Construction and Functional Analysis of Luciferase Reporter Plasmid Containing PCNA Gene Promoter

2014 ◽  
Vol 556-562 ◽  
pp. 257-260
Author(s):  
Tong Cun Zhang ◽  
Yue Wang ◽  
Xing Hua Liao ◽  
Nan Wang ◽  
Hao Zhou
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Transcription Factors ◽  
Transcriptional Activity ◽  
Nuclear Antigen ◽  
Luciferase Reporter ◽  
Reporter Plasmid ◽  
Luciferase Reporter Construct ◽  
Luciferase Reporter Plasmid ◽  
Mcf 7

PCNA (proliferating cell nuclear antigen) is a protein related to tumor development, which has been used extensively in breast cancer diagnosis and prognosis. PCNA has proven to be a useful marker to evaluate cell proliferation and prognosis when combined with other breast cancer markers. Construction of PCNA promoter luciferase reporter plasmid will provide the theory basis for researching the effect of other transcription factors on regulating PCNA transcription. In this study, a human PCNA promoter luciferase reporter construct was generated by PCR amplification of PCNA promoter. The PCR fragment was digested and cloned into pGL3 vector. The promoter sequence was verified by sequencing. The results showed that luciferase reporter plasmids of PCNA promoter were successfully constructed. Then the effects of some key transcription factors, which play important roles in breast cancer cell proliferation, were investigated by luciferase reporter assays in MCF-7 cells. The results showed that ERα can enhance transcriptional activity of PCNA. Furthermore, 17-β-estradiol (E2) also shows an obvious impact in activating PCNA transcription. Our data illuminated that E2 enhances ERα-induced proliferation potential of MCF-7 cells by stimulating the transcriptional activity of PCNA. Our research will provide a model to screen some novel factors in regulating proliferation marker transcription.

scholarly journals MKL1 and STAT3 activate the activity of the luciferase reporter plasmid containing the CAAP1 gene promoter

2019 ◽  
Vol 78 ◽  
pp. 01003
Author(s):  
Jun-Yan Li ◽  
Zhu Yu ◽  
Feng-Yun Wang
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Cells ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Reporter Plasmid ◽  
Transcription Activity ◽  
Luciferase Reporter Plasmid ◽  
The Relationship ◽  
Mcf 7

Breast cancer is the leading cause of cancer death in women worldwide. The etiology of the disease is not yet clear. We know that MKL1 and STAT3 play an important part in the development and progression of breast cancer. CAAP1 is a ubiquitous and highly conserved protein that is closely related to the apoptotic process of tumors. However, the definitive transcriptional mechanism of the CAAP1 gene is still unclear. In our study, we constructed a luciferase reporter plasmid for the human CAAP1 gene promoter. Then one or both of the two overexpression vectors of MKL-1 and STAT3 were co-transfected into MCF-7 cells with CAAP1 promoter plasmid, and we then tested activation of the CAAP1 promoter by luciferase reporter assay. The results show that compared with the transfected pcDNA3.1 group, MKL1 can evidently increase the transcription activity of the CAAP1 gene promoter, while the STAT3 group can slightly upregulate the transcription activity of the CAAP1 gene promoter. Our research will further reveal the relationship between CAAP1 and the occurrence and development of breast cancer cells, and provide a new idea and direction for the cures of breast cancer.

scholarly journals miR-3188 Regulates Cell Proliferation, Apoptosis, and Migration in Breast Cancer by Targeting TUSC5 and Regulating the p38 MAPK Signaling Pathway

2018 ◽  
Vol 26 (3) ◽  
pp. 363-372 ◽  
Author(s):  
Xiaowen Chen ◽  
Jianli Chen
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Molecular Mechanisms ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
Luciferase Reporter ◽  
And Migration ◽  
Mcf 7

This study intended to investigate the effects of miR-3188 on breast cancer and to reveal the possible molecular mechanisms. miR-3188 was upregulated and TUSC5 was downregulated in breast cancer tissues and MCF-7 cells compared to normal tissue and MCF-10 cells. After MCF-7 cells were transfected with miR-3188 inhibitor, cell proliferation and migration were inhibited, whereas apoptosis was promoted. Luciferase reporter assay suggested that TUSC5 was a target gene of miR-3188. In addition, miR-3188 overexpression increased the p-p38 expression, while miR-3188 suppression decreased the p-p38 expression significantly. miR-3188 regulated breast cancer progression via the p38 MAPK signaling pathway. In conclusion, miR-3188 affects breast cancer cell proliferation, apoptosis, and migration by targeting TUSC5 and activating the p38 MAPK signaling pathway. miR-3188 may serve as a potential therapeutic agent for the treatment of breast cancer.

