The role of miR-99b in mediating hepatocellular carcinoma invasion and migration

C.-J. Liu, J.-H. Yang, F.-Z. Huang, J.-H. Yang, C.-P. Liu, X.-H. Mao, W.-M. Yi, X.-B. Shen, C. Peng, M.-F. Chen, B. Jiang, J.-S. Wu

Department of Hepatobiliary Surgery, Hunan People’s Hospital, Changsha, China. huangfeizhous@sina.com

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• Oncology

OBJECTIVE: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer in adults with a high rate of malignancy. The potent invasion and migration of HCC mainly impact the prognosis and recurrence of the disease. Our previous study found that miR-99b was highly expressed in HCC, and its expression was associated with vascular invasion. It was speculated that miR-99b may play a role in HCC invasion and migration, while the specific mechanism remains unclear.

MATERIALS AND METHODS: qRT-PCR was applied to detect expressions of miR-99b and KAI1 genes in L02, HepG2, and MHCC97H cells. HepG2 cells were transfected with miR-99b inhibitor, miR-99b mimic, and NC. Flow cytometry was used to test cell cycle and apoptosis. Dual-luciferase reporter gene assay was adopted to validate the target gene of miR-99b. Wound healing assay was used to detect cell migration. Transwell assay was performed to detect cell invasion. Western blot was performed to detect KAI1, E-cadherin, and N-cadherin expressions. Immunofluorescence assay was adopted to test Vimentin expression.

RESULTS: The level of miR-99b was reduced in L02 while up-regulated in MHCC97H. By contrast, the expression of KAI1 was increased in L02 but declined in MHCC97H. The transfection of miR-99b mimic inhibited HepG2 apoptosis and accelerated cell cycle. MiR-99b suppressed KAI gene expression through targeting its 3’-UTR. MiR-99b mimic or si-KAI1 transfection promoted cell invasion and migration, while their simultaneous action significantly enhanced cell invasion and migration. The overexpression of miR-99b or knockdown of KAI1 significantly weakened HepG2 cell adhesion, reduced E-cadherin expression, upregulated N-cadherin and Vimentin, and promoted cell epithelial-mesenchymal transition (EMT).

CONCLUSIONS: MiR-99b contributes to promoting function in HCC migration and invasion through inhibiting KAI1 expression.

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