scholarly journals Overexpression of miR-221-3p effecting cell proliferation, apoptosis and inflammation by targeting Toll-like receptors 4 in propofol-induced rat astrocytes

2021 ◽  
Author(s):  
Shuang Qi ◽  
Zinan Li ◽  
Shanshan Yu
Keyword(s):  
Neuronal Cell ◽  
Cell Number ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Reporter Assay ◽  
Toll Like Receptors ◽  
Rat Astrocytes ◽  
Dual Luciferase Reporter Assay

IntroductionGrowing evidence indicated that propofol has neurotoxic effects on the brains of developing rodents, leading to neuronal cell death, neurodegeneration, and brain injury.Material and methodsEctopic miR-221-3p was transfected into rat astrocytes, and Cell Counting Kit-8 assay and flow cytometry were performed to evaluate cell growth and apoptosis. The mRNA levels of Toll-like receptors 4 (TLR4), nuclear factor kappa B, interleukin-6, interleukin-1β, myeloid differentiation primary response 88 (MyD88), caspase-3, caspase-12, STAT3, and GRP78 were detected using quantitative real-time polymerase chain reaction. The proteins of TLR4 and MyD88 were determined using Western blotting. The association between miR-221-3p and TLR4 was measured using Dual-Luciferase Reporter Assay (Promega Corporation, Wisconsin, USA). Then, siTLR4 was transfected with 293T cells to study the role of TLR4 in astrocytes with propofol treatment.ResultsThe miR-221-3p expression in rat astrocytes was markedly suppressed by propofol treatment. The miR-221-3p mimics transfection in propofol-treated astrocytes effectively reduced the suppressive effect of propofol on astrocyte growth, repressed the propofol-induced apoptosis in rat astrocytes, and decreased the cell number during the G2–M phase. The expression of MyD88 and TLR4 was induced by propofol, whereas the transfection of miR-221-3p mimics dramatically reduced these genes expression at the mRNA and protein expression. After that, TLR4 was found to be target of miR-221-3p using Dual-Luciferase Reporter Assay. Furthermore, knockdown of TLR4 could suppress the apoptosis rate in propofol-treated astrocytes.ConclusionsThis study revealed that miR-221-3p might prevent astrocytes from propofol-induced damage by targeting TLR4.

scholarly journals miRNA-1290 Promotes Aggressiveness in Pancreatic Ductal Adenocarcinoma by Targeting IKK1

2018 ◽  
Vol 51 (2) ◽  
pp. 711-728 ◽  
Author(s):  
Na Ta ◽  
Xiaoyi Huang ◽  
Kailian Zheng ◽  
Yunshuo Zhang ◽  
Yisha Gao ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Western Blotting ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Low Grade ◽  
Reporter Assay ◽  
Dual Luciferase Reporter Assay

Background/Aims: MicroRNAs (miRNAs) are a group of non-coding RNAs that play diverse roles in pancreatic carcinogenesis. In pancreatic ductal adenocarcinoma (PDAC), NF-kB is constitutively activated in most patients and is linked to a mutation in KRAS via IkB kinase complex 1 (IKK1, also known as IKKa). We investigated the link between PDAC aggressiveness and miR-1290. Methods: We used miRCURYTM LNA Array and in situ hybridization to investigate candidate miRNAs and validated the findings with PCR. The malignant behavior of cell lines was assessed with Cell Counting Kit-8, colony formation, and Transwell assays. A dual-luciferase reporter assay was used to evaluate the interaction between miR-1290 and IKK1. Protein expression was observed by western blotting. Results: In this study, 36 miRNAs were dysregulated in high-grade pancreatic intraepithelial neoplasia (PanIN) and PDAC tissues compared with low-grade PanIN tissues. The area under the curve values of miR-1290 and miR-31-5p were 0.829 and 0.848, respectively (95% confidence interval, 0.722–0.936 and 0.749–0.948, both P < 0.001). There was a significant correlation between miR-1290 and histological differentiation (P = 0.029), pT stage (P = 0.006), and lymph node metastasis (P = 0.001). In addition, the in vitro work showed that miR-1290 promoted PDAC cell proliferation, invasion, and migration. Western blotting and the dual-luciferase reporter assay showed that miR-1290 promoted cancer aggressiveness by directly targeting IKK1. The synergist effect of miR-1290 on the proliferation and metastasis of PDAC cells was attenuated and enhanced by IKK1 overexpression and knockdown, respectively. Consistent with the in vitro results, a subcutaneous tumor mouse model showed that miR-1290 functioned as a potent promoter of PDAC in vivo. Conclusion: MiR-1290 may act as an oncogene by directly targeting the 3’-untranslated region of IKK1, and the miR-1290/IKK1 pathway may prove to be a novel diagnostic and therapeutic target for PDAC.

