The effect of E2F7 expression in prostate cancer on apoptosis and cell cycle of prostate cancer cells.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e16568-e16568 ◽  
Author(s):  
Ying Wang
Keyword(s):  
Prostate Cancer ◽  
Cell Cycle ◽  
Molecular Mechanisms ◽  
The United States ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
G1 Phase ◽  
Reporter Assay ◽  
Phase Percentage

e16568 Background: Prostate cancer (PCa) is a leading cause of illness and death among men in the United States and Western Europe. However, the molecular mechanisms by which PCa developed has not been fully illustrated. Methods: Here, the expression of E2F7 in PCa was examined using IHC method and western blotting assay, and the expression of miR-30c was measured using qRT-PCR method. The cell cycle and apoptosis was determined by FACS. Cell Counting Kit-8 was used to testify cell proliferation. miR-30c mimics, miR-30c inhibitors and E2F7 siRNA transfections were performed to study the loss- and gain-function. Duel-luciferase reporter assay was performed to verify that E2F7 was one of the potential targets of miR-30c. Results: It was found that E2F7 was overexpressed in PCa cells and tissues compared with normal ones. Inhibition of E2F7 in PCa cells led to significant decreased proliferation rates, increased G1 phase percentage, and higher apoptosis rates of PCa. Duel-luciferase reporter assay showed that E2F7 was one of the targets of miR-30c. Moreover, introduction of miR-30c resulted in decreased proliferation rates, increased G1 phase percentage, and higher apoptosis rates of PCa cells while miR-30c inhibitor led to lower apoptosis rates of PCa cells. Conclusions: In conclusion, our study demonstrated that E2F7 may play a tumorigenesis promoting role of PCa cells, and that E2F7 was regulated via miR-30c, indicating that E2F7 and miR-30c might be viable therapeutic targets for PCa.

scholarly journals MiR-424-5p regulates cell cycle and inhibits proliferation of hepatocellular carcinoma cells by targeting E2F7

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242179
Author(s):  
Yichao Zhao ◽  
Chaoqian Zhu ◽  
Qing Chang ◽  
Peng Peng ◽  
Jie Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Ectopic Expression ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
G1 Phase ◽  
Reporter Assay ◽  
Hcc Cells ◽  
Dual Luciferase Reporter Assay

Objective This study aims to explore the mechanism of the miR-424-5p/E2F7 axis in hepatocellular carcinoma (HCC) and provide new ideas for targeted therapy of HCC. Methods Bioinformatics analysis was used to identify the target differentially expressed miRNA in HCC and predict its target gene. qRT-PCR was employed to verify the expression of miR-424-5p and E2F7 mRNA in HCC cells. Western blot was performed to detect the effect of miR-424-5p ectopic expression on the protein expression of E2F7. CCK-8 was used to detect proliferative activity of HCC cells and flow cytometry was carried out for analyzing cell cycle distribution. Dual luciferase reporter assay was conducted to verify the direct targeting relationship between miR-424-5p and E2F7. Results We observed that miR-424-5p was down-regulated in HCC cells. CCK-8 showed that overexpression of miR-424-5p inhibited cell proliferation, and flow cytometry showed that miR-424-5p could block cells in G0/G1 phase. E2F7 was up-regulated in HCC cells, and E2F7 overexpression could facilitate the proliferative ability of HCC cells and promote the cell cycle progressing from G0/G1 to S phase. Furthermore, dual-luciferase reporter assay indicated that miR-424-5p could directly down-regulate E2F7 expression. Analysis on cell function demonstrated that miR-424-5p inhibited the proliferation of HCC cells and blocked cell cycle at G0/G1 phase by targeting E2F7. Conclusion Our results proved that E2F7 was a direct target of miR-424-5p, and miR-424-5p could regulate cell cycle and further inhibit the proliferation of HCC cells by targeting E2F7.

