scholarly journals Polymorphisms in the osteopontin promoter affect its transcriptional activity

2004 ◽  
Vol 20 (1) ◽  
pp. 87-96 ◽  
Author(s):  
Francesca Giacopelli ◽  
Renato Marciano ◽  
Angela Pistorio ◽  
Paolo Catarsi ◽  
Silvana Canini ◽  
...  
Keyword(s):  
Cell Lines ◽  
Reporter Gene ◽  
Molecular Mechanisms ◽  
Association Studies ◽  
Susceptibility Gene ◽  
Organ Damage ◽  
Luciferase Reporter ◽  
Regulation Of Transcription ◽  
Luciferase Reporter Gene ◽  
Encoding Gene

Understanding the molecular mechanisms that underlie regulation of transcription of the human osteopontin encoding gene (OPN) may help to clarify several processes, such as fibrotic evolution of organ damage, tumorigenesis and metastasis, and immune response, in which OPN overexpression is observed. With the aim to evaluate variants with functional effect on transcription, we have analyzed the promoter region and focused our investigation on three common variants present in the first 500 bp upstream of the transcription start site. Transfection of constructs carrying the four most frequent haplotypes relative to variants at −66, −156, and −443 fused to the luciferase reporter gene in a panel of different cell lines showed that one haplotype conferred a significantly reduced level of reporter gene expression in all tested cell lines. We describe that the −66 polymorphism modifies the binding affinity for the SP1/SP3 transcription factors, the −156 polymorphism is included in a yet uncharacterized RUNX2 binding site, and the −443 polymorphism causes differential binding of an unknown factor. The finding of differential effects of various combination of variants in haplotypes may contribute to explain data of association studies reported in several already published articles. Future association studies using haplotypes instead of single OPN variants will allow to achieve more accurate results referable to differential expression of OPN in several common diseases, in which OPN is considered a candidate susceptibility gene.

scholarly journals Hsa-miR-105-1 Regulates Cisplatin-Resistance in Ovarian Carcinoma Cells by Targeting ANXA9

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Xinxin Kou ◽  
Hui Ding ◽  
Lei Li ◽  
Hongtu Chao
Keyword(s):  
Ovarian Carcinoma ◽  
Cell Lines ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Cisplatin Resistance ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Qrt Pcr ◽  
Gene Assay ◽  
Increased Sensitivity

Purpose. Cisplatin is one of the most effective drugs for treating ovarian carcinoma (OC), which is among the most lethal types of carcinoma. However, the chemoresistance to cisplatin that develops over time leads to a poor clinical outcome for many OC patients. Therefore, it is necessary to clearly understand the molecular mechanisms of chemoresistance. In this study, we examined how Hsa-miR-105-1 functions in cisplatin-resistant OC cells. Methods. The levels of Hsa-miR-105-1 expression in cisplatin-sensitive and resistant OC cell lines were detected by qRT-PCR. The target gene of Hsa-miR-105-1 was predicted by using the TargetScan and Starbase databases and verified by the double luciferase reporter gene assay. The target gene of Hsa-miR-105-1 was identified as ANXA9, and ANXA9 expression was evaluated by qRT-PCR, western blotting, and immunofluorescence. To validate the function of Hsa-miR-105-1 in OC cells, we silenced or overexpressed Hsa-miR-105-1 in cisplatin-sensitive or resistant OC cell lines, respectively. Furthermore, the expression levels of several apoptosis-related proteins, including P53, P21, E2F1, Bcl-2, Bax, and caspase-3, were examined by western blot analysis. Results. The levels of Hsa-miR-105-1 expression were abnormally downregulated in cisplatin-resistant OC cells, while ANXA9 expression was significantly upregulated in those cells. Treatment with an Hsa-miR-105-1 inhibitor promoted the expression of ANXA9 mRNA and protein, enhanced the resistance to cisplatin, and attenuated the cell apoptosis induced by cisplatin in cisplatin-sensitive OC cells. Moreover, treatment with Hsa-miR-105-1 mimics inhibited ANXA9 expression, which further increased the levels of P53, P21, and Bax expression and decreased the levels of E2F1 and Bcl-2 expression, finally resulting in an increased sensitivity to cisplatin in cisplatin-resistant OC cells. Conclusion. We found that a downregulation of Hsa-miR-105-1 expression enhanced cisplatin-resistance, while an upregulation of Hsa-miR-105-1 restored the sensitivity of OC cells to cisplatin. The Hsa-miR-105-1/ANXA9 axis plays an important role in the cisplatin-resistance of OC cells.

