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 hsa-miR-33-5p as a Therapeutic Target Promotes Apoptosis of Breast Cancer Cells via Selenoprotein T

2021 ◽  
Vol 8 ◽  
Author(s):  
Wei Zhuang ◽  
Jianhui Liu ◽  
Wenjin Li
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Therapeutic Target ◽  
Cell Apoptosis ◽  
Breast Cancer Cells ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Selenoprotein T ◽  
Cancer Tissues ◽  
Mcf 7

Objective: Increasing evidence suggests that microRNA (miRNA) participates in regulating tumor cell apoptosis. We aimed to observe the effect of hsa-miR-33-5p on the apoptosis of breast cancer cells and to explore its regulatory relationship with selenoprotein T (SelT).Methods: RT-qPCR was used to examine the expression of hsa-miR-33-5p and SelT both in breast cancer tissues and cells. MCF-7 and MDA-MB-231 cells were transfected with hsa-miR-33-5p mimics or si-SelT. Then, a flow cytometry assay was carried out to examine the apoptosis of cells. Furthermore, SelT and apoptosis-related proteins including caspase-3, caspase-8, caspase-9, Bax, and Bcl-2 were detected via RT-qPCR and western blot. A luciferase reporter assay was utilized for assessing whether SelT was targeted by hsa-miR-33-5p.Results: Downregulated hsa-miR-33-5p was found both in breast cancer tissues and cells. After its overexpression, MCF-7 cell apoptosis was significantly promoted. Furthermore, our data showed that miR-33-5p elevated apoptosis-related protein expression in MCF-7 cells. Contrary to hsa-miR-33-5p, SelT was upregulated both in breast cancer tissues and cells. SelT expression was significantly inhibited by hsa-miR-33-5p overexpression. The luciferase reporter assay confirmed that SelT was a direct target of hsa-miR-33-5p. SelT overexpression could ameliorate the increase in apoptosis induced by hsa-miR-33-5p mimics.Conclusion: Our findings revealed that hsa-miR-33-5p, as a potential therapeutic target, could accelerate breast cancer cell apoptosis.

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.

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.

scholarly journals MiR-940 promotes malignant progression of breast cancer by regulating FOXO3

2020 ◽  
Vol 40 (9) ◽  
Author(s):  
Huayao Zhang ◽  
Jingwen Peng ◽  
Jianguo Lai ◽  
Haiping Liu ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Colony Formation ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Poor Survival ◽  
High Level ◽  
The Relationship ◽  
Proliferation And Invasion

Abstract Breast cancer (BC) is a common cancer with poor survival. The present study aimed to explore the effect of miR-940 on the process of BC cells and its target gene FOXO3. The expression of miR-940 was assessed in BC tissues and cells using qRT-PCR. Furthermore, the correlation between miR-940 and prognosis of BC patients from the TCGA database was analyzed. CCK8 assays and colony formation assays were used to explore the effect of miR-940 on BC cell proliferation. The invasion abilities were detected by transwell assays. Luciferase reporter assay was performed to scrutinize the relationship between miR-940 and FOXO3. Finally, rescue experiments were performed through FOXO3 down-regulation and miR-940 inhibitors by using CCK8 assays, colony formation assays and transwell assays. miR-940 was significantly up-regulated in BC cells and tissues. In addition, the high level of miR-940 correlated with poor survival of BC patients (P=0.023). CCK8 assays, colony formation assays and transwell assays indicated that miR-940 promoted the proliferation and invasion abilities of BC cells. The luciferase reporter assay suggested that miR-940 directly targeted FOXO3. Moreover, we found that the effect of si-FOXO3 was rescued by miR-940 inhibitors in BC cells. miR-940 may promote the proliferation and invasion abilities of BC cells by targeting FOXO3. Our study suggested that miR-940 could be a novel molecular target for therapies against BC.

scholarly journals Construction and functional analysis of luciferase reporter plasmid containing connexin43 gene promoter

2017 ◽  
Vol 8 ◽  
pp. 01007
Author(s):  
Hui Li ◽  
Jiapeng Li ◽  
Weilin Shi ◽  
Xiaoyu Zhang ◽  
Yuan Xiang ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Reporter Plasmid ◽  
Luciferase Reporter Plasmid

scholarly journals 17β-Estradiol Activates Glucose Uptake via GLUT4 Translocation and PI3K/Akt Signaling Pathway in MCF-7 Cells

Endocrinology ◽  
2013 ◽  
Vol 154 (6) ◽  
pp. 1979-1989 ◽  
Author(s):  
Pablo Garrido ◽  
Javier Morán ◽  
Ana Alonso ◽  
Segundo González ◽  
Celestino González
Keyword(s):  
Breast Cancer ◽  
Glucose Uptake ◽  
Signaling Pathway ◽  
Breast Cancer Cells ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Increase Glucose Uptake ◽  
17Β Estradiol ◽  
The Relationship ◽  
Mcf 7

Abstract The relationship between estrogen and some types of breast cancer has been clearly established. However, although several studies have demonstrated the relationship between estrogen and glucose uptake via phosphatidylinositol 3-kinase (PI3K)/Akt in other tissues, not too much is known about the possible cross talk between them for development and maintenance of breast cancer. This study was designed to test the rapid effects of 17β-estradiol (E2) or its membrane-impermeable form conjugated with BSA (E2BSA) on glucose uptake in a positive estrogen receptor (ER) breast cancer cell line, through the possible relationship between key components of the PI3K/Akt signaling pathway and acute steroid treatment. MCF-7 human breast cancer cells were cultured in standard conditions. Then 10 nM E2 or E2BSA conjugated were administered before obtaining the cell lysates. To study the glucose uptake, the glucose fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose was used. We report an ER-dependent activation of some of the key steps of the PI3K/Akt signaling pathway cascade that leads cells to improve some mechanisms that finally increase glucose uptake capacity. Our data suggest that both E2 and E2BSA enhance the entrance of the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, and also activates PI3K/Akt signaling pathway, leading to translocation of glucose transporter 4 to the plasma membrane in an ERα-dependent manner. E2 enhances ER-dependent rapid signaling triggered, partially in the plasma membrane, allowing ERα-positive MCF-7 breast cancer cells to increase glucose uptake, which could be essential to meet the energy demands of the high rate of proliferation.

scholarly journals MiR-218-5p promotes breast cancer progression via LRIG1

2021 ◽  
Author(s):  
Mingping Qian ◽  
Hui Xu ◽  
Hongming Song ◽  
Hao Xi ◽  
Lin Fang
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Human Breast Cancer ◽  
Breast Cancer Cells ◽  
Luciferase Reporter ◽  
Human Breast ◽  
Egfr Expression ◽  
Breast Tissues ◽  
Mcf 7

Abstract Background : MiR-218-5p is a small non-coding RNA acting as either oncogenes or tumor suppressor genes in human cancer. The expression levels of some miRNAs in human breast cancer plays a potential role in disease pathogenesis. Methods : Thirty pairs of invasive ductal carcinoma and adjacent specimens were included in the study. Breast tissues cell lines MCF-7 and MDA-MB-231 were identified as a breast cancer research cell line. MiR-218-5p mimics, miR-218-5p inhibitor, or negative controls were transfected. Specific antibodies were probed with LRIG1, ErbB2, and EGFR. Proliferation, migration, cell cycle and apoptosis, dual-luciferase reporter assay and immunohistochemistry were used to analyze miR-218-5p、LRIG1 and so on. Results : It was shown that miR-218-5p expression was higher in 30 breast cancer specimens than adjacent normal breast tissues. In human breast cancer cells MCF-7 and MDA-MB-231, restoring miR-218-5p promoted cell proliferation and migration and inhibited cell apoptosis and cell cycle arrest in the G1 stage. Luciferase assays indicated miR-218-5p could bind with its putative target site in the 3'-untranslated region (3'-UTR) of LRIG1. RT-qPCR, western blot, and immunocytochemistry analyses all indicated miR-218-5p overexpression results in LRIG1 downregulation at the mRNA and protein levels. ErbB2 and EGFR were found to be downstream effectors of miR-218-5p. Conclusion : MiR-218-5p promotes ErbB2 and EGFR expression by inhibiting LRIG1 in breast cancer cells, which suggests miR-218-5p and LRIG1 may act as an oncogene in breast cancer and it could be used as a therapeutic target for breast cancer treatments. Keywords: Breast cancer; miR-218-5p; LRIG1; Oncogene

scholarly journals miR-105-3p acts an oncogene to promote proliferation and metastasis of breast cancer cell by targeting GOLIM4

2020 ◽  
Author(s):  
Bo Lin ◽  
Enyi Shi ◽  
Qiu Jin ◽  
Wenhui Zhao ◽  
Juan Wang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Proliferation And Metastasis ◽  
Cancer Tissues ◽  
Mcf 7

Abstract Background:Dysregulation of miRNAs is involved in carcinogenesis of breast and may be used as prognostic biomarkers and therapeutic targets during cancer process. The purpose of this study was to explore the effect of miR-105-3p on tumourigenicity of breast cancer and its underlying molecular mechanisms.Methods:Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was applied to detect the expression of miR-105-3p in breast cancer tissues and cell lines. The impacts of miR-105-3p on proliferation, migration, invasion and apoptosis of human breast cancer cells (MCF-7 and ZR-75-30) were evaluated by CCK-8, transwell chamber assay, TUNEL assay and western blot assay, respectively. Besides, bioinformatics and luciferase reporter assay were used to find out the target genes of miR-105-3p.Results:The expression of miR-105-3p was elevated in breast cancer tissues and increased along with tumor severity. Downregulation of miR-105-3p could inhibit cell proliferation, suppress cell migration/invasion, and promote cell apoptosis in MCF-7 and ZR-75-30 cells. Furthermore, Golgi integral membrane protein 4 (GOLIM4) was identified to be the direct target gene of miR-105-3p by bioinformatics and luciferase reporter assay. In addition, silencing of GOLIM4 could restore the anti-breast cancer effects induced by miR-105-3p downregulation.Conclusions:miR-105-3p acts an oncogene to promote proliferation and metastasis of breast cancer cell by targeting GOLIM4, which provides a new target for the prevention and treatment of breast cancer.

Sign in / Sign up

Export Citation Format

Share Document