Study on the mechanism of inhibiting invasion of human laryngeal squamous cell carcinoma Hep-2 and TU212 cells after the downregulation of miRNA-106b

Abstract

Objective To investigate the effect of miRNA-106b (miR-106b) on human laryngeal squamous cell carcinoma Hep-2 and TU212 cells and its mechanism. Methods Hep-2 and TU212 cells were divided into miR-106b inhibitory sequence transfected group (the experimental group), miR-106b competitive negative sequence transfected group (the negative control group) and non-intervention group (the blank group). The inhibitory effect of miR-106b inhibitory sequence on the expression of miR-106b was verified by using reverse transcription quantitative polymerase chain reaction (qRT-PCR). Whether phosphatase and tensin homolog (PTEN) was the target gene of miR-106b was analyzed by using bioinformatics and luciferase report vector. PTEN small interfering RNA (siRNA) was used to inhibit the expression of PTEN in Hep-2 and TU212 cells. Transwell method and Western blot were used to detect the change of invasion ability of Hep-2 and TU212 cells after miR-106b silencing or the PTEN intervening, and the expression change of PTEN, epithelial cadherin and vimentin. Results The relative expression levels of miR-106b in Hep-2 and TU212 cells in the experimental group were 0.110 ± 0.037 and 0.074 ± 0.009, respectively, which were lower than those in the negative control group (1.013±0.059 and 1.035±0.062, respectively; all P < 0.05). In Transwell experiments, the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group was less than that in the negative control group [(37.09±4.02) vs. (95.65±4.77), (29.16±2.49) vs. (103.19±6.08), all P < 0.05]. The bioinformatics analysis results showed that 3'-UTR region of PTEN mRNA was complementary to 3'-UTR region of miR-106b. Dual-luciferase reporter system analysis showed that the luciferase reporter activity of wild-type PTEN gene transfected with miR-106b was decreased to (22.84±2.68)%, and that of mutant PTEN gene transfected with miR-106b was almost unchanged [(92.08±3.44)%], and the difference was statistically significant (P < 0.001). The expression level of PTEN protein of Hep-2 and TU212 cells in the experimental group was higher than that in the negative control group. Transwell method showed that the number of invasive cells in each field of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression was more than that in the experimental group without the inhibition of PTEN expression [(65.08±3.57) vs. (26.72±2.58), (57.38±4.96) vs. (31.81±2.97), all P < 0.05]. Western blot showed that the expression level of epithelial-cadherin was up-regulated and vimentin was down-regulated of Hep-2 and TU212 cells in the experimental group with the inhibition of PTEN expression. Conclusions The human laryngeal squamous cell carcinoma Hep-2 and TU212 cell miR-106b can influence the downstream invasion-related protein of PTEN and change the cell invasion ability through the targeted regulation of PTEN expression. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Neoplasm invasiveness; RNA interference; miRNA-106b; Phosphatase and tensin homolog