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 Identification of a cAMP response element within the glucose- 6-phosphatase hydrolytic subunit gene promoter which is involved in the transcriptional regulation by cAMP and glucocorticoids in H4IIE hepatoma cells

1999 ◽  
Vol 338 (2) ◽  
pp. 457-463 ◽  
Author(s):  
Dieter SCHMOLL ◽  
Christina WASNER ◽  
Carolyn J. HINDS ◽  
Bernard B. ALLAN ◽  
Reinhard WALTHER ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Hepatoma Cells ◽  
Luciferase Reporter ◽  
Regulation Of Transcription ◽  
Dibutyryl Camp ◽  
Luciferase Reporter Gene ◽  
Camp Response Element ◽  
Response Element

The expression of a luciferase reporter gene under the control of the human glucose 6-phosphatase gene promoter was stimulated by both dexamethasone and dibutyryl cAMP in H4IIE hepatoma cells. A cis-active element located between nucleotides -161 and -152 in the glucose 6-phosphatase gene promoter was identified and found to be necessary for both basal reporter-gene expression and induction of expression by both dibutyryl cAMP and dexamethasone. Nucleotides -161 to -152 were functionally replaced by the consensus sequence for a cAMP response element. An antibody against the cAMP response element-binding protein caused a supershift in gel-electrophoretic-mobility-shift assays using an oligonucleotide probe representing the glucose 6-phosphatase gene promoter from nucleotides -161 to -152. These results strongly indicate that in H4IIE cells the glucose 6-phosphatase gene-promoter sequence from -161 to -152 is a cAMP response element which is important for the regulation of transcription of the glucose 6-phosphatase gene by both cAMP and glucocorticoids.

scholarly journals Circ_0000005 Facilitates Proliferation, Apoptosis, Migration and Invasion of Acute Myeloid Leukemia Cells via Modulating miR-139-5p/Tspan3 Axis

2020 ◽  
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Changcheng Zheng ◽  
Xiaoyu Zhu ◽  
Xiang Wan ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Qrt Pcr ◽  
Gene Assay

Abstract Background: Accumulating circular RNAs (circRNAs) are reported to be abnormally expressed in diverse cancers, hematologic malignancies included. This study aimed to investigate the biological function and underlying mechanisms of circ_0000005 in acute myeloid leukemia (AML).Materials and methods: Bone marrow samples were enrolled from AML patients with normal samples as controls. Circ_0000005, miR-139-5p and tetraspanin 3 (Tspan3) were detected by qRT-PCR and Western blot, respectively. AML cell lines (KG1 and HL60) were used as cell models. CCK-8, Transwell and flow cytometry assays were adopted to study the biological functions of circ_0000005 on AML cells in vitro. The interrelation between circ_0000005 and miR-139-5p was detected by bioinformatics, qRT-PCR, luciferase reporter gene assay, RNA pull-down assay, and RNA-binding protein immunoprecipitation (RIP) assays. Ultimately, Western blot, qRT-PCR, luciferase reporter gene assay were adopted to corroborate the interrelation between miR-139-5p and its target Tspan3. Results: Circ_0000005 was demonstrably elevated in both AML clinical samples and cell lines. Circ_0000005 overexpression promoted the viability, migration and invasion of AML cells, and repressed the apoptosis of AML cells, while silencing circ_0000005 showed opposite biological effects. Circ_0000005 interacted with miR-139-5p and repressed its expression, and Tspan3 was proved to be negatively regulated by miR-139-5p. Circ_0000005 could promote the expression of Tspan3 via repressing miR-139-5p, and the oncogenic functions of circ_0000005 were dependent on its regulatory function on miR-139-5p/Tspan3 axis.Conclusion: Circ_0000005 facilitates the malignant phenotypes of AML cells via miR-139-5p/Tspan3 axis. Circ_0000005 may serve as a potential therapeutic target in AML.

scholarly journals Long non-coding RNA BCYRN1 exerts an oncogenic role in colorectal cancer through regulating miR-204-3p/KRAS axis

2020 ◽  
Author(s):  
Liu Yang ◽  
Yinan Zhang ◽  
Jun Bao ◽  
Ji-Feng Feng
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Reporter Gene ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Luciferase Reporter Gene ◽  
Tumor Tissues ◽  
Dual Luciferase Reporter Assay

