scholarly journals Overexpression of miRNA-21 Promotes the Proliferation and Invasion in Hepatocellular Carcinoma Cells via Suppressing SMAD7

2019 ◽  
Vol 18 ◽  
pp. 153303381987868 ◽  
Author(s):  
Yan Wang ◽  
Ping Zhang ◽  
Mei Yuan ◽  
Xiaojie Li
Keyword(s):  
Hepatocellular Carcinoma ◽  
Family Member ◽  
Reporter Gene ◽  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Hepatoma Cell ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Proliferation And Invasion

Purpose: This study aimed to explore the molecular mechanism of microRNA-21 and smad family member 7 in hepatocellular carcinoma. Method: A total of 57 participants were divided into control group (healthy participants, n = 10) and hepatocellular carcinoma group (hepatocellular carcinoma patients, n = 37). The expression of microRNA-21 levels were first detected in these two groups. Cell transfection was performed on hepatoma cell lines, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assay to reveal proliferation and invasion ability. Furthermore, the relation between microRNA-21 and smad family member 7 was revealed by luciferase reporter gene and RNA immunoprecipitation assay. Finally, a transplantation tumor model of breast cancer in mice was constructed. Results: The serum indicators including α-alanine aminotransferase, aspartate aminotransferase, and albumin were differentially expressed between hepatocellular carcinoma group and control group. Compared to the control group, there was a high expression of microRNA-21 in hepatocellular carcinoma group. Low expression of microRNA-21 inhibited the proliferation and invasion of HepG2.2.15 and Huh7-1.3 cells. Luciferase reporter gene and RNA innumoprecipitation assay showed that smad family member 7 was the target gene of microRNA-21. Moreover, mice model analysis showed that microRNA-21 might regulate the growth of the transplanted tumors in mice by targeting smad family member 7. Conclusion: The upregulated microRNA-21 might participate in the proliferation and migration in cells of hepatocellular carcinoma via suppression of smad family member 7. Furthermore, serum indicators such as alanine aminotransferase, aspartate aminotransferase, and albumin might be used as serum diagnostic markers for hepatocellular carcinoma.

Interfering with Long Non-Coding RNA FBXL19-AS1 Inhibits the Proliferation, Invasion and Migration of Hepatoma Carcinoma Cells via Targeting miR-541-5p

2021 ◽  
Vol 11 (11) ◽  
pp. 2120-2127
Author(s):  
Weijun Lu ◽  
Qun Wang ◽  
Changbo Fu
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cell Migration ◽  
Cell Lines ◽  
Reporter Gene ◽  
Malignant Tumors ◽  
Human Life ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and the morbidity and mortality of HCC rate in the first few malignant tumors, seriously threatening the safety of human life. LncRNA is a hot topic in tumor research in recent years. The abnormal expression of LncRNA FBXL19-AS1 and its potential target as a tumor diagnostic marker have been confirmed in colon cancer, breast cancer and lung cancer, etc. However, the study on LncRNA FBXL19-AS1 in HCC has not been reported. Rt-qPCR was used to detect the expression of FBXL19-AS1 and miR-541-5p in HCC cell lines, and luciferase reporter gene was used to detect whether there were binding sites between LncRNA FBXL19-AS1 and miR-541-5p. Interfered with FBXL19-AS1 and overexpressed miR-541-5p were detected by cell transfection. Then CCK-8 and colony formation assay were used to detect cell viability and cell proliferation. Wound healing detected the rate of cell migration and Transwell detected the rate of cell invasion. Western blot was used to detect the expression of proteins related to cell migration and invasion. The expression of FBXL19-AS1 in HCC cell lines was significantly higher than that in normal liver cells (LO2). Moreover, FBXL19-AS1 can promote HCC cell proliferation, migration and invasion. Luciferase reporter gene confirmed the binding site between LncRNA FBXL19-AS1 and miR-541-5p. After interfering with the expression of FBXL19-AS1, miR-541-5p was significantly increased. Subsequently, overexpression of miR-541-5p can inhibit the expression of lncRNA FBXL19-AS11 and promote proliferation, migration and invasion of hepatocellular carcinoma. So we can conclude that lncRNA FBXL19-AS1 promoted the proliferation, migration and invasion of HCC cells through targeting miR-541-5p.

scholarly journals Transcriptional Regulation of CYP3A4/2B6/2C9 Mediated via Nuclear Receptor PXR by Helicid and Its Metabolites

2015 ◽  
Vol 2015 ◽  
pp. 1-9
Author(s):  
Qun Chen ◽  
Hai-tang Xie ◽  
Yan Li ◽  
Guo Wang ◽  
Zhe Xu ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Receptor ◽  
Reporter Gene ◽  
Negative Control Group ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Expression Plasmid

