scholarly journals Suppression of CRLF1 promotes the chondrogenic differentiation of bone marrow-derived mesenchymal stem and protects cartilage tissue from damage in osteoarthritis via activation of miR-320

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Hao Xu ◽  
Changrong Ding ◽  
Cuicui Guo ◽  
Shuai Xiang ◽  
Yingzhen Wang ◽  
...  
Keyword(s):  
Chondrogenic Differentiation ◽  
Medial Meniscus ◽  
Joint Disease ◽  
Cartilage Tissue ◽  
Luciferase Reporter ◽  
Biological Functions ◽  
Over Expression ◽  
Primary Chondrocytes ◽  
Reporter Assays ◽  
Direct Target

Abstract Background Osteoarthritis (OA) is the most prevalent chronic joint disease, and is hard to be cured at present. Cytokine receptor-like factor 1 (CRLF1) has been identified as an upregulated gene in OA cartilage. However, the precise identities and functions of CRLF1 in OA progression have remained to be fully elucidated. Methods We used a murine model of injury-induced OA (destabilization of medial meniscus, DMM) and BMSCs to investigate the specific biological functions and mechanisms of CRLF1. Results We found that CRLF1 was significantly increased in the DMM surgery-induced OA model and was down-regulated during chondrogenic differentiation of BMSCs. Luciferase reporter assays showed that CRLF1 was a direct target of miR-320 in BMSCs. miR-320 can reverse the effect of CRLF1 on cell proliferation, apoptosis and chondrogenic differentiation of BMSCs. Furthermore, knockdown of CRLF1 or over-expression of miR-320 can inhibit the apoptosis of primary chondrocytes. Conclusion Suppression of CRLF1 promotes the chondrogenic differentiation of BMSCs and protects cartilage tissue from damage in osteoarthritis via activation of miR-320.

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 Hierarchical Action of Mulberry miR156 in the Vegetative Phase Transition

2021 ◽  
Vol 22 (11) ◽  
pp. 5550
Author(s):  
Hongshun Li ◽  
Yiwei Luo ◽  
Bi Ma ◽  
Jianqiong Hu ◽  
Zhiyuan Lv ◽  
...  
Keyword(s):  
Phase Transition ◽  
Arabidopsis Thaliana ◽  
Woody Plants ◽  
Luciferase Reporter ◽  
Vegetative Phase ◽  
Promoter Sequences ◽  
Over Expression ◽  
Degradome Sequencing ◽  
Pathway Expression ◽  
Reporter Assays

The vegetative phase transition is a prerequisite for flowering in angiosperm plants. Mulberry miR156 has been confirmed to be a crucial factor in the vegetative phase transition in Arabidopsis thaliana. The over-expression of miR156 in transgenic Populus × canadensis dramatically prolongs the juvenile phase. Here, we find that the expression of mno-miR156 decreases with age in all tissues in mulberry, which led us to study the hierarchical action of miR156 in mulberry. Utilizing degradome sequencing and dual-luciferase reporter assays, nine MnSPLs were shown to be directly regulated by miR156. The results of yeast one-hybrid and dual-luciferase reporter assays also revealed that six MnSPLs could recognize the promoter sequences of mno-miR172 and activate its expression. Our results demonstrate that mno-miR156 performs its role by repressing MnSPL/mno-miR172 pathway expression in mulberry. This work uncovered a miR156/SPLs/miR172 regulation pathway in the development of mulberry and fills a gap in our knowledge about the molecular mechanism of vegetative phase transition in perennial woody plants.

scholarly journals Oncoprotein LAMTOR5 activates GLUT1 via upregulating NF-κB in liver cancer

Open Medicine ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. 264-270 ◽  
Author(s):  
Jing Zhou ◽  
Yajun Li ◽  
Danhua Li ◽  
Zhi Liu ◽  
Jie Zhang
Keyword(s):  
Liver Cancer ◽  
Cancer Cells ◽  
Western Blotting ◽  
Glucose Transporter ◽  
Luciferase Reporter ◽  
Pcr Assay ◽  
Glucose Transporter 1 ◽  
Over Expression ◽  
Liver Cancer Cells ◽  
Reporter Assays

