https://orcid.org/0000-0002-0961-3618
Figures & data
Figure 1. MiR-6734-3p and ZEB2 were aberrantly expressed in NSCLC clinical specimens and cells. The levels of (a) miR-6734-3p and (b) ZEB2 mRNA in NSCLC tissues were examined by real-time qPCR. (c) Eight patients were randomly selected, and ZEB2 protein levels in NSCLC tissues were measured by Western Blot. (d) The correlations between miR-6734-3p and ZEB2 mRNA in NSCLC tissues were analyzed by using the pearson correlation analysis. The correlations of (e) miR-6734-3p and (f) ZEB2 mRNA with patient prognosis were analyzed. The expressions of (g) miR-6734-3p and (h) ZEB2 mRNA were determined in NSCLC cells. (i) ZEB2 expressions at protein levels in NSCLC cells were examined. Individual experiment had 3 repetitions, and *P < 0.05
Figure 2. The effects of miR-6734-3 on the malignant phenotypes in NSCLC cells. (a) Delivery of miR-6734-3p mimic and inhibitor into the NSCLC cells. (b, c) At 0 h, 24 h, 48 h and 72 h post-culture, CCK-8 assay was conducted to evaluate cell proliferation. (d) Colony formation assay was performed to reflect cell growth. (e, f) The transwell assay and wound scratch assay were performed to evaluated cell mobility. (g, h) The protein levels of EMT associated biomarkers were examined by Western Blot. (i) Measurement of cell apoptosis ratio by FCM. In vivo animal experiments validated that miR-6734-4p inhibited (j) tumor weight and (k, l) volume in animal models. (m) Localization and expressions of Ki67 protein were examined by IHC in mice tumor tissues. Individual experiment had 3 repetitions, and *P < 0.05
Figure 3. The regulatory mechanisms of miR-6734-3p and ZEB2. (a) The targeting sites in miR-6734-3p and ZEB2 mRNA were predicted. (b, c) dual-luciferase reporter gene system assay and (d, e) RNA pull-down assay was utilized to validate the binding sites of miR-6734-3p and ZEB2. The (f) mRNA and (g) protein levels of ZEB2 could be negatively regulated by miR-6734-3p. (h) The overexpression and silencing vectors for ZEB2 were delivered into NSCLC cells, and (i) manipulation of ZEB2 had little effects on miR-6734-3p expressions in NSCLC cells. Individual experiment had 3 repetitions, and *P < 0.05
Figure 4. Targeting the miR-6734-3p/ZEB2 axis hampered cancer progression in NSCLC. (a, b) CCK-8 assay and (c) colony formation assay was used to evaluated cell growth in vitro. Cell mobility was evaluated by using the (d) Transwell assay and (e) wound scratch assay. (f, g) The protein levels of EMT associated signatures were determined by Western Blot. (h) Cell apoptosis ratio in NSCLC cells was measured by FCM assay. Individual experiment had 3 repetitions, and *P < 0.05
Figure 5. Overexpression of miR-6734-3p sensitized NSCLC cells to cisplatin treatment. The CS-NSCLC and CR-NSCLC cells were challenged by high-dose cisplatin. (a, b) Cell proliferation was determined by CCK-8 assay. (c) FCM was performed to examine cell apoptosis. (d) Downregulated miR-6734-3p, and (e, f) upregulated ZEB2 were observed in CR-NSCLC cells, in contrast with the corresponding CS-NSCLC cells. (g, h) Cisplatin-induced inhibition of NSCLC cell proliferation was enhanced by miR-6734-3p overexpression and ZEB2 ablation. (i) Targeting miR-6734-3p and ZEB2 increased cell apoptosis ratio in CR-NSCLC cells stimulated by high-dose cisplatin. Individual experiment had 3 repetitions, and *P < 0.05
