MIR-147B regulated proliferation and apoptosis of gastric cancer cells by targeting CPEB2 via the PTEN pathway

The five Gastric Cancer cell lines, i.e., SGC-7901, MGC-803, BGC-823, AGS and MKN-45 and normal gastric tissue cell lines were obtained from the sample bank at Shanxi Tumor Hospital. The specific information of 50 pairs of gastric cancer specimens in the biological sample bank is related to gastric body cancer patients who underwent radical gastrectomy at the Shanxi Tumor hospital from January 2014 to January 2015. Of these patients, 29 were male and 21 female. The age ranged from 43 to 70 years. The TNM system was used for clinical staging. There were 20 cases of stage I+II and 30 cases of stage III.

Inclusion criteria:

Exclusion criteria

The tissue samples taken in this study were obtained from the patients after their written consent was obtained. This study has been duly approved by the medical ethics committee of the Beijing Friendship Hospital, Capital Medical University.

The GC tissues of 50 patients with gastric cancer with complete data and the adjacent tissues > 5cm were divided into an experimental group and a control group in order to determine the expression level of the target gene miR-147b in the selected tissues. BGC-823, SGC-7901, AGS, MGC-803 and MKN-45 GC cell lines served as markers for the experimental group, whereas normal gastric tissue cell lines serve as the control group to quantify the expression of miR-147b. Two groups of cell lines with high expression of miR-147b were selected for transfection experiment: 20 ~ 50 nM miR-147b inhibitor was used as the experimental group and miR-147b NCas was used as the negative control group. We measured the cell cycle and colony formation after 24h of transfection and cell migration after 48h of transfection of the two groups of transfected cell lines. The proliferation rate of GC cells at 0, 24, 48, 60, and 72 hours after transfection was determined by the CCK8 method.

All cell lines were maintained in complete Dulbecco’s Modified Eagle Medium, supplemented with 10% (vol./ vol.) fetal calf serum, penicillin G (100 μg/mL) and streptomycin (100 μg/mL). All cell lines were grown at 37°C in humidified 5% CO₂ and 95% air atmosphere. All cell lines were incubated for 24h prior to experiments.

Three pairs of gastric cancer tissues were randomly selected for tissue microarray to detect the expression of the target and related genes in gastric cancer tissues. The expression of the target gene in 50 pairs of gastric cancer tissues was also determined. Total RNA was extracted from GC cell lines, normal tissues, and tissues using Trizol reagent (Invitrogen, USA) to determine the expression of miR-147b gene. The expression of miR-147b in the cells of each group was analyzed and compared. miR-147b was amplified by the upstream primer 5’-GGGGTGTGTGGAAAT-3’ and reverse primer 5’- AACTGGTGTCGTGGAGTCGGC-3’, and reverse transcribed into cDNA using RNA to cDNA premix. QRT-PCR was performed with SYBR Green master mix according to the protocol provided (all from vii7 Q-PCR system). The concentration of RNA was quantified by Nanodrop-2000 (Nanodrop, USA). The relative expression of miR-147b was calculated by 2^-ΔΔ^Ct10.

After the PCR quantitative analysis, two groups of cell lines with high expression of miR-147b were selected for the transfection experiment. 20 ~ 50 nM miR-147b inhibitor was used as the experimental group and miR-147b NC serves as the control group. The cells were cultured at ambient temperature and humidity at 5 % CO₂ for 36 ~ 48h. The clone with the highest expression of foreign protein in monoclonal cells was used in Western blot, and scratch test, apoptosis test, clone formation test and cell proliferation were detected.

Exponentially, cells that were growing were trypsinized and seeded at a density of 200,000 cells per well into 12-well plate for a 24h incubation (~90% confluence). The scratch wounds were made by a sterile 1 mL pipette tip through a pre-marked line. After removal of the resulting debris from the five lineal scratches, the cell monolayer was subsequently rinsed three times with PBS, followed by incubation with sample for 24h. The wound areas were displayed by taking images just above the interchanges between scratched wound areas and pre-marked lines. The effect of the sample on the wound closure was determined microscopically (Olympus IX - 7) after 0, 24, and 48h,of incubation. The effect of the sample on the wound closure was captured using MRI imaging software.