AbstractObjectiveAccumulating reports reveal that serving as an oncogenic factor LAMTOR5 is involved in the progression of many specific cancers. Glucose transporter 1 (GLUT1) is frequently identified in many cancers. However, it remains unexplored whether GLUT1 plays a role in LAMTOR5-enhanced liver cancer. Here, we aim to decipher the function of LAMTOR5 in the regulation of GLUT1 in liver cancer.MethodsThe effect of LAMTOR5 on GLUT1 was analyzed using Western blotting and RT-PCR assay. Dose-increased over-expression or silencing of LAMTOR5 was performed through transient transfection. LAMTOR5-activated GLUT1 promoter was revealed by luciferase reporter assay. The regulation of GLUT1 by LAMTOR5/NF-κB was examined via Western blotting and luciferase reporter assays.ResultsThe data showed that in liver cancer cells under the administration with dose-increased LAMTOR5, the level of mRNA and protein of GLUT1 was obviously raised. Our data revealed that the activities of GLUT1 promoter were induced by LAMTOR5. Then, we found that the elevation of GLUT 1 mediated by LAMTOR5 slowed when the inhibitor or siRNAs of NF-κB was introduced into the liver cancer cells. Conclusion. LAMTOR5 is responsible for the activation of GLUT1 via transcription factor NF-κB in liver cancer.

scholarly journals Bax Targeted by miR-29a Regulates Chondrocyte Apoptosis in Osteoarthritis

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Guiqiang Miao ◽  
Xuehui Zang ◽  
Huige Hou ◽  
Hui Sun ◽  
Lihui Wang ◽  
...  
Keyword(s):  
Cartilage Degeneration ◽  
Degenerative Joint Disease ◽  
Joint Disease ◽  
Luciferase Reporter ◽  
Chondrocyte Apoptosis ◽  
Reporter Assay ◽  
Proapoptotic Protein ◽  
Regulation Mechanisms ◽  
Direct Target

Osteoarthritis (OA) is a chronic degenerative joint disease, where chondrocyte apoptosis is responsible for cartilage degeneration. Bax is a well-known proapoptotic protein of the Bcl-2 family, involved in a large number of physiological and pathological processes. However, the regulation mechanisms of Bax underlying chondrocyte apoptosis in OA remain unknown. In the present study, we determined the role of Bax in human OA and chondrocyte apoptosis. The results showed that Bax was upregulated in chondrocytes from the articular cartilage of OA patients and in cultured chondrocyte-like ATDC5 cells treated by IL-1β. Bax was identified to be the direct target of miR-29a by luciferase reporter assay and by western blotting. Inhibition of miR-29a by the mimics protested and overexpression by miR-29a inhibitors aggravated ATDC5 apoptosis induced by IL-1β. These data reveal that miR-29a/Bax axis plays an important role in regulating chondrocyte apoptosis and suggest that targeting the proapoptotic protein Bax and increasing expression levels of miR-29a emerge as potential approach for protection against the development of OA.

scholarly journals CRP Stimulates GDF15 Expression in Endothelial Cells through p53

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Yoonseo Kim ◽  
Nicole Noren Hooten ◽  
Michele K. Evans
Keyword(s):  
Endothelial Cells ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Aortic Endothelial Cells ◽  
Reactive Protein ◽  
Direct Target Gene ◽  
Reporter Assays ◽  
Direct Target ◽  
Human Aortic Endothelial Cells

