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. 2019 Nov 21:19:308.
doi: 10.1186/s12935-019-1026-4. eCollection 2019.

MicroRNA-1269 promotes cell proliferation via the AKT signaling pathway by targeting RASSF9 in human gastric cancer

Wen-Li Liu #  1 , Hu-Xia Wang #  2 , Cheng-Xin Shi  3 , Fei-Yu Shi  3 , Ling-Yu Zhao  4 , Wei Zhao  3 , Guang-Hui Wang  3
Affiliations

MicroRNA-1269 promotes cell proliferation via the AKT signaling pathway by targeting RASSF9 in human gastric cancer

Wen-Li Liu et al. Cancer Cell Int. .

Abstract

Background: MicroRNAs (miRNAs) play key roles in tumorigenesis and progression of gastric cancer (GC). miR-1269 has been reported to be upregulated in several cancers and plays a crucial role in carcinogenesis and cancer progression. However, the biological function of miR-1269 in human GC and its mechanism remain unclear and need to be further elucidated.

Methods: The expression of miR-1269 in GC tissues and cell lines was detected by quantitative real-time PCR (qRT-PCR). Target prediction programs (TargetScanHuman 7.2 and miRBase) and a dual-luciferase reporter assay were used to confirm that Ras-association domain family 9 (RASSF9) is a target gene of miR-1269. The expression of RASSF9 was measured by qRT-PCR and Western blotting in GC tissues. MTT and cell counting assays were used to explore the effect of miR-1269 on GC cell proliferation. The cell cycle and apoptosis were measured by flow cytometry. RASSF9 knockdown and overexpression were used to further verify the function of the target gene.

Results: We found that miR-1269 expression was upregulated in human GC tissues and cell lines. The overexpression of miR-1269 promoted GC cell proliferation and cell cycle G1-S transition and suppressed apoptosis. The inhibition of miR-1269 inhibited cell growth and G1-S transition and induced apoptosis. miR-1269 expression was inversely correlated with RASSF9 expression in GC tissues. RASSF9 was verified to be a direct target of miR-1269 by using a luciferase reporter assay. The overexpression of miR-1269 decreased RASSF9 expression at both the mRNA and protein levels, and the inhibition of miR-1269 increased RASSF9 expression. Importantly, silencing RASSF9 resulted in the same biological effects in GC cells as those induced by overexpression of miR-1269. Overexpression of RASSF9 reversed the effects of miR-1269 overexpression on GC cells. Both miR-1269 overexpression and RASSF9 silencing activated the AKT signaling pathway, which modulated cell cycle regulators (Cyclin D1 and CDK2). In contrast, inhibition of miR-1269 and RASSF9 overexpression inhibited the AKT signaling pathway. Moreover, miR-1269 and RASSF9 also regulated the Bax/Bcl-2 signaling pathway.

Conclusions: Our results demonstrate that miR-1269 promotes GC cell proliferation and cell cycle G1-S transition by activating the AKT signaling pathway and inhibiting cell apoptosis via regulation of the Bax/Bcl-2 signaling pathway by targeting RASSF9. Our findings indicate an oncogenic role of miR-1269 in GC pathogenesis and the potential use of miR-1269 in GC therapy.

Keywords: Apoptosis; Gastric cancer; Proliferation; RASSF9; miR-1269.

