  
Zhongzhu Li ◽  
Qingwu Tian ◽  
Qing Wang ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Morphogenetic Protein ◽  
Bone Morphogenetic Protein 9 ◽  
Reporter Assays ◽  
Direct Target

Abstract Background Bone morphogenetic protein 9 (BMP9) has been identified as a crucial inducer of osteoblastic differentiation in mesenchymal stem cells (MSCs). Although microRNAs (miRNAs) are known to play a role in MSC osteogenesis, the mechanisms of action of miRNAs in BMP9-induced osteoblastic differentiation remain poorly understood. Methods In this study, we investigate the possible role of the miR17-92 cluster in the BMP9-induced osteogenic differentiation of MSCs by using both in vitro and in vivo bone formation assays. Results The results show that miR-17, a member of the miR17-92 cluster, significantly impairs BMP9-induced osteogenic differentiation. This impairment is effectively rescued by a miR-17 sponge, an antagomiR sequence against miR-17. Using TargetScan and the 3′-untranslated region luciferase reporter assays, we show that the direct target of miR-17 is the retinoblastoma gene (RB1), a gene that is pivotal to osteoblastic differentiation. We also confirm that RB1 is essential for the miR-17 effects on osteogenesis. Conclusion Our results indicate that miR-17 expression impairs normal osteogenesis by downregulating RB1 expression and significantly inhibiting the function of BMP9.

scholarly journals Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids

2021 ◽  
Vol 22 (11) ◽  
pp. 5590
Author(s):  
Clément Veys ◽  
Abderrahim Benmoussa ◽  
Romain Contentin ◽  
Amandine Duchemin ◽  
Emilie Brotin ◽  
...  
Keyword(s):  
Luciferase Reporter ◽  
Functional Screening ◽  
Western Blots ◽  
Egfr Expression ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis ◽  
First Time

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.

scholarly journals Let-7a regulates EV secretion and mitochondrial oxidative phosphorylation by targeting SNAP23 in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
You Dong Liu ◽  
Xiao Peng Zhuang ◽  
Dong Lan Cai ◽  
Can Cao ◽  
Qi Sheng Gu ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Mitochondrial Oxidative Phosphorylation ◽  
In Vivo Assays ◽  
Reporter Assays ◽  
Extracellular Mirna

Abstract Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

scholarly journals MicroRNA-29c Increases the Chemosensitivity of Pancreatic Cancer Cells by Inhibiting USP22 Mediated Autophagy

2018 ◽  
Vol 47 (2) ◽  
pp. 747-758 ◽  
Author(s):  
Limin Huang ◽  
Chaoquan Hu ◽  
Hui Cao ◽  
Xiaoliang Wu ◽  
Rongpin Wang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
Annexin V ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis

Background/Aims: Pancreatic cancer (PC) is an aggressive malignancy with a poor survival rate. Despite advances in the treatment of PC, the efficacy of therapy is limited by the development of chemoresistance. Here, we examined the role of microRNA-29c (miR-29c) and the involvement of autophagy and apoptosis in the chemoresistance of PC cells in vivo and in vitro. Methods: We employed qRT-PCR, western blot and immunofluorescence to examine the expression level of miR-29c, USP22 and autophagy relative protein. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Luciferase reporter assays confirmed the relationship between USP22 and miR-29c. Results: miR-29c overexpression in the PC cell line PANC-1 enhanced the effect of gemcitabine on decreasing cell viability and inducing apoptosis and inhibited autophagy, as shown by western blotting, immunofluorescence staining, colony formation assays, and flow cytometry. Ubiquitin specific peptidase (USP)-22, a deubiquitinating enzyme known to induce autophagy and promote PC cell survival, was identified as a direct target of miR-29c. USP22 knockdown experiments indicated that USP22 suppresses gemcitabine-induced apoptosis by promoting autophagy, thereby increasing the chemoresistance of PC cells. Luciferase reporter assays confirmed that USP22 is a direct target of miR-29c. A xenograft mouse model demonstrated that miR-29c increases the chemosensitivity of PC in vivo by downregulating USP22, leading to the inhibition of autophagy and induction of apoptosis. Conclusions: Taken together, these findings reveal a potential mechanism underlying the chemoresistance of PC cells mediated by the regulation of USP22-mediated autophagy by miR-29c, suggesting potential targets and therapeutic strategies in PC.

scholarly journals PAX8-AS1 knockdown facilitates cell growth and inactivates autophagy in osteoblasts via the miR-1252-5p/GNB1 axis in osteoporosis

2021 ◽  
Author(s):  
Caiqiang Huang ◽  
Runguang Li ◽  
Changsheng Yang ◽  
Rui Ding ◽  
Qingchu Li ◽  
...  
Keyword(s):  
Antisense Rna ◽  
Bioinformatic Analysis ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Protein Subunit ◽  
Reporter Assays

