Background and objective: microRNAs (miRs) are small noncoding RNAs that modulate a variety of cellular processes by regulating multiple targets, which can promote or inhibit the development of malignant behaviors. Accumulating evidence suggests that microRNA-221 (miR-221) plays important roles in human carcinogenesis. It has recently found that miR-221 was overexpressed in hepatocellular carcinoma (HCC), and overexpression of miR-221 has a bad prognosis in these patients. Thus, down-regulation of miR-221 expression in HCC would provide new treatment approaches. This study aimed to study the role of miR-221 on HCC cell growth, apoptosis, invasion and metastasis in vitro and vivo, and explored the possible mechanisms involved.
Methods: Effects of miR-221 upregulation or miR-221 downregulation by miR-221 inhibitor (anti-miR-221) or miR-221 mimic (miR-221) transfection on growth, apoptosis and invasion of HepG2 cells in vitro was detected. Using p65 siRNA and p65 cDNA transfection to examine the NFκB signaling pathway. A subcutaneously implanted tumor model of HepG2 cells in nude mouse was used to assess the effects of anti-miR-221 or miR-221 overexpression on tumorigenesis development. Using an intravenously injected tumor model of HepG2 cells to assess the effects of anti-miR-221 or miR-221 overexpression on lung metastasis. The signaling pathway was analyzed in vivo.
Results: Anti-miR-221 inhibited growth, invasion and induced apoptosis of HepG2 cells in vitro. This was accompanied by concomitant attenuation of NFκB, and downregulation of NFκB downstream genes such as Bcl-2, VEGF and MMP-9. In addition, miR-221 overexpression promoted growth and invasion of HepG2 cells in vitro, and accompanied by activation of NFκB, and upregulation of NFκB downstream genes Bcl-2, VEGF and MMP-9. Targeting P65 or P65 overexpression reversed the effect of miR-221, and inhibited or induced miR-221 expression, creating a positive feedback loop in human HepG2, respectively. Morever, stable overexpression of anti-miR-221 in HepG2 cells inhibits establishment of xenografts and lung metastasis in nude mice; Stable overexpression of miR-221 in HepG2 cells promotes establishment of xenografts and lung metastasis in nude mice.
Conclusions: Therapies targeting the miR-221 signaling pathway may be more effective to prevent primary tumor formation and organ metastasis. The ability of this therapy to decrease tumorigenesis and metastasis may be related to NFκB signals.
Keywords: Hepatocellular carcinoma; NFκB; apoptosis; metastasis; microRNA-221.