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

Abstract

Background: Metastasis-associated in colon cancer 1 (MACC1) is demonstrated to be up-regulated in several types of cancer, and can serve as biomarker for cancer invasion and metastasis. To investigate the relations between MACC1 and biological processes of ovarian cancer, MACC1 specific small hairpin RNA (shRNA) expression plasmids were used to investigate the effects of MACC1 inhibition on ovarian carcinoma OVCAR-3 cells.

Methods: Expressions of MACC1 were detected in different ovarian tissues by immunohistochemistry. MACC1 specific shRNA expression plasmids were constructed and transfected into OVCAR-3 cells. Then, expressions of MACC1 were examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation was observed by MTT and monoplast colony formation assay. Flow cytometry and TUNEL assay were used to measure cell apoptosis. Cell migration was assessed by wound healing and transwell migration assay. Matrigel invasion and xenograft model assay were performed to analyze the potential of cell invasion. Activities of Met, MEK1/2, ERK1/2, Akt, cyclinD1, caspase3 and MMP2 protein were measured by Western blot.

Results: Overexpressions of MACC1 were detected in ovarian cancer tissues. Expression of MACC1 in OVCAR-3 cells was significantly down-regulated by MACC1 specific small hairpin RNA. In OVCAR-3 cells, down-regulation of MACC1 resulted in significant inhibition of cell proliferation, migration and invasion, meanwhile obvious enhancement of apoptosis. As a consequence of MACC1 knockdown, expressions of Met, p-MEK1/2, p-ERK1/2, cyclinD1 and MMP2 protein decreased, level of cleaved capase3 was increased.

Conclusions: RNA interference (RNAi) against MACC1 could serve as a promising intervention strategy for gene therapy of ovarian carcinoma, and the antitumor effects of MACC1 knockdown might involve in the inhibition of HGF/Met and MEK/ERK pathways.

Figure 1

Immunohistochemistry analysis of MACC1 expression in different ovarian tissues. Normal ovary (A) and benign ovarian tumor (B) showed a lower staining of MACC1, but ovarian cancer (C) showed higher density staining (DAB staining, × 400). (D): Bar graphs show the positive rates of MACC1 protein. *P < 0.05 versus normal and benign ovarian tissues.

Figure 2

Transfection of MACC1-shRNA into ovarian carcinoma OVCAR-3 cells. (A): Normal OVCAR-3 cells under incandescent light (× 200). (B): After transfection 24 h, OVCAR-3-s3 cells under fluorescent light (× 100). (C): Monoplast colony of OVCAR-3-s3 cells selected by G418 for three weeks (× 200). (D): G418 resistant OVCAR-3-s3 cell line (× 100).

Figure 3

Down-regulation of MACC1 by MACC1-shRNA in ovarian carcinoma cells. The best inhibitory effects of MACC1 were identified in OVCAR-3-s3 cells by RT-PCR (A) and Western blot (C), which were both performed for three times independently. Bar graphs show the relative expression levels of MACC1 mRNA (B) and protein (D).*P < 0.05 versus control groups.

Figure 4

Suppression of proliferation by MACC1 RNAi in ovarian carcinoma cells measured by MTT assay. Obviously inhibitory effect of cell proliferation was observed from the second day after MACC1 knockdown.*P < 0.05 versus control groups.

Figure 5

MACC1-shRNA inhibited the monoplast colony formation of ovarian carcinoma cells. Monoplast colony in 50-cells wells of each group. (A): OVCAR-3 cells. (B): OVCAR-3-neo cells. (C): OVCAR-3-NC cells. (D): OVCAR-3-s3 cells (Hematoxylin staining, × 100). Bar graphs show the average rates of monoplast colony formation.*P < 0.05 versus control groups.

Figure 6

Apoptosis induced by MACC1 RNAi in ovarian carcinoma cells. After MACC1 inhibition, cell apoptosis was obviously induced in ovarian carcinoma cells measured by flow cytometry assay.

Figure 7

MACC1-shRNA increased the apoptosis rate of ovarian carcinoma cells. TUNEL assay was used to measure the apoptosis rate in OVCAR-3 cells (A), OVCAR-3-neo cells (B), OVCAR-3-NC cells (C), and OVCAR-3-s3 cells (D). DAB staining, × 400. Bar graphs show the rates of apoptosis.*P < 0.05 versus control groups.

Figure 8

Knockdown of MACC1 by RNAi suppressed the migration ability of ovarian carcinoma cells. Wound healing assay was used for monolayer cell migration assay (Hematoxylin staining, × 100).

Figure 9

Bar graph of the wound healing assay. Each bar represents the value of wound healing assay. *P < 0.05 versus control groups.

Figure 10

Inhibition of MACC1 by RNAi suppressed the migration ability of ovarian carcinoma cells. Transwell migration assay was used for cell migration ability assay. (A): OVCAR-3 cells. (B): OVCAR-3-neo cells. (C): OVCAR-3-NC cells. (D): OVCAR-3-s3 cells (Hematoxylin staining, × 400). Each bar represents the cell numbers adherent on lower membrane.*P < 0.05 versus control groups.

Figure 11

Inhibition of invasion by MACC1 RNAi in ovarian carcinoma cells. Cell invasive ability was assessed by Matrigel invasion assay. (A): OVCAR-3 cells. (B): OVCAR-3-neo cells. (C): OVCAR-3-NC cells. (D): OVCAR-3-s3 cells (Hematoxylin staining, × 400). Each bar represents the cell numbers adherent on lower membrane.*P < 0.05 versus control groups.

Figure 12

Xenograft tumor growth of ovarian carcinoma cells was retarded by MACC1 RNAi. On the 35th day, volumes of subcutaneous tumor in OVCAR-3-s3 group were remarkably smaller than those of control groups. Line curves represent the tumor volumes of xenograft models. *P < 0.05 versus control groups.

Figure 13

Activities of HGF/Met and MEK/ERK signaling in ovarian carcinoma cells after MACC1 knockdown. After MACC1 inhibition, down-regulations of Met, p-MEK1/2, p-ERK1/2 were observed in ovarian carcinoma cells analyzed by Western blot.

Figure 14

Activity of PI3K/Akt signaling in ovarian carcinoma cells after MACC1 knockdown. After MACC1 inhibition, none obvious changes of Akt and p-Akt expression were detected in ovarian carcinoma cells by Western blot analysis.

Figure 15

Expressions of cyclinD1, cleaved caspase3 and MMP2 in ovarian carcinoma cells after MACC1 knockdown. After MACC1 inhibition, expressions of cyclinD1 and MMP2 decreased, level of cleaved caspase3 was increased in ovarian carcinoma cells by Western blot analysis.