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Updated: May 2, 2026
An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
miR-638 suppresses cell proliferation in gastric cancer by targeting Sp2
Ling Yu Zhao1, Yu Yao, Jia Han
1Department of Genetics and Cell Biology, Environment and Genes Related to Diseases Key Laboratory of Education Ministry, College of Medicine, Xi'an Jiaotong University, Xi'an, 710061, People's Republic of China, lingyuzhao@aliyun.com.
Background:
MicroRNAs play important roles in the development and progression of various cancers. Recent studies have shown that miR-638 was downregulated in several tumors; however, its role in gastric cancer (GC) has not been investigated in detail.
Aims:
The purpose of this study was to determine the role of miR-638 and to elucidate its regulatory mechanism in GC.
Methods:
The expression levels of miR-638 and specificity protein 2 (Sp2) were detected by real-time PCR and Western blotting in GC. After pcDNA6.2-GW/EmGFP-miR-638 vector, miR-638 inhibitor and Sp2-siRNA transfection, the AGS cell proliferation was investigated by MTT assay and cell cycle, and apoptosis was detected using the Annexin V/PI. In addition, the regulation of Sp2 by miR-638 was evaluated by real-time RT-PCR, Western blot and luciferase reporter assays; cyclin D1 expression was measured by Western blotting.
Results:
The expression of miR-638 is dramatically down-regulated and Sp2 expression is remarkably up-regulated in GC tissues. Luciferase assays revealed that miR-638 inhibited Sp2 expression by targeting the 3'-UTR of Sp2 mRNA. Overexpression of miR-638 and Sp2-siRNA reduced Sp2 expression at both the mRNA and protein levels in vitro, and inhibition of miR-638 increased Sp2 expression. Moreover, we found that miR-638 overexpression and Sp2-siRNA markedly suppressed cell proliferation with decreasing expression of cyclin D1 and inducing G1-phase cell-cycle arrest in vitro; inhibition of miR-638 significantly promoted cell proliferation by increasing expression of cyclin D1 and leading more cells into the S and G2/M phase.
Conclusions:
Our results demonstrated that miR-638 suppressed GC cell proliferation by targeting Sp2 with influence on the expression of cyclin D1. We suggest that miR-638 might be a candidate predictor or an anticancer therapeutic target for GC patients.
Insights
MicroRNA 638 (miR-638) is downregulated in gastric cancer (GC), inhibiting tumor cell proliferation by targeting Sp2 and cyclin D1. This suggests miR-638 as a potential therapeutic target for GC.
Area of Science:
- Oncology
- Molecular Biology
- Gene Regulation
Background:
- MicroRNAs (miRNAs) are crucial in cancer development.
- miR-638 is downregulated in multiple cancers.
- Its role in gastric cancer (GC) requires detailed investigation.
Purpose of the Study:
- To investigate the role of miR-638 in gastric cancer.
- To elucidate the regulatory mechanism of miR-638 in GC.
- To assess miR-638 as a potential therapeutic target for GC.
Main Methods:
- Real-time PCR and Western blotting to measure miR-638 and Sp2 expression in GC.
- Cell proliferation, cell cycle, and apoptosis assays (MTT, Annexin V/PI) post-transfection.
- Luciferase reporter assays to confirm miR-638 targeting of Sp2 mRNA.
Main Results:
- miR-638 expression was significantly downregulated, while Sp2 expression was upregulated in GC tissues.
- miR-638 directly targets Sp2 mRNA, inhibiting its expression.
- miR-638 overexpression suppressed GC cell proliferation by downregulating cyclin D1 and inducing G1 arrest.
Conclusions:
- miR-638 suppresses GC cell proliferation by targeting Sp2 and influencing cyclin D1 expression.
- miR-638 demonstrates potential as a predictive biomarker or therapeutic target for GC.
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