| UCHL1, short for ubiquitin carboxyl-terminal hydrolase L1, is a de-ubiquitinating enzyme (DUB) and a member of ubiquitin C-terminal Hydrolases (UCHs). UCHL1is found to be expressed in brain and testis/ovary and its expression is especially high in brain where it account for almost2%of the total protein. Low expression or mutations in specific position of the gene often leads to neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Recently more and more researches discovered its altered expression in various malignant tumors, indicating its role in carcinogenesis. Yet UCHL1plays different roles in different kinds of cancers. In non-small cell lung cancer and renal cell carcinoma, etc, UCHL1enhances cell proliferation and migration; however, in breast cancer and prostate cancer, etc, UCHL1inhibits cell proliferation while induces apoptosis, playing the role as a tumor suppressor gene. In ovarian cancer, only one paper reported there’s promoter hypermethylation of UCHL1. Yet its detailed role in ovarian cancer is still unclear and remains to be identified.In the first section, we found that both of two ovarian cancer cell lines A2780and IGROV1showed enhanced cell proliferation ability and decreased apoptosis level after UCHL1knockdown. There was no statistical significance of apoptosis level between IGROV1-shUCHL1and IGROV1-control, however, IGROV1-shUCHL1still showed a similar trend. In addition, after induction of apoptosis by treatment with cisplatin, A2780-shUCHLl and IGROV1-shUCHLl showed less apoptosis than their controls. We further demonstrated that UCHL1knocked down cells showed higher cisplatin resistance (IC50value). To confirm the relationship between UCHL1expression level and cisplatin resistance, we detected UCHL1expression level and cisplatin resistance in a panel of7ovarian cancer cell lines. It showed that the higher UCHL1expression level the more sensitive the cell is; the lower UCHL1expression level the more resistant the cell is. The UCHL1expression level and cisplatin resistance was negatively correlated with correlation coefficient-0.96(p＜0.001). This is consistent with a UCHL1expression at the GEO database showing that UCHL1was reduced to no expression in the tissues from carboplatin (a parental compound of cisplatin) resistant ovarian cancer patients. Moreover, UCHL1expression level is related to its promoter methylation status. Cell lines with high UCHL1expression showed less or no methylation in its promoter. On the contrary, cell lines with low UCHL1expression showed heavy methylation in its promoter. Treatment with demethylation compound could reverse UCHL1silencing, confirming that UCHL1silence was due to promoter hypermethylation.In the second section, we further investigated the mechanisms underneath the biological change after UCHL1knockdown. Firstly, we performed microarray assay to study the gene expression differences between A2780-shUCHL1and A2780-control. After analyzing the microarray data, we found that genes with fold change>2enriched in cell apoptosis and cell death related gene ontology (GO) terms, indicating that UCHL1played a role in regulating apoptosis. Further analysis showed a list of apoptosis related genes regulated by UCHL1, in which the up-regulation of BCL2was most noticeable. Also, we detected that another member of BCL2family Bax was down regulated in protein level. As a key regulator of apoptosis, p53showed no significant alteration of expression level. Knockdown of BCL2led to decreased cell proliferation potential and cisplatin resistance. In addition, phosphor AKT level was up-regulated in UCHL1knockdown cells, suggesting hyper-activation of AKT pathway in UCHL1knockdown cells. Taking together, we provide strong evidence that UCHL1knockdown induced cisplatin resistance might be mediated through up-regulation of BCL2and activation of the AKT pathway and targeting these pathway might be a promising strategy to overcome cisplatin resistance in ovarian caner. |
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