scholarly journals Mechanism of MicroRNA-146a/Notch2 Signaling Regulating IL-6 in Graves Ophthalmopathy

2017 ◽  
Vol 41 (4) ◽  
pp. 1285-1297 ◽  
Author(s):  
Ning Wang ◽  
Feng-E Chen ◽  
Zi-Wen Long
Keyword(s):  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Relative Expression ◽  
Over Expression ◽  
Graves Ophthalmopathy ◽  
Gene Assay ◽  
Elevated Expression ◽  
Orbital Connective Tissue

Background/Aims: We intended to investigate the significance of microRNA-146a, Notch2 and IL-6 on Graves ophthalmopathy (GO) and the relationships among them. Methods: About 27 GO patients were incorporated in this study, including 13 patients with inactive GO and14 patients with active GO. Another 15 patients who had previously received strabismus orthopedics or ophthalmectomy due to trauma were selected as the control population. QRT-PCR assay was used to detect microRNA-146a and Notch2 expression levels in plasma. MTT assay and flow cytometry were respectively used to assess the viability and mitosis of the fibroblasts isolated from orbital connective tissue. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was employed to detect serum IL-6 levels. The dual luciferase reporter gene assay was used to verify the targeting relationship between microRNA-146a and Notch2. Results: Compared with the control group, the relative expression of miR-146a was significantly increased whereas the relative expression of Notch2 was significantly decreased (all P < 0.05) in GO patients compare with the control. Notch2 can be directly targeted by microRNA-146a. The over-expression of miR-146a markedly facilitated Orbital Fibroblasts (OFs) viability and mitosis whereas markedly suppressed cell apoptosis (all P < 0.05). Exogenous microRNA-146a mimics could down-regulat the expression of Notch2 and up-regulate IL-6 (P < 0.05). The inhibition of microRNA-146 resulted in the elevated expression of Notch2 and decreased expression of IL-6 (P < 0.05). Conclusion: MicroRNA-146a may increase the IL-6 levels and exacerbate GO by directly targeting Notch2.

scholarly journals MiR-206 regulates the progression of osteoporosis via targeting HDAC4

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Zhiyuan Lu ◽  
Dawei Wang ◽  
Xuming Wang ◽  
Jilong Zou ◽  
Jiabing Sun ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Diagnostic Value ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Mineral Density ◽  
Gene Assay

Abstract Background More and more studies have confirmed that miRNAs play an important role in maintaining bone remodeling and bone metabolism. This study investigated the expression level of miR-206 in the serum of osteoporosis (OP) patients and explored the effect and mechanism of miR-206 on the occurrence and development of osteoporosis. Methods 120 postmenopausal women were recruited, including 63 cases with OP and 57 women without OP. The levels of miR-206 were determined by qRT-PCR technology. Spearman correlation coefficient was used to evaluate the correlation of miR-206 with bone mineral density (BMD). An ROC curve was used to evaluate the diagnostic value of miR-206 in osteoporosis. The effects of miR-206 on cell proliferation and cell apoptosis of hFOBs were measured by CCK-8 assay and flow cytometry, respectively. Luciferase reporter gene assay was used to confirm the interaction of miR-206 and the 3′UTR of HDAC4. Results Serum miR-206 had low expression level in osteoporosis patient group compared with control group. The expression level of serum miR-206 had diagnostic value for osteoporosis, and the serum miR-206 levels were positively correlated with BMD. The down-regulated miR-206 could inhibit cell proliferation and promote cell apoptosis. Luciferase analysis indicated that HDAC4 was the target gene of miR-206. Conclusions MiR-206 could be used as a new potential diagnostic biomarker for osteoporosis, and in in vitro cell experiments, miR-206 may regulate osteoblast cell proliferation and apoptosis by targeting HDAC4.

scholarly journals Circular RNA hsa_circ_0000282 contributes to osteosarcoma cell proliferation by regulating miR-192/XIAP axis

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Houkun Li ◽  
Limin He ◽  
Yuan Tuo ◽  
Yansheng Huang ◽  
Bing Qian
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Control Group ◽  
Osteosarcoma Cell ◽  
Luciferase Reporter Gene ◽  
Apoptosis Protein ◽  
Gene Assay

