Effects of metastasis-associated in colon cancer 1 inhibition by small hairpin RNA on ovarian carcinoma OVCAR-3 cells

Paraffin-embedded 20 specimens of normal ovary, 19 specimens of benign ovarian tumor and 52 specimens of ovarian cancer tissues were obtained from Department of Pathology of Zhengzhou University. Rabbit-anti-human polyclonal MACC1 antibody (Sigma, USA) was used for immunohistochemistry assay, which was performed following the protocol of Universal SP kit (Zhongshan Goldenbridge Biotechnology, Peking, China). Positive staining of MACC1 protein presents brown in cytoplasm, partly in nucleus. Semi-quantitative counting method was used to determine positive staining described as following: Selected 10 visual fields under high power lens (× 400) randomly, counted the numbers of positive cells in 100 cells per field, calculated the average positive rate. Positive rate less than 1/3 scored as 1, more than 1/3 and less than 2/3 scored as 2, more than 2/3 scored as 3, without positive cell scored as 0. Cells without brown staining scored as 0, with mild brown staining scored as 1, with moderate brown staining scored as 2, with intense brown staining scored as 3. The final positive scores = positive rate score × staining intensity score, 0 score was negative staining (-), 1~4 scores were positive staining (+), more than 4 scores was strong positive (++).

Single shRNA strands were 5'-GATCCCC-N21-TTCAAGAGA-N'21-TTTTTGGA-AA-3' (sense) and 5'-AGCTTTTCCAAAAA-N21-TCTCTTGAAN'21-GGG-3' (antisense). N21 was the sense sequence of MACC1 target oligonucleotides, N'21 was antisense sequence of MACC1 target oligonucleotides. Three different template oligonucleotides targeting MACC1 [GeneBank, NM_182762.3] were as follow: MACC1-s1, 5'-AAAGACAGAAGGAGAAAGGAA-3'; MACC1-s2, 5'-AATCAAC-

TGTCTGCTTCTAAC-3'; MACC1-s3, 5'-AATTATATGCCAGGACAGCTT-3'. As a negative control, one scrambled sequence 5'-AACAGTTATCTATGCGACAGT-3' (corresponding to MACC1-s3) was designed. These sequences were submitted to BLAST against human genome sequence to ensure that only MACC1 gene was targeted. All single shRNA strands were synthesized at Sangon Biotechnology Co., Ltd (Shanghai, China), and were annealed and ligated into the BglII and HindIII sites of linearized psuper-EGFP plasmid. The four shRNAs inserted vectors were named as psuper-EGFP-s1, psuper-EGFP-s2, psuper-EGFP-s3, and psuper-EGFP-NC respectively.

Human ovarian carcinoma OVCAR-3 cells (with high level of MACC1 expression measured in our preliminary study) were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China), and cultured in DMEM medium (HyClone, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C with 5% CO₂. Cells were harvested in logarithmic phase of growth for all experiments described below. Cell transfection was performed following the protocol of Lipofectamine 2000 (Invitrogen, USA). The untransfected cells, empty vector (psuper-EGFP-neo) transfected cells, and nonspecific shRNA (psuper-EGFP-NC) transfected cells were used as controls. Stably transfected OVCAR-3 cells were selected with 800 μg/ml G418 (Sigma, USA) after tansfection 48 h. After 12 days, resistant colonies were trypsinized and cultured in selective medium. Names of the stably transfected cells were OVCAR-3-neo, OVCAR-3-NC, OVCAR-3-s1, OVCAR-3-s2, and OVCAR-3-s3 respectively.

Cell total RNA was isolated using Trizol Reagent (Invitrogen, USA), and first strand cDNA was synthesized from 1 μg total RNA according to the protocol of RevertAid first strand cDNA synthesis kit (Fermentas, EU). Primers used in RT-PCR were as follow: MACC1, 5'-CCTTCGTGGTAATAATGCTTCC-3' (sense) and 5'-AGGGCTTCCATTGTATTGAGGT-3' (antisense); β-actin, 5'-ACGCACC- CCAACTACAACTC-3' (sense) and 5'-TCTCCTTAATGTCACGCACGA-3' (antisense). PCR cycling parameters (19 cycles) were: denaturation (94°C, 30s), annealing (56°C, 30s) and extension (72°C, 30s). Equal amounts of PCR products were electrophoresed on 1.2% agarose gels and visualized by ethidium bromide staining. The specific bands of PCR products were analyzed by Image-Pro Plus 6.0 system, β-actin was used as a control for normalization. RT-PCR was performed for three times independently.