The cells were seeded in 12-well plates (2×105 cells/ well). The cells were cultured and incubated (with 5% CO₂ and 95% air) at 37°C. Various concentrations of test drugs were dissolved in DMSO and incubated with the cells for 48h. DMSO in the culture medium never exceeded 0.1% (v/v), the concentration known not to affect cell proliferation. The Annexin V-FITC/PI apoptosis kit (Keygen Biotech, China) was used. For this purpose, the cells were incubated with 5 μl Annexin V-FITC and 5 μl PI for 5 min in the dark. Data were collected with moflow (Beckman Coulter, USA).

One thousand cells were plated in a 35 mm Petri dish after a different treatment was applied for 24h and allowed to grow for two weeks. The medium was changed twice per week. Then, the colonies were fixed with 4% para-formaldehyde, stained with methyl violet. The colonies were observed under a microscope. Colony inhibition rate = [(1-number of colonies in experimental group / control group) ×100%; colony formation eficiency = 1-colony inhibition rate.

Cell suspensions of the two cell lines were inoculated into 96 well plates, incubated at ambient temperature and humidity at 5 % CO₂, and further cultured after adding 10μL CCK-8 reagent (Dojindo, Japanese) to each well. CCK-8, being nonradioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. It works on the principle of highly water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt] produces a water-soluble formazan dye upon reduction in the presence of an electron mediator. WST8 is reduced by dehydrogenases in cells to give an orange colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. The absorbance values of each well were measured at 450 nm and 630 nm at 0, 24, 48, 60 and 72 hours after transfection. The experiments were conducted three times. Cell viability has been calculated using the formula * (%) = [a (dosing) - A (blank)] / [A (0 dosing) - A (blank)] × 100.

The target scan database was selected to search for the target gene that can be combined with miR-147b and map the base pairs that cross the 3 ‘untranslated region of miR-147b and the predicted target gene. CPEB2 plasmid was constructed by the pGL3 promoter vector: wtCPEB2 (5′-GUUUCUGUAUAGAAUCGCACAAG-3′) and mutant mutCPEB2 (5′-GUUUCUGUAUAGAAUTTCTCGAG3′). MGC-803 cells were cultured until the cell density grew to 80% - 90%. The cells were divided into an experimental group and a control group. Lipofectamine™ 2000 transfection reagent was used for co-transfection of the MGC-803 cells with the wt-CPEB2 plasmid and scramble or miR-147b inhibitor. The second group was divided into an experimental group and a control group. Lipofectamine™ 2000 transfection reagent was used for co-transfection of MGC-803 cells with mut-CPEB2 plasmid and scramble or miR-147b inhibitor were co transfected into MGC-803 cells by using. Double glo stop and glo reagent were added into 24 well plate cells at 48h. After full reaction at room temperature, the fluorescence intensity was detected according to the instructions of double luciferase reporting system (Promega, USA) and the luciferase activity in MGC-803 cells was analyzed.

MGC-803 cells were co-transfected with miR-147b NC, miR-147binhibitor and miR-147b NC+CPEB2 and were further categorized into two groups to identify the expression of CPEB2 and GAPDH (control group).MGC-803 and BGC-823 cells were separated into two groups after the culture. One group was added with an anti-CPEB2 antibody (Abcam, ab222070) to establish the si-CPEB2 and si-control experimental group and control group. After loading (25 μg/lane), the protein concentration was estimated using the BCA Kit and protein was separated using SDS-PAGE. Thereafter, the western transfer was performed with PVDF membranes (Millipore, Bedford, MA, USA) using the primary and secondary antibodies as per the earlier reported procedure. 9

Routine H&E staining and immunohistochemical analysis was conducted to determine the expression of CPEB2 in the gastric cancer tissues and was observed using a Zeiss UV LSM confocal microscope.

Statistical analysis was evaluated using a two tailed t-test and one-way ANOVA with SPSS packaging program (SPSS17.0, USA). The Pearsonχ2 test was used to compare the differences between groups. If the conditions were not met, continuity correction χ2 analysis was further used. Statistical significance was set as P < 0.05.