scholarly journals MicroRNA-219c-5p regulates bladder smooth muscle cell fibrosis by targeting FN1

2020 ◽  
Author(s):  
Bowen Liu ◽  
Yafei Ding ◽  
Peng Li ◽  
Tao Wang ◽  
Zhankui Jia ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Bladder Smooth Muscle ◽  
Muscle Fibrosis ◽  
Dual Luciferase Reporter Assay

Abstract Background We found that the bladders of multiple sclerosis mice were significantly fibrotic. This study aimed to investigate the relationship between fibronectin 1 (FN1) and bladder fibrosis, as well as the microRNAs involved in FN1 regulation. Methods The degree of bladder smooth muscle fibrosis was observed by immunohistochemistry. In addition, we used quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting to determine FN1 expression in bladders with different grades of fibrosis. Bioinformatics analysis revealed that miR-199a-3p, miR-219c-5p and miR-3572-3p could inhibit FN1 synthesis. Therefore, miR-199a-3p, miR-219c-5p and miR-3572-3p were overexpressed or knocked down in bladder smooth muscle cells (BSMCs), and the respective transfection and FN1 knockdown efficiencies were detected by RT-qPCR. Only miR-219c-5p overexpression and knockdown produced the expected results. A dual luciferase reporter assay was used to determine the targeting relationship between miR-219c-5p and FN1. Flow cytometry and Cell Counting Kit 8 (CCK8) experiments confirmed that miR-219c-5p reduced FN1 expression and affected the biological activity of smooth muscle cells. Results With increasing bladder fibrosis, FN1 expression increased, while miR-199a-3p, miR-219c-5p, and miR-3572-3p expression levels decreased. The RT-qPCR results after transfection showed that only miR-219c-5p could regulate FN1. Indeed, the dual luciferase reporter assay results indicated that miR-219c-5p targeted FN1 directly. CCK8 and cell cycle assays showed that miR-219c-5p overexpression inhibited BSMC proliferation, while miR-219c-5p knockdown promoted BSMC proliferation. An apoptosis assay showed that miR-219c-5p overexpression promoted apoptosis, while miR-219c-5p knockdown inhibited BSMC apoptosis. Conclusions Our findings indicate that FN1 upregulation and miR-219c-5p downregulation play an important role in the development of bladder fibrosis, and miR-219c-5p participates in the fibrosis of BSMCs by regulating FN1 expression. Thus, a novel antifibrotic function of miR-219c-5p is proposed, which may represent a potential target for the diagnosis and treatment of bladder fibrosis.

scholarly journals MicroRNA-330-3p Expression Indicates Good Prognosis and Suppresses Cell Proliferation by Targeting Bmi-1 in Osteosarcoma

2018 ◽  
Vol 46 (2) ◽  
pp. 442-450 ◽  
Author(s):  
Zhenxin Zheng ◽  
Feng Bao ◽  
Xuhong Chen ◽  
Hongbin Huang ◽  
Xiangfeng Zhang
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Colorimetric Assay ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Biological Behavior ◽  
Luciferase Reporter ◽  
Osteosarcoma Cell ◽  
Reporter Assay ◽  
Dual Luciferase Reporter Assay

Background/Aims: Growing evidence has shown that miR-330-3p is closely related to the biological behavior of cancer, including proliferation, metastasis, and prognosis. However, there have been no reports on miR-330-3p expression and function in osteosarcoma. Methods: Expression of miR-330-3p in osteosarcoma tissues and cell lines was examined by quantitative PCR. Effects of miR-330-3p on osteosarcoma cell proliferation were investigated in vitro with the Cell Counting Kit-8 colorimetric assay. Targets of miR-330-3p were identified by dual-luciferase reporter assay. Results: The results showed that expression of miR-330 decreased in osteosarcoma tissues and cell lines. Prognosis of patients with high miR-330-3p expression was much better than that of those with low expression (P=0.001), and multivariate analysis suggested that miR-330-3p is an independent prognostic factor for osteosarcoma. In addition, miR-330-3p overexpression significantly inhibited the growth of MG-63 and U2OS osteosarcoma cells. Dual-luciferase reporter assay demonstrated that Bmi-1 was a direct target gene of miR-330-3p, and in a recovery experiment, miR-330-3p suppressed osteosarcoma cell proliferation by directly targeting Bmi-1. Conclusion: Our results suggest that miR-330-3p acts as a tumor suppressor by regulating Bmi-1 expression in osteosarcoma. Thus, miR-330-3p may represent a novel therapeutic target for the treatment of osteosarcoma.

