Silencing of LINC00467 inhibits cell proliferation in testicular germ cell tumors cells Running Title: Silencing of LINC00467 regulates cell proliferation

Abstract Our previous study showed that LINC00467 expression decreased significantly in testicular germ cell tumors (TGCTs) compared with adjacent tissue. LINC00467 expression in TGCTs was upregulated at stages II/III compared with stage I, and was negatively correlated with the 5-year overall survival and 5-year disease-free survival. However, the role of LINC00467 in the development of TGCTs remains to be elucidated further. LINC00467 expression was silenced in the NCCIT and TCAM-2 cell lines using small interfering RNA (siRNA). The expression level of genes was validated using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The MTT and CCK8 assays were used to detect cell proliferation. The effects on cell cycle were evaluated using flow cytometry. Western blotting analysis was used to detect the expression level of proteins, while RNA-sequencing and bioinformatics methods were used to explore the mechanism of action of LINC00467 in TGCTs. The silencing of LINC00467 expression decreased cell proliferation, induced S phase arrest, and downregulated the cell cycle-related protein PCNA expression while upregulating the expression of P21. Dihydrotestosterone (DHT) stimulation experiments showed that DHT could upregulate the expression of LINC00467 and that the silencing of LINC00467 could reverse the effect of testosterone on cell proliferation. The Gene Set Enrichment Analysis (GSEA) showed that the P53 signaling pathway was associated with LINC00467. Our study reported that LINC00467 regulated cell proliferation and induced S phase arrest in TGCTs cells through the cell cycle-related proteins, PCNA, and P21. These enriched the mechanism of non-coding RNAs in the development of TGCTs.