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

Journal of Experimental & Clinical Cancer Research Effects of metastasis-associated in colon cancer 1 inhibition by small hairpin RNA on ovarian carcinoma OVCAR-3 cells Ruitao Zhang 0 Huirong Shi 0 Zhimin Chen 0 Qinghua Wu 0 Fang Ren 0 Haoliang Huang 0 0 Department of Obstetrics and Gynecology, First Affiliated Hospital, Zhengzhou University , NO.1 Jianshe Road, Zhengzhou, Henan, 450052 , P.R. China 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. Ovarian carcinoma OVCAR-3 cells; Metastasis-associated in colon cancer 1; Small hairpin RNA; Therapy target - Background Ovarian cancer is one of malignant tumors in female genital system, but is the leading cause of death from gynecological cancer in the world [1]. Despite improvements in the application of aggressive cytoreductive surgery and combination chemotherapy, ovarian cancer has the most unfavorable prognosis due to its insidious onset, diagnosis at late stage, dissemination, relapse, and tendency to develop chemotherapy resistance. Though considerable efforts aim at elucidating the tumorigenesis of ovarian carcinoma, its molecular mechanism has not been completely explained. Recently, MACC1 has been identified as a prognosis biomarker for colon cancer, which promotes proliferation, invasion and hepatocyte growth factor (HGF)induced scattering of colon cancer cells in vitro and in vivo [2]. MET, which encodes Met protein, has been proven to be a transcriptional target of MACC1. MACC1 controls the activity and expression of MET, and regulates HGF/Met signal pathway [2]. HGF/Met pathway plays key roles in carcinogenesis, aberrant activation of Met leads to enhancement of cell proliferation, invasion and metastasis, and Met is essential for metastatic potential of many malignances [3]. Once activated by HGF, Met transmits intracellular signals and activates downstream Ras-mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt pathways, which promote cell survival, migration, invasion, and suppress apoptosis [4]. MACC1 was demonstrated to be associated with poor prognosis and high risk of metastasis in colon cancer, gastric carcinoma, lung cancer, and hepatocellular carcinoma [5-8]. However, the mechanism of MACC1 implicates in ovarian cancer is still unclear. Small interfering RNA can specifically silence particular genes, and is used as a powerful tool to research gene functions and as a genetic therapy strategy for carcinoma [9]. In present study, expressions of MACC1 were detected in different ovarian tissues by immunohistochemistry, effects of MACC1 inhibition on OVCAR-3 cells were observed by RNA interference, and the possible antitumor mechanisms of MACC1 knockdown in ovarian carcinoma cells were discussed. Materials and methods Immunohistochemistry and evaluation 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 (++). ShRNAs synthesis and plasmids construction Single shRNA strands were 5-GATCCCC-N21-TTCAAGAGA-N21-TTTTTGGA-AA-3 (sense) and 5-AGCTT TTCCAAAAA-N21-TCTCTTGAAN21-GGG-3 (antisense). N21 was the sense sequence of MACC1 target oligonucleotides, N21 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 psuperEGFP-s1, psuper-EGFP-s2, psuper-EGFP-s3, and psuperEGFP-NC respectively. Ce (...truncated)