scholarly journals miR-190 promotes malignant transformation and progression of human urothelial cells through CDKN1B/p27 inhibition

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shirui Huang ◽  
Xiaohui Hua ◽  
Mengjiao Kuang ◽  
Junlan Zhu ◽  
Haiqi Mu ◽  
...  
Keyword(s):  
Malignant Transformation ◽  
Cell Transformation ◽  
Soft Agar ◽  
Luciferase Reporter ◽  
Mechanistic Study ◽  
Potential Contribution ◽  
Urothelial Cells ◽  
Soft Agar Assay ◽  
Specific Pcr ◽  
Dual Luciferase Reporter Assay

Abstract Background Although miR-190 has been reported to be related to human diseases, especially in the development and progression of cancer, its expression in human bladder cancer (BC) and potential contribution to BC remain unexplored. Methods RT-qPCR was used to verify the expression level of miR-190 and CDKN1B. Flow cytometry (FCM) assays were performed to detect cell cycle. Soft agar assay was used to measure anchorage-independent growth ability. Methylation-Specific PCR, Dual-luciferase reporter assay and Western blotting were used to elucidate the potential mechanisms involved. Results Our studies revealed that downregulation of the p27 (encoded by CDKN1B gene) protein is an important event related to miR-190, promoting the malignant transformation of bladder epithelial cells. miR-190 binds directly to CDKN1B 3’-UTR and destabilizes CDKN1B mRNA. Moreover, miR-190 downregulates TET1 by binding to the TET1 CDS region, which mediates hypermethylation of the CDKN1B promoter, thereby resulting in the downregulation of CDKN1B mRNA. These two aspects led to miR-190 inhibition of p27 protein expression in human BC cells. A more in-depth mechanistic study showed that c-Jun promotes the transcription of Talin2, the host gene of miR-190, thus upregulating the expression of miR-190 in human BC cells. Conclusions In this study, we found that miR-190 plays an important role in the development of BC. Taken together, these findings indicate that miR-190 may promote the malignant transformation of human urothelial cells by downregulating CDKN1B, which strengthens our understanding of miR-190 in regulating BC cell transformation.

scholarly journals MiR-190 Promotes Malignant Transformation and Progression of Human Urothelial Cells through CDKN1B Inhibition

2020 ◽  
Author(s):  
Shirui Huang ◽  
Xiaohui Hua ◽  
Mengjiao Kuang ◽  
Junlan Zhu ◽  
Haiqi Mu ◽  
...  
Keyword(s):  
Malignant Transformation ◽  
Cell Transformation ◽  
Soft Agar ◽  
Luciferase Reporter ◽  
Mechanistic Study ◽  
Potential Contribution ◽  
Urothelial Cells ◽  
Soft Agar Assay ◽  
Specific Pcr ◽  
Dual Luciferase Reporter Assay

Abstract BackgroundAlthough miR-190 has been reported to be related to human diseases, especially in the development and progression of cancer, its expression in human bladder cancer (BC) and potential contribution to BC remain unexplored.MethodsqRT-PCR was used to verify the expression level of miR-190 and CDKN1B. Flow cytometry (FCM) assays were performed to detect cell cycle. Soft agar assay was used to measure anchorage-independent growth ability. Methylation-Specific PCR, Dual-luciferase reporter assay and Western blotting were used to elucidate the potential mechanisms involved.ResultsOur studies revealed that downregulation of the CDKN1B/p27 protein is an important event related to miR-190, promoting the malignant transformation of bladder epithelial cells. miR-190 binds directly to CDKN1B 3’-UTR and destabilizes CDKN1B mRNA. Moreover, miR-190 downregulates TET1 by binding to the TET1 CDS region, which mediates hypermethylation of the CDKN1B promoter, thereby resulting in the downregulation of CDKN1B mRNA. These two aspects led to miR-190 inhibition of p27 protein expression in human BC cells. However, the increased expression of miR-190 in BC and its upstream regulatory mechanism remain unclear. A more in-depth mechanistic study showed that c-Jun promotes the transcription of Talin2, the host gene of miR-190, thus upregulating the expression of miR-190 in human BC cells.ConclusionIn this study, we found that miR-190 plays an important role in the development of BC. Taken together, these findings indicate that miR-190 may promote the malignant transformation of human urothelial cells by downregulating CDKN1B, which strengthens our understanding of miR-190 in regulating BC cell transformation.