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Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
miR-1269 is upregulated in human GC tissues and cell lines. a qRT-PCR showed that miR-1269 expression was significantly increased in GC tissues compared with normal gastric tissues. b miR-1269 expression was significantly increased in GC cell lines (BGC-823, SGC-7901, MKN-45 and AGS) compared with a normal gastric epithelial cell line (GES-1). Wilcoxon test, *p < 0.01
Fig. 2
Fig. 2
miR-1269 promotes human GC AGS/MKN-45 cell proliferation and inhibits apoptosis. a miR-1269 expression was examined in AGS/MKN-45 cells after miR-1269 overexpression. b miR-1269 expression was detected in AGS/MKN-45 cells after anti-miR-1269 treatment. c MTT assay revealed that miR-1269 overexpression promoted cell activity at 48 and 72 h after transfection. d MTT assay showed that anti-miR-1269 suppressed cell activity at 48 and 72 h after transfection. e Cell counting assay showed that miR-1269 overexpression increased GC cell proliferation. f Cell counting assays revealed that anti-miR-1269 suppressed GC cell growth. g Flow cytometry analysis of the cell cycle. The histogram represents the proportion of cells in the G0/G1, S and G2/M phases after miR-1269 overexpression. h The ratio of cells in the G0/G1, S and G2/M phases after anti-miR-1269 transfection. i Flow cytometry analysis of cell apoptosis. The data showed the ratios of early and late apoptosis after miR-1269 overexpression. j The data revealed the proportions of cells in early and late apoptosis after anti-miR-1269 transfection. Wilcoxon test, *p < 0.01, n = 3
Fig. 3
Fig. 3
miR-1269 directly targets the RASSF9 gene. a Bioinformatics analysis predicted interactions between miR-1269 and its binding sites in the 3′-UTR of RASSF9. b Luciferase activity was measured by the dual-luciferase assay. c RASSF9 mRNA expression in human GC tissues. d RASSF9 protein levels were examined by Western blotting (n = 10). e miR-1269 and RASSF9 levels were negatively correlated. The 2−∆∆Ct values of miR-1269 and negatively were subjected to a Spearman correlation analysis (n = 73, r = − 0.7222, p < 0.0001). Wilcoxon test, *p < 0.01
Fig. 4
Fig. 4
miR-1269 regulates the AKT and Bcl-2/Bax signaling pathways in human GC cells by targeting RASSF9. a RASSF9 mRNA and protein expression levels were significantly decreased in GC cell lines (BGC-823, SGC-7901, MKN-45 and AGS) compared with a normal gastric epithelial cell line (GES-1). b RASSF9 mRNA was examined in AGS/MKN-45 cells after miR-1269 overexpression. c RASSF9 mRNA was determined in AGS/MKN-45 cells after anti-miR-1269 treatment. d miR-1269 overexpression promoted the expression of the p-AKT, Cyclin D1, CDK2 and Bcl-2 proteins and inhibited RASSF9 and Bax expression in AGS/MKN-45 cells. e Anti-miR-1269 inhibited p-AKT, Cyclin D1, CDK2 and Bcl-2 protein expression and increased RASSF9 and Bax expression. Wilcoxon test, *p < 0.01
Fig. 5
Fig. 5
RASSF9 siRNAs promote the proliferation of human GC cells. a The results showed the knockdown efficiency of RASSF9 siRNA-1/2 in AGS/MKN-45 cells at 24 h after transfection. b MTT assay showed that RASSF9 siRNA-1/2 increased the activity of AGS/MKN-45 cells at 48 and 72 h. c Cell counting assay revealed that RASSF9 siRNA-1/2 promoted AGS/MKN-45 cell proliferation at 48 and 72 h. d Flow cytometric analysis showed the percentage of AGS/MKN-45 cells in the G0/G1, S and G2/M phases. The proportion of G0/G1 phase cells decreased after RASSF9 siRNA-1/2 transfection, and the proportion of S and G2/M phase cells increased. e The data showed the percentage of early and late apoptosis after RASSF9 siRNA-1/2 transfection. f RASSF9, p-AKT, Cyclin D1, CDK2, Bcl-2 and Bax protein expression levels were measured 48 h after RASSF9 siRNA-1/2 transfection. Wilcoxon test, *p < 0.01, n = 3
Fig. 6
Fig. 6
RASSF9 overexpression inhibits the proliferation of human GC cells. a RASSF9 overexpression promoted RASSF9 mRNA levels in AGS/MKN-45 cells. b MTT assay showed that RASSF9 overexpression decreased AGS/MKN-45 cell activity at 48 and 72 h. c Cell counting assay showed that RASSF9 overexpression inhibited AGS/MKN-45 cell growth. d The cell cycle was measured in AGS/MKN-45 cells at 48 h. e Apoptosis was examined in AGS/MKN-451 cells at 48 h. f RASSF9, p-AKT, AKT, Cyclin D1, CDK2, Bcl-2 and Bax expression levels were detected after transfection with the RASSF9 overexpression vector. Wilcoxon test, *p < 0.01, n = 3
Fig. 7
Fig. 7
RASSF9 overexpression rescues the miR-1269-induced cellular phenotypes in GC cells. a RASSF9 overexpression rescued RASSF9 mRNA expression levels reduced by miR-1269. b RASSF9 overexpression rescued RASSF9 protein expression levels reduced by miR-1269. c MTT assay was performed to examine the growth of AGS/MKN-45 cells after cotransfection with RASSF9 and miR-1269 expression vectors. d Cell counting assay was used to measure AGS/MKN-45 cell proliferation following cotransfection with RASSF9 and miR-1269. One-way ANOVA, *p < 0.01, compared with the control group; #p < 0.01, compared with the miR-1269 overexpression group, n = 3
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