scholarly journals MicroRNA-219c-5p Regulates Bladder Fibrosis by Targeting FN1

2020 ◽  
Author(s):  
Bowen Liu ◽  
Yafei Ding ◽  
Peng Li ◽  
Siyuan He ◽  
Tao Wang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Bladder Smooth Muscle ◽  
Muscle Fibrosis ◽  
Dual Luciferase Reporter Assay

Abstract BackgroundWe found that the bladders of multiple sclerosis mice were significantly fibrotic. This study aimed to investigate the relationship between fibronectin 1 (FN1) and bladder fibrosis, as well as the microRNAs involved in FN1 regulation. Methods The degree of bladder smooth muscle fibrosis was observed by immunohistochemistry. In addition, we used quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting to determine FN1 expression in bladders with different grades of fibrosis. Bioinformatics analysis revealed that miR-199a-3p, miR-219c-5p and miR-3572-3p could inhibit FN1 synthesis. Therefore, miR-199a-3p, miR-219c-5p and miR-3572-3p were overexpressed or knocked down in bladder smooth muscle cells (BSMCs), and the respective transfection and FN1 knockdown efficiencies were detected by RT-qPCR. Only miR-219c-5p overexpression and knockdown produced the expected results. A dual luciferase reporter assay was used to determine the targeting relationship between miR-219c-5p and FN1. Flow cytometry and Cell Counting Kit 8 (CCK8) experiments confirmed that miR-219c-5p reduced FN1 expression and affected the biological activity of smooth muscle cells. Agomir and anagomir of miR-219c-5p were transfected in vivo to observe the change of bladder fibrosis in mice.ResultsWith increasing bladder fibrosis, FN1 expression increased, while miR-199a-3p, miR-219c-5p, and miR-3572-3p expression levels decreased. The RT-qPCR results after transfection showed that only miR-219c-5p could regulate FN1. Indeed, the dual luciferase reporter assay results indicated that miR-219c-5p targeted FN1 directly. CCK8 and cell cycle assays showed that miR-219c-5p overexpression inhibited BSMC proliferation, while miR-219c-5p knockdown promoted BSMC proliferation. An apoptosis assay showed that miR-219c-5p overexpression promoted apoptosis, while miR-219c-5p knockdown inhibited BSMC apoptosis. The agomir and anagomir transfected with miR-219c-5p in vivo found that the bladder fibrosis of the mice in the agomir group was reduced, and the anagomir group was worse.ConclusionsOur findings indicate that FN1 up-regulation and miR-219c-5p down-regulation play an important role in the development of bladder fibrosis, and miR-219c-5p participates in bladder fibrosis by regulating FN1 expression. Thus, a novel antifibrotic function of miR-219c-5p is proposed, which may represent a potential target for the diagnosis and treatment of bladder fibrosis.

scholarly journals MicroRNA-219c-5p regulates bladder fibrosis by targeting FN1

BMC Urology ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Bowen Liu ◽  
Yafei Ding ◽  
Peng Li ◽  
Tao Wang ◽  
Siyuan He ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Bladder Smooth Muscle ◽  
Dual Luciferase Reporter Assay

Abstract Background We found that the bladders of multiple sclerosis mice were significantly fibrotic. This study aimed to investigate the relationship between fibronectin 1 (FN1) and bladder fibrosis, as well as the microRNAs involved in FN1 regulation. Methods The degree of bladder smooth muscle fibrosis was observed by immunohistochemistry. In addition, we used quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting to determine FN1 expression in bladders with different grades of fibrosis. Bioinformatics analysis revealed that miR-199a-3p, miR-219c-5p and miR-3572-3p could inhibit FN1 synthesis. Therefore, miR-199a-3p, miR-219c-5p and miR-3572-3p were overexpressed or knocked down in bladder smooth muscle cells (BSMCs), and the respective transfection and FN1 knockdown efficiencies were detected by RT-qPCR. Only miR-219c-5p overexpression and knockdown produced the expected results. A dual luciferase reporter assay was used to determine the targeting relationship between miR-219c-5p and FN1. Flow cytometry and Cell Counting Kit 8 (CCK8) experiments confirmed that miR-219c-5p reduced FN1 expression and affected the biological activity of smooth muscle cells. Agomir and anagomir of miR-219c-5p were transfected in vivo to observe the change of bladder fibrosis in mice. Results With increasing bladder fibrosis, FN1 expression increased, while miR-199a-3p, miR-219c-5p, and miR-3572-3p expression levels decreased. The RT-qPCR results after transfection showed that only miR-219c-5p could regulate FN1. Indeed, the dual luciferase reporter assay results indicated that miR-219c-5p targeted FN1 directly. CCK8 and cell cycle assays showed that miR-219c-5p overexpression inhibited BSMC proliferation, while miR-219c-5p knockdown promoted BSMC proliferation. An apoptosis assay showed that miR-219c-5p overexpression promoted apoptosis, while miR-219c-5p knockdown inhibited BSMC apoptosis. The agomir and anagomir transfected with miR-219c-5p in vivo found that the bladder fibrosis of the mice in the agomir group was reduced, and the anagomir group was worse. Conclusions Our findings indicate that FN1 up-regulation and miR-219c-5p down-regulation play an important role in the development of bladder fibrosis, and miR-219c-5p participates in bladder fibrosis by regulating FN1 expression. Thus, a novel antifibrotic function of miR-219c-5p is proposed, which may represent a potential target for the diagnosis and treatment of bladder fibrosis.