scholarly journals MiR-190 Promotes Malignant Transformation and Progression of Human Urothelial Cells through CDKN1B Inhibition

2021 ◽  
Author(s):  
Shirui Huang ◽  
Xiaohui Hua ◽  
Mengjiao Kuang ◽  
Junlan Zhu ◽  
Haiqi Mu ◽  
...  
Keyword(s):  
Malignant Transformation ◽  
Cell Transformation ◽  
Soft Agar ◽  
Luciferase Reporter ◽  
Mechanistic Study ◽  
Potential Contribution ◽  
Urothelial Cells ◽  
Soft Agar Assay ◽  
Specific Pcr ◽  
Dual Luciferase Reporter Assay

Abstract BackgroundAlthough miR-190 has been reported to be related to human diseases, especially in the development and progression of cancer, its expression in human bladder cancer (BC) and potential contribution to BC remain unexplored. MethodsqRT-PCR was used to verify the expression level of miR-190 and CDKN1B. Flow cytometry (FCM) assays were performed to detect cell cycle. Soft agar assay was used to measure anchorage-independent growth ability. Methylation-Specific PCR, Dual-luciferase reporter assay and Western blotting were used to elucidate the potential mechanisms involved. ResultsOur studies revealed that downregulation of the CDKN1B/p27 protein is an important event related to miR-190, promoting the malignant transformation of bladder epithelial cells. miR-190 binds directly to CDKN1B 3’-UTR and destabilizes CDKN1B mRNA. Moreover, miR-190 downregulates TET1 by binding to the TET1 CDS region, which mediates hypermethylation of the CDKN1B promoter, thereby resulting in the downregulation of CDKN1B mRNA. These two aspects led to miR-190 inhibition of p27 protein expression in human BC cells. However, the increased expression of miR-190 in BC and its upstream regulatory mechanism remain unclear. A more in-depth mechanistic study showed that c-Jun promotes the transcription of Talin2, the host gene of miR-190, thus upregulating the expression of miR-190 in human BC cells. ConclusionIn this study, we found that miR-190 plays an important role in the development of BC. Taken together, these findings indicate that miR-190 may promote the malignant transformation of human urothelial cells by downregulating CDKN1B, which strengthens our understanding of miR-190 in regulating BC cell transformation.

scholarly journals MicroRNA-384 Inhibits the Progression of Papillary Thyroid Cancer by Targeting PRKACB

2020 ◽  
Vol 2020 ◽  
pp. 1-11 ◽  
Author(s):  
Yongxia Wang ◽  
Beixi Wang ◽  
Hong Zhou ◽  
Xiangnan Zhang ◽  
Xinlai Qian ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Papillary Thyroid Cancer ◽  
Target Gene ◽  
Underlying Mechanism ◽  
Soft Agar ◽  
Luciferase Reporter ◽  
Papillary Thyroid ◽  
Soft Agar Assay ◽  
Spearman’S Correlation ◽  
Dual Luciferase Reporter Assay

Background. Growing evidence shows that dysregulation of miRNAs plays a significant role in papillary thyroid cancer (PTC) tumorigenesis and development. The abnormal expression of miR-384 has been acknowledged in the proliferation or metastasis of some cancers. However, the function and the underlying mechanism of miR-384 in PTC progression remain largely unknown. Methods. Real-time PCR was conducted to detect miR-384 expression in 58 cases of PTC and their adjacent noncancerous tissues. MTT, soft agar assay Transwell assay, and wound-healing assay were carried out to explore the biological function of miR-384 in PTC cell lines of BCPAP and K1. Bioinformatics analysis, dual-luciferase reporter assay, western blot, and functional complementation analysis were conducted to explore the target gene of miR-384. Moreover, Spearman’s correlation analysis was conducted to reveal the correlation between miR-384 and PRKACB mRNA in PTC. Results. The expression of miR-384 decreased obviously in PTC, especially in the tumors with lymph node metastasis or larger tumor size. The ectopic upregulation of miR-384 significantly suppressed PTC progression, and the inhibition of miR-384 had the opposite effects. Moreover, PRKACB gene was confirmed as the target of miR-384. Conclusion. The study suggests that miR-384 serves as a tumor suppressor in PTC progression by directly targeting the 3′-UTR of PRKACB gene.