Objective. This study aims at establishing and validating an in vitro system to screen drug inducers of CYPs mediated via hPXR, as well as studying transcriptional regulation of CYPs mediated via hPXR by helicid and its two metabolites.Methods. Cloning the nuclear receptor hPXR and the promoters of CYP3A4, CYP2B6, CYP2C9, and inserting the trans-element to the upstream of firefly luciferase reporter gene of the pGL4.17 vectors, then cotransfecting the report vectors and hPXR expression plasmid to HepG2 cell line. After 24 hours, the transfected cells were treated with helicid (0.004, 0.04, and 0.4 μmol/L) and its metabolite I and metabolite II (0.0004, 0.004, and 0.04 μmol/L) for 48 h, while rifampin (10 μmol/L) was included as the positive control and 0.1% DMSO as the negative control group. Cells were lysized and luciferase activity was determined using a dual luciferase reporter assay kit.Results. Helicid and its metabolites did not significantly increase promoter activities of CYP3A4, CYP2B6, and CYP2C9 in HepG2 cells transfected with PXR expression plasmid (P>0.05).Conclusion. PXR-expressed CYP3A4, CYP2B6, and CYP2C9 dual luciferase reporter gene platforms were successfully established, and helicid and its metabolites I, II do not significantly induce the transcription of CYP3A4, CYP2B6, and CYP2C9.

scholarly journals Hsa_circ_0011385 knockdown represses cell proliferation in hepatocellular carcinoma

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Chuangye Ni ◽  
Shikun Yang ◽  
Yang Ji ◽  
Yunfei Duan ◽  
Wenjie Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

AbstractCircular RNAs (circRNAs), continuous loops of single-stranded RNA, regulate gene expression during the development of various cancers. However, the function of circRNAs in hepatocellular carcinoma (HCC) is rarely discussed. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the mRNA levels of circ_0011385, miR-361-3p, and STC2 in 96 pairs of HCC tissues (tumor tissues and adjacent normal tissues), HCC cell lines, and L02 (human normal liver cell line) cells. The relationships between circ_0011385 expression and clinical features of HCC were evaluated. Functional experiments in vitro or in vivo were used to evaluate the biological function of circ_0011385. Bioinformatics analysis was performed to predict miRNAs and mRNAs sponged by circ_0011385. RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assays were used to elucidate the interactions among circ_0011385, miR-361-3p, and STC2 (stanniocalcin 2). ChIP and dual-luciferase reporter gene assays were used to identify the upstream regulator of circ_0011385. High expression of circ_0011385 was observed in HCC tissues and cell lines and was significantly associated with tumor size, TNM stage, and prognosis. In addition, inhibition of circ_0011385 expression prevented the proliferation of HCC cells in vitro and in vivo. Circ_0011385 sponged miR-361-3p, thereby regulating the mRNA expression of STC2. In addition, the transcription of circ_0011385 was regulated by SP3. Circ_0011385 knockdown suppressed cell proliferation and tumor activity in HCC. Circ_0011385 may therefore serve as a new biomarker in the diagnosis and treatment of HCC.

scholarly journals miR-16-5p Regulates PTPN4 and Affects Cardiomyocyte Apoptosis and Autophagy Induced by Hypoxia/Reoxygenation

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Zheng Cao ◽  
Jinglan Liu ◽  
Zhanqing Zhao ◽  
Qiao Wang
Keyword(s):  
Reporter Gene ◽  
Cell Apoptosis ◽  
Cell Damage ◽  
Negative Control Group ◽  
Negative Control ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Hypoxia Reoxygenation