scholarly journals Identification of Differentially Expressed microRNAs in Metastatic Ovarian Cancer

2020 ◽  
Author(s):  
Yang Gu ◽  
Shulan Zhang
Keyword(s):  
Cell Cycle ◽  
Ovarian Cancer ◽  
Molecular Mechanisms ◽  
Cell Function ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter Assay ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Dual Luciferase Reporter Assay

Abstract Background: The molecular mechanisms of ovarian cancer (OC) remain unclear. We sought to comprehensively identify microRNAs (miRNAs) that are aberrantly expressed in metastatic OC. Methods: Differentially expressed miRNAs were screened from six pairs of primary and metastatic OC tissues; their possible functions were analyzed and target genes were predicted by bioinformatics. Then gene expression profiling results were established by reverse transcription quantitative polymerase chain reaction and western blot. Targeting relationship between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. CCK-8, Transwell assay, scratch test and flow cytometry were used for cell function detection after miR-7-5p overexpression. Results: Twelve miRNAs and 10 target mRNAs were differentially expressed in primary and metastatic OC tissues. ITGB3, TGFβ2 and TNC correlated to miRNA function in metastatic OC. Among all 7 miRNAs, expression of hsa-miR-141-3p, hsa-miR-7-5p, hsa-miR-187-5p, hsa-miR-200a-3p, and hsa-miR-200b-3p in metastatic OC tissues was obviously lower than that in primary OC tissues ( p < 0.05). Moreover, there was a significant correlation between hsa-miR-7-5p and TGFβ2 in OC tissues. Dual-luciferase reporter assay confirmed that hsa-miR-7-5p negatively targeted TGFβ2. After miR-7-5p overexpression, the OC cell viability and invasion were reduced, the cell cycle was blocked ( p < 0.05). Conclusions: Hsa-miR-141-3p, hsa-miR-187-5p, hsa-miR-7-5p, hsa-miR-200a-3p, and hsa-miR-200b-3p expression was prominently lower in metastatic OC than in primary OC, while TGFβ2 expression was markedly increased in metastatic OC tissues. Hsa-miR-7-5p bound to TGFβ2 3’-UTR to inhibit its expression. Hsa-miR-7-5p targeted TGFβ2 to inhibit cell proliferation, invasion and cell cycle entry.

scholarly journals MiR-196b-5p regulates the proliferation of drug-resistant hepatocellular carcinoma cell lines by activating NFκB/ABCB1 signaling pathway

2020 ◽  
Vol 19 (1) ◽  
pp. 39-44
Author(s):  
Bangming Pu ◽  
Yong Cao ◽  
Yan Li ◽  
Li Tang ◽  
Jiyi Xia ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Hepg2 Cells ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Drug Resistant ◽  
Reporter Assay

Purpose: To explore the molecular function of miR-196b-5p in hepatocellular carcinoma (HCC).Methods: MiR-196b-5p expression levels in HCC tissue samples were assessed by qRT-PCR. MiR-196b-5p was knocked-down or over-expressed in HepG2 cells by transfecting the cells with plasmids expressing either a miR-196b-5p inhibitor or mimic, respectively, while cell proliferation was  assessed by MTT assay. The interaction of miR-196b-5p with target molecules was confirmed using luciferase reporter assay. Cell cycle was investigated by flow cytometry, while NFκBIA expression was assessed by western blotting.Results: MiR-196b-5p was over-expressed in HCC, and miR-196b-5p expression levels in patients with HCC were related to tumor grade. MiR-196b-5p over-expression promoted cell proliferation and colony formation and suppressed cell cycle arrest and apoptosis. The results of luciferase reporter assay showed that miR-196b-5p reduced NFκBIA expression in HepG2 cells by binding to a response element in the 3′ UTR of NFκBIA. Further investigation showed that NFκBIA interacts with NFκB1 and reduces the concentration of NFκB1 in HepG2 cells. The promoter of ATP-binding cassette sub-family B member 1 (ABCB1) was also targeted and bound by NFκB1, which altered the expression of ABCB1 in HepG2 cells.Conclusion: MiR-196b-5p regulates cell proliferation in drug-resistant HCC cell lines via activation of the NFκB/ABCB1 signaling pathway. Keywords: Hepatocellular carcinoma, miR-196b-5p, NFκBIA, NFκB1, ABCB1

