Genetic and epigenetic changes can lead to tumorigenesis and development.
Substantial evidences have verified that epigenetic modifications, such as DNA methylation alterations, nucleosome remodeling, and histone modification are involved in the biological process of maintaining normal development and gene expression . DNA methylation alteration is one of the most common events in tumorigenesis and plays an important role in the initiation and development of tumors . It has been recognized that ZNF family proteins are frequently decreased by DNA methylation in various types of human cancer .For example, ZNF471 is one of the most significantly hypermethylated with low mRNA expression in esophageal cancer as compared with normal tissues . It has been confirmed that ZNF671 functions as a tumor suppressor in multiple carcinomas due to DNA methylation [21–22].
Previous studies have demonstrated that DNA methylation alterations participate in tumorigenesis and are significantly correlated with the clinicopathological features of ccRCC . Dysregulated of gene methylation has been shown to increase ccRCC cell proliferation and invasion, and may contribute to the progression and recurrence of ccRCC [24–25]. ZNF677, a member of the ZNFs family, recently has been reported that downregulated in several different types of cancer due to promoter hypermethylation [13–15].The present study demonstrated that,combining with the online data, ZNF677 acted as a tumor suppressor gene with significantly higher methylation in ccRCC tumor tissues compared to normal tissues. Clinical data showed that ZNF677 methylation was significantly positively associated with clinicopathologic features. In addition, hypermethylation was negatively correlated with mRNA expression, thereby suggesting that ZNF677 silenced may be due to aberrant methylation. Moreover, drug demethylation experiments showed that treated with the demethylation drug Aza combined with the HDAC inhibitor trichostatin A restored the expression of ZNF677 in 786-0 cells. These results indicate that the hypermethylation of ZNF677 regulates its downregulation expression in ccRCC, which is consistent with previous studies in thyroid cancer,lung cancer, and gastric cancer, implying that ZNF677 methylation may be a common event in tumorigenesis [13–15]. Furthermore, we used online data analysis to explore the potential prognostic value of ZNF677 in ccRCC. We found that low expression of ZNF677 predicted poor survival in ccRCC. These data suggested that ZNF677 may be an unfavorable prognostic factor in ccRCC. To further estimate the potential biological function of ZNF677 in ccRCC, we carried out a series of experiments about proliferation, apoptosis, and invasion on 786-O cells. And the results showed that ectopic expression of ZNF677 could significantly inhibit cell proliferation, invasion and induce apoptosis. Therefore, we infer that ZNF677 may serve as a potential therapeutic target to improve the prognosis of ccRCC.
Metastasis is the main cause of death of ccRCC. Epithelial Mesenchymal transition (EMT) is an essential process of tumor metastasis . Recent studies have proposed that EMT is a key event in cancer cell development, invasion, and metastasis. EMT can enhance the mobility and reduce the adhesion of tumor cells,and then promotes tumor metastasis and invasion . The decreased expression of epithelial marker genes and the increased expression of mesenchymal marker genes are the markers of EMT. Therein, the reduced expression of E-cadherin is what marks the threshold of the EMT process . The influence of ZNFs factors on EMT and cancer invasion has been confirmed in various numbers of cancers. ZNF703 has been reported that induced EMT by inhibiting E-cadherin expression and enhanced breast cancer cell invasion and resist to sorafenib . ZNF281 has been proved to play a crucial role in controlling cellular stemness expression and EMT by EMT induction and regulation of EMT-associated gene expression in colorectal cancer . ZNF471 suppressed cervical cancer cell invasion through modulating EMT by negatively regulating Wnt/ β-catenin signaling pathway . In our study, we observed that ZNF677 significantly reduced 786-0 cell invasion. Furthermore, ectopic expression of ZNF677 significantly elevated E-cadherin protein expression and reduced vimentin protein expression in 786-0 cells, indicating that ZNF677 decreased 786-0 cell invasion by reversing EMT. The finding was consistent with other researchers in thyroid cancer, thus supporting the concept that ZNF677 suppressed cancer metastasis and invasion through regulating EMT.
EMT can be triggered by many signaling pathways. Aberrant activation of AKT signaling pathway has been revealed in many cancers that attribute to cancer cell metastasis and EMT [32–34]. The precise molecular mechanisms of ZNF677 involved in tumor invasion and EMT are currently elusive, but activation of the PI3K/AKT signaling pathway has been reported [15, 17]. Siraj AK etal. demonstrated that overexpression of ZNF677 inhibited thyroid cancer cell invasion, decreased E-cadherin expression, and increased N-cadherin expression. Whereas, knockdown PI3K/AKT signaling pathway reduced the expressions of N-cadherin, Twist, and Zeb1, with an accompanying expression of E-cadherin in PTC cell lines. This result proposed that ZNF677 mediated thyroid cancer cell invasion and EMT through AKT signaling pathway-dependent. Therefore, we sought to determine whether PI3K/AKT signaling pathway was involved in ZNF677 regulated ccRCC cell EMT. Western blot analysis showed that ectopic expression of ZNF677 significantly elevated E-cadherin protein expression and reduced vimentin protein expression in 786-0 cells, accompanied with inhibition of cell invasion. Meanwhile,the expression of p-AKT protein was decreased. Thus, the PI3K/AKT signaling pathway may play a role in ZNF677 promoted 786-0 cell EMT. However, additional studies are still required to explore the exact molecular mechanism underlying the effects of ZNF677 and ccRCC cell EMT.
Some limitations in our study should not be ignored. First, there are no in vivo experiments in our study, all results are based on public data and cellular experiments, and require to further validate by in vivo experiments. Second,because there is only 786-0 cell line in our laboratory, only one cell line was examined in the functional experiment.Third,the exact mechanism of ZNF677 inactivating AKT signaling pathway remains unclear. Given that ZNGF677 is a putative transcription factor, we speculate that it may bind to downstream target genes and then regulate the PI3K/AKT signaling pathway. However, the specific mechanism needs to be further investigated.
In summary, this is the first report to evaluate the expression and function of ZNF677 and ccRCC. Our findings reveal that ZNF677 is hypermethylated in ccRCC, and functional assays suggested that ZNF677 inhibits tumor cell proliferation, invasion and induces apoptosis. Further prognosis analysis indicated that low expression of ZNF677 was associated with shorter overall survival (OS) and ZNF677 suppressed invasion and EMT of 786-0 cells through inactivating PI3K/AKT signaling pathway, but the mechanism remains unknown. Further studies are needed to clarify this issue. Our results confirm that ZNF677 can be used as an adverse prognostic biomarker and a potential therapeutic target for ccRCC.
and Consent to participate
Ethical consent was granted from the Committee for Ethical Review of Research of First Hospital of Guangxi Medical University.