Abstract Background Circular RNAs (circRNAs) have emerged as a novel category of non-coding RNA, which exhibit a pivotal effect on regulating gene expression and biological functions, yet how circRNAs function in osteosarcoma (OSA) still demands further investigation. This study aimed at probing into the function of hsa_circ_0000282 in OSA. Methods The expressions of circ_0000282 and miR-192 in OSA tissues and cell lines were examined by quantitative real-time polymerase chain reaction (qRT-PCR), and the correlation between the expression level of circ_0000282 and clinicopathological features of OSA patients was analyzed. The expressions of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in OSA cells were assayed by Western blot. The proliferation and apoptosis of OSA cells were examined by CCK-8, BrdU and flow cytometry, respectively. Bioinformatics analysis, dual-luciferase reporter gene assay and RIP experiments were employed to predict and validate the targeting relationships between circ_0000282 and miR-192, and between miR-192 and XIAP, respectively. Results Circ_0000282 was highly expressed in OSA tissues and cell lines, which represented positive correlation with Enneking stage of OSA patients and negative correlation with tumor differentiation degree. In vitro experiments confirmed that overexpression of circ_0000282 markedly facilitated OSA cell proliferation and repressed cancer cell apoptosis in comparison to control group. Besides, knockdown of circ_0000282 repressed OSA cell proliferation and promoted apoptosis. Additionally, the binding relationships between circ_0000282 and miR-192, and between miR-192 and XIAP were validated. Circ_0000282 indirectly up-regulated XIAP expression by adsorbing miR-192, thereby playing a role in promoting cancer in OSA. Conclusion Circ_0000282 was a novel oncogenic circRNA in OSA. Circ_0000282/miR-192/XIAP axis regulated OSA cell proliferation apoptosis with competitive endogenous RNA mechanism.

scholarly journals miR-138-5p inhibits the malignant progression of prostate cancer by targeting FOXC1

2020 ◽  
Author(s):  
Dapeng Zhang ◽  
Xiaodong Liu ◽  
Qingwei Zhang ◽  
Xin Chen
Keyword(s):  
Cell Lines ◽  
Bioinformatics Analysis ◽  
Malignant Progression ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Over Expression ◽  
High Gleason Score ◽  
Gene Assay ◽  
Proliferation And Metastasis

Abstract Background: This study aimed to uncover the effect of miR-138-5p on the proliferation and metastasis of PCa cell lines, and further explore the potential regulatory mechanisms via regulating FOXC1.Methods: 60 pairs cancer tissues and corresponding paracancerous ones from PCa patients were collected to assess the expression level of miR-138-5p by qRT-PCR. Subsequently, over-expression of miR-138-5p were established to explore the proliferation and metastasis of miR-138-5p in PCa cell lines was analyzed by CCK-8, Tranwell assay and Wounding healing assay, respectively. Bioinformatics analysis and luciferase reporter gene assay were performed to search for the target genes of miR-138-5p, and FOXC1 was selected. Finally, the biological role of miR-138-5p and FOXC1 in the progression of PCa was clarified by a series of rescue experiments. Results: The results of qRT-PCR revealed that miR-138-5p was lowly expressed in PCa tissues and cell lines. Besdies, the PCa patients with low-miR-138-5p had a high Gleason score, lymph node metastasis and poor prognosis of PCa, compared with these patients with high-miR-138-5p. Over-expression of miR-138-5p inhibited the proliferative, migratory and invasive capacities of PC-3 and DU-145 cells. Bioinformatics analysis and luciferase reporter gene assay suggested that FOXC1 was predicted to be the target gene of miR-138-5p. Moreover, FOXC1 expression level was negatively correlated to that of miR-138-5p in PCa tissues. Importantly, Over-expression of FOXC1 could reverse miR-138-5p mimic induced-inhibition of PCa malignant progression.Conclusions: Downregulated miR-138-5p was closely associated with high Gleason score, more lymph node metastasis and poor prognosis of PCa patients. In addition, miR-138-5p alleviated the malignant progression of PCa by targeting and downregulating FOXC1.

scholarly journals Effects of Long Noncoding RNA HOTAIR Targeting miR-138 on Inflammatory Response and Oxidative Stress in Rat Cardiomyocytes Induced by Hypoxia and Reoxygenation

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Guofeng Wang ◽  
Qi Wang ◽  
Weixue Xu
Keyword(s):  
Inflammatory Response ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Rat Cardiomyocytes ◽  
Luciferase Reporter Gene Assay ◽  
Expression Levels ◽  
Gene Assay ◽  
And Oxidative Stress