scholarly journals Unique Roles of p160 Coactivators for Regulation of Breast Cancer Cell Proliferation and Estrogen Receptor-α Transcriptional Activity

Endocrinology ◽  
2008 ◽  
Vol 150 (4) ◽  
pp. 1588-1596 ◽  
Author(s):  
Sudipan Karmakar ◽  
Estrella A. Foster ◽  
Carolyn L. Smith
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Estrogen Receptor ◽  
Mrna Expression ◽  
Cancer Cells ◽  
Small Interfering Rna ◽  
Breast Cancer Cells ◽  
Transcriptional Activity ◽  
Interfering Rna ◽  
Mcf 7

Each of the three members of the p160 steroid receptor coactivator (SRC) family of coactivators (SRC-1, SRC-2 and SRC-3) stimulates estrogen receptor (ER)-α function in trans-activation assays. Consequently, we sought to elucidate their contributions to the ER-regulated processes of cell proliferation, apoptosis, and the expression of ERα target genes in MCF-7 breast cancer cells. The small interfering RNA depletion of SRC-2 or SRC-3 but not SRC-1 inhibited growth of MCF-7 cells, and this was reflected in decreased cell cycle progression and increased apoptosis in SRC-2- or SRC-3-depleted cells as well as a reduction in ERα transcriptional activity measured on a synthetic reporter gene. However, only SRC-3 depletion blocked estradiol stimulated cell proliferation. Depletion of SRC-1 did not affect these events, and together this reveals functional differences between each of the three SRC family coactivators. Regulation of the endogenous ERα target gene, c-myc was not affected by depletion of any of the p160 coactivators although depletion of each of them decreased pS2 mRNA expression in estradiol-treated MCF-7 cells. Moreover, progesterone receptor and cyclin D1 gene expression were decreased in SRC-3 small interfering RNA-treated cells. Expression of mRNA and protein levels for the antiapoptotic gene, Bcl-2 was dependent on SRC-3 expression, whereas Bcl-2 protein but not mRNA expression also was sensitive to SRC-1 depletion. Together these data indicate that the closely related p160 coactivators are not functionally redundant in breast cancer cells because they play gene-specific roles in regulating mRNA and protein expression, and they therefore are likely to make unique contributions to breast tumorigenesis.

scholarly journals Compounds from Cynomorium songaricum with Estrogenic and Androgenic Activities Suppress the Oestrogen/Androgen-Induced BPH Process

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Xueni Wang ◽  
Rui Tao ◽  
Jing Yang ◽  
Lin Miao ◽  
Yu Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Immunofluorescence Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Lncap Cells ◽  
Mcf 7

Objective. To investigate the phytoestrogenic and phytoandrogenic activities of compounds isolated from CS and uncover the role of CS in prevention of oestrogen/androgen-induced BPH. Methods. Cells were treated with CS compounds, and immunofluorescence assay was performed to detect the nuclear translocation of ERα or AR in MCF-7 or LNCaP cells; luciferase reporter assay was performed to detect ERs or AR transcriptional activity in HeLa or AD293 cells; MTT assay was performed to detect the cell proliferation of MCF-7 or LNCaP cells. Oestrogen/androgen-induced BPH model was established in rat and the anti-BPH, anti-estrogenic, and anti-androgenic activities of CS in vivo were further investigated. Results. The nuclear translocation of ERα was stimulated by nine CS compounds, three of which also stimulated AR translocation. The transcriptional activities of ERα and ERβ were induced by five compounds, within which only ECG induced AR transcriptional activity as well. Besides, ECG stimulated the proliferation of both MCF-7 cells and LNCaP cells. CS extract suppressed oestrogen/androgen-induced BPH progress in vivo by downregulation of E2 and T level in serum and alteration of the expressions of ERα, ERβ, and AR in the prostate. Conclusion. Our data demonstrates that compounds from CS exhibit phytoestrogenic and phytoandrogenic activities, which may contribute to inhibiting the oestrogen/androgen-induced BPH development.