AbstractOsteoporosis (OP) is the most common systematic bone disorder among elderly individuals worldwide. Long noncoding RNAs (lncRNAs) are involved in biological processes in various human diseases. It has been previously revealed that PAX8 antisense RNA 1 (PAX8-AS1) is upregulated in OP. However, its molecular mechanism in OP remains unclear. Therefore, we specifically designed this study to determine the specific role of PAX8-AS1 in OP. We first established a rat model of OP and then detected PAX8-AS1 expression in the rats with RT-qPCR. Next, to explore the biological function of PAX8-AS1 in osteoblasts, in vitro experiments, such as Cell Counting Kit-8 (CCK-8) assays, flow cytometry, western blotting and immunofluorescence (IF) staining, were conducted. Subsequently, we performed bioinformatic analysis and luciferase reporter assays to predict and identify the relationships between microRNA 1252-5p (miR-1252-5p) and both PAX8-AS1 and G protein subunit beta 1 (GNB1). Additionally, rescue assays in osteoblasts clarified the regulatory network of the PAX8-AS1/miR-1252-5p/GNB1 axis. Finally, in vivo loss-of-function studies verified the role of PAX8-AS1 in OP progression. The results illustrated that PAX8-AS1 was upregulated in the proximal tibia of OP rats. PAX8-AS1 silencing promoted the viability and inhibited the apoptosis and autophagy of osteoblasts. PAX8-AS1 interacted with miR-1252-5p. GNB1 was negatively regulated by miR-1252-5p. In addition, the impacts of PAX8-AS1 knockdown on osteoblasts were counteracted by GNB1 overexpression. PAX8-AS1 depletion suppressed OP progression by inhibiting apoptosis and autophagy in osteoblasts. In summary, PAX8-AS1 suppressed the viability and activated the autophagy of osteoblasts via the miR-1252-5p/GNB1 axis in OP.

scholarly journals Knockdown of lncRNA LINC00958 inhibits the proliferation and migration of NSCLC cells by miR-204-3p/KIF2A axis

2020 ◽  
Author(s):  
Guan-Bin Qi ◽  
Lei Li
Keyword(s):  
Cell Proliferation ◽  
Tumor Growth ◽  
Luciferase Reporter ◽  
Cell Proliferation And Migration ◽  
Reporter Assays ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background: LINC00958, a newly identified lncRNA, has been reported to be closely linked to tumorigenesis in multiple cancers. However, its specific role in non-small cell lung cancer (NSCLC) remains unclear.Methods: The expression of LINC00958 was determined by RT-qPCR analysis. Cell proliferation and migration were evaluated by CCK-8 and transwell assays, respectively. Xenograft tumor models were established to examine the effect of LINC00958 on tumor growth in vivo. Luciferase reporter assays were performed to determine the interaction between LINC00958 and miR-204-3p and the interaction between miR-204-3p and KIF2A.Results: We found that LINC00958 was up-regulated in NSCLC tissues and cell lines. Down-regulation of LINC00958 inhibited cell proliferation and migration in vitro and suppressed tumor growth in vivo. Mechanically, we revealed that LINC00958 influenced NSCLC progression partly by sponging miR-204-3p and regulating KIF2A expression.Conclusions: Our study provided new insights into the role of LINC00958 as a promising prognostic biomarker and a therapeutic target for NSCLC.

scholarly journals RETRACTED ARTICLE: Inhibition of miR-296-5p protects the heart from cardiac hypertrophy by targeting CACNG6

Gene Therapy ◽  
2019 ◽  
Author(s):  
Wei Wang ◽  
Nian Liu ◽  
Li Xin ◽  
Yanfei Ruan ◽  
Xin Du ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Hypertrophic Growth ◽  
Retracted Article ◽  
Reporter Assays ◽  
Clinical Potential

AbstractHeart often undergoes mal-remodeling and hypertrophic growth in response to pathological stress. MiRNAs can regulate the cardiac function and participate in the regulation of cardiac hypertrophy. The present study aims at identifying the role of miR-296-5p in cardiac hypertrophy and further the underlying mechanism in hypertrophic cascades. Mice with cardiac hypertrophy were established by transverse aortic constriction (TAC). Cardiac hypertrophy in cardiomyocytes was induced by angiotensin II. Expression of miR-296-5p and its target gene CACNG6 was examined in cardiomyocytes transfected by miRNA. The expression of miR-296-5p was upregulated in mice with TAC surgery. The inhibition of miR-296-5p attenuated cardiac hypertrophy both in vitro and in vivo. And dual-luciferase reporter assays showed CACNG6 was the direct target of miR-296-5p, which modulated the expression of calcium signaling. MiR-296-5p was found to aggravate cardiac hypertrophy by targeting CACNG6, which suggests inhibition of miR-296-5p might have clinical potential to suppress cardiac hypertrophy and heart failure.

scholarly journals miR-129-5p Promotes Osteogenic Differentiation of BMSCs and Bone Regeneration via Repressing Dkk3

