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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
MiR-130b can suppress proliferation of glioma cells through targeting PTEN to regulate AKT pathway
Haitao Niu1, Yang Liu, Yanzhou Wang
1Department of Neurosurgery, Cangzhou Central Hospital, Cangzhou, China.
Purpose:
To study the mechanism of action of micro ribonucleic acid (miR)-130b in the proliferation and apoptosis of glioma cells, and to determine whether it regulates the target gene phosphatase and tensin homolog deleted on chromosome ten (PTEN).
Methods:
The endogenous expression of miR-130b was silenced via transfection with the miR-130b inhibitor. The effects of miR-130b silencing on the proliferation and apoptosis of LN229 cells were detected using cell counting kit-8 (CCK-8) assay, colony formation assay and flow cytometry. Whether miR-130b binds to the target gene PTEN was detected via luciferase reporter assay. The changes in the mRNA level of PTEN after miR-130b silencing were determined through quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The effects of miR-130b on protein kinase B (AKT) signaling pathway-related proteins were determined through Western blotting, and the roles of miR-130b and PTEN in the proliferation of glioma cells were detected via CCK-8 assay.
Results:
Compared with that in normal human astrocytes, the expression of miR-130b was significantly up-regulated in the three kinds of glioma cell lines (p<0.05). Silencing of miR-130b reduced the proliferation (p<0.05) and the colony formation of LN229 cells (p<0.05), and obviously increased their apoptosis (p<0.05), suggesting that silenced miR-130b is a growth inhibitor of glioma cells in vitro. The luciferase reporter assay confirmed that miR-130b directly bound to the 3'-untranslated region (3'UTR) of PTEN to suppress its expression. After transfection with the miR-130b inhibitor, both mRNA and protein expressions of PTEN were up-regulated (p<0.05). Moreover, after silencing of miR-130b, the phosphorylation of AKT was remarkably inhibited, while the cancer suppressor gene p27 was up-regulated.
Conclusions:
The carcinogenic effect of miR-130b in glioma was clarified in this study. Silencing of miR-130b may inhibit the AKT signaling pathway through up-regulating PTEN, thereby suppressing the proliferation of glioma cells.
Insights
MicroRNA-130b promotes glioma cell growth by inhibiting PTEN and activating the AKT pathway. Silencing miR-130b suppresses proliferation and induces apoptosis in glioma cells, offering a potential therapeutic strategy.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- Glioma is a common primary brain tumor with complex molecular mechanisms.
- MicroRNAs (miRNAs) play crucial roles in cancer development and progression.
- Dysregulation of miR-130b and PTEN has been implicated in various cancers.
Purpose of the Study:
- To investigate the role of microRNA (miR)-130b in glioma cell proliferation and apoptosis.
- To determine if miR-130b targets the phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene.
- To elucidate the underlying molecular mechanisms of miR-130b action in glioma.
Main Methods:
- miR-130b expression was silenced using miR-130b inhibitors in LN229 glioma cells.
- Cell proliferation and apoptosis were assessed using CCK-8, colony formation, and flow cytometry assays.
- PTEN targeting was confirmed via luciferase reporter assays, qRT-PCR, and Western blotting; AKT pathway modulation was also analyzed.
Main Results:
- miR-130b expression was significantly upregulated in glioma cell lines compared to normal astrocytes.
- Silencing miR-130b inhibited LN229 cell proliferation and colony formation while increasing apoptosis.
- miR-130b directly targeted PTEN's 3'-UTR, suppressing its expression; PTEN upregulation upon miR-130b inhibition led to decreased AKT phosphorylation and increased p27.
Conclusions:
- miR-130b exhibits carcinogenic effects in glioma by promoting cell proliferation and inhibiting apoptosis.
- Silencing miR-130b suppresses glioma cell growth by upregulating PTEN, subsequently inhibiting the AKT signaling pathway.
- Targeting miR-130b represents a potential therapeutic strategy for glioma treatment.
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