Construction and Functional Analysis of Luciferase Reporter Plasmid Containing NF-H Gene Promoter

2014 ◽  
Vol 915-916 ◽  
pp. 942-946
Author(s):  
Tong Cun Zhang ◽  
Yao Meng ◽  
Nan Wang ◽  
Feng Lin ◽  
Tao Qin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Reporter Plasmid ◽  
Extracellular Signal Regulated Kinase ◽  
Luciferase Reporter Plasmid ◽  
Extracellular Signal ◽  
Mature Neurons ◽  
Reporter Assays ◽  
Transcription 3

NF-H (a member of neurofilaments) is a protein widely expressed in all kinds of neurons after birth and can be used as one of the symbols of mature neurons. Constuction of NF-H promoter luciferase reporter plasmid will provide the theory basis for researching the effect of other transcription factors on regulating NF-H transcription. Here, human NF-H promoter luciferase reporter plasmid were successfully constructed. Then the effects of some key transcription factors were investigated by luciferase reporter assays in COS-7 cells. The results showed that retinoid X receptor α (RXRα) can enhance transcriptional activity of NF-H. Furthermore, ERK 1/2 (extracellular signal-regulated kinase 1/2) and STAT3 (signal transducer and activator of transcription 3) also show obvious impact in activating NF-H transcription.

scholarly journals Regulatory roles of lncRNA PANDAR in breast cancer cell proliferation

2021 ◽  
Vol 15 (6) ◽  
pp. 285-291
Author(s):  
Qinnuan Sun ◽  
Xiumei Wang
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Marker Gene ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Cell Marker ◽  
Mesenchymal Transition ◽  
Mcf 7

Abstract Background Breast cancer represents the second most deadly malignancy in women, and long noncoding RNAs (lncRNAs) have crucial functions in its development. Objective To investigate effects of the promoter of CDKN1A antisense DNA damage-activated RNA (PANDAR) on epithelial-mesenchymal transition (EMT) in breast cancer cells and their proliferation. Methods lncRNAs potentially regulating the transcriptional activity of the E-cadherin (E-cad, an epithelial cell marker) gene promoter were screened using a dual-luciferase reporter assay. PANDAR was overexpressed in Michigan cancer foundation 7 (MCF-7) breast cancer cells. E-cad and N-cadherin (N-cad, a mesenchymal cell marker) levels were detected by immunoblotting. Cell viability was assessed using a cell counting kit-8. Results PANDAR and TCONS00068220/LOC105375819 conservatively regulated the promoter activity of E-cad. PANDAR overexpression in MCF-7 inhibited E-cad expression, but upregulated N-cad. The enhanced expression of PANDAR promoted cell proliferation. Conclusion PANDAR is a key transcriptional repressor of E-cad and has regulatory effects on the promotion of cell proliferation. PANDAR is an oncogene in breast cancer, potentially facilitating the EMT process and promoting cell proliferation.

Function Analysis of Luciferase Reporter Plasmid with AQP1 Gene Promoter

2011 ◽  
Vol 396-398 ◽  
pp. 1486-1489
Author(s):  
Yong Jiang ◽  
Zhe Sun ◽  
Tong Cun Zhang
Keyword(s):  
Transcriptional Regulation ◽  
Function Analysis ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Reporter Plasmid ◽  
Reporter Construct ◽  
Luciferase Reporter Construct ◽  
Transcription Activity ◽  
Luciferase Reporter Plasmid ◽  
Precise Role

The aquaporins (AQP) are a family of homologous water transporting proteins that are expressed in many epithelial, endothelial and other tissues. Myocardin is important for SMC differentiation, but its precise role in regulating the initiation of AQP1 transcription activity is less clear. Function analysis of AQP1 promoter luciferase reporter plasmid will provide the theory basis for researching the function of transcription activity. In this study, the rat and human myocardin promoter luciferase reporter construct were successfully constructed. Then to determine whether AQP1 transcription activity were regulated by myocardin, luciferase repoeter assays were performed in COS-7 cells. The results illustrated that myocardin significantly activated rat and human AQP1 promoter. AQP1 promoter transactivity was inhibited by △Q. The present study provided the first evidence that myocardin may have an influence on the expression of AQP1 and reveal a basis of the mechanism transcriptional regulation of AQP1.