Objective. To investigate the effects of HOX transcript antisense RNA (HOTAIR) and miR-138 on inflammatory response and oxidative stress (OS) induced by IRI in rat cardiomyocytes. Methods. H9C2 cells were divided into the control group, H/R group, H/R+siRNA NC group, H/R+si-HOTAIR group, and H/R+si-HOTAIR+inhibitor group. Expression levels of HOTAIR, miR-138, and inflammatory factors were detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The double luciferase reporter gene assay was used to detect the targeting relationship between HOTAIR and miR-138. Results. Compared with the control group, the level of miR-138 and SOD in the H/R group was obviously reduced, while the expression levels of the HOTAIR, MDA, and NF-κB pathway were obviously increased. Compared with the H/R group, the level of miR-138 and SOD in the H/R+si-HOTAIR group was obviously increased, and the expression levels of the HOTAIR, MDA, and NF-κB pathway were obviously decreased. Compared with the H/R+si-HOTAIR group, the level of SOD in the H/R+si-HOTAIR+inhibitor group decreased; MDA content and the NF-κB pathway expression level increased. In the double luciferase reporter gene assay, compared with the HOTAIR wt+NC group, the luciferase activity of the HOTAIR wt+miR-138 mimic group was obviously decreased. Conclusions. Silent HOTAIR can promote the expression of miR-138 and inhibit H/R-induced inflammatory response and OS by regulating the NF-κB pathway, thus protecting cardiomyocytes.

scholarly journals MiR-539-5p inhibits the inflammatory injury in septic H9c2 cells by regulating IRAK3

2021 ◽  
Author(s):  
Xiaochen Hu ◽  
Hongjun Miao
Keyword(s):  
Reporter Gene ◽  
Luciferase Reporter ◽  
Septic Cardiomyopathy ◽  
H9c2 Cells ◽  
Luciferase Reporter Gene ◽  
Inflammation Response ◽  
Over Expression ◽  
Qrt Pcr ◽  
Gene Assay ◽  
Proliferation And Apoptosis

Abstract Background MicroRNAs (miRNAs) have been confirmed to play a potential role in sepsis, but little is known about their role in sepsis-induced cardiomyopathy (SIC). Methods The model of septic cardiomyopathy was constructed with H9c2 cells induced by lipopolysaccharide (LPS), and the expression of miR-539-5p was detected by qRT-PCR assay. ELISA, CCK-8, EdU TUNEL analysis were performed to evaluate the role of miR-539-5p in inflammation response, viability, proliferation and apoptosis of LPS-treated H9c2 cells. Moreover, miRWalk and TargetScan prediction, and dual-luciferase reporter gene assays were carried out to predict and confirm the target of miR-539-5p. Furthermore, the effects of target on inflammation response, proliferation and apoptosis of LPS-induced H9c2 cells mediated by miR-539-5p was further explored. Results The expression of miR-539-5p was obviously down-regulated in LPS-induced H9c2 cells. In addition, over-expression of miR-539-5p significantly inhibited the inflammation response, promoted viability and proliferation, and suppressed apoptosis of LPS-treated H9c2 cells. Moreover, interleukin-1 receptor-associated kinase 3 (IRAK3) was verified as a target of miR-539-5p by dual-luciferase reporter gene assay. Besides, IRAK3 was highly expressed in H9c2 cells transfected with miR-539-5p inhibitor detected with qRT-PCR and western blot assays. Furthermore, over-expression of IRAK3 partially weakened the effects of miR-539-5p mimic on the inflammation response, proliferation and apoptosis of LPS-induced H9c2 cells. Conclusions MiR-539-5p potentially plays an important role in the pathogenesis of LPS-induced sepsis by targeting IRAK3, suggesting that miR-539-5p may be a potential new target for the treatment of LPS-induced sepsis.

scholarly journals MicroRNA-486 promotes a more catabolic phenotype in chondrocyte-like cells by targeting SIRT6

2021 ◽  
Vol 10 (7) ◽  
pp. 459-466
Author(s):  
Jie Yang ◽  
Yunping Zhou ◽  
Xiaojun Liang ◽  
Bingfei Jing ◽  
Zandong Zhao
Keyword(s):  
Western Blot ◽  
Messenger Rna ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Luciferase Reporter Gene ◽  
Western Blot Assay ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Gene Assay ◽  
A Disintegrin And Metalloproteinase

