Fig 2: Piwi-like RNA-mediated gene silencing (PIWIL2) regulates the proliferation of the oral squamous cell carcinoma (OSCC) cell line TCA8113. (a) qPCR detection confirmed the effect of PIWIL2 knockdown using two PIWIL2-targeting short hairpin RNAs (shRNAs) in Tca8113 cells. (b) Cell Counting Kit-8 (CCK8) assays demonstrated repressed proliferation in Tca8113 cells with PIWIL2 knockdown. (c) Colony formation analysis showed inhibited cancer cell clone formation ability in Tca8113 cells with PIWIL2 knockdown. The statistics of the colony counts are shown in the right panel. (d) Flow cytometry analysis showed arrested cell cycle progression in Tca8113 cells following PIWIL2 knockdown. The cell population increased in the G0/G1-phase and decreased in the G2/M-phase. (e) Western blot analysis showed that PIWIL2 knockdown resulted in downregulated proliferating cell nuclear antigen (PCNA), cyclin D1, and cyclin dependent kinase 2 (CDK2) protein expression and upregulated p53 protein expression. The statistics of the protein levels are shown in right panel. Data are representative of three independent experiments. Values are presented as the mean ± standard deviation (SD). #P < 0.05, **P < 0.01 versus the corresponding control.

Fig 3: Piwi-like RNA-mediated gene silencing (PIWIL2) modulates oral cancer cell migration. (a) Wound healing assays showed that knockdown of PIWIL2 inhibited Tca8113 cell migration. The statistics of the migration rate experiments are shown in the right panel. (b) Transwell migration assays demonstrated inhibited migration of Tca8113 cells when PIWIL2 was silenced. The statistics of migrated cells per microscopical field of vision are shown in the right panel. (c) Transwell invasion assays revealed impaired invasion in Tca8113 cells with PIWIL2 knockdown. The statistics of invaded cells per microscopical field are shown in the right panel. (d) Western blot analyses showed that the protein expression levels of cell migration and invasion-related genes N-cadherin, vimentin, and matrix metalloproteinase 9 (MMP9) were downregulated, while epithelial gene E-cadherin was upregulated, following PIWIL2 knockdown. The bar graph shows the protein levels normalized to GAPDH protein levels. Data are representative of three independent experiments. Values are presented as the mean ± standard deviation (SD). **P < 0.01 versus the corresponding control.

Fig 4: Piwi-like RNA-mediated gene silencing (PIWIL2) knockdown induces apoptosis in oral cancer cells. (a) Silencing PIWIL2 induced apoptosis in Tca8113 cells, as detected by Annexin V/propidium iodide (PI) staining. The statistics of the percentage of apoptotic cells are shown in the right panel. (b) The anti-apoptotic protein BCL-2 was downregulated, while cleaved caspase-3 and cleaved caspase-9 were activated, when PIWIL2 was silenced in Tca8113 cells. The statistics of the protein level quantification are shown in the right panel. Data are representative of three independent experiments. Values are presented as the mean ± standard deviation (SD). **P < 0.01 versus the corresponding control.