Collaboration of AR and ERα in conferring resistance to an aromatase inhibitor.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 579-579
Author(s):  
Yassine Rechoum ◽  
Domenico Iacopetta ◽  
Ines Barone ◽  
Daniela Rovito ◽  
Sebastiano Ando' ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Aromatase Inhibitor ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Transcriptional Activity ◽  
Single Agent ◽  
Luciferase Reporter ◽  
Breast Cancers ◽  
Estrogen Synthesis ◽  
Mcf 7

579 Background: We have previously shown a role for AR overexpression in tamoxifen resistance in ERα-positive MCF-7 breast cancer cells; here we hypothesized that AR overexpression might similarly be involved in resistance to the aromatase inhibitor anastrazole (Anas). Methods: MCF-7 cells were transfected to express the aromatase gene (MCF-7 Arom), or the aromatase and AR (MCF-7 AR Arom cells). Western blot analysis was used to evaluate protein levels, MTT and soft agar assays to evaluate proliferation, luciferase reporter assays to evaluate transcriptional activities and confocal microscopy was used for localization. Results: Anas inhibited androstendione (AD)-stimulated growth in MCF-7 Arom cells but not in MCF-7 AR Arom cells. Similarly, Anas did not inhibit ERα transcriptional activity in MCF-7 AR Arom cells. Enhanced activation of pIGF-1R, pIRS-1, pAKT, and pMAPK were also observed in AR Arom cells, suggesting constitutive activation of nongenomic signaling in these cells. Consistent with activation of these potential treatment “escape” mechanisms, inhibitors of AKT and IGF-1R restored sensitivity to Anas. Sensitivity to Anas was also restored using the AR antagonist MDV3100, however use of Abiraterone acetate as a single agent most effectively blocked proliferation of AR-overexpressing cells. These results suggest that both AR and ERα must be blocked to restore sensitivity to hormonal therapies in AR overexpressing ERα-positive breast cancers. Unexpectedly, AR contributed to ERα transcriptional activity in MCF-7 AR Arom cells, as shown by inhibition with the AR antagonist bicalutamide. AR and ERα co-localized both in the cytoplasm and in the nucleus of AD+Anas-treated cells, suggesting potential activation of both non-genomic and nuclear-mediated effects when AR is overexpressed in ERα-positive cells. We confirmed these findings in breast cancer cells with acquired resistance to tamoxifen. Conclusions: These results show the necessity to block both AR and ER in patients whose tumors express elevated levels of AR. In addition, inhibitors to the AKT/IGF-1R signaling pathways or direct inhibition of androgen/estrogen synthesis provide alternative approaches to restore hormone sensitivity in resistant breast tumors.

SOX4 Serves an Oncogenic Role in the Tumourigenesis of Human Breast Adenocarcinoma by Promoting Cell Proliferation, Migration and Inhibiting Apoptosis

2020 ◽  
Vol 15 (1) ◽  
pp. 49-58
Author(s):  
Junhe Zhang ◽  
Shujie Chai ◽  
Xinyu Ruan
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Caspase 3 ◽  
Breast Adenocarcinoma ◽  
Migration Ability ◽  
Expression Levels ◽  
Apoptosis Rate ◽  
And Migration ◽  
Mcf 7 ◽  
Proliferation And Migration

Background: Breast cancer is among the most common malignant cancers worldwide, and breast adenocarcinoma in glandular tissue cells has excessive metastasis and invasion capability. However, little is known on the molecular process by which this disease develops and progresses. Objective: In this study, we explored the effects of sex-determining region Y-box 4 (SOX4) protein on proliferation, migration, apoptosis and tumourigenesis of breast adenocarcinoma and its possible mechanisms. Methods: The SOX4 overexpression or knockdown Michigan Cancer Foundation-7 (MCF-7) cell lines were established. Among the SOX4 overexpression or MCF-7 knockdown cell lines, proliferation, migration ability and apoptosis rate were detected. The expression levels of apoptosis-related proteins (Bax and Cleaved caspase-3) were analysed using Western blot. The effect of SOX4 on tumourigenesis was analysed using the clone formation assay in vitro and tumour xenograft experiment in nude mice. Results: Compared with the overexpression of control cells, proliferation and migration ability of SOX4 overexpression cells significantly increased, the apoptosis rate significantly decreased in addition to the expression levels of Bax and Cleaved caspase-3 (P < 0.05). Compared with the knockdown of control cells, proliferation and migration ability of SOX4 knockdown cells significantly decreased, and the apoptosis rate and expression levels of Bax and Cleaved caspase-3 significantly increased (P < 0.05). Clone formation and tumour growth abilities of SOX4 overexpression cells were significantly higher than those of the control cells (P < 0.05), whereas SOX4 knockdown cells had the opposite effect. Conclusion: SOX4 plays an oncogenic role in breast adenocarcinoma tumourigenesis by promoting cell proliferation, migration and inhibiting apoptosis. It can be used as a potential molecular target for breast cancer gene therapy.

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