scholarly journals MicroRNA-34a Attenuates Paclitaxel Resistance in Prostate Cancer Cells via Direct Suppression of JAG1/Notch1 Axis

2018 ◽  
Vol 50 (1) ◽  
pp. 261-276 ◽  
Author(s):  
Xiaobing Liu ◽  
Xing Luo ◽  
Yuqi Wu ◽  
Ding Xia ◽  
Wei Chen ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Tumor Growth ◽  
Western Blot ◽  
Target Genes ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Qrt Pcr

Background/Aims: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically centered on paclitaxel-based chemotherapy. In this study, we aimed to evaluate whether miR-34a attenuates chemoresistance to paclitaxel by regulating target genes associated with drug resistance. Methods: We used data from The Cancer Genome Atlas to compare miR-34a expression levels in prostate cancer (PC) tissues with normal prostate tissues. The effects of miR-34a inhibition and overexpression on PC proliferation were evaluated in vitro via Cell Counting Kit-8 (CCK-8) proliferation, colony formation, apoptosis, and cell-cycle assays. A luciferase reporter assay was employed to identify the interactions between miR-34a and specific target genes. To determine the effects of up-regulation of miR-34a on tumor growth and chemo-resistance in vivo, we injected PC cells overexpressing miR-34a into nude mice subcutaneously and evaluated the rate of tumor growth during paclitaxel treatment. We examined changes in the expression levels of miR-34a target genes JAG1 and Notch1 and their downstream genes via miR-34a transfection by quantitative reverse transcription PCR (qRT-PCR) and western blot assay. Results: miR-34a served as an independent predictor of reduced patient survival. MiR-34a was down-regulated in PC-3PR cells compared with PC-3 cells. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to paclitaxel while miR-34a down-regulation resulted in chemoresistance to paclitaxel in vitro. A study of gain and loss in a series of functional assays revealed that PC cells expressing miR-34a were chemosensitive. Furthermore, the overexpression of miR-34a increased the sensitivity of PC-3PR cells to chemotherapy in vivo. The luciferase reporter assay confirmed that JAG1 and Notch1 were directly targeted by miR-34a. Interestingly, western blot analysis and qRT-PCR confirmed that miR-34a inhibited the Notch1 signaling pathway. We found that miR-34a increased the chemosensitivity of PC-3PR cells by directly repressing the TCF1/ LEF1 axis. Conclusion: Our results showed that miR-34a is involved in the development of chemosensitivity to paclitaxel. By regulating the JAG1/Notch1 axis, miR-34a or its target genes JAG1 or Notch1 might serve as potential predictive biomarkers of response to paclitaxel-based chemotherapy and/or therapeutic targets that will help to overcome chemoresistance at the mCRPC stage.

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 CDK6 and miR-320c Co-Regulate Chondrocyte Catabolism Through NF-κB Signaling Pathways

2018 ◽  
Vol 51 (2) ◽  
pp. 909-923 ◽  
Author(s):  
Hao Sun ◽  
Zhiyu Huang ◽  
Peihui Wu ◽  
Zongkun Chang ◽  
Weiming  Liao ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Late Stage ◽  
Chondrogenic Differentiation ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Inflammatory Factor ◽  
Reporter Assay ◽  
Middle Stage ◽  
Qrt Pcr