The differentially expressed miR-147b was screened out in 3 pairs of samples by the miRNA chip (Fig. 1A), which was found three times higher than that of adjacent tissues in three random pairs of gastric cancer samples (Fig. 1B). Q-PCR further detected the expression of miR-147b in 50 pairs of gastric cancer and adjacent tissues, and miR-147bwas-found to be highly expressed in the gastric cancer tissues (0.53±0.02 vs 1.35±0.03, P<0.05) (Fig. 1C). The expression of miR-147b in each GC cell line was recorded as Normal cell0.99±0.01, SGC-7901: 1.55±0.03, BGC-8231.74±0.12, AGS: 1.47±0.02, MGC-803, 1.8±0.04, MKN-45, 1.44±0.03, P<0.05) (Fig. 1D, table 1). BGC-823 and MGC-803 cell lines with increased expression level of miR-147b were selected for further analysis. After transfection, the relative gene expression of miR-147b nc in the control group and miR-147b inhibitor group were found to be 1.00±0.09, and 0.23±0.07 respectively, with a significant difference (t = 3.07, P < 0.05), indicating successful transfection (Fig. 1D).

Figure 1

Scratch analysis showed that the miR-147b inhibitor group inhibited GC cell growth and reduced cell migration compared with miR-147b NC (Fig. 1E). In addition, by flow cytometry, we have also analyzed the apoptotic effect of miR-147b. As compared with miR-147b NC, miR-147b inhibitor increased the early apoptosis of MGC-803 cells [(61.8±0.2)% vs (55.4±0.1)% ]. It increased the early apoptosis of BGC-823 cells [(35.5 ± 0.2)% vs. (23.1 ± 0.2)%] (Fig. 2A). On the other hand, clone formation experiments showed that themiR-147b inhibitor drastically restricts the proliferation of BGC-823 and MGC-803 cells (Fig. 2B, table 2). CCK-8 proliferation experiment showed that themiR-147b inhibitor significantly inhibited the growth of BGC-823 and MGC-803 cells, and inhibition rate was more prominent as the time increases (t = 2.78, P < 0.05) (Fig. 2C and D, table 3).

Figure 2

We searched the target gene CPEB2 that can bind to miR-147b through the target scan database, and we mapped the base pairs that miR-147b crosses with the 3 ‘untranslated region of the predicted target gene CPEB2 (Fig. 3A). We selected MGC-803 with relatively high endogenous CPEB2 content to detect whether the miR-147b inhibitor of miR-147b NC affects CPEB2 expression. The double luciferase reporter vectors of CPEB2 wild-type and mutant were further constructed (Fig.3A). The above vectors were co-transfected with NC and miR-147b inhibitor into MGC-803 cells, respectively. It was found that CPEB2 was the target gene directly affected by miR-147b. In the presence of CPEB23 ‘- UTR, this activity produced about 20% (Fig. 3B), indicating that the expression of miR-147b inhibitor accelerated the activity of CPEB23’-UTR. Western blot showed that the expression of CPEB2 in MGC-803 cells in miR-147b inhibitor group and miR-147b NC+CPEB2 group showed higher concentration as compared to negative control group (Fig. 3C). These results suggest that CPEB2 is the target of miR-147b and is negatively regulated by it.

Figure 3

Quantitative PCR (qPCR) analysis of 50 pairs of clinical GC tissues and adjoining normal tissue samples showed that CPEB2 mRNA expression in tumor tissues was down regulated in 40 patients (80%) (Fig. 4A). The non-parametric test results of the relationship between CPEB2 expression and clinicopathological factors showed that the lower CPEB2 level was related to the GC pTNM stage (P < 0.05, stages I-II and III-IV, FIG. 4B). In addition, GC patients with lower CPEB2 expression showed lower survivability and improved prognosis (Fig. 4C). Immunohistochemical analysis showed that CPEB2 staining increased significantly in adjacent normal tissues (Fig. 4D). H & E staining showed more metastatic nodules in GC tissues compared with adjacent normal tissues (Fig. 4D). CPEB2 was found highly expressed in GC cells.

Figure 4

Western blot results showed that the expression of PTEN protein decreased significantly after reducing CPEB2 in 803 and 823 cell lines. At the same time, the expression of tumor suppressor genes p27 and E-cadherin was inhibited, while the expression of Ki67 and p21 reflecting tumor activity index increased significantly (Fig. 4E).