Abstract Background: It has been well documented that long non-coding RNAs (lncRNAs) regulate numerous characteristics of cancer, including proliferation, migration, metastasis, apoptosis, even metabolism. LncRNA BCYRN1 (BCYRN1) is a newly identified brain cytoplasmic lncRNA with 200-nucleotide, which was discovered highly expressed in tumor tissues of cancer, including hepatocellular carcinoma, gastric cancer and lung cancer. However, the roles of BCYRN1 in colorectal cancer (CRC) remain obscure. This study was designed to reveal the acts of BCYRN1 in the occurrence and progression of CRC.Methods: RT-PCR was used to detect the expression level of BCYRN1 in tumor tissues and CRC cell lines. Knock down BCYRN1 in CRC cells, evaluate cell proliferation changes by CCK-8 test, EdU test, and Ki-67 and PCNA expression; evaluate cell migration and invasion changes by wound healing assay, Transwell assay and invasion-related protein expression . Through flow cytometry analysis to assess whether BCYRN1 regulates apoptosis of CRC cells. The dual luciferase reporter gene detects the competitive binding of BCYRN1 to miR-204-3p. In vivo experiments to evaluate the effect of BCYRN1 on tumor development. TargetScan analysis and dual luciferase reporter gene detect the target gene of miR-204-3p. Rescue experiments verified the effect of BCYRN1 on CRC by regulating the effect of miR-204-3p on KRAS.Results: We found that compared with normal tissues and human intestinal epithelial cells (HIECs), BCYRN1 levels were significantly increased in tumor tissues and cell lines of CRC. We further determined that knockdown of BCYRN1 inhibited proliferation, migration, invasion, and promoted apoptosis of CRC cells. In addition, bioinformatics analysis and dual luciferase reporter assay showed that BCYRN1 served as a competitive endogenous RNA (ceRNA) to regulate the development of CRC through competitively binding to miR-204-3p. Further studies proved that overexpression of miR-204-3p reversed the effects of BCYRN1 on CRC. Next, TargetScan analysis and dual luciferase reporter assay indicated that KRAS was a target gene of miR-204-3p and negatively regulated by miR-204-3p. A series of rescue experiments showed that BCYRN1 affected the occurrence and development of CRC by regulating the effects of miR-204-3p on KRAS. In addition, tumorigenic experiments in CRC model mice confirmed that down-regulated BCYRN1 effectively inhibited tumor growth. Conclusions: Our findings suggested that BCYRN1 plays a carcinogenic role in CRC by regulating the miR-204-3p/KRAS axis.

scholarly journals CircMED13L_012 promotes lung adenocarcinoma progression by upregulation of MAPK8 mediated by miR-433-3p

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wenshu Chen ◽  
Guanying Zheng ◽  
Jianyuan Huang ◽  
Lihuan Zhu ◽  
Wujin Li ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Nsclc Cell ◽  
Disease Stage ◽  
Luciferase Reporter ◽  
Clinical Samples ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

Abstract Background Metastasis and disease refractoriness remain as major challenges for non-small cell lung cancer (NSCLC) treatment and understanding the underlying molecular mechanisms is of scientific and clinical value. Therefore, in this study, we aimed to explore the effects of circMED13L_012 on the proliferation, migration, invasion and drug-resistance of NSCLC tumor cells. Methods In this study, we utilized clinical samples and NSCLC cell lines to explore the association between circMED13L_012 expressions and tumor cell metastasis and chemo resistance. CCK8 and transwell assay were conducted to explore the impact of circMED13_012 on NSCLC tumor proliferation and migrative capabilities. Dual-luciferase reporter gene assay was conducted to validate the circMED13L_012 interaction network. Results Our results demonstrated that circMED13L_012 exhibited significantly elevated average level in our clinical samples of NSCLC, compared with normal tissues. circMED13L_012 level was positively correlated with disease stage and metastatic status. Increased circMED13L_012 expression was associated with the enhanced migration, proliferation and chemo resistance of NSCLC cell lines. Further experiments indicated that circMED13L_012 promoted malignant behavior of NSCLC tumor cells by targeting MAPK8 through modulation miR-433-3p expression. Conclusions Our study for the first time demonstrated that circMED13L_012–miR-433-3p–MAPK8 axis played important role for NSCLC pathogenesis, which could be potential therapeutic target for the development of future NSCLC treatment.