scholarly journals miR-520c-3p regulates IL-1β-stimulated human chondrocyte apoptosis and cartilage degradation by targeting GAS2

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Le Peng ◽  
Ming Deng ◽  
Yonggang Ma ◽  
Wei Hu ◽  
Fan Liang
Keyword(s):  
Real Time ◽  
Bioinformatic Analysis ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Chondrocyte Apoptosis ◽  
Reporter Assay ◽  
Oa Chondrocytes ◽  
Apoptosis And Inflammation ◽  
Dual Luciferase Reporter Assay

Abstract Background MicroRNAs (miRNAs) have been shown to be associated with osteoarthritis (OA) progression. This study aimed to explore the role of miR-520c-3p in OA progression. Methods Expression levels of miR-520c-3p and Growth arrest-specific 2 (GAS2) were detected using quantitative real-time PCR. The proliferation and apoptosis of cells were measured using cell counting kit 8 (CCK8) assay and flow cytometry. Furthermore, the protein levels of apoptosis-related markers, extracellular degradation markers, inflammatory response markers, and GAS2 were tested using quantitative real-time polymerase chain reaction (RT-PCR) and western blot (WB) analysis. In addition, the interaction between miR-520c-3p and GAS2 was examined using dual luciferase reporter assay. Results GAS2 was highly expressed, and miR-520c-3p was lowly expressed in OA cartilage tissues. miR-520c-3p could promote the proliferation and inhibit the apoptosis and inflammation of OA chondrocytes. miR-520c-3p could be sponged by GAS2, and its inhibitor could reverse the regulation of GAS2 on the biological functions of OA chondrocytes. GAS2 was a target of miR-520c-3p, which was identified by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of GAS2 could inhibit the proliferation and promoted the apoptosis and inflammation of OA chondrocytes. Conclusion Our data showed that miR-520c-3p might regulate the GAS2 to inhibit the progression of OA.

scholarly journals MicroRNA-513b-5p targets COL1A1 and COL1A2 associated with the formation and rupture of intracranial aneurysm

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zheng Zheng ◽  
Yan Chen ◽  
Yinzhou Wang ◽  
Yongkun Li ◽  
Qiong Cheng
Keyword(s):  
Cell Death ◽  
Intracranial Aneurysm ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Control Group ◽  
Type I ◽  
Mrna Levels ◽  
Reporter Assay ◽  
Over Expression ◽  
Tnf Α

