Targeting of YAP1 by microRNA-15a and microRNA-16-1 exerts tumor suppressor function in gastric adenocarcinoma

Abstract

Background: MicroRNAs (miRNAs) have been reported to play an important role in tumorigenesis. In this study, the role of miR-15a and miR-16-1 in gastric adenocarcinoma (GAC) was investigated.

Methods: The expression of miR-15a and miR-16-1 in cell lines and primary tumors was examined by miRNA qRT-PCR. Proliferative assays, colony formation, cell invasion and migration, flow cytometry analysis and in vivo study were performed by ectopic expression of miR-15a and miR-16-1. The putative target genes of miR-15a and miR-16-1 were explored by TargetScan and further validated.

Results: We found that miR-15a and miR-16-1 were down-regulated in GAC cell lines and primary tumor samples compared with normal gastric epithelium. Functional study demonstrated that ectopic expression of miR-15a and miR-16-1 suppressed cell proliferation, monolayer colony formation, invasion and migration, and xenograft formation in vivo. In addition, miR-15a and miR-16-1 induced G0/G1 cell cycle arrest which was further confirmed by Western blot and qRT-PCR of related cell cycle regulators. YAP1 was confirmed to be a functional target of miR-15a and miR-16-1 in GAC. YAP1 re-expression partly abrogated the inhibitory effect of miR-15a and miR-16-1 in GAC cells. In clinical samples, YAP1 protein expression shows negative correlation with miR-15a and miR-16-1 expression.

Conclusion: In conclusion, targeting YAP1 by tumor suppressor miRNA miR-15a and miR-16-1 plays inhibitory effect and this might have a therapeutic potential in GAC.

Figure 1

miR-15a and miR-16-1 show decreased expression in GAC. (A) The expression of miR-15a and miR-16-1 in 10 GAC cell lines compared with GES-1 cells. The standard deviations (SDs) were achieved by the values in triplicate wells. (B) Expression of miR-15a and miR-16-1 in paired primary GAC samples (n = 60; miR-15a, P = 0.011; miR-16-1, P < 0.001).

Figure 2

miR-15a and miR-16-1 exert tumor suppressor function in GAC. (A) MTT proliferation results of ectopic miR-15a and miR-16-1 expression in AGS, MKN1 and MGC-803 cells (**, P < 0.001). The P-Value was calculated by the 575 nm absorbance readings in Day 5. (B) Ectopic expression of miR-15a and miR-16-1 inhibited monolayer colony formation in AGS, MKN1 and MGC-803 cells (**, P < 0.001). The experiment was performed in triplicate wells to get SDs. (C) Flow cytometry analysis of miR-15a and miR-16-1 transfectants compared with scramble miRNA transfectants. Two independent experiments were performed and the representative one was shown. (D) miR-15a and miR-16-1 suppressed cell invasion through matrigel (**, P < 0.001). Representative images of cell invasion were also shown. The invaded cells in 3 random vision fields were counted and calculated to get SDs. (E) miR-15a-MGC-803 and miR-16-1-MGC-803 formed smaller tumors than scramble-miRNA-MGC-803 (**, P < 0.001). The tumor sizes in the Day 24 were calculated for the P-Value. (F) Western blot analysis of CCND3, CCNE1, CDK6, p21, p27, p-Rb and cleaved-PARP after miR-15a and miR-16-1 transfection.

Figure 3

YAP1 is a target of miR-15a and miR-16-1 in GAC. (A) The miR-15a and miR-16-1 binding sites in YAP1 3′UTR as TargetScan predicted (www.targetscan.org). (B) The YAP1 mRNA expression after ectopic miR-15a and miR-16-1 expression in AGS, MKN1, MGC-803 and NCI-N87 cells (**, P < 0.001). (C) miR-15a and miR-16-1 transfection down-regulated YAP1 protein expression in GAC cells. (D) miR-15a and miR-16-1 suppressed the luciferase activities in the constructs containing wild-type binding sites in YAP1 3′UTR, but for the constructs containing the deletion binding sites, miR-15a and miR-16-1 had no suppressive effect on the luciferase activities. (E1) miR-15a showed negative correlation with YAP1 protein expression (P = 0.048). (E2) miR-16-1 expression was negatively correlated with YAP1 protein expression (P = 0.010).

Figure 4

siRNA-mediated YAP1 knockdown suppressed GAC cell proliferation in vitro and in vivo . (A) YAP1 knockdown suppressed cell proliferation in MGC-803, NCI-N87 and SGC-7901 cells (**, P < 0.001). The 575 nm absorbance readings in Day 6 were calculated for the P-Value. (B) siYAP1-MGC-803 formed smaller xenografts than siScramble-MGC-803 control in nude mice (**, P < 0.001). The tumor sizes in the Day 28 were calculated for the P-Value. (C) YAP1 knowdown decreased the expression of CCND3, CDK6 and p-Rb, but increased the expression of p21 and p27. The cleaved-PARP showed activated expression in AGS and MGC-803 cells upon YAP1 knockdown.

Figure 5

YAP1 re-expression partly abrogated the inhibitory effect of miR-15a and miR-16-1 in NCI-N87 and MGC-803. (A) YAP1 re-expression increased cell proliferation compared with miR-15a or miR-16-1 alone (*, P < 0.05; **, P < 0.001). The SDs were achieved by the 575 nm absorbance readings in 6 wells of each item. (B) Monolayer colony formation assays revealed YAP1 re-expression abolished proliferation-inhibition of miR-15a and miR-16-1 partially (*, P < 0.05; **, P < 0.001). The representative colony formation pictures were shown in the bottom. The experiments was performed in triplicate wells to get SDs. (C) The suppressed cell invasion abilities was partially alleviated by YAP1 re-expression in NCI-N87 and MGC-803 cells (*, P < 0.05; **, P < 0.001). The cells were counted in 3 random vision fields of each item to get SDs.