A High-Throughput Soft Agar Assay for Identification of Anticancer Compound

2007 ◽  
Vol 12 (7) ◽  
pp. 938-945 ◽  
Author(s):  
Steven N. Anderson ◽  
Danli L. Towne ◽  
David J. Burns ◽  
Usha Warrior
Keyword(s):  
Kinase Inhibitors ◽  
Cell Transformation ◽  
Growth Factor Receptor ◽  
Soft Agar ◽  
Anticancer Compounds ◽  
3 Dimensional ◽  
Soft Agar Assay ◽  
Epithelial Growth Factor ◽  
Highly Sensitive ◽  
Egfr Tyrosine Kinase Inhibitors

A 384-well soft agar assay was developed to identify potential novel anticancer compounds. Normally used to detect cell transformation, the assay is used here to quantitate cell proliferation in a 3-dimensional (3-D) anchorage-independent format. HCC827 cells, which are highly sensitive to epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors, were used to develop the method and a set of 9600 compounds used to validate the assay. Results were compared to a monolayer assay using the same compound set. The assay provides a robust method to discover compounds that could be missed using traditional monolayer formats. ( Journal of Biomolecular Screening 2007:938-945)

scholarly journals Proapoptotic and Anticarcinogenic Activities of Leviusculoside G from the Starfish Henricia leviuscula and Probable Molecular Mechanism

2008 ◽  
Vol 3 (10) ◽  
pp. 1934578X0800301 ◽  
Author(s):  
Sergey N. Fedorov ◽  
Larisa K. Shubina ◽  
Alla A. Kicha ◽  
Natalia V. Ivanchina ◽  
Jong Y. Kwak ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Mouse Skin ◽  
Soft Agar ◽  
Probable Mechanism ◽  
Luciferase Assay ◽  
Human Leukemia ◽  
Soft Agar Assay ◽  
A Cell ◽  
Induced Apoptosis ◽  
Mts Assay

Proapoptotic and anticarcinogenic properties of leviusculoside G (LSG) isolated from the starfish Henricia leviuscula and a probable mechanism of these effects were investigated in human leukemia HL-60, THP-1, and mouse skin JB6 Cl41 cells, using a variety of assessments, including a cell viability (MTS) assay, flow cytometry, anchorage-independent soft agar assay, luciferase assay, and western-blotting. Application of LSG at nontoxic doses induced apoptosis in cancer cells and decreased cell transformation. This compound exerted its actions, at least in part, through the induction of p53-dependent apoptosis and inhibition of AP-1, NF-κB, and ERKs activities.

scholarly journals Upregulation of miR-150-5p alleviates LPS-induced inflammatory response and apoptosis of RAW264.7 macrophages by targeting Notch1

2020 ◽  
Vol 15 (1) ◽  
pp. 544-552
Author(s):  
Xiaoyan Deng ◽  
Zhixing Lin ◽  
Chao Zuo ◽  
Yanjie Fu
Keyword(s):  
Inflammatory Response ◽  
Underlying Mechanism ◽  
Notch Receptor ◽  
Raw264.7 Cells ◽  
Luciferase Reporter ◽  
Raw264.7 Macrophages ◽  
Factor Α ◽  
Anti Inflammation ◽  
Necrosis Factor ◽  
Dual Luciferase Reporter Assay

AbstractCirculating miR-150-5p has been identified as a prognostic marker in patients with critical illness and sepsis. Herein, we aimed to further explore the role and underlying mechanism of miR-150-5p in sepsis. Quantitative real-time-PCR assay was performed to detect the expression of miR-150-5p upon stimulation with lipopolysaccharide (LPS) in RAW264.7 cells. The levels of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β were measured by ELISA assay. Cell apoptosis was determined using flow cytometry. Western blot was used to assess notch receptor 1 (Notch1) expression in LPS-induced RAW264.7 cells. Dual-luciferase reporter assay was employed to validate the target of miR-150-5p. Our data showed that miR-150-5p was downregulated and Notch1 was upregulated in LPS-stimulated RAW264.7 cells. miR-150-5p overexpression or Notch1 silencing alleviated LPS-induced inflammatory response and apoptosis in RAW264.7 cells. Moreover, Notch1 was a direct target of miR-150-5p. Notch1 abated miR-150-5p-mediated anti-inflammation and anti-apoptosis in LPS-induced RAW264.7 cells. miR-150-5p alleviated LPS-induced inflammatory response and apoptosis at least partly by targeting Notch1 in RAW264.7 cells, highlighting miR-150-5p as a target in the development of anti-inflammation and anti-apoptosis drugs for sepsis treatment.