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 Long non-coding RNA BCYRN1 exerts an oncogenic role in colorectal cancer through regulating miR-204-3p/KRAS axis

2020 ◽  
Author(s):  
Liu Yang ◽  
Yinan Zhang ◽  
Jun Bao ◽  
Ji-Feng Feng
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Reporter Gene ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Luciferase Reporter Gene ◽  
Dual Luciferase Reporter Assay

Abstract Background: It has been well documented that long non-coding RNAs (lncRNAs) regulate numerous characteristics of cancer, including proliferation, migration, metastasis, apoptosis, and even metabolism. LncRNA BCYRN1 (BCYRN1) is a newly identified brain cytoplasmic lncRNA with 200 nucleotides that was discovered to be highly expressed in tumour tissues, including those of hepatocellular carcinoma, gastric cancer and lung cancer. However, the roles of BCYRN1 in colorectal cancer (CRC) remain obscure. This study was designed to reveal the role of BCYRN1 in the occurrence and progression of CRC.Methods: RT-PCR was used to detect the expression level of BCYRN1 in tumour tissues and CRC cell lines. BCYRN1 was knocked down in CRC cells, and cell proliferation changes were evaluated by cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), and Ki-67 and proliferating cell nuclear antigen (PCNA) expression assays. Cell migration and invasion changes were evaluated by wound healing, Transwell and invasion-related protein expression assays. Flow cytometry analysis was used to assess whether BCYRN1 regulates the apoptosis of CRC cells. The dual luciferase reporter gene detects the competitive binding of BCYRN1 to miR-204-3p. In vivo experiments were performed to evaluate the effect of BCYRN1 on tumour development. TargetScan analysis and dual luciferase reporter gene assays were applied to detect the target gene of miR-204-3p. Rescue experiments verified that BCYRN1 affects CRC by regulating the effect of miR-204-3p on KRAS.Results: We found that compared with normal tissues and human intestinal epithelial cells (HIECs), CRC tumour tissues and cell lines had significantly increased BCYRN1 levels. We further determined that knockdown of BCYRN1 inhibited the proliferation, migration, and invasion and promoted the apoptosis of CRC cells. In addition, bioinformatics analysis and dual luciferase reporter assay showed that BCYRN1 served as a competitive endogenous RNA (ceRNA) to regulate the development of CRC through competitively binding to miR-204-3p. Further studies proved that overexpression of miR-204-3p reversed the effects of BCYRN1 on CRC. Next, TargetScan analysis and dual luciferase reporter assay indicated that KRAS is a target gene of miR-204-3p and is negatively regulated by miR-204-3p. A series of rescue experiments showed that BCYRN1 affected the occurrence and development of CRC by regulating the effects of miR-204-3p on KRAS. In addition, tumorigenesis experiments in a CRC mouse model confirmed that BCYRN1 downregulation effectively inhibited tumour growth.Conclusions: Our findings suggest that BCYRN1 plays a carcinogenic role in CRC by regulating the miR-204-3p/KRAS axis.

PPARβ-Mediated Suppression of the Growth and Survival in Human Myeloma Cells Conteracting NF-kB Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5053-5053
Author(s):  
Ken-ichiro Otsuyama ◽  
Zi Ma ◽  
Shangqin Liu ◽  
Saeid Abroun ◽  
Hideki Asaoku ◽  
...  
Keyword(s):  
Cell Lines ◽  
Reporter Gene ◽  
Target Genes ◽  
Myeloma Cell ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Human Myeloma Cells

Abstract PPAR(peroxisomal proliferators-activated receptor)β is considered to be involved in the lipid metabolism and regulating the inflammatory response and in human myeloma cells PPARβis predominantly expressed among these PPARs. However, it remains to be clarified what is the functional role of PPARβ in myeloma cells. In order to identify what are the ligands for PPARβ, we constructed the PPREβ-luciferase reporter gene and performed the reporter assay in Hela cells. Since we have already identified several reagents (DHEA (dehydroepiandrosterone), DHEA-S, baicalein, baicalin, dexamethasone) that suppressed the growth or survival of human myeloma cells (Cancer Res65:2269,2005; Blood105:3312,2005), we analyzed whether these reagents augmented the expression of reporter gene. DHEA-S, baicalein and dexamethasone showed the possibility of their PPARβ binding. Furthermore, these reagents could induce or upregulate the expression of PPREβ-target genes such as ILK and COX2 in myeloma cell lines. We’ve already confirmed that DHEA-S inhibited the proliferation and survival of primary myeloma cells as well as myeloma cell lines, and downregulated the activity of NF-kB, also baicalein showed the growth suppression through the downregulation of NF-kB. Therefore, it is possible that PPARβ-binding reagents such as DHEA-S and baicalein could counteract NF-kB activity to suppress the growth or survival in myeloma cells, while the exact mechanism is under investigation.

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