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.
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Data Availability
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
Abbreviations
- GC:
-
Gastric cancer
- mRNA:
-
Messenger RNA
- miRNA:
-
Micro-RNAs
- GCSCs:
-
Gastric cancer stem-like cells
- MMP:
-
Matrix Metalloproteinase enzyme
- NGS:
-
Next generation sequencing
- CSCs:
-
Cancer Stem-like Cells
- DMEM/F12:
-
Dulbecco modified Eagle medium/F12
- FBS:
-
Fetal Bovine Serum
- SBCs:
-
Spheroid body cells
- DMSO:
-
Dimethyl sulfoxide
- PBS:
-
Phosphate-buffered saline
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- cDNA:
-
Complementary DNA
- Ct:
-
Threshold cycle
- HUVECs:
-
Human umbilical vein endothelial cells
- SDS:
-
Sodium dodecyl sulfate
- IDT:
-
Integrated DNA Technologies
- EDTA:
-
Ethylenediaminetetraacetic acid
- PMSF:
-
Phenyl methane sulfonyl fluoride
- PVDF:
-
Polyvinylidene difluoride
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Acknowledgements
With thanks to the Vice-Chancellor of Research Department of Shiraz University of Medical Sciences by Code No 98-01-13-20379 for their financial support. Also, the authors would like to thank Nasrin Shokrpour in the English department for her editorial assistance in the Research Consulting Center (RCC) of Shiraz University of Medical Sciences.
Funding
This work was supported by the Vice-Chancellor of the Research Department of Shiraz University of Medical Sciences by a Code No 98-01-13–20379.
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DBF contributed to cell culture, gene expression, and writing the manuscript. Hassan Akrami designed and directed the investigation and wrote and revised the manuscript, BM conducted a gene interaction network, KM accomplished gene expression and MF contributed to revising the manuscript.
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Farahani, D.B., Akrami, H., Moradi, B. et al. The Effect of hsa-miR-451b Knockdown on Biological Functions of Gastric Cancer Stem-Like Cells. Biochem Genet 59, 1203–1224 (2021). https://doi.org/10.1007/s10528-021-10057-8
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DOI: https://doi.org/10.1007/s10528-021-10057-8
Keywords
- Gastric cancer stem-like cells (GCSCs)
- Micro-RNAs (miRNA)
- Has-miR-451b
- Apoptosis
- Metastasis
- Stemness markers