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Upregulation of LINC01503 expression by SOX9 promotes malignant biological behaviors and tumor stem cell stemness in laryngeal squamous cell carcinoma
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Abstract:
Objective: To investigate the influence of SOX9 on the proliferation, migration, invasion, and stemness of laryngeal squamous cell carcinoma (LSCC) cells by upregulating the expression of long non-coding RNA LINC01503. Methods: Human LSCC cells AMC-HN-8, TU177, TU212, and TU686 were cultured routinely. Control nucleic acids (NC) and knockdown sequences (si-SOX9- NC, si-SOX9#1, si-SOX9#2, si-LINC01503-NC, si-LINC01503#1, si-LINC01503#2) or overexpression plasmids (pcDNA3.1-SOXNC, pcDNA3.1-SOX-oe, pcDNA3.1-LIN01503-NC, and pcDNA3.1-LIN01503-oe) were transfected into TU177 cells or TU686 cells. These groups were named as si-SOX9-NC group, si-SOX9#1 group, si-SOX9#2 group, si-LINC01503-NC group, si-LINC01503#1 group, si-LINC01503#2 group; pcDNA3.1-SOX9-NC group, pcDNA3.1-SOX9-oe group, pcDNA3.1-LINC01503-NC group, pcDNA3.1-LINC01503-oe group, si-SOX9-NC + pcDNA3.1-LINC01503-NC group, and si-SOX9 + pcDNA3.1-LINC01503-oe group. The expression of SOX9 mRNA and LINC01503 in each group of cells was detected by qPCR. Bioinformatics tool was employed to identify the binding site of SOX9 to lncRNA LINC01503 promoter region. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene experiments were carried out to validate the binding between SOX9 and the LINC01503 promoter. SOX9 knockdown efficiency and the influence of LINC01503 on the expression of stem cell markers in TU177 and TU686 cells were detected by WB assay. MTS assay was used to detect cell proliferation, scratch wound healing, and Transwell assays assessed cell migration, while colony formation assays evaluated the ability of cells to form colonies. Results: SOX9 was highly expressed in all LSCC cells (R = 0.12, P < 0.05). Database analysis showed a positive correlation between SOX9 and LINC01503 expression in head and neck squamous cell carcinoma (P = 0.005 9). It was proved that SOX9 directly binds with the LINC01503 promoter and promotes its transcriptional expression (P < 0.05). Knockdown of LINC01503 significantly suppressed the proliferation, migration, and invasion of TU177 cells (all P<0.05). Overexpression of LINC01503 notably enhanced the proliferation, migration, invasion and colony formation of TU686 cells (all P<0.05), as well as the expression of cell stemness markers CD133, OCT4, and SOX2 at both mRNA and protein levels (all P<0.05). However, knockdown of LINC01503 inhibited colony formation and expression of stem cell markers in TU686 cells (P < 0.05). Knockdown of SOX9 significantly suppressed the proliferation, migration, and invasion ability of TU177 cells and reduced the expression of their stem cell markers (all P<0.05). Meanwhile, overexpression of LINC01503 partially reversed the inhibitory effect of SOX9 knockdown on the malignant biological behavior and stem cell marker expression in TU177 cells (all P < 0.05). Conclusion: Both SOX9 and LINC01503 are highly expressed in LSCC cells. SOX9 may promote the proliferation, migration, invasion and stemness of laryngeal squamous cell carcinoma by upregulating LINC01503 expression.
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