scholarly journals Hsa-miR-494-3p attenuates gene HtrA3 transcription to increase inflammatory response in hypoxia/reoxygenation HK2 Cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Qian Gong ◽  
Zhi-ming Shen ◽  
Zhe Sheng ◽  
Shi Jiang ◽  
Sheng-lin Ge
Keyword(s):  
Cardiac Surgery ◽  
Inflammatory Response ◽  
Target Gene ◽  
Kidney Injury ◽  
Human Kidney ◽  
Luciferase Reporter ◽  
The Relationship ◽  
Hk2 Cells ◽  
Dual Luciferase Reporter Assay ◽  
Hypoxia Reoxygenation

AbstractThe occurrence of cardiac surgery-associated acute kidney injury (CSA-AKI) increases hospital stay and mortality. MicroRNAs has a crucial role in AKI. This objective of the current study is to explore the function of hsa-miR-494-3p in inflammatory response in human kidney tubular epithelial (HK2) cells with hypoxia/reoxygenation. According to KDIGO standard, patients after cardiac surgery with cardiopulmonary bypass were divided into two groups: AKI (n = 10) and non-AKI patients (n = 8). HK2 were raised in the normal and hypoxia/reoxygenation circumstances and mainly treated by overexpression ofmiR-494-3p and HtrA3. The relationship between miR-494-3p and HtrA3 was determined by dual-luciferase reporter assay. Our result showed that Hsa-miR-494-3p was elevated in the serum of patients with CSA-AKI, and also induced in hypoxic reoxygenated HK2 cells. Hsa-miR-494-3p also increased a hypoxia-reoxygenation induced inflammatory response in HK2 cells. Moreover, as a target gene of miR-494-3p, overexpression of HtrA3 downregulated the hypoxia-reoxygenation induced inflammatory response in HK2 cells. Overexpression of hsa-miR-494-3p-induced inflammatory response was inhibited by overexpression of HtrA3. Collectively, we identified that hsa-miR-494-3p, a miRNA induced in both circulation of AKI patients and hypoxia-reoxygenation-treated HK2 cells, enhanced renal inflammation by targeting HtrA3, which may suggest a possible role as a new therapeutic target for CSA-AKI.

scholarly journals LINC00942 Promotes Tumor Proliferation and Metastasis in Lung Adenocarcinoma via FZD1 Upregulation

2021 ◽  
Vol 20 ◽  
pp. 153303382097752
Author(s):  
Ronghua Wang ◽  
Xiuyun Wang ◽  
Jingtao Zhang ◽  
Yanpei Liu
Keyword(s):  
Lung Adenocarcinoma ◽  
Wnt Signaling ◽  
Molecular Mechanisms ◽  
Bioinformatic Analysis ◽  
Luciferase Reporter ◽  
Regulatory Relationship ◽  
Potential Biomarker ◽  
Proliferation And Metastasis ◽  
Tumor Proliferation And Metastasis ◽  
Dual Luciferase Reporter Assay

Background: Long non-coding RNAs (lncRNAs) have been reported to play important roles in the progression of human cancers. Herein, bioinformatic analysis identified that LINC00942 was a highly overexpressed lncRNA in lung adenocarcinoma (LUAD). The present study aimed to explore the roles and possible molecular mechanisms of LINC00942 in LUAD. Methods: First, on the basis of TCGA database, the expression and prognosis of LINC00942 were analyzed in LUAD tissues. Then, si-LINC00942 was transfected into A549 and H1299 cells to knockdown the expression of LINC00942. Cell viability was detected by MTT assay. Flow cytometry was used to analyze cell apoptosis. The expressions of PCNA, Bax, Bcl-2, and wnt/β-catenin pathway proteins were detected by western blotting. Dual-luciferase reporter assay was used to evaluate the regulatory relationship between LINC00942 and miR-5006-5p, or miR-5006-5p and FZD1. Results: We discovered that LINC00942 was up-regulated in LUAD tissues compared with adjacent tissues. Besides, we found the increased LINC00942 expression was associated with poor survival. In addition, silencing of LINC00942 suppressed the proliferation, migration, invasion and facilitated the apoptosis of A549 and H1299 cells. Moreover, silencing of LINC00942 repressed the expression of PCNA, Bcl-2, and enhanced Bax expression in A549 and H1299 cells. Mechanically, LINC00942 exerted its effects via enhancing Wnt signaling. LINC00942 functioned as competing endogenous RNA (ceRNA) by binding to miR-5006-5p, upregulating the expression of FZD1, which was a direct target of miR-5006-5p. Conclusion: Our findings indicated that LINC00942/miR-5006-5p/FZD1 axis played important roles in LUAD growth through enhancing Wnt signaling. LINC00942/miR-5006-5p/FZD1 axis might serve as a potential biomarker and therapeutic target for LUAD treatment.

