Abstract: Objective To investigate the effect of microRNA-375 (miR-375) targeting autophagy associated gene 14 (Atg14)-mediated autophagy on the proliferation and metastasis of hepatoma cells (SMMC-7721). Methods SMMC-7721 cells were divided into miR-375 mimics, miR-375 inhibitor and Atg14 interfering RNA treatment groups. Models with hypoxia for 1 h and reoxygenation for 6 h were established, and were divided into miR-375 NC group, miR-375 mimics group, miR-375 inhibitor group, siRNA NC group, Atg14 siRNA group and miR-375+Atg14 siRNA group. Targetscan was used to predict the genetic association between miR-375 and the Atg14. Fluorescence real-time quantitative PCR (QRTPCR) was used to detect the relative expressions of miR-375 and ATG14 mRNA in miR-375 NC group, miR-375 mimics group and miR-375 inhibitor group. Immunocytochemical staining was used to detect the expressions of N-Cadherin and β-catenin in the above three groups. The three groups of cells were transfected with GFP-RFP-LC3 adenovirus, and the formation of autophagosomes was observed by the fluorescence microscope. Plate cloning was used to detect the proliferation of liver cancer cells. The expressions of Atg14, P62, LC3Ⅰ, LC3Ⅱ, Beclin1, N-Cadherin, β-catenin and Vimentin were detected by Western blot assay. Results miR-375 gene was highly associated with the Atg14 gene. qRT-PCR showed that overexpression of miR-375 inhibited Atg14 mRNA expression, and conversely, it promoted Atg14 mRNA expression.Overexpression of miR-375 could inhibit the expression of N-Cadherin, increase the expression of β-catenin, and inhibit the proliferation of hepatoma cells (P＜0.01). On the contrary, the inhibition of miR-375 expression promoted the expression of N-Cadherin, decreased β-catenin expression and increased the proliferation of liver cancer cells (P＜0.01). Overexpression of miR-375 inhibited the expression levels of Atg14, LC3Ⅱ and Beclin1, and promoted the expression of P62 (P＜0.01).Conversely, it promoted the expressions of Atg14, LC3Ⅱ and Beclin1, and inhibited the expression of P62 (P＜0.01). By fluorescence microscope, the inhibition of autophagosome formation with overexpression of miR-375 was observed, while the inhibition of miR-375 expression promoted autophagosome formation (P＜0.01). Interfering with the expression of Atg14 enhanced the inhibitory effect of miR-375 on the proliferation and metastasis of hepatoma cells (P＜0.01). Conclusion The activation of miR-375 can inhibit the proliferation and metastasis of hepatoma cells, while the inhibition of miR-375 can promote the proliferation and metastasis of hepatoma cells. The mechanism may be related to miR-375 activating Atg14 to inhibit hepatocellular autophagy, and further influence the proliferation and metastasis of hepatoma cellsSMMC-7721.

Key words: carcinoma, hepatocellular, autophagy, microRNAs, cell proliferation, neoplasm metastasis