Effects of <i>PTPN6</i> Gene Knockdown in SKM-1 Cells on Apoptosis, Erythroid Differentiation and Inflammations
<b>Objective:</b> Protein tyrosine phosphatase non-receptor type 6 (<i>PTPN6)</i> is a cytoplasmic phosphatase that acts as a key regulatory protein in cell signaling to control inflammation and cell death. In order to investigate the role of <i>PTPN6</i> in hemat...
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| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-10-01
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| Series: | Current Issues in Molecular Biology |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1467-3045/46/11/715 |
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| Summary: | <b>Objective:</b> Protein tyrosine phosphatase non-receptor type 6 (<i>PTPN6)</i> is a cytoplasmic phosphatase that acts as a key regulatory protein in cell signaling to control inflammation and cell death. In order to investigate the role of <i>PTPN6</i> in hematologic tumor myelodysplastic syndrome (MDS), this study infected SKM-1 cell line (MDS cell line) with packaged H_<i>PTPN6</i>-shRNA lentivirus to obtain H_<i>PTPN6</i>-shRNA SKM-1 stable strain. The effect of <i>PTPN6</i> knockdown on apoptosis, erythroid differentiation, and inflammations in SKM-1 cell line was examined. <b>Methods:</b> The stable knockdown SKM-1 cell line was validated using qPCR and Western blot assays. The proliferation activity, apoptosi, erythroid differentiation, and inflammatory cytokines in SKM-1 cells were assessed before and after transfection. <b>Results:</b> qPCR confirmed that the expression level of H_<i>PTPN6</i>-shRNA in SKM-1 cells was significantly reduced, and Western blot showed that the protein expression level of H_<i>PTPN6</i>-shRNA in SKM-1 cells was also significantly reduced. The CCK-8 cell viability assay confirmed that stable gene knockdown did not affect cell viability. Flow cytometry revealed that the apoptosis rate of cells in the <i>PTPN6</i> knockdown group was 0.8%, lower than the 2.7% observed in the empty plasmid group; the expression rate of the erythroid differentiation marker CD235a was 13.2%, lower than the 25.0% observed in the empty plasmid group. The expression levels of the proinflammatory factors IL-6 and IL-8 increased, and the expression levels of the inhibitor factor IL-4 decreased. <b>Conclusions:</b> The <i>PTPN6</i> gene was successfully knocked down using lentivirus-mediated transduction, and the constructed cell line was validated using PCR and Western blot. The CCK-8 cell viability assay confirmed that stable gene knockdown did not affect cell proliferation viability. Flow cytometry analysis of apoptosis and erythroid differentiation indicated that <i>PTPN6</i> knockdown inhibits apoptosis and erythroid differentiation in SKM-1 cells and also alters the level of inflammations in the bone marrow microenvironment. It suggests that the <i>PTPN6</i> gene acts as a tumor suppressor in myelodysplastic syndrome cells, influencing hematopoietic cell apoptosis, erythroid differentiation, and inflammations. This provides a reliable experimental basis for further in-depth studies on the mechanism of <i>PTPN6</i> in MDS and related pharmacological research. |
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| ISSN: | 1467-3037 1467-3045 |