Altered expression of microRNAs in the rat diaphragm in a model of ventilator-induced diaphragm dysfunction after controlled mechanical ventilation

Abstract Background Ventilator-induced diaphragm dysfunction (VIDD) is a common complication of life support by mechanical ventilation observed in critical patients in clinical practice and may predispose patients to severe complications such as ventilator-associated pneumonia or ventilator discontinuation failure. To date, the alterations in microRNA (miRNA) expression in the rat diaphragm in a VIDD model have not been elucidated. This study was designed to identify these alterations in expression. Results Adult male Wistar rats received conventional controlled mechanical ventilation (CMV) or breathed spontaneously for 12 h. Then, their diaphragm tissues were collected for RNA extraction. The miRNA expression alterations in diaphragm tissue were investigated by high-throughput microRNA-sequencing (miRNA-seq). For targeted mRNA functional analysis, gene ontology (GO) analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were subsequently conducted. qRT-PCR validation and luciferase reporter assays were performed. We successfully constructed a model of ventilator-induced diaphragm dysfunction and identified 38 significantly differentially expressed (DE) miRNAs, among which 22 miRNAs were upregulated and 16 were downregulated. GO analyses identified functional genes, and KEGG pathway analyses revealed the signaling pathways that were most highly correlated, which were the MAPK pathway, FoxO pathway and Autophagy–animal. Luciferase reporter assays showed that STAT3 was a direct target of both miR-92a-1-5p and miR-874-3p and that Trim63 was a direct target of miR-3571. Conclusions The current research supplied novel perspectives on miRNAs in the diaphragm, which may not only be implicated in diaphragm dysfunction pathogenesis but could also be considered as therapeutic targets in diaphragm dysfunction.

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Overexpression of miR-340-5p Inhibits Skin Fibroblast Proliferation by Targeting Kruppel-like Factor 2

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190725112304 ◽

2019 ◽

Vol 20 (13) ◽

pp. 1147-1154 ◽

Cited By ~ 2

Author(s):

Ling Chen ◽

Qian Li ◽

Xun Lu ◽

Xiaohua Dong ◽

Jingyun Li

Keyword(s):

Gene Ontology ◽

Skin Fibroblast ◽

Kegg Pathway ◽

Normal Skin ◽

Skin Fibroblasts ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Fibroblast Proliferation ◽

Pathway Analyses ◽

Normal Skin Fibroblast

Objective: MicroRNA (miR)-340-5p has been identified to play a key role in several cancers. However, the function of miR-340-5p in skin fibroblasts remains largely unknown. Methods: Gain of function experiments were performed by infecting normal skin fibroblast cells with a lentivirus carrying 22-bp miR-340-5p. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. To uncover the mechanisms, mRNA-seq was used. Differentially expressed mRNAs were further determined by Gene Ontology and KEGG pathway analyses. The protein levels were analysed by Western blotting. A dual-luciferase reporter assay was used to detect the direct binding of miR-340-5p with the 3'UTR of Kruppel-like factor 2 (KLF2). Results: MiR-340-5p lentivirus infection suppressed normal skin fibroblast proliferation. The mRNAseq data revealed that 41 mRNAs were differentially expressed, including 22 upregulated and 19 downregulated transcripts in the miR-340-5p overexpression group compared with those in the control group. Gene Ontology and KEGG pathway analyses revealed that miR-340-5p overexpression correlated with the macromolecule biosynthetic process, cellular macromolecule biosynthetic process, membrane, and MAPK signalling pathway. Bioinformatics analysis and luciferase reporter assays showed that miR-340-5p binds to the 3'UTR of KLF2. Forced expression of miR-340-5p decreased the expression of KLF2 in normal skin fibroblasts. Overexpression of KLF2 restored skin fibroblast proliferation in the miR-340-5p overexpression group. Conclusion: This study demonstrates that miR-340-5p may suppress skin fibroblast proliferation, possibly through targeting KLF2. These findings could help us understand the function of miR-340-5p in skin fibroblasts. miR-340-5p could be a therapeutic target for preventing scarring.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22115590 ◽

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.

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Faculty Opinions recommendation of Both high level pressure support ventilation and controlled mechanical ventilation induce diaphragm dysfunction and atrophy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.716598016.792003078 ◽

2012 ◽

Author(s):

Christer Sinderby

Keyword(s):

Mechanical Ventilation ◽

Pressure Support Ventilation ◽

Pressure Support ◽

Diaphragm Dysfunction ◽

Support Ventilation ◽

Level Pressure ◽

Controlled Mechanical Ventilation ◽

High Level

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CRP Stimulates GDF15 Expression in Endothelial Cells through p53

Mediators of Inflammation ◽

10.1155/2018/8278039 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 6

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.

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

Bioscience Reports ◽

10.1042/bsr20180695 ◽

2019 ◽

Vol 39 (7) ◽

Cited By ~ 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.