Growth differentiation factor 15 (GDF15) is a multifunctional, secreted protein that is a direct target gene of p53. GDF15 is a prospective biomarker of cardiovascular disease (CVD). C-reactive protein (CRP), like GDF15, is implicated in inflammation and an independent biomarker of CVD. However, the molecular interactions between GDF15 and CRP remain unexplored. In women, we found a significant relationship between hsCRP and GDF15 serum and mRNA levels. In vitro treatment of cultured human aortic endothelial cells (HAECs) with purified CRP or transfection of a CRP plasmid into HAECs induced GDF15 expression. Dual-luciferase reporter assays confirmed that CRP significantly increased the levels of GDF15 promoter luciferase activity, indicating that CRP induces GDF15 transcription. Chromatin immunoprecipitation (ChIP) assays confirmed that p53 was recruited to both p53 binding sites 1 and 2 in the GDF15 promoter in response to CRP. We have uncovered a linkage between CRP and GDF15, a new clue that could be important in the pathogenesis of endothelial inflammation.

scholarly journals miR-20a regulates sensitivity of colorectal cancer cells to NK cells by targeting MICA

2019 ◽  
Vol 39 (7) ◽  
Author(s):  
Siwen Tang ◽  
Hongyu Fu ◽  
Qihua Xu ◽  
Ying Zhou
Keyword(s):  
Colorectal Cancer ◽  
Nk Cells ◽  
Luciferase Reporter ◽  
Normal Tissues ◽  
Histocompatibility Complex ◽  
Colorectal Cancer Cells ◽  
Reporter Assays ◽  
Direct Target ◽  
Causes Of Deaths

Abstract Colorectal cancer (CRC) is one of the leading cancer-related causes of deaths in the world. Recently, microRNAs have been reported to regulate the tumor growth, invasion and the immunosuppression. In the present study, we found that miR-20a was increased in human CRC specimens compared with the healthy normal tissues. However, miR-20a overexpression and knockdown did not impair the CRC cell growth in vitro. Our results indicated that CD107a+ NK cells are increased in CRC group. Furthermore, cytotoxicity assays demonstrated that miR-20a knockdown promoted the CRC cells sensitive to NK cells, whereas miR-20a overexpression showed the opposite results. Our results suggest that the regulation of NK cells by miR-20a depends on NKG2D. Luciferase reporter assays revealed that the NKG2D ligand Major Histocompatibility Complex (MHC) class I-related chain genes A (MICA) is the direct target of miR-20a. Flow cytometry showed the MICA protein level is significantly reduced in miR-20a-overexpressing CRC cells and increased in miR-20a knockdown CRC cells. Taken together, our results suggest that miR-20a regulates sensitivity of CRC cells to NK cells by targeting MICA.

scholarly journals Aip regulates cAMP signalling and GH secretion in GH3 cells

2013 ◽  
Vol 20 (4) ◽  
pp. 495-505 ◽  
Author(s):  
R Formosa ◽  
A Xuereb-Anastasi ◽  
J Vassallo
Keyword(s):  
Gh3 Cells ◽  
Luciferase Reporter ◽  
Interacting Protein ◽  
Over Expression ◽  
Knock Down ◽  
Camp Signalling ◽  
Gh Secretion ◽  
Aryl Hydrocarbon ◽  
Reporter Assays ◽  
Aip Gene

Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been linked to predisposition to pituitary adenomas. However, the mechanism by which this occurs remains unknown. AIP interacts with a number of interesting proteins, including members of the cAMP signalling pathway that has been shown to be consistently altered in pituitary tumours. The functional role of Aip was investigated using both over-expression and knock down of Aip in GH3 cells. cAMP signalling and its downstream effectors, including GH secretion, were then investigated. cAMP signalling was analysed using cAMP assays, cAMP-response element-promoter luciferase reporter assays, real-time PCR and finally secreted GH quantification. Over-expression of wild-type (WT)-Aip reduced forskolin-induced cAMP signalling at the total cAMP level, luciferase reporter activity and target gene expression, when compared with empty vector and the non-functional R304X mutant. Additionally, GH secretion was reduced in WT-Aip over-expressing GH3 cells treated with forskolin. Knock down of endogenous Aip resulted in increased cAMP signalling but a decrease in GH secretion was also noted. Inhibition of phosphodiesterase activity using general and selective inhibitors did not completely ablate the effect of Aip on forskolin-augmented cAMP signalling. A mechanism by which Aip acts as a tumour suppressor, by maintaining a low cAMP signalling and concentration, is suggested. Mutations of Aip render the protein incapable of such activity. This effect appears not to be mediated by the AIP–PDE interaction, suggesting the involvement of other interacting partners in mediating this outcome.