AbstractCollagen-type I alpha 1 chain (COL1A1) and COL1A2 are abnormally expressed in intracranial aneurysm (IA), but their mechanism of action remains unclear. This study was performed to investigate the mechanism of COL1A1 and COL1A2 affecting the occurrence and rupture of IA. Quantitative real-time polymerase chain reaction was used to measure the expression of hsa-miR-513b-5p, COL1A1, COL1A2, TNF-α, IL-6, MMP2, MMP3, MMP9 and TIMP4 in patients with ruptured IA (RA) (n = 100), patients with un-ruptured IA (UA) (n = 100), and controls (n = 100). Then, human vascular smooth muscle cells (HASMCs) were cultured, and dual luciferase reporter assay was performed to analyse the targeting relationship between miR-513b-5p and COL1A1 or COL1A2. The effects of the miR-513b-5p mimic and inhibitor on the proliferation, apoptosis, and death of HASMC and the RIP1-RIP3-MLKL and matrix metalloproteinase pathways were also explored. The effect of silencing and over-expression of COL1A1 and COL1A2 on the role of miR-513b-5p were also evaluated. Finally, the effects of TNF-α on miR-513b-5p targeting COL1A1 and COL1A2 were tested. Compared with those in the control group, the serum mRNA levels of miR-513b-5p, IL-6 and TIMP4 were significantly decreased in the RA and UA groups, but COL1A1, COL1A2, TNF-α, IL-1β, MMP2, MMP3 and MMP9 were significantly increased (p < 0.05). Compared with those in the UA group, the expression of COL1A1, COL1A2, TNF-α, IL-1β and MMP9 was significantly up-regulated in the RA group (p < 0.05). Results from the luciferase reporter assay showed that COL1A1 and COL1A were the direct targets of miR-513b-5p. Further studies demonstrated that miR-513b-5p targeted COL1A1/2 to regulate the RIP1-RIP3-MLKL and MMP pathways, thereby enhancing cell death and apoptosis. Over-expression of COL1A1 or COL1A2, rather than silencing COL1A1/2, could improve the inhibitory effect of miR-513b-5p on cell activity by regulating the RIP1-RIP3-MLKL and MMP pathways. Furthermore, over-expression of miR-513b-5p and/or silencing COL1A1/2 inhibited the TNF-α-induced cell proliferation and enhanced the TNF-α-induced cell death and apoptosis. The mechanism may be related to the inhibition of collagen I and TIMP4 expression and promotion of the expression of RIP1, p-RIP1, p-RIP3, p-MLKL, MMP2 and MMP9. MiR-513b-5p targeted the inhibition of COL1A1/2 expression and affected HASMC viability and extracellular mechanism remodelling by regulating the RIP1-RIP3-MLKL and MMP pathways. This process might be involved in the formation and rupture of IA.

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.

scholarly journals MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

2021 ◽  
Vol 20 ◽  
pp. 153303382098586
Author(s):  
Xuhui Wu ◽  
Gongzhi Wu ◽  
Huaizhong Zhang ◽  
Xuyang Peng ◽  
Bin Huang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Invasion ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

159 In vitro Characterization of Potential Pig Calcium-sensing Receptor (CaSR) Ligands and Cell Signaling Pathways Related to CaSR Activation by a Dual-luciferase Reporter Assay

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 82-83
Author(s):  
Xiaoya Zhao ◽  
Qianru Hui ◽  
Paula Azevedo ◽  
Karmin O ◽  
Chengbo Yang
Keyword(s):  
Signaling Pathways ◽  
Signaling Pathway ◽  
Binding Pocket ◽  
Extracellular Calcium ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Biased Agonism ◽  
Calcium Sensing ◽  
Dual Luciferase Reporter Assay

Abstract The calcium-sensing receptor (CaSR) is a pivotal regulator of calcium homeostasis. Our previous study has found that pig CaSR (pCaSR) is widely expressed in intestinal segments in weaned piglets. To characterize the activation of pCaSR by potential ligands and related cell signaling pathways, a dual-luciferase reporter assay was employed for the ligands screening and molecular docking was utilized to predict the binding mode of identified ligands. Our results showed that the dual-luciferase reporter assay system was well suited for pCaSR research and its ligand screening. The extracellular calcium activated pCaSR in a concentration-dependent manner with a half-maximal effective concentration (EC50) = 4.74 mM through the Gq/11 signaling pathway, EC50 = 2.85 mM through extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation signaling pathway, and EC50 = 2.26 mM through the Ras homolog family member A (RhoA) activation signaling pathway. Moreover, the activation of pCaSR stimulated by extracellular calcium showed biased agonism through three main signaling pathways: ERK1/2 phosphorylation signaling, Gq/11 signaling, and G12/13 signaling. Both L-Tryptophan and α-casein (90–95) could activate the pCaSR in the presence of extracellular calcium. Furthermore, we characterized the L-tryptophan binding pocket formed by pCaSR residues TRP 70, SER 147, ALA168, SER 169, SER 170, ASP 190, GLU 297, ALA 298, and ILE 416, as well as the α-casein (90–95) binding pocket formed by pCaSR residues PRO188, ASN189, GLU191, HIS192, LYS225, LEU242, ASP480, VAL486, GLY487, VAL513, and TYR514. In conclusion, similar to the human CaSR, the pCaSR also shows biased agonism through three main signaling pathways and both α-casein (90–95) and L-tryptophan are agonists for pCaSR. Furthermore, the binding sites of α-casein (90–95) and L-tryptophan are mainly located within the extracellular domain of pCaSR.

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