MACC1 Expression In Human Lung Cancer Tissues And Effects Of The Gene On Lung Cancer Cells

Objective:MACC1(metastasis-associated in colon cancer l,MACC1)gene associated with migration and metastasis in colon cancer, was identified in 2009. According to research,MACC1, an important regulator of HGF/MET pathway signaling is overexpressed in primary and metastatic colon carcinomas ,while the high level of MACC1expression is inversely correlated with prognosis. Moreover,MACC1 target MET promoter to improve motility and proliferation of tumor cells.MACC1 also play an important role in HGF triggering migration and metastasis of tumor cells. As a new gene, the relationship between the level of MACC1 expression with tumorigenicity, development, migration and metastasis of human lung cancer are unknown. We investigated MACC1 gene expression in lung adenocarcinoma tissues and its effects in the growth of lung cancer cells. In our previous study, we have detected that MACC1 is overexpressed in SBC-5 cell line. Therefore, we constructed siRNA eukaryotic expression vector targeting MACC1 and transfected into lung cancer cells (SBC-5). To explore the possibility of MACC1 as an therapeutic target for the treatment of lung cancer, the effects of MACC1 downregulation on in vitro proliferation and migration of SBC-5 cells were evaluated.Methods:(1) The expression of MACC1 in lung adenocarcinoma tissues and lung cancer cells were measured by immunohistochemistry and Western blot, respectively. (2) Hairpin siRNA oligonucleotide sequence was designed and chemically synthesized according to the MACC1 mRNA sequence. Then, contrast the target gene with other genes by BLAST search. The hairpin siRNA oligonucleotide sequence was annealed and inserted between BamHⅠand HindⅢrestricted enzyme sites of pSilencer 4.1-CMV neo vector. The reconstructed vector and negative control vector were named MACC1/shRNA-1, MACC1/shRNA-2, MACC1/shRNA-NC, respectively. (3)The specific reconstructed vector targeting MACC1 gene and negative control vector were transfected into human lung cancer cell line SBC-5 by LipofectamineTM2000, respectively. Followingly, Stable cell lines were selected with G418 (500μg/ml) 48 hours later after transfection. Individual cell clones were isolated and maintained in a medium with G418 (200μg /ml). (4) Expression of MACC1 mRNA and protein in the transfected SBC-5 cells were examined by RT-PCR and Western blot analysis. We chose the stable transfected cells showing downregulated-MACC1 expression for further assays. (5) Proliferation of transfected SBC-5 cells were examined by MTT and colony formation assays. Wound healing assay were performed to evaluate migration of transfected SBC-5 cells in vitro.Results:(1) The positive rate of MACC1 expression in lung adenocarcinoma tissues is 38.3%. MACC1 expression was found in SBC-5 cells but not found in A549, PC14, SBC-3 cells by Western blot method at protein level. (2) The specific reconstructed vectors targeting MACC1 gene were successfully constructed. (3) The specific reconstructed vectors targeting MACC1 gene transfected into SBC-5 cells using Lipofectamine^TM2000. Stable cell lines were selected with G418 and named SBC-5-S1,SBC-5-S2,SBC-5-NC. (4) The levels of MACC1 mRNA and protein expression in SBC-5-S1 cells were decreased by 68.5% and58.5%, respectively. But there were no obvious difference in SBC-5-S2 cells. SBC-5-S1 cells were chose for further assays. (5) MTT results showed that the proliferation of SBC-5-S1 cells was obviously decreased. In the mean time, SBC-5-S1 cells were examined by Wound healing assay showed decreased ability of migration.Conclusion:MACC1 gene overexpression may be a useful marker for predicting metastasis in patients with lung adenocarcinoma. We successfully constructed siRNA eukaryotic expression vector targeting MACC1.The siRNA eukaryotic expression vector targeting MACC1 can significantly inhibit proliferation and migration of SBC-5 cells in vitro. The experiment results showed that MACC1 may serve as a potential target for of lung cancer therapy. Specific silencing of MACC1 gene may provide a new approach for exploiting novel therapeutic target.