Biological Function And Molecular Mechanism Of OLC1 Overexpression In Human Esophageal Squamous Cell Carcinoma (ESCC) Cells

Objective: Esophageal cancer is highly aggressive and the 9th most frequent malignancy. In more than 90% of all patients with esophageal cancer, the tumors were detected in an advanced stage. Over the past research, much has been learned about the molecular biology associated with esophageal cancer. However, the precise mechanisms underlying esophageal cancer remain unresolved. OLC1 (overexpressed in lung cancer) was a functionally unknown gene, which was related to lung cancer. OLC1 was also overexpressed in esophageal squamous cell carcinoma (ESCC), especially in earlier stage. These observations suggest that OLC1 overexpression correlates with the occurrence and development of ESCC. However, the direct evidence for a role of OLC1 in ESCC remains to be defined.Methods: 1. In this study, pEGFP-OLC1 plasmid was stably transfected into KYSE150 cells, and the OLC1-overexpressed cell line was screened and established by Western-bolt experiments. In vitro analysis (growth curve, colony formation and transwell), we evaluated the alterations of cell growth, adhesion and migration in OLC1-transfected ESCC cells. Then we established stable transfectants of EC9706 cells (an endogenously high-level OLC1 cell line) with pGCsi-OLC1, to silence the expression of OLC1 for further verifying the effect of OLC1 on the biological phenotype in ESCC cells. 2. We used a series of apoptosis methods, such as DAPI staining, DNA Ladder, TdT-mediated biotinylated-dUTP nick end labling (TUNEL), Flow cytometry analysis (FAC) to investigate the impact of overexpression of OLC1 on cisplatin- or Ultraviolet radiation (UVR)- induced apoptosis in OLC1 overexpressed cells, and which was also reversely investigated in the OLC1-silenced cell line. Furthermore, we detected the expression change of apoptosis-associated proteins, such as Caspase-3 and Bcl-2 by Western-blot. 3. In EC9706/pGCsi-OLC1 interfering cell line, using small interfering RNAs (siRNA) to silence BRCA1, we further investigated the molecular mechanism of apoptosis related to OLC1.Results: 1. The OLC1-transfected clones promoted ESCC cells growth and increased the adhesion but not migration, when compared with those transfected with vector alone. Also, knockdown of OLC1 induced inhibition of ESCC cells growth. 2. Overexpression of OLC1 inhibited cisplatin- or Ultraviolet radiation (UVR)-induced apoptosis, and this result was also reversely confirmed in OLC1-interfering cell line. . OLC1 overexpression decreased Caspase3 cleavages, and increased Bcl-2 stability. Furthermore, we found that silencing of endogenous OLC1 substantially enhanced the sensitivity to cisplatin- or UV-induced apoptosis, and increased the Caspase3 cleavages, and decreased the Bcl-2 stability in EC9706. 3. In OLC1-overexpressed cell line, BRCA1 was down-regulated by OLC1, and reversely BRCA1 was up-regulated in OLC1 interfering cell line. Our results showed that OLC1 overexpression-induced down-regulation of BRCA1 was through post-transcriptional channel. The direct interaction between OLC1 and BRCA1 was also observed by Pulldown and IP experiments. We further studied the molecular mechanism of apoptosis regulated by OLC1 through interfering BRCA1 expression. The results showed that the apoptotic cells were reduced obviously in BRCA1-silenced group, when compared with the control group. Furthermore, Western-bolt assay indicated that BRCA1-siRNA inhibited BRCA1 protein expression effectively, and also inhibited Caspase3 cleavages and enhanced Bcl-2 stability.Conclusion: Taken together, for the first time, our investigations provided strong evidences that OLC1 promotes proliferation and adhesion, and additionally depressed the apoptotic sensitiveness in ESCC cell line. One of the mechanisms related to OLC1 overexpression-induced inhibition of cell apoptosis might be through the down- regulation of BRCA1 expression. Overexpressed OLC1 enhanced Bcl-2 stability, and inhibited Caspase3 cleavages through down-regulated BRCA1 expression. At the same time, our studies suggested that BRCA1 down-regulated expression might have an impact on BRCA1 interaction with Gadd45a or p53 proteins. These three proteins may be involved in apoptotic inhibition induced by OLC1 overexpression. This study on the imbalance between proliferation and apoptosis of human ESCC induced by abnormal expression of OLC1 indicated that OLC1 plays an important role in the development of human ESCC.