Aims Osteoarthritis (OA) is characterized by persistent destruction of articular cartilage. It has been found that microRNAs (miRNAs) are closely related to the occurrence and development of OA. The purpose of the present study was to investigate the mechanism of miR-486 in the development and progression of OA. Methods The expression levels of miR-486 in cartilage were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN), matrix metalloproteinase (MMP)-13, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) in SW1353 cells at both messenger RNA (mRNA) and protein levels was determined by qRT-PCR, western blot, and enzyme-linked immunosorbent assay (ELISA). Double luciferase reporter gene assay, qRT-PCR, and western blot assay were used to determine whether silencing information regulator 6 (SIRT6) was involved in miR-486 induction of chondrocyte-like cells to a more catabolic phenotype. Results Compared with osteonecrosis, the expression of miR-486 was significantly upregulated in cartilage from subjects with severe OA. In addition, overexpressed miR-486 promoted a catabolic phenotype in SW1353 cells by upregulating the expressions of ADAMTS4 and MMP-13 and down-regulating the expressions of COL2A1 and ACAN. Conversely, inhibition of miR-486 had the opposite effect. Furthermore, overexpression of miR-486 significantly inhibited the expression of SIRT6, confirming that SIRT6 is a direct target of miR-486. Moreover, SW1353 cells were transfected with small interfering RNA (si)-SIRT6 and it was found that SIRT6 was involved in and inhibited miR-486-induced changes to SW1353 gene expression. Conclusion Our results indicate that miR-486 promotes a catabolic phenotype in SW1353 cells in OA by targeting SIRT6. Our findings might provide a potential therapeutic target and theoretical basis for OA. Cite this article: Bone Joint Res 2021;10(7):459–466.

The miR-15a affected the development of sepsis and septic shock by suppression BCL-2

2019 ◽  
Author(s):  
Yunzhen Wu ◽  
Yuanli Xie ◽  
Fangfang Jiao ◽  
Xinlei Liu
Keyword(s):  
Septic Shock ◽  
Western Blot ◽  
Troponin I ◽  
Sepsis Group ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Rt Pcr ◽  
Gene Assay ◽  
Sepsis And Septic Shock

Abstract Background: This study aimed to investigate the mechanism of microRNA-15a (miR-15a) in the development of sepsis and septic shock. Methods: Sepsis and septic shock rat models were constructed by intraperitoneal injection of E.coli endotoxin (LPS). The real-time polymerase chain reaction (RT-PCR), Enzyme-Linked ImmunoSorbent Assay (ELISA), ematoxylin-eosin (HE) and Masson staining, TdT-mediated dUTP Nick-End Labeling (TUNEL), as well as Western blot analysis were performed to reveal the expression of microRNA-15a and changes in sepsis/septic shock myocardial cells or tissue. The rat sepsis model (sepsis group and septic shock group) was successfully established. Results: The results of HE and Mason staining showed that myocardial tissue damage gradually deepened with the progression of sepsis. Moreover, the serum levels of creatinine kinase-mb (CK-MB) and cardiac troponin I (cTnI) in model groups were significantly increased than those in control group. In addition, the RT-PCR analysis showed that miR-15a was up-regulated in model groups. Furthermore, luciferase reporter gene assay showed that 3'-UTR was the binding site of BCL-2 to miR-15a. Finally, the TUNEL and Western blot showed the cardiomyocyte apoptosis in model group. Conclusions: The overexpression of miR-15a might take part in the progression of LPS-induced sepsis and septic shock via suppressing Bcl-2 expression. Furthermore, myocardial markers such as CK-MB and cTnI might be biomarkers for sepsis progression.

scholarly journals microRNA-27a-3p down-regulation inhibits malignant biological behaviors of ovarian cancer by targeting BTG1

Open Medicine ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. 577-585 ◽  
Author(s):  
Enfang Li ◽  
Ke Han ◽  
Xuan Zhou
Keyword(s):  
Ovarian Cancer ◽  
Protein Expression ◽  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Down Regulation ◽  
Over Expression ◽  
Bax Protein ◽  
Gene Assay

AbstractOvarian cancer is the most deadly malignant tumor. MicroRNA-27a-3p (miR-27a-3p) was a tumor oncogene in various cancers. However, the role and mechanism of miR-27a-3p in ovarian cancer are still unknown. In this study, we found that miR-27a-3p over-expression could significantly promote the viability of SK-OV-3 cells, enhance cell migration and invasion, and reduce cell apoptosis. Besides, results from western blot assay showed that miR-27a-3p over-expression could increase Bcl-2 protein expression and decrease Bax protein expression. Furthermore, TargetScan and the dual luciferase reporter gene assay revealed that BTG anti-proliferation factor 1 (BTG1) was a direct target of miR-27a-3p. In addition, we found that miR-27a-3p down-regulation suppressed SK-OV-3 cell viability, migration and invasion, and promoted cell apoptosis. All the effects of miR-27a-3p down-regulation on SK-OV-3 cells were reversed by BTG1-siRNA. Therefore, miR-27a-3p/BTG1 axis may be a new potential target for the treatment of ovarian cancer.

