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. 2016 Aug 12;11(8):e0158961.
doi: 10.1371/journal.pone.0158961. eCollection 2016.

MiR-148a Functions as a Tumor Suppressor by Targeting CCK-BR via Inactivating STAT3 and Akt in Human Gastric Cancer

Beiqin Yu  1 , Xin Lv  1   2 , Liping Su  1 , Jianfang Li  1 , Yingyan Yu  1 , Qinlong Gu  1 , Min Yan  1 , Zhenggang Zhu  1 , Bingya Liu  1
Affiliations

MiR-148a Functions as a Tumor Suppressor by Targeting CCK-BR via Inactivating STAT3 and Akt in Human Gastric Cancer

Beiqin Yu et al. PLoS One. .

Abstract

MicroRNAs (miRNAs) have been widely accepted as a class of gene expression regulators which post-translationally regulate protein expression. These small noncoding RNAs have been proved closely involved in the modulation of various pathobiological processes in cancer. In this research, we demonstrated that miR-148a expression was significantly down-regulated in gastric cancer tissues in comparison with the matched normal mucosal tissues, and its expression was statistically associated with lymph node metastasis. Ectopic expression of miR-148a inhibited tumor cell proliferation and migration in vitro, and inhibited tumor formation in vivo. Subsequently, we identified cholecystokinin B receptor (CCK-BR) as a direct target of miR-148a using western blot and luciferase activity assay. More importantly, siRNA-induced knockdown of CCK-BR elicited similar anti-oncogenic effects (decreased proliferation and migration) as those induced by enforced miR-148a expression. We also found that miR-148a-mediated anti-cancer effects are dependent on the inhibition of STAT3 and Akt activation, which subsequently regulates the pathways involved in cell proliferation and migration. Taken together, our results suggest that miR-148a serves as a tumor suppressor in human gastric carcinogenesis by targeting CCK-BR via inactivating STAT3 and Akt.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. MiR-148a expression was down-regulated in gastric cancer tissues and gastric cancer cell lines compared with the corresponding controls.
(A) MiRNA microarray assay was carried out using 28 surgical specimens of gastric cancer tissues. (B) QRT-PCR was carried out using nine gastric cancer cell lines and one pooled normal gastric mucosa tissue. The mean and standard deviation of miR-148a expression levels are shown. The data represent triplicate measurements from single RNA samples (*, P < 0.05, compared with pooled normal gastric mucosa). (C) QRT-PCR was carried out using 41 surgical specimens of gastric cancer tissues (black bar) and matched normal tissues (white bar). The mean and standard deviation of miR-148a expression levels are shown. The data represent triplicate measurements from single RNA samples (*, P < 0.05).
Fig 2
Fig 2. The effect of miR-148a on the proliferation and migration of NCI-N87, SGC-7901 and MKN-45 cells.
(A) Transfection efficiency was assessed by qRT-PCR. The results showed that miR-148a expression vector (pSilencer/miR-148a) and miR-148a inhibitor could dramatically affect miR-148a expression in gastric cell lines. Results are means of three independent experiments ± S.D. (*, P < 0.05). (B) Cell proliferation was measured by CCK-8. NCI-N87 cells stably transfected with pSilencer/miR-148a grew more slowly than the control or wild type group; SGC-7901 and MKN-45 cells transfected with miR-148a inhibitors grew faster than their respective controls. Points, average of three independent experiments; bars, S.D. (*, P < 0.05). (C) Up-regulation of miR-148a depressed migration of NCI-N87 cells; SGC-7901 and MKN-45 cells migrated faster after miR148a knockdown. Average migratory cell number of three independent experiments ± S.D. (*, P < 0.05).
Fig 3
Fig 3. MiR-148a inhibited tumor growth in vivo.
(A) Photographs of tumors derived from NCI-N87/miR-148a (NCI-N87 cells stably transfected with pSilencer/miR-148a vector), NCI-N87/nc (NCI-N87 cells stably transfected with pSilencer/nc vector) or wild type (parent NCI-N87 cells) in nude mice. (B) Tumor growth curves showed that tumors derived from NCI-N87/miR-148a cells (black line) in nude mice grew more slowly than the control group (dark grey line) or the parental cells (light grey line). Bars, S.D. (C) Average weight of tumors derived from NCI-N87/miR-148a, NCI-N87/nc or wild type NCI-N87 cells. Means ± S.D. are shown (*, P < 0.05).
Fig 4
Fig 4. CCK-BR is a validated target of miR-148a.
(A, B) Putative binding sites of miR-148a in the CCK-BR 3’UTR (white sequences) predicted by TargetScan. (C) MiR-148a mimics depressed the relative luciferase activity control by wild-type CCK-BR 3’UTR, which could be eliminated by nucleotide mutations in BS4 but not BS1, BS2 or BS3 sequences. Means ± S.D. are shown (*, P < 0.05). (D) The relative luciferase activity in NCI-N87 stably transfected with pSilencer/miR-148a was lower than that in NCI-N87 stably transfected with pSilencer/nc. Means ± S.D. are shown. (*, P < 0.05).
Fig 5
Fig 5. MiR-148a expression correlates inversely with CCK-BR protein expression in gastric cancer.
(A) CCK-BR protein was detected in NCI-N87 cells stably transfected by pSilencer/miR-148a (or pSliencer/nc vector) or in SGC-7901 cells transfected by miR-148a inhibitor (or inhibitor control). GAPDH was used as an internal loading control. (B) CCK-BR mRNA was detected in NCI-N87 cells stably transfected by pSilencer/miR-148a or pSliencer/nc vector and SGC-7901 cells transfected by miR-148a inhibitor or inhibitor control. The results are shown as fold changes relative to the control. The data represent triplicate measurements from single RNA samples. (C) QRT-PCR analysis of CCK-BR in four paired tumor/matched normal tissues. The data represent triplicate measurements from single RNA samples. (D) Western blot analysis of CCK-BR in four paired tumor/normal tissues (T/N).
Fig 6
Fig 6. The effect of miR-148a can be rescued by down-expressed CCK-BR.
(A) Western blot was used to monitor the protein level of CCK-BR in NCI-N87 cells 48 h after transfection with CCK-BR siRNA (or negative control). (B) Effects of CCK-BR knockdown on cell proliferation in NCI-N87 cells. Means ± S.D. are shown (*, P < 0.05). (C) Migration assay in NCI-N87 cells with CCK-BR siRNA or negative control. Means ± S.D. are shown (*, P < 0.05).
Fig 7
Fig 7. MiR-148a inhibits activation of STAT3 and Akt in gastric cancer cells.
(A) Western blot indicated alterations of STAT3, p-STAT3, Akt, p-Akt, CCK-BR protein levels in NCI-N87/miR-148a, NCI-N87/nc and NCI-N87 group. (B) Western blot showed changes of STAT3, p-STAT3, Akt, p-Akt, CCK-BR protein levels in SGC-7901/miR-148a inhibitor, NCI-N87/inhibitor control and mock group.
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Grants and funding

This study was supported by grants from National Natural Science Foundation of China (Nos. 81172324, 81272749, 91229106, 81372187), Science and Technology Commission of Shanghai Municipality (Nos. 12XD1403700, 13ZR1425600), Key Projects in the National Science & Technology Pillar Program of China (No. 2014BAI09B03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript.