Related Experiment Video
Updated: Jun 2, 2026
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Published on: May 30, 2012
[miR-145 inhibits lung adenocarcinoma stem cells proliferation by targeting OCT4 gene]
Shuai Zhang1, Yaqin Wu, Dongjie Feng
1Department of Thoracic Surgery, Nanjing Medical University Affiliated Cancer Hospital of Jiangsu Province, Nanjing, China.
Background And Objective:
MiR-145 functions as a protective miRNA identified in tumor tissues of lung adenocarcinoma patients. The aim of this study is to investigate the relationship between miR-145 and proliferation of lung cancer stem cells and involved molecular mechanisms in human lung adenocarcinoma A549 cell line.
Methods:
MicroRNA microarray technology was conducted to compare miRNA signature between tumor and adjacent normal tissue of lung adenocarcinoma. The potential target gene of miR-145 was predicted by online bioinformatic softwares. Pre-miR-145 mimics and anti-miR-145 inhibitor were transfected into A549 cell line by lipofectamine 2000. miR-145 expression in each group was detected by real time PCR. The OCT4 protein level was analyzed by Western blot. The predicted miR-145 binding site in OCT4 3'-untranslated region (UTR) was validated by dual-luciferase reporter gene assay. CCK-8 assay was employed to observe the proliferation activity of A549 cells. The ratio of CD133 positive cells in each group was analyzed by flow cytometry.
Results:
miR-145 expression was significantly down-regulated in lung adenocarcinoma compared with ajacent normal tissue. OCT4 is a potential target gene of miR-145 predicted by miRanda. Compared with control group, miR-145 was significantly up-regulated and down-regulated in the pre-miR-145 mimics and anti-miR-145 inhibitor groups respectively. Overexpression of miR-145 inhibited the proliferation of A549 cells. Both the OCT4 protein level and CD133 positive ratio were remarkably decreased in the pre-miR-145 mimics group, whereas significantly increased in the anti-miR-145 inhibitor group. Dual-luciferase reporter gene assay validated the predicted miR-145 binding site of OCT4 3'UTR.
Conclusions:
MiR-145 can inhibit the proliferation of lung cancer stem cells in A549 cell line via down-regulating OCT4 expression. MiR-145 is a potential protective miRNA of lung cancer.
Insights
MicroRNA-145 (miR-145) inhibits lung cancer stem cell proliferation by down-regulating OCT4. This finding highlights miR-145 as a potential protective factor in lung adenocarcinoma.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- Lung adenocarcinoma is a major cancer type with limited therapeutic options.
- MicroRNAs (miRNAs) play crucial roles in cancer development and progression.
- MiR-145 is implicated as a tumor suppressor in various cancers, including lung adenocarcinoma.
Purpose of the Study:
- To investigate the role of miR-145 in regulating lung cancer stem cell (LCSC) proliferation.
- To elucidate the molecular mechanisms underlying miR-145's function in human lung adenocarcinoma A549 cells.
- To identify potential therapeutic targets for lung adenocarcinoma based on miR-145 activity.
Main Methods:
- MicroRNA microarray analysis to identify differentially expressed miRNAs in lung adenocarcinoma tissues.
- Bioinformatic prediction and experimental validation of miR-145 targets.
- Transfection of A549 cells with miR-145 mimics and inhibitors.
- Quantitative real-time PCR, Western blot, and dual-luciferase reporter assays to assess gene and protein expression.
- CCK-8 and flow cytometry assays to evaluate cell proliferation and stem cell markers.
Main Results:
- MiR-145 expression was significantly downregulated in lung adenocarcinoma tissues compared to normal adjacent tissues.
- OCT4 was identified as a direct target of miR-145, with miR-145 binding to the OCT4 3'-UTR.
- Overexpression of miR-145 inhibited A549 cell proliferation and reduced OCT4 protein levels and CD133+ cell ratios.
- Inhibition of miR-145 led to increased OCT4 expression and promoted proliferation.
Conclusions:
- MiR-145 suppresses lung cancer stem cell proliferation in A549 cells by downregulating OCT4 expression.
- MiR-145 acts as a tumor-suppressive miRNA and holds potential as a therapeutic agent for lung adenocarcinoma.
- Targeting the miR-145/OCT4 pathway may offer a novel strategy for lung cancer treatment.
Related Concept Videos
02:40Cancer Stem Cells and Tumor Maintenance
Cancer stem cells are thought to originate from tissue-specific normal stem cells or progenitor cells. The normal stem cells usually reside in...
02:40Cancer Stem Cells and Tumor Maintenance
Cancer stem cells are thought to originate from tissue-specific normal stem cells or progenitor cells. The normal stem cells usually reside in...
01:22MicroRNAs