Objectives. To explore the effects of miR-16-5p and PTPN4 on the apoptosis and autophagy of AC16 cardiomyocytes after hypoxia/reoxygenation treatment. Methods. AC16 cells were divided into the control group (NC), hypoxia/reoxygenation group (H/R), knockdown miR-16-5p negative control group (NC inhibitor), knockdown miR-16-5p group (miR-16-5p inhibitor), overexpression miR-16-5p negative control group (NC mimics), overexpression miR-16-5p group (miR-16-5p mimics), silent PTPN4 negative control group (sh-NC), silent PTPN4 group (sh-PTPN4), and silent PTPN4 + knockdown miR-16-5p group (sh-PTPN4 + miR-16-5p inhibitor). Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting (WB) were used to measure the expression level of miR-16-3p, miR-16-5p, protein tyrosine phosphatase nonreceptor type 4 (PTPN4), and autophagy-related proteins (beclin-1, LC3 II/I, and P26) in AC16 cells. The apoptosis level of AC16 cells in each group was measured by flow cytometry and TUNEL. The dual-luciferase reporter gene experiment was also used to verify the targeting relationship between miR-16-5p and PTPN4. Results. After H/R treatment, the levels of myocardial injury markers including LDH and CK-MB in AC16 cells were increased significantly ( P < 0.05 ), and the levels of cell apoptosis and autophagy also increased significantly ( P < 0.05 ). The level of miR-16-3p in AC16 cells did not change significantly after H/R treatment, whereas the level of miR-16-5p was increased significantly ( P < 0.05 ). After miR-16-5p was knocked down, the levels of LDH and CK-MB in AC16 cells treated with H/R were significantly reduced ( P < 0.05 ), and the rates of cell apoptosis and autophagy were also significantly reduced ( P < 0.05 ). miR-16-5p negatively regulated the expression level of PTPN4 protein in AC16 cells ( P < 0.05 ), and the dual-luciferase reporter gene experiment confirmed that PTPN4 was the downstream target of miR-16-5p. Silencing of PTPN4 significantly increased the damage of AC16 cells induced by H/R treatment ( P < 0.05 ), but simultaneously inhibiting the expression of PTPN4 and miR-16-5p reversed the protective effect of miR-16-5p knockdown on AC16 cells ( P < 0.05 ). Conclusions. The expression of miR-16-5p is upregulated in AC16 cells after H/R treatment and the knockdown which can protect AC16 cells from H/R-induced cell damage that may be due to its regulation on the expression of PTPN4.

scholarly journals Construction of Novel lncRNA–miRNA–mRNA Network Associated With Recurrence and Identification of Immune-Related Potential Regulatory Axis in Hepatocellular Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Tian Zhan ◽  
Xiang Gao ◽  
Guoguang Wang ◽  
Fan Li ◽  
Jian Shen ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Correlation Analysis ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Immune Infiltration ◽  
Gene Assay

Hepatocellular carcinoma (HCC) is one of the most common malignant diseases globally. Despite continuous improvement of treatment methods, high postoperative recurrence rate remains an urgent problem. In order to determine the mechanism underlying recurrence of liver cancer and identify prognostic genes, data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were integrated and analyzed. Differentially expressed genes (DEGs) between HCC tissue and normal liver tissue were identified, and a protein–protein interaction network was constructed to find hub genes. Clinical correlation analysis and disease-free survival (DFS) analysis were performed using the R language and GEPIA to identify relapse-related genes. Correlation analysis was used to identify a potential regulatory axis. Dual-luciferase reporter gene assay was used to confirm the reliability of the long non-coding RNA (lncRNA)–microRNA (miRNA)–mRNA regulatory axis. Immune infiltration analysis was performed using the TIMER database. Correlations between immune gene markers and ASF1B were verified using quantitative real-time polymerase chain reaction (RT-qPCR). In this work, we found that nine lncRNAs and five mRNAs were significantly overexpressed in HCC tissues from patients with recurrence. SNHG3, LINC00205, ASF1B, AURKB, CCNB1, CDKN3, and DTL were also closely related to HCC grade and stage. Survival analysis showed that these seven DEGs were significantly correlated with poor DFS. Correlation analysis identified SNHG3–miR-214-3p–ASF1B as a potential regulatory axis. Dual-luciferase reporter gene assay showed that SNHG3 and ASF1B directly bound to miR-214-3p. ASF1B was negatively regulated by miRNA-214-3p, and overexpression of SNHG3 could inhibit the expression of miRNA-214-3p. In addition, ASF1B was positively correlated with immune infiltration. A reduction in ASF1B could markedly inhibit the expression of CD86, CD8, STAT1, STAT4, CD68, and PD1 in HCC cells. Flow cytometry showed that SNHG3 promoted the PD-1 expression by regulating ASF1B. Meanwhile, elevated ASF1B predicted poor prognosis of HCC patients in subgroups with decreased B cells, CD8+ T cells, or neutrophils, and those with enriched CD4+ T cells. In conclusion, we found that a novel lncRNA SNHG3/miR-214-3p/ASF1B axis could promote the recurrence of HCC by regulating immune infiltration.

