Unveiling ER-Phagy Receptors in Thyroid Cancer: A Combined Approach of Public Data Mining and In Vitro Validation

<h4>Background</h4>The incidence of papillary thyroid cancer (PTC) has risen sharply over the past several decades. Early identification of high-risk PTC is crucial to mitigate the societal burden associated with its overdiagnosis. ER-phagy, a selective form of autophagy targeting the endoplasmic reticulum, is categorized into macro-ER-phagy and micro-ER-phagy. To date, the role of ER-phagy receptors and their impact on risk stratification and progression in thyroid cancer remain unclear.<h4>Methods</h4>This study employed an integrative approach combining bioinformatics analysis with wet-lab molecular biology experiments. Transcriptomic and clinical data from TCGA were processed using Xiantaoxueshu, GSCA, TIMER, and TISIDB platforms to assess differential expression, survival, ROC characteristics, and KEGG/GO enrichment, as well as immune infiltration. In vitro, shRNAs targeting RTN3, SEC62, or ATL3 were transfected into TPC-1 and CAL-62 cells to silence gene expression, validated by RT-qPCR and Western Blot. Cell proliferation, migration, and invasion were assessed by CCK-8, EdU, colony formation, wound healing, and Transwell assays. All experiments were performed in at least three independent replicates, and the resulting data were subjected to statistical analysis. Data were analyzed using SPSS 18.0 and GraphPad Prism 9.3.0. Quantitative results are presented as mean ± SEM. Independent-samples t-tests, univariate/multivariate Cox regression and LASSO Cox regression were used; P < 0.05 was considered significant.<h4>Results</h4>Comprehensive analysis of all ER-phagy receptors using public databases revealed that several receptors significantly impact the diagnosis and prognosis of thyroid cancer. We also characterized the mutation and methylation pattern of these receptors. Furthermore, these genes were also closely associated with immune infiltration in thyroid cancer. Of note, RTN3, SEC62 and ATL3 appeared to have distinct importance in thyroid cancer. All three genes were downregulated and their reduced expression was significantly related to poor survival in thyroid cancer. Receiver Operating Characteristic (ROC) curve analysis demonstrated that these markers hold substantial promise for thyroid cancer diagnosis, particularly RTN3 and SEC62. Immune-related analysis indicated a strong correlation with immune infiltration, implying a potential role in modulating the immune landscape of thyroid cancer. Functional assays demonstrated that knockdown of RTN3, SEC62, and ATL3 promoted proliferation and metastasis of thyroid cancer cells in vitro. Western blot analysis indicated that this process is likely mediated by the Epithelial-Mesenchymal Transition (EMT).<h4>Conclusion</h4>In summary, our findings indicate that ER-phagy represents a promising avenue for thyroid cancer diagnosis. Specifically, RTN3, SEC62, and ATL3 were found to suppress the proliferation and metastasis of thyroid cancer cells, underscoring their pivotal roles in disease progression.