The MicroRNA-217 Functions as a Potential Tumor Suppressor in Gastric Cancer by Targeting GPC5

<div><p>Gastric cancer (GC) is one of the most common malignancies worldwide. Emerging evidence has shown that aberrant expression of microRNAs (miRNAs) plays important roles in cancer progression. However, little is known about the potential role of miR-217 in GC. In this study, we investigated the role of miR-217 on GC cell proliferation and invasion. The expression of miR-217 was down-regulated in GC cells and human GC tissues. Enforced expression of miR-217 inhibited GC cells proliferation and invasion. Moreover, Glypican-5 (GPC5), a new ocncogene, was identified as the potential target of miR-217. In addition, overexpression of miR-217 impaired GPC5-induced promotion of proliferation and invasion in GC cells. In conclusion, these findings revealed that miR-217 functioned as a tumor suppressor and inhibited the proliferation and invasion of GC cells by targeting GPC5, which might consequently serve as a therapeutic target for GC patients.</p></div

The expression of miR-217 is downregulated in GC cell lines.

<p>(A) The expression level of miR-217 in four human GC cell lines (SGC-7901, HGC-27, MGC-803, MKN-45) and GES-1 was quantified using Northern blot.(B) The expression of miR-217 was assessing in GC cell lines (SGC-7901, HGC-27, MGC-803, MKN-45)andGES-1using Quantitative RT–PCR.</p

Restoration of miR-217 inhibits GPC5-mediated GC cell proliferation and invasion.

<p>(A) The cell growth in HGC-27co-transfected with either miR-217 mimic or 2.0 μg pEZ-GPC5 or pCDNA empty vector using CCK-8 proliferation assay.(B) The cell growth in HGC-27co-transfected with either miR-217 inhibitor or 2.0 μg shGPC5 (knocks down GPC5) or pCDNA empty vector using CCK-8 proliferation assay.(C) The cell invasive in HGC-27co-transfected with either miR-217 mimic or 2.0 μg pEZ-GPC5 or pCDNA empty vector using invasion assay.(D) The cell invasive in HGC-27co-transfected with either miR-217inhibitor or 2.0 μg shGPC5 or pCDNA empty vector using invasion assay. *p<0.05, ** p<0.01, and ***p<0.001.</p

miR-217 inhibits GC cell proliferation and invasion.

<p>(A) Real-time RT-PCR analysis of miR-217 in HGC-27 cells upon transfection ofmiR-217 mimic. The expression of miR-217 in HGC-27 cells transfected with miR-217 mimics was up-regulated.U6 snRNA was used as internal control. (B) The expression of miR-217 in HGC-27 cells transfected with miR-217inhibitor was down-regulated.U6 snRNA was used as internal control. (C) Ectopic miR‑217 expression significantly inhibited cell proliferation, as demonstrated by CCK8 assay. (D) Inhibition of miR-217 expression significantly promoted cell proliferation, as demonstrated by CCK8 assay. (E) Invasion analysis of HGC-27cells after treatment withmiR-217 mimics, inhibitors or scramble or control; the relative ratio of invasive cells per field is shown below, *p<0.05, ** p<0.01, and ***p<0.001.</p

MiR-217 is downregulated in GC tissues.

<p>(A) The tissues were histological confirmed using H&E staining. (Original magnification, ×100). (B) miR-217 was detected in 50 GC patients by real-time PCR. Data are presented as log 2 T/N of fold change of GC tissues relative to non-tumor adjacent tissues. (C) The expression of miR-217 in GC tissues compared with normal tissues. The expression of miR-217 was normalized to U6 snRNA. (D) The statistical analysis of the association between miRNA level and pTNM stage (I, II, III and IV). *p<0.05, and **p<0.01, ***p<0.001. One-way ANOVA was performed for analysis.</p

GPC5 is upregulated in GC cell lines and specimens.

<p>(A) The mRNA expression of CPG5 was assessing in GC cell lines (SGC-7901, HGC-27, MGC-803, MKN-45) and GES-1 using Quantitative RT–PCR. (B) The protein level of CPG5 in eight human GC tissues and its adjacent normal controls was quantified using western blot.</p

miR-217 posttranscriptional reduces GPC5 expression by directly targeting its 3’UTR.

<p>(A) The 3'-UTR of GPC5 mRNA contains the binding sequences of miR-217. (B) Relative luciferase activity of the indicated GPC5reporter construct in HGC-27cells is shown. Firefly luciferase values were normalized to Renilla luciferase activity and plotted as relative luciferase activity. (C) miR-217 overexpression significantly reduced the GPC5 mRNA levels in the HGC-27 cells and miR-217 inhibitor significantly enhanced the GPC5 mRNA levels in the HGC-27 cells. (D) Western blotting was performed to examine the effects of miR-217 on the expression of GPC5. GAPDH was also detected as a loading control.</p

Immunohistochemical staining for apoptosis-related protein in BGC-823 xenografts.

<p>Immunohistochemical staining for apoptosis-related proteins: Bcl-2, Bcl-xl, Bax, Activated caspase-3, and Activated capase-9. Magnification is 200×. Representative microphotographs of three groups are shown. DAB stained immunoreactive cells (dark brown).</p

Tetrandrine inhibits tumor growth in vivo.

<p>BGC-823 cells were subcutaneously inoculated into BALB/c nude mice to establish the xenograft model. (A) Tumor growth was monitored at the indicated time points, and the tumor volume was measured every 3 days. The starting day of drug treatment was defined as day 0. Data represent means ± SD of the relative tumor volume for each group. (B) Tumor weight was measured at the end of the experiment. Data represent means ± SD of the tumor weight for each group. ***<i>P</i> < 0.001 versus the control group.</p

Apoptosis of BGC-823 cells induced by tetrandrine.

<p>A PI-Annexin V-FITC binding assay was used to detect apoptosis of BGC-823 cells. (A) Cells treated with tetrandrine for 24 h at 0, 6, 8, and 10 μg/ml. (C) Cells treated with 8 μg/ml tetrandrine for 0, 12, 24, and 48 h. (B, D) Columns represent the means ± SD of apoptotic cells obtained from three independent experiments. **<i>P</i> < 0.01; ***<i>P</i> < 0.001 versus the control group.</p