Luciferase Reporter Assays to Study Transcriptional Activity of Hedgehog Signaling in Normal and Cancer Cells

2015 ◽  
pp. 71-79
Author(s):  
Silvia Pandolfi ◽  
Barbara Stecca
Keyword(s):  
Cancer Cells ◽  
Hedgehog Signaling ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Reporter Assays

scholarly journals Oncoprotein LAMTOR5 activates GLUT1 via upregulating NF-κB in liver cancer

Open Medicine ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. 264-270 ◽  
Author(s):  
Jing Zhou ◽  
Yajun Li ◽  
Danhua Li ◽  
Zhi Liu ◽  
Jie Zhang
Keyword(s):  
Liver Cancer ◽  
Cancer Cells ◽  
Western Blotting ◽  
Glucose Transporter ◽  
Luciferase Reporter ◽  
Pcr Assay ◽  
Glucose Transporter 1 ◽  
Over Expression ◽  
Liver Cancer Cells ◽  
Reporter Assays

AbstractObjectiveAccumulating reports reveal that serving as an oncogenic factor LAMTOR5 is involved in the progression of many specific cancers. Glucose transporter 1 (GLUT1) is frequently identified in many cancers. However, it remains unexplored whether GLUT1 plays a role in LAMTOR5-enhanced liver cancer. Here, we aim to decipher the function of LAMTOR5 in the regulation of GLUT1 in liver cancer.MethodsThe effect of LAMTOR5 on GLUT1 was analyzed using Western blotting and RT-PCR assay. Dose-increased over-expression or silencing of LAMTOR5 was performed through transient transfection. LAMTOR5-activated GLUT1 promoter was revealed by luciferase reporter assay. The regulation of GLUT1 by LAMTOR5/NF-κB was examined via Western blotting and luciferase reporter assays.ResultsThe data showed that in liver cancer cells under the administration with dose-increased LAMTOR5, the level of mRNA and protein of GLUT1 was obviously raised. Our data revealed that the activities of GLUT1 promoter were induced by LAMTOR5. Then, we found that the elevation of GLUT 1 mediated by LAMTOR5 slowed when the inhibitor or siRNAs of NF-κB was introduced into the liver cancer cells. Conclusion. LAMTOR5 is responsible for the activation of GLUT1 via transcription factor NF-κB in liver cancer.

scholarly journals miR-128 Targets the SIRT1/ROS/DR5 Pathway to Sensitize Colorectal Cancer to TRAIL-Induced Apoptosis

2018 ◽  
Vol 49 (6) ◽  
pp. 2151-2162 ◽  
Author(s):  
Bo Lian ◽  
Dongxiang Yang ◽  
Yanlong Liu ◽  
Gang Shi ◽  
Jibin Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cancer Cells ◽  
Cell Lines ◽  
Sirtuin 1 ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Reporter Assays ◽  
Induced Apoptosis

Background/Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an ideal anti-tumor drug because it exhibits selective cytotoxicity against cancer cells. However, certain cancer cells are resistant to TRAIL, and the potential mechanisms are still unclear. The aim of this study was to reduce the resistance of colorectal cancer (CRC) cells to TRAIL. Methods: Quantitative real-time PCR analysis was performed to detect the expression of microRNA-128 (miR-128) in tissues from patients with CRC and CRC cell lines. MTT assays were used to evaluate the effect of miR-128 on TRAIL-induced cytotoxicity against CRC cell lines. The distribution of death receptor 5 (DR5) and the production of reactive oxygen species (ROS) were detected by flow cytometry analysis. Western blot, flow cytometry, and luciferase reporter assays were performed to evaluate the potential mechanism and pathway of miR-128-promoted apoptosis in TRAIL-treated CRC cells. Results: MiR-128 expression was downregulated in tumor tissues from patients with CRC as well as in CRC cell lines in vitro. The enforced expression of miR-128 sensitized CRC cells to TRAIL-induced cytotoxicity by inducing apoptosis. Mechanistically, bioinformatics, western blot analysis, and luciferase reporter assays showed that miR-128 directly targeted sirtuin 1 (SIRT1) in CRC cells. miR-128 overexpression suppressed SIRT1 expression, which promoted the production of ROS in TRAIL-treated CRC cells. This increase of ROS subsequently induced DR5 expression, and thus increased TRAIL-induced apoptosis in CRC cells. Conclusion: The combination of miR-128 with TRAIL may represent a novel approach for the treatment of CRC.

