UBE2C Directly Targeted by mirR-548-5p Increases the Cellular Growth and Invasive Abilities of Cancer Cells Interacting with the EMT Marker Protein ZEB1/2 in NSCLC

Abstract

Background: Recent evidence indicates that miR-548-5p dysregulation plays a vital role in tumor progression. Aberrant miR-5485p expression may be linked to tumorigenesis since miRNAs play important roles in various cellular processes and cancer metastasis. UBE2C is a type of ubiquitination enzyme catalyzing the degradation of proteins into smaller polypeptides and amino acids. UBE2C participates in carcinogenesis by regulating the cell cycle, apoptosis, metastasis and transcriptional processes. However, the molecular mechanism of UBE2C as a growth factor regulating the tumorigenesis and metastasis of lung cancer is not clearly. EMT progression is mediated by the EMT-inducing transcriptional factors ZEB1/2, which is an important characteristic for cancer metastasis. What molecules regulate the expression of ZEB1/2 and then regulate the EMT in the lung cancer is also not clearly. We found that miR-548-5p, UBE2C, ZEB1/2 have the similar functions in tumor progression and metastasis. However, the relationship between miR-548-5p, UBE2C and ZBE1/2 in the tumorigenesis and development of lung cancer is still unknown. Methods: the mRNA expression of miR-548-5p, UBE2C was analyzed by RT-PCR assay. The protein level of UBE2C and ZEB1/2 was analyzed by western blot and Immunofluorescent staining. The cellular proliferation was detected by CCK8 and MTT. The cell migration and invasion growth were analyzed by wound healing assay and transwell assay. The activities of promoter and gene transcription were analyzed by luciferase reporter assay. Chromatin immunoprecipitation detected the combining ability of UBE2C and 5'UTRZEB1/ 2. Results: In our study, we found that miR-548-5p was significantly downregulated in lung cancer tissue specimens. Our objective was to elucidate the functions, direct target genes, and molecular mechanisms of miR-548-5p in lung cancer tissue. We found that miR-548-5p downregulated the expression of its direct substrate, 4,5-ubiquitin-conjugating enzyme E2C (UBE2C). UBE2C is a proto-oncogene that is activated in lung cancer. However, its role in lung cancer metastasis is unclear. We found here that UBE2C expression was higher in non-small-cell lung cancer (NSCLC) than in the normal tissue adjacent to it and was associated with increased cell proliferation and invasion. UBE2C enhanced NSCLC progression and metastasis by arresting the cell cycle and inhibiting apoptosis. We used reverse transcription (RT)-PCR assay to determine miR548-5p and UBE2C expression in lung cancer patients. Overexpression of miR-548-5p and UBE2C were established in lung cancer cells. Methyl thiazolyl tetrazolium (MTT)- and colony formation assays were performed to evaluate their effects on cell proliferation. We also performed cell migration- and invasion assays on lung cancer cells overexpressing miR548-5p and under expressing UBE2C. The results indicated that the aforementioned treatments significantly suppressed lung cancer cell proliferation, migration, and invasion. Moreover, luciferase reporter and Chromatin immunoprecipitation assay indicated that miR-548-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. Using RT-PCR and western blot (WB), we also assessed epithelial-mesenchymal transition (EMT). We found that UBE2C knockdown downregulated the expression of the mesenchymal marker vimentin. In contrast, the epithelial marker E-cadherin was upregulated. Therefore, UBE2C promoted EMT. Bioinformatics assays coupled with WB and luciferase assays revealed that UBE2C directly binds to the 5′-untranslated region of the E-cadherin repressor, ZEB1/2 and promotes EMT in lung cancer cells. Conclusion: the miR-548-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. UBE2C is an oncogene promoting EMT in lung cancer cells by directly targeting the 5'-UTR of E-cadherin repressor ZEB1/2. The miR-548-5p, UBE2C, and ZEB1/2 constitute the miR548-5p-UBE2C-ZEB1/2 signal axis which enhances cancer cell invasiveness by directly interacting with the EMT maker protein. We believe that the miR-548-5p-UBE2C-ZEB1/2 signal axis may be a suitable diagnostic marker and a potential target in lung cancer therapy Funding: This work was supported by the Science and Technology Development Foundation of Yantai (2015ZH082), Natural Science Foundation of Shandong Province (ZR2018QH004, ZR2016HB55, ZR2017PH067, ZR2017MH125 and ZR2015PH004), and Research Foundation of Binzhou Medical University (BY2015KYQD25 and BY2015KJ14). Declaration of Interest: The authors declare no competing financial interests. Ethical Approval: The experimental protocol was approved by the Research Ethics Committee of Binzhou Medical University, China (No. 2017-016-01 for human lung cancer specimen and No. 2017-009-09 for mouse experiments in vivo) and the written informed consent was obtained from all subjects. Informed consent was obtained from all individual participants included in the study. All patients were staged based on the criteria of the 7th Edition of the AJCC Cancer Staging Manual: Stomach (2010)