Effect of LINC00320 Regulating the Expression of PLEKHA1 through the Transcription Factor MYC in Glioma Cell Proliferation, Migration, Invasion and Apoptosis In vivo and In vitro

2021 ◽  
Author(s):  
Jianyong Ji ◽  
Pengfei Xue ◽  
Juan Zheng ◽  
Rongrong Li ◽  
Jinyue Fu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Over Expression ◽  
Gene Assay ◽  
Rip Assay

Abstract Aim: This study was carried out to explore the mechanism and function of LINC00320 in the development of glioma by regulating PLEKHA1 expression through transcription factor MYC.Methods: By searching LINCDISEASE database and through difference analysis of glioma chip, glioma related lncRNAs were screened, and lncRNA-transcription factor-mRNA triplet was predicted through lncMAP database. The expressions of LINC00320 and PLEKHA1 were detected in glioma and normal controls, followed by the detection of the proliferation, invasion, migration, and apoptosis of glioma cells by using CCK-8 method, Transwell assay, and flow cytometry, respectively. In addition, the expression patterns of MMP9 and cleaved-Caspase 3 were detected with Western Blot. Furthermore, the possible mechanism of LINC00320 was predicted in gliomas by LncMAP. RIP assay was performed to verify the interaction between LINC00320 and MYC, and ChIP assay was applied to validate the binding of MYC and PLEKHA1 promoter. The existence of binding site between MYC and PLEKHA1 promoter were determined by dual luciferase reporter gene assay. Lastly, in vivo test was conducted by using nude mice as the objects of study for verification of the results obtained by in vitro tests.Results: LINC00320 was found to be significantly down-expressed in glioma, and patients with low expression levels of LINC00320 exhibited an even worse prognostic outcome. Over-expression of LINC00320 in glioma cells brought about a significant reduction in cell proliferation, migration, invasion, and promoted apoptosis. There was a significant decrease in the protein expression of MMP9 but remarkable increase in that of cleaved-Caspase 3 after LINC00320 over-expression. LncRNA-transcription factor-mRNA triplet prediction showed that LINC00320 regulated the expression of PLEKHA1 through MYC. RIP assay demonstrated that MYC could significantly enrich LINC00320, Chip assay showed that MYC bound with the PLEKHA1 promoter, and dual luciferase reporter gene assay further confirmed the presence of binding site between MYC and PLEKHA1 promoter. Cell function experiment verified that PLEKHA1 could reverse the effect of LINC00320 over-expression.Conclusion: Over-expression of LINC00320 can attenuate the binding of MYC with PLEKHA1 by recruiting MYC, and ultimately inhibit the proliferation, migration and invasion, and promote the apoptosis of glioma cells.

scholarly journals miRNA-210 promotes the progression of hepatitis B cirrhosis to liver cancer via targeting inhibition of EGR3

2019 ◽  
Author(s):  
Xiaojie Li ◽  
Mei Yuan ◽  
Lu Song ◽  
Yan Wang
Keyword(s):  
Liver Cancer ◽  
Hepatitis B ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Blank Group ◽  
Cancer Group ◽  
Gene Assay ◽  
Liver Tissues

Abstract Background: This paper was aimed to research the mechanism of miRNA-210 in the progression of hepatitis B cirrhosis to liver cancer. Methods: Health examiners liver tissues (Control group), liver tissues of patients with hepatitis B cirrhosis (Cirrhosis group) and liver cancer tissues of patients induced by hepatitis B virus infection (Liver cancer group) were collected. HL-7702, HepG2 and HepG2.2.15 cells were cultured. HepG2 and HepG2.2.15 cells were transfected by miRNA-210 inhibitor (miRNA-210 Inhibitor group) and its negative control (miRNA-210-NC group). Normal HepG2 and HepG2.2.15 cells were named Blank group. Cells proliferation and apoptosis was analyzed. qRT-PCR technology, Western blot analysis and dual luciferase reporter gene assay were performed. Results: Tissues of Liver cancer group had higher miRNA-210 and lower EGR3 expression than Cirrhosis group (P < 0.05). Increased miRNA-210 and decreased EGR3 expression in HepG2.2.15 cells was presented when compared with those in HepG2 cells (P < 0.05). Compared to Blank group and miRNA-210-NC group, HepG2 and HepG2.2.15 cells of miRNA-210 Inhibitor group had lower A590 value, higher apoptosis rate and EGR3 expression (P < 0.05). EGR3 was directly inhibited by miRNA-210. Conclusions: miRNA-210 might promote the progression of hepatitis B cirrhosis to liver cancer via targeting inhibition of EGR3.

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