Primary antibodies used in Western blot, following manufacturer's protocols, were anti-MACC1 (Sigma, USA), anti-Met, anti-p-MEK1/2(ser212/ser218), anti-MEK1/2, anti-p-ERK1/2(Thr202/Tyr204), anti-ERK1/2 and anti-MMP2 (Santa Cruz, USA), anti-Akt, anti-p-Akt(Thr308), anti-cyclinD1, anti-cleaved caspase3 and anti-β-actin (Beyotime Biotechnology, Jiangsu, China). Total protein was extracted using Cell Lysis Buffer for Western and IP (Beyotime Biotechnology, Jiangsu, China), and protein concentration was determined using Bradford assay. Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The detection of hybridized protein was performed by enhanced chemiluminescence kit (Zhongshan Goldenbridge Biotechnology, Peking, China), β-actin was used as a control for normalization. The specific bands were analyzed by Image-Pro Plus 6.0 system.

Planted 2 × 10⁴ cells per well into 96-well plates, and added 100 μl medium containing 10% FBS into each well. Five duplicate wells were set up for each group. Cultured cells continuously for 7 days, added 20 μl MTT reagent (5 mg/ml, Sigma, USA) into each well, incubated for another 4 h then aspirated former medium and added 150 μl DMSO. The absorbance of sample was measured by Microplate spectrophotometer (Thermo, USA) at 492 nm. All experiments were done in triplicate. Cell growth curve was plotted versus time by origin 8 software.

Prepared single cell suspension, seeded about 50, 100, 200 cells of each group into 6-well plates respectively. Added 2 ml medium containing 10% FBS into each well, cultured cells continuously for one week. Fixated cells with methanol for 5 min, stained cells by hematoxylin for 30 min, counted the numbers of colony (more than 10 cells per colony) under low power lens (× 100) of inverted microscope (OLYMPUS, IX71, Japan), and calculated the rate of colony formation.

About 1 × 10⁶ cells were treated into single cell suspension with PBS solution, and were prepared following manufacture's protocol of Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, Jiangsu, China). Then, rates of apoptosis were analyzed with FACScan system (BD, USA).

Dripped single cell suspension onto microscopic slides, incubated cells for 4 h till cells were adherent. Three duplicate slides were set up for each group. Fixated cells by 4% paraformaldehyde for 30 min, blocked cells by 0.3% H₂O₂ for 30 min, incubated cells with 0.1% Triton X-100 for 2 min, then performed following manufacture's protocol of In situ cell death detection kit (Roche, German). Selected five visual fields under high power lens (× 400) randomly, counted the numbers of apoptotic body in 100 cells, calculated the rate of apoptosis.

About 5 × 10⁴~1 × 10⁵ cells were seeded into each well of 6-well plates, three duplicate wells were set up for each group, monolayer cells were obtained after cells confluence. Scratched monolayer cells with 200 μl pipette tip, washed cells 3 times with PBS, and added 2 ml medium without FBS into each well. The values of scratch were measured at 0 h and 24 h after scratching by Image Pro-Plus 6.0 system.

Transwell chambers (8 μm pore size; Millipore, USA) were also used to measure cell migration. Seeded 2 × 10⁵ cells into each upper chamber with 200 μl fresh medium without FBS, added 500 μl medium with 20% FBS into each lower chamber, three duplicate wells were set up for each group. After 12 h, fixated cells with methanol for 5 min, and stained cells by hematoxylin for 30 min. Cleaned upper chamber and inverted the chamber, counted cell numbers on the lower membrane under high power lens (× 400) in five random visual fields.

Transwell chamber (8 μm pore size; Millipore, USA) covered with 100 μl of 1 mg/ml Matrigel (BD, USA) was used to measure cell invasive ability. Seeded 1 × 10⁵ cells into each upper chamber with 200 μl fresh medium without FBS, added 500 μl medium with 20% FBS into each lower chamber, three duplicate wells were set up for each group. After 12 h, fixated cells with methanol for 5 min, and stained cells by hematoxylin for 30 min. Cleaned upper chamber and inverted the chamber, counted cell numbers on the lower membrane under high power lens (× 400) in five random visual fields.