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Downregulation of miR-193a-3p is involved in the pathogenesis of hepatocellular carcinoma by targeting CCND1

PeerJ ◽

10.7717/peerj.8409 ◽

2020 ◽

Vol 8 ◽

pp. e8409 ◽

Cited By ~ 2

Author(s):

Shi-shuo Wang ◽

Zhi-guang Huang ◽

Hua-yu Wu ◽

Rong-quan He ◽

Li-hua Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Correlation Analysis ◽

Kegg Pathway ◽

Pearson Correlation ◽

Luciferase Reporter ◽

Kegg Pathway Analysis ◽

Reporter Assays ◽

Pearson Correlation Analysis ◽

Hcc Cells

Background Hepatocellular carcinoma (HCC) is the second-highest cause of malignancy-related death worldwide, and many physiological and pathological processes, including cancer, are regulated by microRNAs (miRNAs). miR-193a-3p is an anti-oncogene that plays an important part in health and disease biology by interacting with specific targets and signals. Methods In vitro assays were performed to explore the influences of miR-193a-3p on the propagation and apoptosis of HCC cells. The sequencing data for HCC were obtained from The Cancer Genome Atlas (TCGA), and the expression levels of miR-193a-3p in HCC and non-HCC tissues were calculated. The differential expression of miR-193a-3p in HCC was presented as standardized mean difference (SMD) with 95% confidence intervals (CIs) in Stata SE. The impact of miR-193a-3p on the prognoses of HCC patients was determined by survival analysis. The potential targets of miR-193a-3p were then predicted using miRWalk 2.0 and subjected to enrichment analyses, including Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Protein-Protein Interaction (PPI) network analysis. The interaction between miR-193a-3p and one predicted target, Cyclin D1 (CCND1), was verified by dual luciferase reporter assays and Pearson correlation analysis. Results MiR-193a-3p inhibited the propagation and facilitated the apoptosis of HCC cells in vitro. The pooled SMD indicated that miR-193a-3p had a low level of expression in HCC (SMD: −0.88, 95% CI [−2.36 −0.59]). Also, HCC patients with a higher level of miR-193a-3p expression tended to have a favorable overall survival (OS: HR = 0.7, 95% CI [0.43–1.13], P = 0.14). For the KEGG pathway analysis, the most related pathway was “proteoglycans in cancer”, while the most enriched GO term was “protein binding”. The dual luciferase reporter assays demonstrated the direct interaction between miR-193a-3p and CCND1, and the Pearson correlation analysis suggested that miR-193a-3p was negatively correlated with CCND1 in HCC tissues (R = − 0.154, P = 0.002). Conclusion miR-193a-3p could suppress proliferation and promote apoptosis by targeting CCND1 in HCC cells. Further, miR-193a-3p can be used as a promising biomarker for the diagnosis and treatment of HCC in the future.

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Hsa_circRNA_103124 Upregulation in Crohn’s Disease Promotes Cell Proliferation and Inhibits Autophagy by Regulating the Hsa-miR-650/AKT2 Signaling Pathway

Frontiers in Genetics ◽

10.3389/fgene.2021.753161 ◽

2021 ◽

Vol 12 ◽

Author(s):

Juan Yin ◽

Fuyi Tong ◽

Yulan Ye ◽

Tong Hu ◽

Lijuan Xu ◽

...

Keyword(s):

Cell Proliferation ◽

Crohn’S Disease ◽

Crohn's Disease ◽

Signaling Pathway ◽

Luciferase Reporter ◽

Circular Rnas ◽

Downstream Target ◽

Reverse Transcription Pcr ◽

Reporter Assays ◽

Pathway Analyses

Circular RNAs (circRNAs) play important roles in the pathogenesis of Crohn’s disease (CD). We discovered that hsa_circRNA_103124 was upregulated in CD patients in our previous study. Nonetheless, the function of hsa_circRNA_103124 is unclear. In this study, hsa_circRNA_103124 was predicted to interact with hsa-miR-650. Gene Ontology (GO) and pathway analyses identified AKT serine/threonine kinase 2 (AKT2) as the downstream target protein of hsa-miR-650. Activated AKT2 inhibits autophagy, but promotes cell proliferation. Recent studies suggest that the inhibition of autophagy is one of the mechanisms of CD pathogenesis. Therefore, we inferred that hsa_circRNA_103124 might regulate autophagy and proliferation by targeting AKT2 as a sponge for hsa-miR-650. Here, quantitative reverse transcription PCR (RT-QPCR) results revealed that upregulated hsa_circRNA_103124 expression in patients with CD was negatively correlated with hsa-miR-650 expression but positively correlated with the white blood cell count and calprotectin levels. TSC complex subunit 1 (TSC1), one of the proteins upstream of autophagy was downregulated in patients with CD. Consisting with the bioinformatics prediction, it was verified that hsa_circRNA_103124 targeted to hsa-miR650 by fluorescence in situ hybridization (FISH) and luciferase reporter assays. A hsa-miR-650 inhibitor reversed the promotion of rapamycin-induced autophagy and the inhibition of cell proliferation by the hsa_circRNA_103124 siRNA. However, hsa-miR-650 mimics reversed the inhibition of rapamycin-induced autophagy and the promotion of cell proliferation through hsa_circRNA_103124 overexpression. These results indicate that hsa_circRNA_103124 upregulation in patients with CD promotes cell proliferation and inhibits autophagy by regulating the hsa-miR-650/AKT2 signaling pathway.

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

10.21203/rs.3.rs-25032/v1 ◽

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.

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

Cellular Physiology and Biochemistry ◽

10.1159/000490027 ◽

2018 ◽

Vol 47 (2) ◽

pp. 747-758 ◽

Cited By ~ 26

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.

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