X-F Wang1, J-Y Liu, J-L Li, F-Z Wang, B-L Sun. 1. Department of Hepatobiliary Surgery, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China. SUNBL6612@outlook.com.
Abstract
OBJECTIVE: To explore the role of hsa-microRNA-99b/let-7e/microRNA-125a cluster in the progression of liver cancer and its possible regulatory mechanisms. PATIENTS AND METHODS: Ten liver cancer tissues were randomly selected and matched with normal liver tissue samples. The mRNA expression levels of miR-99b, let-7e and miR-125a were detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The three miRNA mimics were transfected alone or together into hepatoma cells SMMC-7721; at the same time, the knockdown of the three miRNAs was also performed. Then the cell invasive and migratory abilities were examined through transwell assay. Bioinformatics analysis was used to detect the potential transcription factors that bind to the hsa-microRNA-99b/let-7e/microRNA-125a promoter sequence, and the binding of the two was verified by the Luciferase reporting assay. The level of hsa-microRNA-99b/let-7e/microRNA-125a mature mRNA was detected after ZEB1 was inhibited by ZEB1 siRNA. Meanwhile, after interfering with Drosha and ZEB1, the expression level of hsa-microRNA-99b/let-7e/microRNA-125a of the primary transcript was examined. Rescue experiments were carried out to assess the role of hsa-microRNA-99b/let-7e/microRNA-125a in the ZEB1 regulation of invasive cell capacity. RESULTS: The mRNA levels of microRNA-99b, let-7e, and microRNA-125a in 10 selected hepatocarcinoma tissues were significantly higher than those in the matched paracancerous tissues. After overexpressing the three miRNA mimics either alone or together, cell invasive and migratory abilities were extensively enhanced, and vice versa. It was found that there is a binding site in the upstream sequence of the promoter region of the hsa-microRNA-99b/let-7e/microRNA-125a cluster for ZEB1. The Luciferase reporter gene results showed an increase in the Luciferase activity of the cells transfected with E-box element wild-type sequence, while mutant E-box element group did not change. After knocking out ZEB1, the levels of mature microRNA-99b, let-7e and microRNA-125a were reduced. However, when DROSHA was knocked out, the levels of immature microRNA-99b, let-7e and microRNA-125a were increased, while simultaneous knocking out ZEB1 reversed this effect. Besides, the invasive ability of SMMC-7721 cells decreased after KEB1 was knocked down, while the opposite result was observed after transfection of hsa-microRNA-99b/let-7e/microRNA-125a alone or together. CONCLUSIONS: Hsa-microRNA-99b/let-7e/microRNA-125a cluster is highly-expressed in hepatocarcinoma, and its expression can be regulated by ZEB1. In addition, the overexpression of this cluster can promote the invasion of liver cancer cells and advance liver cancer progression.
OBJECTIVE: To explore the role of hsa-microRNA-99b/let-7e/microRNA-125a cluster in the progression of liver cancer and its possible regulatory mechanisms. PATIENTS AND METHODS: Ten liver cancer tissues were randomly selected and matched with normal liver tissue samples. The mRNA expression levels of miR-99b, let-7e and miR-125a were detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The three miRNA mimics were transfected alone or together into hepatoma cells SMMC-7721; at the same time, the knockdown of the three miRNAs was also performed. Then the cell invasive and migratory abilities were examined through transwell assay. Bioinformatics analysis was used to detect the potential transcription factors that bind to the hsa-microRNA-99b/let-7e/microRNA-125a promoter sequence, and the binding of the two was verified by the Luciferase reporting assay. The level of hsa-microRNA-99b/let-7e/microRNA-125a mature mRNA was detected after ZEB1 was inhibited by ZEB1 siRNA. Meanwhile, after interfering with Drosha and ZEB1, the expression level of hsa-microRNA-99b/let-7e/microRNA-125a of the primary transcript was examined. Rescue experiments were carried out to assess the role of hsa-microRNA-99b/let-7e/microRNA-125a in the ZEB1 regulation of invasive cell capacity. RESULTS: The mRNA levels of microRNA-99b, let-7e, and microRNA-125a in 10 selected hepatocarcinoma tissues were significantly higher than those in the matched paracancerous tissues. After overexpressing the three miRNA mimics either alone or together, cell invasive and migratory abilities were extensively enhanced, and vice versa. It was found that there is a binding site in the upstream sequence of the promoter region of the hsa-microRNA-99b/let-7e/microRNA-125a cluster for ZEB1. The Luciferase reporter gene results showed an increase in the Luciferase activity of the cells transfected with E-box element wild-type sequence, while mutant E-box element group did not change. After knocking out ZEB1, the levels of mature microRNA-99b, let-7e and microRNA-125a were reduced. However, when DROSHA was knocked out, the levels of immature microRNA-99b, let-7e and microRNA-125a were increased, while simultaneous knocking out ZEB1 reversed this effect. Besides, the invasive ability of SMMC-7721 cells decreased after KEB1 was knocked down, while the opposite result was observed after transfection of hsa-microRNA-99b/let-7e/microRNA-125a alone or together. CONCLUSIONS: Hsa-microRNA-99b/let-7e/microRNA-125a cluster is highly-expressed in hepatocarcinoma, and its expression can be regulated by ZEB1. In addition, the overexpression of this cluster can promote the invasion of liver cancer cells and advance liver cancer progression.