scholarly journals Etv5, an ETS transcription factor, is expressed in granulosa and cumulus cells and serves as a transcriptional regulator of the cyclooxygenase-2

2008 ◽  
Vol 198 (2) ◽  
pp. 281-290 ◽  
Author(s):  
Jinwon Eo ◽  
Kyuyong Han ◽  
Kenneth M Murphy ◽  
Haengseok Song ◽  
Hyunjung Jade Lim
Keyword(s):  
Transcriptional Activity ◽  
Cumulus Cells ◽  
Cyclooxygenase 2 ◽  
Luciferase Reporter ◽  
Ets Transcription Factors ◽  
Cellular Events ◽  
Reporter Assays ◽  
Ets Transcription Factor ◽  
Cell Niche ◽  
Rate Limiting

Etv4, Etv1, and Etv5 are members of Etv4 subfamily of E26 transformation-specific (Ets) transcription factors that are known to influence a host of biological processes. We previously showed that Etv5, expressed in Sertoli cells, plays a crucial role in maintaining spermatogonial stem cell niche in the mouse testis. However, it is not yet known whether Etv4 family members are expressed in the ovary or play any role in ovarian functions. Here, we show that Etv5 and Etv4 are expressed in mouse ovaries in granulosa and cumulus cells during folliculogenesis. Both Etv5 and Etv4 mRNAs are also detected in cumulus–oocyte complexes (COCs) and denuded oocytes. Notably, Etv4 is highly expressed in the cumulus cells of ovulated COCs at 16-h post-human chorionic gonadotropin. Cyclooxygenase-2 (PTGS2), a rate-limiting enzyme for prostaglandin synthesis, is critical for oocyte maturation and ovulation. Since several putative Ets-binding sites are present in the PTGS2 promoter, we examined whether Etv5 influences Ptgs2 transcriptional activity. Indeed, we found that addition of Etv5 increases the transcriptional activity of the 3.2-kb mouse Ptgs2 promoter by 2.5-fold in luciferase reporter assays. Collectively, the results show that Etv4 and Etv5 are expressed in granulosa and cumulus cells during folliculogenesis and ovulation, suggesting that they influence cellular events in the ovary by regulating downstream genes such as Ptgs2.

scholarly journals HOX Transcript Antisense RNA HOTAIR Abrogates Vasculogenic Mimicry by Targeting the AngiomiR-204/FAK Axis in Triple Negative Breast Cancer Cells

2020 ◽  
Vol 6 (2) ◽  
pp. 19
Author(s):  
Allan Lozano-Romero ◽  
Horacio Astudillo-de la Vega ◽  
María Cruz del Rocío Terrones-Gurrola ◽  
Laurence A. Marchat ◽  
Daniel Hernández-Sotelo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Migration ◽  
Cancer Cells ◽  
Triple Negative Breast Cancer ◽  
Breast Cancer Cells ◽  
Triple Negative ◽  
Vasculogenic Mimicry ◽  
Bioinformatic Analysis ◽  
Luciferase Reporter ◽  
Reporter Assays