Background/Aims: Cyclin-dependent kinase 6 (CDK6) regulates inflammatory response and cell differentiation. This study sought to determine whether CDK6 and miR-320c co-regulate chondrogenesis and inflammation. Methods: Utilizing quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC), CDK6 and miR-320c expression were assessed in a micromass culture of human bone mesenchymal stem cells that underwent chondrogenesis in vitro as well as in chondrocytes from E16.5 mouse forelimbs. Normal chondrocytes were transfected with miR-320c mimic, miR-320c inhibitor, or CDK6-siRNA. Luciferase reporter assay results confirmed that miR-320c directly targets CDK6 by interacting with the 3′-untranslated region (3′-UTR) of its mRNA. qRT-PCR, Western blotting, and Cell Counting Kit-8 were subsequently used to evaluate the effects of miR-320c overexpression and CDK6 inhibition on inflammatory factor expression, as well as to investigate the effects of NF-kB and MAPK signaling pathway activation on IL-1β-induced chondrocyte inflammation. Results: Our results show that miR-320c expression increased during the middle stage and decreased during the late stage of hBMSC chondrogenic differentiation. In contrast, CDK6 expression decreased during the middle stage and increased during the late stage of hBMSC chondrogenic differentiation. Moreover, CDK6 expression increased in severe OA cartilage and in hypertrophic chondrocytes of mouse forelimbs at E16.5. Results of the luciferase reporter assay showed that miR-320c modulated CDK6 expression by binding to the 3′-UTR of its mRNA. miR-320c overexpression and CDK6 inhibition repressed IL-1β-induced expression of inflammatory factors and regulated the NF-kB signaling pathway. Conclusion: CDK6 and miR-320c co-regulate hBMSC chondrogenesis and IL-1β-induced chondrocyte inflammation through the NF-kB signaling pathway, suggesting that miR-320c and CDK6 inhibitors can be used to repress catabolism in human chondrocytes.

scholarly journals Long Non­coding RNA SNHG16 Functions as Tumor Activator by Sponging hsa-miR-373-3p to Regulate TGFBR2/SMAD Pathway in Prostate Cancer

2020 ◽  
Author(s):  
WuBin Weng ◽  
ChangMing Liu ◽  
GuoMin Li ◽  
QiongFang Ruan ◽  
HuiZhang Li ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
Gleason Score ◽  
Cell Apoptosis ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Smad3 Expression ◽  
Dual Luciferase Reporter Assay

