MiR-29a inhibits cell proliferation and induces cell cycle arrest through the downregulation of p42.3 in human gastric cancer

Abstract

As a newly identified and characterized gene, p42.3 is associated with cell proliferation and tumorigenicity. The expression of p42.3 is upregulated in human gastric cancer (GC), but its underlying mechanisms of action are not well understood. MicroRNAs (miRNAs) are known to play vital regulatory roles in many cellular processes. Here we utilized bioinformatics and experimental approaches to investigate the regulatory relationship between miRNAs and the p42.3 gene. We showed that miR-29a could repress p42.3 expression at both the mRNA and protein levels via directly binding to its 3'UTR. Furthermore, an inverse relationship was observed between miR-29a and p42.3 expression in gastric cancer cell lines and GC tissue samples, especially in cases where p42.3 was downregulated. Taken together, we have elucidated previously unrecognized roles of miR-29a and indicated that miR-29a may function, at least partially, by targeting the p42.3 gene in human GC.

Figure 1. MiR-29a targets a putative binding site in p42.3–3’UTR.

A. The sequence of miR-29a with the putative binding site in the human p42.3 gene. The putative binding site with the mutant is shown in the lower panel. B. Regulation of reporter gene expression by miR-29a in MKN-45 cells co-transfected with the pri-miR-29a and reporter gene containing the putative binding site. **: P<0.01.

Figure 2. Expression of endogenous p42.3 and miR-29a in GC cells.

A. Expression of p42.3 mRNA and mature miR-29a in six GC cell lines (SGC-7901, MKN-28, MKN-45, MGC-803, SNU-1, and AGS) normalized to the normal gastric epithelium cell line (GES-1) using quantitative real-time RT-PCR. Endogenous references were GAPDH and U6 small nuclear RNA, respectively. *: P<0.05, **: P<0.01. B and C. Expression of p42.3 protein in GC and normal gastric epithelial cell lines analyzed by western blotting (B) and shown as mean ± SD (normalized; C). *: P<0.05, **: P<0.01.

Figure 3. Regulation of endogenous p42.3 expression by miR-29a.

A. Effect of miR-29a mimics on the endogenous expression of p42.3 mRNA. Mature miR-29a was exogenously upregulated while the endogenous expression of p42.3 mRNA was significantly downregulated in MKN-45 cells. **: P<0.01. B. Effect of miR-29a inhibitors on the endogenous expression of p42.3 mRNA. Mature miR-29a was exogenously downregulated, while the endogenous expression of p42.3 mRNA was significantly upregulated in MKN-45 cells. *: P<0.05, **: P<0.01. C and D. Effect of miR-29a mimics on the endogenous expression of p42.3, cyclin B1 and CHK2 proteins were similar to those of si-p42.3. The expression levels of p42.3 and cyclin B1 protein decreased significantly after MKN-45 cells were treated with si-p42.3 or miR-29a mimics while the level of CHK2 protein increased dramatically. Data are shown as mean ± SD (normalized; D). *: P<0.05. E and F. Effect of miR-29a inhibitors on the endogenous expression of p42.3, cyclin B1 and CHK2 protein. MiR-29a inhibitors reversed the expression changes seen in C and D. *: P<0.05, **: P<0.01.

Figure 4. Effect of miR-29a on cell proliferation in the MKN-45 cell line.

A. MiR-29a mimics inhibited cell proliferation and the inhibiting rate at 48 h was approximately 20% and approximately 40% at 72 h, while p42.3 siRNA inhibited cell proliferation by approximately 15% at 48 h and 72 h. **: P<0.01. B. MiR-29a inhibitors promoted cell growth by approximately 10% at 48 h, but the effect weakened at 72 h. *: P<0.05.

Figure 5. Effect of miR-29a on cell cycle in the MKN-45 cell line.

Flow cytometric analysis confirmed that both p42.3 siRNA and miR-29a mimics induced G1 phase arrest in the MKN-45 cell line.

Figure 6. The scatterplot of the expression of p42.3 protein and miR-29a in 60 gastric cancer tissues.

There was an inverse relationship between the expression of p42.3 protein and miR-29a and the equation of their relationship was y = −0.3458x+2.8126 (y: the expression of miR-29a, x: the expression of p42.3 protein).