scholarly journals miR-4319 Reduces Tumorigenicity of Cervical Cancer Cells by Targeting TUFT1

2021 ◽  
Author(s):  
Lijun Zheng ◽  
Qiongzhen Ren ◽  
Weipei Zhu ◽  
Xiaomin Tao ◽  
Liangsheng Guo
Keyword(s):  
Cell Viability ◽  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Tumor Suppressor Function ◽  
Over Expression ◽  
Anti Cancer ◽  
Cervical Cancer Cells ◽  
Direct Target ◽  
Dual Luciferase Reporter Assay

Abstract Background: In the present study, a new tumor suppressor function of miR-4319 was disclosed in CC. Methods: Up-regulation of miR-4319 suppressed cell viability, proliferation, migration, invasion, and induced cell apoptosis in CC cells were measured by cell transfection, CCK-8, colony formation, EdU, flow cytometer, wound healing, transwell migration and invasion and western blot assays. Moreover, Tuftelin 1 (TUFT1) was verified as a direct target of miR-4319 by binding its 3’-UTR, confirmed by dual-luciferase reporter assay. Result: The expression of miR-4319 was obviously decreased in clinical CC tissues and CC cell lines.TUFT1 was remarkably increased in clinical CC tissues and CC cell lines, and was negatively associated with miR-4319 expression. Furthermore, over-expression of TUFT1 partially restored the effects of miR-4319 mimic on cell viability, proliferation, migration, invasion, and cell apoptosis in CC cells. Conclusion: miR-4319 played an anti-cancer role in the occurrence and development of CC, which might be achieved by targeting TUFT1.

scholarly journals Long non-coding RNA SNHG6 promotes tumorigenesis of nasopharyngeal carcinoma by competitively binding to miR-944 with RGS17

2020 ◽  
Author(s):  
Yu’e Han ◽  
Xing Liu ◽  
Guangling Li ◽  
Xia Ju ◽  
Zhongyi Song
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Regulatory Mechanism ◽  
Suppressive Effect ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Non Coding Rna ◽  
Reporter Assays ◽  
Direct Target ◽  
Long Non Coding Rna

Abstract Background Previous studies have shown that many long noncoding RNAs (lncRNAs) are involved in the pathogenesis of nasopharyngeal carcinoma (NPC). However, the regulatory mechanism of lncRNA SNHG6 remains unknown. Therefore, this study was design to preliminarily elucidate the role of SNHG6 in NPC. Methods The mRNA expression was detected by RT-qPCR. CCK-8, Transwell and dual luciferase reporter assays were used to investigate the function of SNHG6 in NPC. Results Upregulation of SNHG6 and downregulation of miR-944 were identified in NPC and were associated with TNM stage and distant metastasis in NPC patients. Additionally, SNHG6 acts as a molecular sponge of miR-944. More importantly, SNHG6 promoted NPC cell proliferation, migration and invasion by downregulating miR-944. Further, RGS17 was confirmed to be a direct target of miR-944. MiR-944 restrained NPC progression by targeting RGS17. Besides that, knockdown of RGS17 was found to block NPC progression. Upregulation of SNHG6 weakened the suppressive effect of RGS17 knockdown in NPC. Conclusion LncRNA SNHG6 promotes tumorigenesis of NPC by competitively binding to miR-944 with RGS17.

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.

Sign in / Sign up

Export Citation Format

Share Document