scholarly journals Melatonin promotes bone marrow mesenchymal stem cell osteogenic differentiation and prevents osteoporosis development through modulating circ_0003865 that sponges miR-3653-3p

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xudong Wang ◽  
Taiqiu Chen ◽  
Zhihuai Deng ◽  
Wenjie Gao ◽  
Tongzhou Liang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Biological Processes ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Dual Luciferase Reporter Assay

Abstract Background Little is known about the implications of circRNAs in the effects of melatonin (MEL) on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoporosis (OP) progression. The aim of our study was to investigate circRNAs in MEL-regulated BMSC differentiation and OP progression. Methods BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red, and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade was validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on OP development were tested in murine OP model. Results MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 had no effect on proliferation while promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSC osteogenic differentiation induced by MEL, but proliferation of BMSCs induced by MEL had no change whether circ_0003865 was overexpression or not. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSC proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing, but had no effect on proliferation. Finally, circ_0003865 silencing repressed OP development in mouse model. Conclusion MEL promotes BMSC osteogenic differentiation and inhibits OP pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

scholarly journals Micro-RNA-338-3p Promotes the Development of Atherosclerosis by Targeting Desmin and Promoting Proliferation

2021 ◽  
Author(s):  
Shiran Yan ◽  
Jing Chen ◽  
Teng Zhang ◽  
Jian Zhou ◽  
Ge Wang ◽  
...  
Keyword(s):  
Rat Model ◽  
Luciferase Reporter Assay ◽  
Immunofluorescent Staining ◽  
Luciferase Reporter ◽  
Cell Phenotype ◽  
Reporter Assay ◽  
Synthetic Cell ◽  
Incorporation Assay ◽  
Morphologic Changes ◽  
Dual Luciferase Reporter Assay

AbstractAtherosclerosis (AS) is a dynamic and multi-stage process that involves various cells types, such as vascular smooth muscle cells (VSMCs) and molecules such as microRNAs. In this study, we investigated how miR-338-3p works in the process of AS. To determine how miR-338-3p was expressed in AS, an AS rat model was established and primary rat VSMCs were cultured. Real-time polymerase chain reaction was performed to detect miR-338-3p expression. Markers of different VSMC phenotypes were tested by Western blot. Immunofluorescent staining was employed to observe the morphologic changes of VSMCs transfected with miR-338-3p mimics. A dual luciferase reporter assay system was used to verify that desmin was a target of miR-338-3p. To further identify the role of miR-338-3p in the development of AS, VSMC proliferation and migration were evaluated by EdU incorporation assay, MTT assay, and wound healing assay. miR-338-3p expression was upregulated in the aortic tissues of an AS rat model and in primary rat VSMCs from a later passage. The transfection of miR-338-3p mimics in VSMCs promoted the synthetic cell phenotype. Bioinformatics analysis proposed desmin as a candidate target for miR-338-3p and the dual luciferase reporter assay confirmed in vivo that desmin was a direct target of miR-338-3p. The MTT and EdU incorporation assay revealed increased cell viability when miR-338-3p mimics were transfected. The increased expression of PCNA was a consistent observation, although a positive result was not obtained with respect to VSMC mobility. In AS, miR-338-3p expression was elevated. Elevated miR-338-3p inhibited the expression of desmin, thus promoting the contractile-to-synthetic VSMC phenotypic transition. In addition to morphologic changes, miR-338-3p enhanced the proliferative but not mobile ability of VSMCs. In summary, miR-338-3p promotes the development of AS.

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