HOX transcript antisense RNA (HOTAIR) is an oncogenic long non-coding RNA frequently overexpressed in cancer. HOTAIR can enhance the malignant behavior of tumors by sponging microRNAs with tumor suppressor functions. Vasculogenic mimicry is a hypoxia-activated process in which tumor cells form three-dimensional (3D) channel-like networks, resembling endothelial blood vessels, to obtain nutrients. However, the role of HOTAIR in vasculogenic mimicry and the underlying mechanisms are unknown in human cancers. In the current study, we investigated the relevance of HOTAIR in hypoxia-induced vasculogenic mimicry in metastatic MDA-MB-231 and invasive Hs-578t triple negative breast cancer cells. Analysis of The Cancer Genome Atlas (TCGA) database using cBioPortal confirmed that HOTAIR was upregulated in clinical breast tumors relative to normal mammary tissues. Our quantitative RT-PCR assays showed a significant increase in HOTAIR levels after 48 h hypoxia relative to normoxia in breast cancer cell lines. Remarkably, knockdown of HOTAIR significantly abolished the hypoxia-induced vasculogenic mimicry which was accompanied by a reduction in the number of 3D channel-like networks and branch points. Likewise, HOTAIR silencing leads to reduced cell migration abilities of cancer cells. Bioinformatic analysis predicted that HOTAIR has a potential binding site for tumor suppressor miR-204. Luciferase reporter assays confirmed that HOTAIR is a competitive endogenous sponge of miR-204. Congruently, forced inhibition of HOTAIR in cells resulted in augmented miR-204 levels in breast cancer cells. Further bioinformatic analysis suggested that miR-204 can bind to the 3′ untranslated region of focal adhesion kinase 1 (FAK) transcript involved in cell migration. Western blot and luciferase reporter assays confirmed that FAK is a novel target of miR-204. Finally, silencing of HOTAIR resulted in low levels of cytoplasmic FAK protein and alterations in the organization of cellular cytoskeleton and focal adhesions. In summary, our results showed, for the first time, that HOTAIR mitigates cell migration and vasculogenic mimicry by targeting the miR-204/FAK axis in triple negative breast cancer cells.

Molecular Mechanism of circRAPGEF5 in Regulating the Molecular Axis of microRNA-4712-5p/Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Epsilon Affecting Cellular Proliferation and Apoptosis in Mammary Cancer

2021 ◽  
Vol 11 (6) ◽  
pp. 1053-1058
Author(s):  
Tao Chen ◽  
Shengrong Sun
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Mammary Cancer ◽  
Caspase 3 ◽  
Cellular Proliferation ◽  
Molecular Axis ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Proliferation And Apoptosis ◽  
Reporter Assays

To understand the molecular mechanism of circRAPGEF5, its effect on the proliferation and apoptosis of mammary cancer cells, and its regulatory effect on the molecular axis of miRNA-4712-5p/YWHAE. qRT-PCR and Western blot were used to test circRAPGEF5, miRNA-4712-5p, and YWHAE expression in mammary cancer and paracancerous tissues. The human mammary cancer cell, MDA-MB-231, was cultured in vitro, and pcDNA-NC, pcDNA-circRAPGEF5, anti-miRNA-NC, anti-miRNA-4712-5p, pcDNA-circRAPGEF5, and miRNA-NC, pcDNA-circRAPGEF5 were transfected into MDA-MB-231 cells with miRNA-4712-5p mimics. qRT-PCR and Western blot were employed to detect circRAPGEF5, miRNA-4712-5p, and YWHAE expression in cells. The CCK-8 methodand plate clone formation experiment were conducted to test cellular proliferation ability. Flow cytometry was performed to detect apoptosis rate. Dual luciferase reporter assays were used to test the targeting association between circRAPGEF5 and miRNA-4712-5p, and the targeting association between miRNA-4712-5p and YWHAE. Western blot was utilized to detect Bcl-2, Bax, and Cleared Caspase-3 protein expression. In comparison with paracancerous tissues, circRAPGEF5 and YWHAE expression levels in mammary cancer tissues were significantly reduced (P < 0.05), and miRNA-4712-5p expression levels were significantly increased (P < 0.05). Transfection of pcDNA-circRAPGEF5 or trans-anti-miRNA-4712-5p could reduce the optical density (OD) value, Bcl-2 protein level and clonal formation number to a significant extent (P < 0.05), and it increases Bax and Cleaved Caspase-3 apoptosis rate and protein levels (P < 0.05). Dual luciferase reporter assays confirmed that there was target binding between circRAPGEF5 and miRNA-4712-5p and between miRNA-4712-5p and YWHAE. Co-transfection of pcDNA-circRAP GEF5 and miRNA-4712-5p could greatly reduce transfection of pcDNA-circRAP GEF5 and its effect on the proliferation and apoptosis of MDA-MB-231 cells. Overexpression of circRAPGEF5 can inhibit the proliferation of mammary cancer cells and induce apoptosis by regulating the molecular axis of miRNA-4712-5p/YWHAE.