Abstract Background: Long noncoding RNAs (lncRNAs) are one of the major causes of tumorigenesis. However, the roles and mechan­­isms of lncRNA SNHG16 in prostate cancer (PCa) remain unknown. The purpose of this study was to elucidate the mech­­anisms of lncRNA SNHG16 in the proliferation and metastasis of human PCa cells.Material and Methods: First, the quantitative polymerase chain reaction (qPCR) was used to measure SNHG16 expression in PCa tissues and adjacent normal tissues (n=80). Down-regulate and over-express SNHG16 in human PCa DU-145 cell. Then cell proliferation was detected by CCK8 assay, cell apoptosis was analyzed by flow cytometry, cell migration were determined by wound healing, and cell invasion was examined by transwell. Western blot assays were used to examine the expression of the TGFBR2, c-MYC, E2F4, SMAD2, p-SMAD2, SMAD3, and p-SMAD3. Second, the targeting relationship between SNHG16 and hsa-miR-373-3p was verified by dual-luciferase reporter assay and rescue experiments. Third, the targeting relationship between hsa-miR-373-3p and TGFBR2 was verified by dual-luciferase reporter assay and rescue experiments. Results: The expression of SNHG16 was significant increase in PCa tissues (Z=-8.405, P<0.001), and with significant correlation with patient's age (<60 and ≥60 years old, P=0.007). Silencing SNHG16 inhibited DU-145 cell proliferation, migration, and invasion, while induced cell apoptosis significantly (P<0.01, respectively). Overexpressing SNHG16 promoted cell proliferation, migration and invasion, and reduced cell apoptosis rate (P<0.05, respectively). SNHG16 overexpression observably increased TGFBR2, c-MYC, E2F4, p-SMAD2, and p-SMAD3 expression (P<0.001, respectively), but SNHG16 inhibition was opposite. However, SNHG16 did not regulate SMAD2 and SMAD3 expression. Next, hsa-miR-373-3p was found down-regulated in PCa tissues (Z=-8.344, P<0.001), and the down-regulation of hsa-miR-373-3p were closely linked to Gleason score (Gleason score: <7 and >7, P = 0.024). Hsa-miR-373-3p expression of hsa-miR-373-3p was negatively correlated with SNHG16 (r=-0.544, P<0.001). The result of dual-luciferase reporter assay and qPCR test revealed that hsa-miR-373-3p was a target of SNHG16. Hsa-mir-373-3p inhibitor could rescue sh-SNHG16-inhibited cell proliferation, migration and invasion by promoting TGFBR2, C-MYC, E2F4, P-Smad2, and P-smad3 expression. Finally, we found that TGFBR2 may be the target gene of hsa-mir-373-3p through TargetScan and starbase. Further research found that TGFBR2 was markedly up-regulated in PCa tissues (Z=-5.945, P<0.001), and the expression of TGFBR2 was negatively correlated with hsa-miR-373-3p (r=-0.627, P<0.001). Dual-luciferase reporter assay and qPCR test showed that TGFBR2 was a target of hsa-miR-373-3p. TGFBR2 knockdown could inhibit hsa-mir-373-3p inhibitor-induced cell proliferation, migration and invasion, and reversed the effect of hsa-mir-373-3p inhibitor on cell apoptosis. Based on the data, sh-TGFBR2 partially disabled hsa-mir-373-3p inhibitor effect. Conclusion: LncRNA SNHG16 might act as a ceRNA to regulate the proliferation and migration of DU-145 cells by modulating the hsa-miR-373-3p/TGFBR2/SMAD axis.

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 Brucine Inhibits Proliferation of U87 Glioblastoma Cells by Targeting the G-quadruplexes in the c-Myb Promoter

2020 ◽  
Author(s):  
Qiaochu Liu ◽  
Di Huo ◽  
Qunhui Wang ◽  
Chuanqi Lv ◽  
Ziqiang Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Promoter Region ◽  
Luciferase Reporter Assay ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Ionization Mass ◽  
Reporter Assay ◽  
U87 Cells ◽  
Dual Luciferase Reporter Assay

Abstract Background: The proto-oncogene c-Myb plays an important role in the proliferation of cells and its upregulation affects the development of glioblastomas. G-quadruplexes are secondary structures of DNA or RNA that usually form in the promoter region of oncogenes, including c-Myb, and regulate the expression of these genes. The traditional Chinese medicine brucine is a ligand of G-quadruplexes located in the promoter region of c-Myb. In this study, the U87 cell line was used both in vitro and in vivo to investigate the therapeutic effect and mechanism of action of brucine. Methods: MTT assay and flow cytometry were used to determine the effect of brucine on the cell cycle, viability, and apoptosis of U87 cells. The effects of brucine on transcription and expression of c-Myb were determined through RT-PCR and western blotting. Dual-luciferase reporter assay and electrospray ionization-mass spectrometry were used to investigate whether brucine acts directly and binds G-quadruplexes in the promoter region of c-Myb, respectively. Results: The results showed that brucine suppressed the growth of U87 cells in vitro by arresting the cell cycle and reducing the expression of c-Myb. Through the dual luciferase reporter assay, brucine was found to inhibit the expression of c-Myb by targeting the guanine-rich sequence that forms G-quadruplexes in the c-Myb promoter. Moreover, U87 tumors were suppressed by brucine in a tumor xenograft nude mice model. Conclusion: The findings of the study indicate that brucine is a potentially effective medicine for treatment of glioblastomas.

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.

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