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

Planta Medica ◽  
2009 ◽  
Vol 75 (09) ◽  
Author(s):  
S Vogl ◽  
P Picker ◽  
N Fakhrudin ◽  
A Atanasov ◽  
E Heiß ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plant Extracts ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

Investigation of Targeting Relationship between Micro-Rna-22 and Vegfr3 in Lung Squamous Cell Carcinoma

2020 ◽  
Vol 23 ◽  
Author(s):  
Zheng Dong ◽  
Qing-Hua Xu ◽  
Yuan-Bin Zhu ◽  
Yong-Feng Wang ◽  
Jie Xiong ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Lung Squamous Cell Carcinoma ◽  
Luciferase Reporter ◽  
Control Group ◽  
Vascular Endothelial ◽  
Luciferase Reporter Gene ◽  
Endothelial Growth Factor

Aims : The present study explored the clinical significance of microRNA-22 (miR-22) expression in lung squamous cell carcinoma and to explore the targeting relationship with vascular endothelial growth factor receptor 3 (VEGFR3). Methods: A total of 49 patients with lung squamous cell carcinoma who underwent surgical treatment was selected. The expression of miR-22 was detected by fluorescence quantitative real-time PCR (qPCR), the expression of VEGFR3 was detected by Western blotting assays, and D240 labeled microlymphatic vessels density (MLVD) was detected immunohistochemistry (IHC). Lung squamous cell carcinoma cell line SK-MES-1 was selected and the targeting relationship between miR-22 and VEGFR3 was analyzed by double luciferase reporter gene assay. Western blotting assays were used to detect the expression of vascular endothelial growth factor-D (VEGF-D) and D240 in the blank control group, empty vector transfection group, miR-22 transfection group, miR-22 and VEGFR3 co-transfection group. Results: The expression range of miR-22 in lung squamous cell carcinoma was 0.8-3.5. The expression of miR-22 in lung squamous cell carcinoma was significantly different by tumor maximum diameter, lymph node metastasis, vascular invasion and TNM stage. The expression of miR-22 was linked to survival time. There was a negative correlation between miR-22 and VEGFR3, miR-22 and MLVD. Double luciferase reporter gene assays showed that miR-22 reduced the luciferase activity of pGL3-VEGFR3-WT transfected cells. Compared with the control group, the expression of VEGF-D and D2-40 in the miR-22 transfection group was significantly decreased. However, VEGF-D and D240 in the miR-22 and VEGFR3 cotransfection group reversed the changes. Conclusion: We assumed that the abnormal expression of miR-22 in lung squamous cell carcinoma may be involved in the development and progression of lung squamous cell carcinoma. MiR-22 negatively regulated the target gene VEGFR3 to mediate lymphangiogenesis. The expression of miR-22 may also be linked to the prognosis of the disease.

scholarly journals KLF7/VPS35 axis contributes to hepatocellular carcinoma progression through CCDC85C-activated β-catenin pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yarong Guo ◽  
Bao Chai ◽  
Junmei Jia ◽  
Mudan Yang ◽  
Yanjun Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Luciferase Reporter ◽  
Aberrant Expression ◽  
Proliferation And Invasion ◽  
Hcc Cells

Abstract Objective Dysregulation of KLF7 participates in the development of various cancers, but it is unclear whether there is a link between HCC and aberrant expression of KLF7. The aim of this study was to investigate the role of KLF7 in proliferation and migration of hepatocellular carcinoma (HCC) cells. Methods CCK8, colony growth, transwell, cell cycle analysis and apoptosis detection were performed to explore the effect of KLF7, VPS35 and Ccdc85c on cell function in vitro. Xenografted tumor growth was used to assess in vivo role of KLF7. Chip-qPCR and luciferase reporter assays were applied to check whether KLF7 regulated VPS35 at transcriptional manner. Co-IP assay was performed to detect the interaction between VPS35 and Ccdc85c. Immunohistochemical staining and qRT-PCR analysis were performed in human HCC sampels to study the clinical significance of KLF7, VPS35 and β-catenin. Results Firstly, KLF7 was highly expressed in human HCC samples and correlated with patients’ differentiation and metastasis status. KLF7 overexpression contributed to cell proliferation and invasion of HCC cells in vitro and in vivo. KLF7 transcriptional activation of VPS35 was necessary for HCC tumor growth and metastasis. Further, co-IP studies revealed that VPS35 could interact with Ccdc85c in HCC cells. Rescue assay confirmed that overexpression of VPS35 and knockdown of Ccdc85c abolished the VPS35-medicated promotion effect on cell proliferation and invasion. Finally, KLF7/VPS35 axis regulated Ccdc85c, which involved in activation of β-catenin signaling pathway, confirmed using β-catenin inhibitor, GK974. Functional studies suggested that downregulation of Ccdc85c partly reversed the capacity of cell proliferation and invasion in HCC cells, which was regulated by VPS35 upregulation. Lastly, there was a positive correlation among KLF7, VPS35 and active-β-catenin in human HCC patients. Conclusion We demonstrated that KLF7/VPS35 axis promoted HCC cell progression by activating Ccdc85c-medicated β-catenin pathway. Targeting this signal axis might be a potential treatment strategy for HCC.

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.

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