scholarly journals Resveratrol Activated Sonic Hedgehog Signaling to Enhance Viability of NIH3T3 Cells in Vitro via Regulation of Sirt1

2018 ◽  
Vol 50 (4) ◽  
pp. 1346-1360 ◽  
Author(s):  
Shuang Guo ◽  
Hongyan Liao ◽  
Jie Liu ◽  
Jing Liu ◽  
Fanren Tang ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Hedgehog Signaling ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Shh Signaling ◽  
Nih3t3 Cells ◽  
Cord Injury ◽  
Fibrotic Scar ◽  
Gli 1

Background/Aims: Injuries of the brain and spinal cord result in the formation of glial (reactive gliosis) and fibrotic (formed by fibroblasts) scars. Recent studies have shown that the fibrotic scar was much more important for hindering regeneration after brain or spinal cord injury than the astrocytic scar. However, it has been given much less attention for effects and mechanism of fibroblasts during formation of the fibrotic scar. Resveratrol may be a potential anti-scarring agent in burn-related scarring and keloid fibroblasts. However, it is unclear whether and how resveratrol affects formation of the fibrotic scar after brain or spinal cord injury. Earlier studies have shown that the activated Shh signaling has anti-apoptosis, anti-oxidation, anti-inflammation properties. Moreover, resveratrol can activate the Shh signaling. However, it is unclear how resveratrol activates the Shh signaling. Resveratrol is a activator of Sirt1. It is unknown whether resveratrol activates the Shh signaling via Sirt1. Methods: NIH3T3 cells, a fibroblast cell line, were used as model cells and treated with drugs. Cell viability was assessed by Cell Counting Kit 8. The expressions and activity of Shh signaling pathway proteins were evaluated by immunocytochemistry and Western blotting. Transcriptional activity of Gli-1 was detected with Dual-Luciferase Reporter Gene Assay Kit. Results: Resveratrol, Sirt1 agonist STR1720 and recombinant mouse Shh protein, an activator of hedgehog signaling, enhanced the viability of NIH3T3 cells, promoted Smo to translocated to the primary cilia and Gli-1 entered into the nuclei from cytoplasm, and upregulated expressions of Shh, Ptc-1, Smo, and Gli-1 proteins, which can be reversed by Smo antagonist cyclopamine and Sirt1 antagonist Sirtinol. Additionally, resveratrol increased transcriptional activity of Gli-1. Conclusion: We indicate in the first time that it may be mediated by Sirt1 for resveratrol activating the Shh signaling to enhance viability of NIH3T3 cells, and Sirt1 may be a regulator for upstream of the Shh signaling pathway.This study provides a basis for further investigating effects and mechanism of resveratrol during the formation of fibrous scar after brain or spinal cord injury.

scholarly journals Berberine inhibited metastasis through miR-145/MMP16 axis in vitro

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Jie Li ◽  
Songlin Zhang ◽  
Lei Wu ◽  
Meili Pei ◽  
Yu Jiang
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Ovarian Cancer Cells ◽  
Inhibition Of Proliferation ◽  
Reporter Assays ◽  
Polymerase Chain ◽  
Effective Component

