As an important cis-acting element, super-enhancers are rich in transcription factors and cofactors, such as high-density BRD4[21, 22], which greatly enhance gene expression[23]. Accumulating studies indicate that super-enhancers play a pivotal role in regulating target genes, fostering the malignant progression of tumors[24–27]. The LINC00963 gene is situated on human chromosome 9q34.11, and research indicates that its disorders are commonly involved in tumor invasion, metastasis, and disease deterioration. [28–30]. However, it remained unclear whether LINC00963 is associated with any super enhancers. In our study, we comprehensively characterized the super-enhancer landscape in GC by analyzing uploaded H3K27ac ChIP-seq data from GC cell lines (GSE75595). Subsequently, we identified LINC00963 as a super-enhancer-associated gene. It has been reported that transcripts sensitive to JQ1 are potential super-enhancer-related genes[31]. Our results demonstrated that low doses of JQ1 inhibited the transcription of LINC00963 in GC cell lines. These findings indirectly suggest that LINC00963 is regulated by super-enhancers. Furthermore, we observed high expression of LINC00963 in both GC cell lines and the peripheral blood of GC patients, implying a potential role for LINC00963 in the initiation and progression of GC.
The types of proteins in humans have been shown to be far greater than the number of genes, due in part to multiple alternative splicing in genes translate proteins with different functions. We found multiple alternative splicing variants in GC, which may have a crucial role in the malignant progression of tumors. Our results showed that LINC00963 has at least three variants not included in the NCBI database, while the level of wild-type transcript (NR 038955.1) expression was very low. In several studies involving the alternative splicing of the LINC00963 gene, the newly discovered splicing variants is the deletion of the 5’end sequence[32, 33]. Our research results showed that the LINC00963 alternative splicing was mainly caused by the 59 base increase in the 5’ end of the second exon and the deletion of the third exon, which may be due to the splicing variants produced by different malignant tumors. Notably, we did not perform a 5’ RACE test to verify whether there is a deletion of the RNA 5’ end of the LINC00963 gene. Because there are multiple splicing variants in LINC00963 and the LINC00963-V1 expression was the highest, it is still unknown whether the variable splicing event of LINC00963 was related to the mutation or deletion of some sites of LINC00963, which affected the post-transcriptional modification of RNA, this will be the focus of our further research. The LINC00963 expression was downregulated in both AGS and MKN45 cells. Although bioinformatics analysis showed that LINC00963 was the target gene of the super-enhancer in AGS and MKN45 cell lines, the reason for this result might be the high degree of differentiation between AGS and MKN45. The differences caused by the different sources of cancer cells when the cells were constructed may also be caused by some mutations in the continuous culture of cell lines, which may result in the low expression of LINC00963 or the weakened function of super-enhancers. In conclusion, the high expression of LINC00963 and its different splicing variants in peripheral blood indicates that LINC00963 may be a potential molecular marker of GC, which implys a new insight for LINC00963 studies.
At present, accumulated studies have shown that EMT is closely related to the invasion and metastasis of tumors, and involves the signal transmission of Wnt/β-catenin, TGF-β, and other related pathways [30, 34–37]. After the knockout of LINC00963-V1 in vitro, the invasion and migration ability of GC cells were greatly weakened, the EMT-related proteins changed, and the expression of β-catenin was decreased. As β-catenin is an important molecular marker in the Wnt/β-catenin signaling pathway, the Wnt/β-catenin signaling pathway has been associated with EMT in multiple tumor studies[17, 38]. Numerous studies have shown that LINC00963 has an oncogene role in tumor occurrence and development, promoting the metastasis and invasion of cancer cells[39–41], which is consistent with our results. We also found that the LINC00963 gene fosters EMT of GC via the Wnt/β-catenin signaling pathway, thereby promoting the invasion and migration of GC cells.
In summary, this study has unveiled multiple previously unreported alternative splicing variants of LINC00963 and identified the underlying mechanisms by which LINC00963 is involved in tumor invasion and migration relying on super-enhancer. Specifically, LINC00963 mediates the regulation of the Wnt/β-catenin signaling pathway through super-enhancer, promoting epithelial-mesenchymal transition of gastric cancer. These findings offer new insights into LINC00963 as a novel biomarker for GC metastasis and a potential therapeutic target in gastric cancer treatment.