DLGAP1-AS2 knockdown increases chemosensitivity to vandetanib and synergistically inhibits metastatic activity of papillary thyroid cancer cells: experimental and computational approaches

Papillary thyroid carcinoma (PTC) progression is closely associated with the regulatory roles of long non-coding RNAs (lncRNAs). The role of DLGAP1-AS2 has been investigated as a potentially oncogenic lncRNA (oncoLnc) in several malignancies; nevertheless, its specific tumorigenic activity in PTC remains largely unexplored. The relative expression of DLGAP1-AS2 was evaluated using FASTQ files and read count data obtained from thyroid cancer (THCA) samples accessible via the SRA and TCGA repositories. Additionally, siRNA transfection was employed to efficiently inhibit DLGAP1-AS2 activity. The effects of DLGAP1-AS2 and vandetanib on the proliferation and metastatic capability of PTC cells were evaluated using in vitro experiments (MTT, apoptosis analysis, DAPI staining, cell cycle assessment, and scratch & colony formation assays). The gene expressions of Caspase-3, Caspase-9, Bcl2, Bax, p53, CDK1, MMP-3, MMP-9, CD44, and CD133, WNT9B, MAPK10, and CACNA1D were determined using quantitative real-time PCR. Transfection with si-DLGAP1-AS2 resulted in a marked downregulation of DLGAP1-AS2 expression in B-CPAP cells. Treatment with si-DLGAP1-AS2 or vandetanib alone exerted limited effects on B-CPAP cells’ viability, whereas their combination exerted marked toxicity against these cells and decreased the IC50 of vandetanib from 44.96 µg/ml to 27.36 µg/ml, reflecting the higher sensitivity of B-CPAP cells to this chemotherapeutic when combined with si-DLGAP1-AS2. Flow cytometric analysis demonstrated that the combination therapy substantially augmented apoptosis rate, surpassing the level observed in si-DLGAP1-AS2 or vandetanib-treated groups. The sub-G1 cell population increased after si-DLGAP1-AS2 and vandetanib individual treatments, yet combination treatment led to a higher sub-G1 ratio. The combined use of si-DLGAP1-AS2 and vandetanib markedly impeded cell migration, accompanied by MMP-9 and MMP-3 downregulation. There was a down-expression of stemness-associated markers of CD44 and CD133 measured by qRT-PCR. Pro-apoptotic genes, including Caspase-3, Caspase-9 and Bax, were upregulated while Bcl2, WNT9B, MAPK10, and CACNA1D genes were substantially downregulated in the combined treatment group vs. control group. In conclusion, si-DLGAP1-AS2 and vandetanib combination promoted cell cycle arrest by regulating key genes involved in the process. DLGAP1-AS2 seems to be a potential prognostic biomarker involved in the molecular pathogenesis of PTC and a promising candidate for targeted therapy development. This work aimed to investigate the role of DLGAP1-AS2 as a possible therapeutic target in thyroid cancer. Targeted suppression of this lncRNA was achieved utilizing siRNA-loaded lipofectamine. Prior to the experimental phase, the potential involvement of DLGAP1-AS2 in oncogenic pathways was ascertained through bioinformatic analyses. Following these computational findings, the functional consequences of DLGAP1-AS2 silencing were confirmed through a series of in vitro assays. Expression of genes involved in tumorigenic pathways were investigated via real-time PCR; cellular proliferation and migration were examined through colony formation and scratch assays, and cell viability and programmed cell death were measured using MTT and apoptosis assays, respectively, to understand the oncogenic mechanisms regulated by DLGAP1-AS2.