AbstractOvarian cancer is the first leading cause of death in gynecological cancers. The continuous survival and metastasis of cancer cells are the main causes of death and poor prognosis in patients with ovarian cancer. Berberine is an effective component extracted from the rhizomes of coptis chinensis and phellodendron chinensis. In our study, we aim to explore the molecular mechanism underlying the regulation of proliferation, migration and invasion by berberine in ovarian cancer cells. CCK8 assay was used for detection of proliferative capacity of SKOV3 and 3AO cells. Wound healing assay was used to estimate cell migration and transwell assay was used to assess cell invasion. The mRNA expression of miR-145 and MMP16 were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The protein level of MMP16 was detected by western blot analysis. In addition, luciferase reporter assays were used to demonstrate MMP16 was a target of miR-145. The results demonstrated berberine inhibited proliferation, migration and invasion, promoted miR-145 expression, and decreased MMP16 expression in SKOV3 and 3AO cells. MMP16 was a target of miR-145. Moreover, downregulation of MMP16 contributed to the inhibition of proliferation, migration and invasion by berberine. Together, our results revealed that berberine inhibited proliferation, migration and invasion through miR-145/MMP16 in SKOV3 and 3AO cells, highlighting the potentiality of berberine to be used as a therapeutic agent for ovarian cancer.

scholarly journals Restore of miR-541 resensitizes pancreatic cancer cells to gemcitabine-induced apoptosis through suppression of HAX-1

2020 ◽  
Author(s):  
Hong Liu ◽  
Xuemei Gan ◽  
Jun Zhang ◽  
Xingdiao Zhang ◽  
Jie Xiong ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Potential Strategy ◽  
Induced Apoptosis ◽  
Mtt Assays

Abstract Background: MiR-541 acts as a tumor suppressor in some cancers. However, the role of miR-541 in regulating the chemosensitivity to cancer cells is still unclear. The aim of this study is to explore the effect of miR-541 on chemoresistance of pancreatic cancer (PCa) cells to gemcitabine-induced apoptosis.Methods: Gemcitabine-resistant Panc-1 and Capan-2 PCa cell lines (Panc-1/R and Capan-2/R) were established through long term exposure to gemcitabine. Effect of miR-541 on changing the sensitivity of Panc-1/R and Capan-2/R to gemcitabine-induced cytotoxicity was evaluated by MTT assays. Regulation of miR-541 on HAX-1 was confirmed by bioinformatics, western blot analysis and luciferase reporter assays. Cell apoptosis and mitochondrial membrane potential (MMP) was measured by flow cytometry analysis.Results: Comparison with Panc-1 and Capan-2, downregulation of miR-541 was observed in Panc-1/R and Capan-2/R cells. Overexpression of miR-541 was found to increase the cytotoxicity of gemcitabine to Panc-1/R and Capan-2/R cells. However, transfection with HAX-1 plasmid can abolish the effect of miR-541 on gemcitabine-induced cytotoxicity against Panc-1/R and Capan-2/R.Conclusion: Downregulation of miR-541 is responsible for development of gemcitabine resistance in PCa. Overexpression of miR-541 may represent a potential strategy to reverse the chemoresistance of PCa.

scholarly journals miR-186 Regulates the Initiation and Development of Breast Cancer via SHP-1 Methylation

2020 ◽  
Author(s):  
Kun Wang ◽  
Chunxia Zhou ◽  
Di Wang ◽  
Bin Zhang ◽  
Yongjian Zheng ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Promoter Region ◽  
Breast Cancer Cells ◽  
Methylation Status ◽  
Luciferase Reporter ◽  
Molecular Diagnostic ◽  
Double Labeling ◽  
Reporter Assays ◽  
Mcf 7

