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Updated: Nov 12, 2025
Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells
Published on: May 9, 2020
The Effect of hsa-miR-451b Knockdown on Biological Functions of Gastric Cancer Stem-Like Cells
Diba Borzabadi Farahani1, Hassan Akrami2, Behrouz Moradi1
1Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran.
Abstract:
Numerous researches have extensively studied factors such as microRNAs that lead to cancer. Thus, the current study's purpose is to investigate the biological consequences of hsa-miR-451b inhibition on the properties and functions of gastric cancer stem-like cells. First, gastric cancer stem-like cells were transfected by hsa-miR-451b inhibitor then we used real-time RT-PCR to evaluate its effect on the expression of hsa-miR-451b and two of its direct target genes, Stemness markers such as KLF4, SOX2, CD44, OCT3/4 and NANOG genes and finally Akt, PI3K, Bcl-2, Bax, CASP3 and PCNA genes involved in apoptosis. Here, we conducted a DNA Laddering assay to investigate apoptosis. The level of the MMP-2 and -9 Activities and Migration were examined by Zymography and Transwell invasion assay. HUVEC cells were used to investigate angiogenesis. The outcomes revealed that the level of the MMP-2 and -9 Activities, migration and angiogenesis decreased, but apoptosis was induced by inhibiting hsa-miR-451b. Evaluating KREMEN1 and CASK expression showed that the former increased, and the latter dropped under hsa-miR-451b inhibition. Also, upregulation of the KLF4 and SOX2 and downregulation of the CD44, OCT3/4, and NANOG decreased Self-renewal ability of gastric cancer stem cells under hsa-miR-451b inhibition. Even, under hsa-miR-451b inhibition, downregulation of Akt, PI3K, Bcl-2 and PCNA as well as upregulation of Bax and CASP3 revealed a movement towards apoptosis in MKN-45 stem-like cells. In summary, hsa-miR-451b is an oncomir in the carcinogenesis of gastric cancer stem-like cells and may be suggested as an appropriate therapeutic target for future gastric cancer treatment.
Insights
Inhibiting hsa-miR-451b suppresses gastric cancer stem-like cell growth by inducing apoptosis and reducing migration. This microRNA acts as an oncomir, making it a potential therapeutic target for gastric cancer treatment.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- MicroRNAs (miRNAs) play critical roles in cancer development.
- Gastric cancer stem-like cells (GCSCs) are crucial for tumor initiation and progression.
- Understanding miRNA roles in GCSCs is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the biological effects of inhibiting hsa-miR-451b on GCSCs.
- To assess the impact of hsa-miR-451b inhibition on stemness, apoptosis, invasion, and angiogenesis.
- To explore hsa-miR-451b as a potential therapeutic target in gastric cancer.
Main Methods:
- Transfection of GCSCs with hsa-miR-451b inhibitor.
- Real-time RT-PCR to analyze gene expression (hsa-miR-451b, target genes, stemness markers, apoptosis-related genes).
- DNA Laddering assay, Zymography, Transwell invasion assay, and HUVEC co-culture for apoptosis, MMP activity, migration, and angiogenesis assessment.
Main Results:
- Inhibition of hsa-miR-451b decreased MMP-2/-9 activity, migration, and angiogenesis.
- Apoptosis was induced in GCSCs upon hsa-miR-451b inhibition.
- Stemness markers (KLF4, SOX2) were upregulated, while others (CD44, OCT3/4, NANOG) were downregulated, reducing self-renewal.
- Expression of apoptosis-related genes (Bax, CASP3) increased, while pro-survival genes (Akt, PI3K, Bcl-2, PCNA) decreased.
Conclusions:
- hsa-miR-451b functions as an oncomir in gastric carcinogenesis.
- Inhibiting hsa-miR-451b effectively suppresses GCSC properties and induces apoptosis.
- hsa-miR-451b inhibition presents a promising therapeutic strategy for gastric cancer.