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Changming Zhao ◽  
Yulin Gu ◽  
Yan Wang ◽  
Qiaozhen Qin ◽  
Ting Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Bone Regeneration ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Alizarin Red ◽  
Gene Assay

Objective. Accumulating evidence indicates that microRNAs (miRNAs) play crucial roles in osteogenic differentiation. However, the associated mechanisms remain elusive. This paper is aimed at exploring the role of miR-129-5p in regulating bone marrow mesenchymal stem cell (BMSC) differentiation and bone regeneration in vivo and in vitro. Methods. BMSCs were transduced by miR-129-5p mimic, miR-129-5p inhibitor, and negative control lentivirus. The ability of BMSC differentiation to osteoblast was tested by alkaline phosphatase (ALP) and alizarin red staining (ARS). The expression of osteogenic genes (Runx2, Bmp2, and OCN) was examined via quantitative RT-PCR and western blot. A mouse model of calvaria defect was investigated by Micro-CT, immunohistochemistry, and histological examination. The luciferase reporter gene assay was performed to confirm the binding between Dkk3 and miR-129-5p. For the transfection experiments, lipofectamine 3000 was used to transfect pcDNA-Dkk3 into BMSCs to overexpress Dkk3. Coimmunoprecipitation and immunofluorescent localization assay were included for exploring the role of Dkk3 and β-catenin. Results. miR-129-5p was induced in BMSCs and MSC cell line C3H10T1/2 cells under osteogenic medium. Overexpression of miR-129-5p significantly promoted osteogenic differentiation of BMSCs in vitro. Moreover, BMSCs transduced with miR-129-5p mimic exhibited better bone regeneration compared with BMSCs transduced with control counterpart in vivo. Luciferase and western blot data showed that Dickkopf3 (Dkk3) is a target gene of miR-129-5p and the expression of Dkk3 was inhibited in BMSCs transduced with miR-129-5p mimic but enhanced in BMSCs transduced with miR-129-5p inhibitor. In addition, Dkk3 interacted with β-catenin directly. Conclusions. miR-129-5p promotes osteogenic differentiation of BMSCs and bone regeneration, and miR-129-5p/Dkk3 axis may be new potential targets for the treatment of bone defect and bone loss.

scholarly journals Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zizhen Si ◽  
Lei Yu ◽  
Haoyu Jing ◽  
Lun Wu ◽  
Xidi Wang
Keyword(s):  
Colorectal Cancer ◽  
Rna Binding ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Cancer Proliferation ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

scholarly journals KLF7/VPS35 axis contributes to hepatocellular carcinoma progression through CCDC85C-activated β-catenin pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yarong Guo ◽  
Bao Chai ◽  
Junmei Jia ◽  
Mudan Yang ◽  
Yanjun Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Luciferase Reporter ◽  
Aberrant Expression ◽  
Proliferation And Invasion ◽  
Hcc Cells

Abstract Objective Dysregulation of KLF7 participates in the development of various cancers, but it is unclear whether there is a link between HCC and aberrant expression of KLF7. The aim of this study was to investigate the role of KLF7 in proliferation and migration of hepatocellular carcinoma (HCC) cells. Methods CCK8, colony growth, transwell, cell cycle analysis and apoptosis detection were performed to explore the effect of KLF7, VPS35 and Ccdc85c on cell function in vitro. Xenografted tumor growth was used to assess in vivo role of KLF7. Chip-qPCR and luciferase reporter assays were applied to check whether KLF7 regulated VPS35 at transcriptional manner. Co-IP assay was performed to detect the interaction between VPS35 and Ccdc85c. Immunohistochemical staining and qRT-PCR analysis were performed in human HCC sampels to study the clinical significance of KLF7, VPS35 and β-catenin. Results Firstly, KLF7 was highly expressed in human HCC samples and correlated with patients’ differentiation and metastasis status. KLF7 overexpression contributed to cell proliferation and invasion of HCC cells in vitro and in vivo. KLF7 transcriptional activation of VPS35 was necessary for HCC tumor growth and metastasis. Further, co-IP studies revealed that VPS35 could interact with Ccdc85c in HCC cells. Rescue assay confirmed that overexpression of VPS35 and knockdown of Ccdc85c abolished the VPS35-medicated promotion effect on cell proliferation and invasion. Finally, KLF7/VPS35 axis regulated Ccdc85c, which involved in activation of β-catenin signaling pathway, confirmed using β-catenin inhibitor, GK974. Functional studies suggested that downregulation of Ccdc85c partly reversed the capacity of cell proliferation and invasion in HCC cells, which was regulated by VPS35 upregulation. Lastly, there was a positive correlation among KLF7, VPS35 and active-β-catenin in human HCC patients. Conclusion We demonstrated that KLF7/VPS35 axis promoted HCC cell progression by activating Ccdc85c-medicated β-catenin pathway. Targeting this signal axis might be a potential treatment strategy for HCC.

Sign in / Sign up

Export Citation Format

Share Document