The experimental protocol was approved by Zhengzhou University Ethics Committee for Animal Experimentation. Female BALB/c nu/nu mice (4-5 weeks old, 13-17 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd (Peking, China), and were randomly assigned into four groups with 4 mice per group. About 1 × 10⁷ cells were suspended in 0.2 ml PBS and injected subcutaneously into one mouse. The tumors were monitored every 5 days beginning at day 5 by measuring two perpendicular diameters with a caliper. The mice were sacrificed on the 35th day after injection, tumors were dissected and measured, and tumor volume in mm³ was calculated by the formula: volume = (width)² × length/2 [10].

Average values were expressed as mean ± standard deviation (SD). Count data were analyzed by χ² test. Measurement data were analyzed by one-way ANOVA and Bonferroni test using SPSS 17.0 software package. Difference was considered significant when P value was less than 0.05.

The positive rates of MACC1 in normal ovary, benign ovarian tumor and ovarian cancer tissues were detected by immunohistochemistry (Table 1). Compared to normal ovary and benign ovarian tumor, expressions of MACC1 were obviously up-regulated in ovarian cancer tissues (Figure 1), which showed abnormal expression of MACC1 might be associated with ovarian cancer.

After transfection 48 h, transfected cells with green fluorescence under fluorescence microscopy were observed (Figure 2). Expressions of MACC1 in stably transfected cells, which were selected by G418, were measured by RT-PCR and Western blot. Compared to control cells, levels of MACC1 mRNA and protein were significantly down-regulated in OVCAR-3-s1, OVCAR-3-s2 and OVCAR-3-s3 cells, especially in OVCAR-3-s3 cells (Figure 3). According to these results, OVCAR-3-s3 cells which showed the highest inhibitory rate of MACC1 were used for further assay described below.

According to Figure 4, the proliferation of OVCAR-3-s3 cells was obviously inhibited from the second day, when compared with control cells. There were no differences among OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells. In addition, OVCAR-3-s3 cells had lower rate of colony formation than control groups as shown in Figure 5. Thus, knockdown of MACC1 by RNAi could inhibit the growth of ovarian carcinoma cells.

Cell apoptosis rate measured by flow cytometer (Figure 6) in OVCAR-3-s3 cells was markedly increased to 24.13%, higher than 3.37% for OVCAR-3, 7.82% for OVCAR-3-neo, and 7.19% for OVCAR-3-NC cells (P < 0.05). Furthermore, TUNEL assay showed numbers of apoptosis body were increased in OVCAR-3-s3 cells (Figure 7). The results of apoptosis assay indicated the inhibitory effect of cell growth might due to the enhancement of apoptosis by MACC1 RNAi.

Compared with control groups, OVCAR-3-s3 cells showed suppressed capacity of impaired migration (Figure 8 and 9). Moreover, numbers of cell adherent on lower membranes of transwell chamber were sharply decreased in OVCAR-3-s3 group, which were shown in Figure 10. These results suggested MACC1 RNAi could suppress migration capability of ovarian carcinoma cells.

The numbers of cell, assessed in Matrigel invasion assay, were remarkably decreased in OVCAR-3-s3 group (Figure 11). On the other hand, the volumes of xenograft tumors removed from nude mice were retarded apparently in OVCAR-3-s3 group after 35 days. As shown in Figure 12, the growth of xenograft tumors in OVCAR-3-s3 group obviously fell behind other groups. Results of invasion assay indicated invasive potential of ovarian carcinoma cells could be retarded by MACC1 RNAi.

Expressions of Met, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, Akt and p-Akt were measured by Western blot in OVCAR-3, OVCAR-3-neo, OVCAR-3-NC and OVCAR-3-s3 cells. As a result of MACC1 knockdown, significant reductions of Met and p-MEK1/2 and p-ERK1/2 expression were observed in OVCAR-3-s3 cells. However, none obvious changes were detected on levels of total MEK1/2, total ERK1/2, total Akt and p-Akt (Figure 13 and 14). In addition, expressions of cyclinD1 and MMP2 decreased, level of cleaved caspase3 was increased after MACC1 inhibition (Figure 15).