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The Effect of hsa-miR-451b Knockdown on Biological Functions of Gastric Cancer Stem-Like Cells

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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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Contributions

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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Correspondence to Hassan Akrami.

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The authors declare that they have no conflicts of interest.

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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

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