Abstract Background Change in the methylation status of genomic DNA, especially in CpG islands in the promoter region, is considered to be an early event in tumor initiation, leading to silencing of gene expression, subsequent abnormalities in gene structure and function, and malignant transformation of the cell. Due to the abnormal expression of miR-186 and SHP-1 in breast cancer tissues and cells, we propose that miR-186 is closely related to the methylation of SHP-1Method Using 5-azacytidine as a de-methylation agent and Validating with Setylation-specific polymerase chain reaction (MSP) after treatment. Measurement of the viability of breast cancer cells using the CCK-8 method Measurement of the apoptotic rate of breast cancer cells using annexin V-FITC/PI double labeling. Cell metastasis were measured by wound healing assay. Luciferase reporter assays was used to confirm the target of MiR-186. SHP-1 and miR-186 expression was measured by RT-PCR and western blot.Results In the present study, we found that SHP-1 expression was reduced to various degrees in all 5 cell lines (UACC-812, MDA-MB-213, MDA-MB-468, SK-RB-3 and MCF-7). 5-azacytidine can remove the methylation from the SHP-1 promoter region. Apoptosis was observed in MCF-7 cells after demethylation of the SHP-1 gene promoter region by 5-azacytidine, and the effect was time- and concentration-dependent. Luciferase reporter assays showed that miR-186 promotes methylation through binding with the 3’ UTR of the SHP-1 promoter region.Western blot showed miR-186 regulates the initiation and development of tumor cells through the SHP-1-JAK-STAT axis. In animal models, low expression of miR-186 can cause significantly limited tumor growth.Conclusion The low SHP-1 expression may be an important factor in the initiation of breast cancer, and that miR-186 could serve as an excellent molecular diagnostic marker and a possible therapeutic target.

scholarly journals LncRNA OTUD6B-AS1 Induces Cisplatin Resistance in Cervical Cancer Cells Through Up-Regulating Cyclin D2 via miR-206

2021 ◽  
Vol 11 ◽  
Author(s):  
Hui Hou ◽  
Rong Yu ◽  
Haiping Zhao ◽  
Hao Yang ◽  
Yuchong Hu ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Cisplatin Resistance ◽  
Luciferase Reporter ◽  
Cyclin D2 ◽  
Aberrant Expression ◽  
Qrt Pcr ◽  
Cervical Cancer Cells ◽  
Reporter Assays ◽  
Underlying Mechanisms

Cervical cancer is one of the most common gynecological cancers. Cisplatin resistance remains a major hurdle in the successful treatment of cervical cancer. Aberrant expression of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are implicated in cisplatin resistance. However, the regulatory functions of lncRNAs and miRNAs in cervical cancer cisplatin resistance and the underlying mechanisms are still elusive. Our qRT-PCR assays verified that miR-206 levels were down-regulated in cisplatin-resistant cervical cancer cells. The introduction of miR-206 sensitized cisplatin-resistant cervical cancer cells to cisplatin. Our qRT-PCR and luciferase reporter assays showed that Cyclin D2 (CCND2) was the direct target for miR-206 in cervical cancer cells. The cisplatin-resistant cervical cancer cells expressed higher CCND2 expression than the parental cells, whereas inhibition of CCND2 could sensitize the resistant cells to cisplatin treatment. Furthermore, we demonstrated that lncRNA OTUD6B-AS1 was up-regulated in cisplatin-resistant cervical cancer cells, and knocking down OTUD6B-AS1 expression induced re-acquirement of chemosensitivity to cisplatin in cervical cancer cells. We also showed that OTUD6B-AS1 up-regulated the expression of CCND2 by sponging miR-206. Low miR-206 and high OTUD6B-AS1 expression were associated with significantly poorer overall survival. Taken together, these results suggest that OTUD6B-AS1-mediated down-regulation of miR-206 increases CCND2 expression, leading to cisplatin resistance. Modulation of these molecules may be a therapeutic approach for cisplatin-resistant cervical cancer.

Sign in / Sign up

Export Citation Format

Share Document