Cyclin D1 SiRNA Inhibits Proliferation, Invasion And Metastasis Of A549 Lung Adenocarcinoma Cell Lines In Vitro And In Vivo

Background and Objective Lung cancer causes high morbidity in China. Although in the last decade several new chemotherapeutic agents have been introduced into clinical practice, the prognosis of advanced non–small cell lung cancer (NSCLC) remains poor, 5-year survival rates is about 10%. Cyclin D1,critical in the G1-S transition in the cell cycle,is amplified or overexpressed in many cancers. Cyclin D1 overexpression is predominantly associated with tumorigenesis and cellular metastasis. In this study,we created stably transfected A549 cell lines carrying a Cyclin D1 siRNA plasmid vector to simultaneously down-regulate Cyclin D1. We observed if Cyclin D1 siRNA-mediated inhibition of Cyclin D1 may be a promising anti-growth and anti-metastatic strategy for lung cancer gene therapy.Materials and Methods1. The expression of Cyclin D1,NF-?Bp65 and P27 was detected in 58 cases of NSCLC and 15 cases of benign pulmonary disease tissues by immunohistochemistry S-P method.2. Three designed DNA sequences of hCCND11,hCCND12,hCCND13,and one scrambled sequence of hK with small hairpin structure were cloned into the transcripted carrier Pgenesil-1 in order to construct recombinants. The recombinant vectors were transfected into the A549 cells.At 48h after transfection,the expression of Cyclin D1 at the level of mRNA and protein was detected by RT-PCR and western blot.3. we created stably transfected A549 cells carrying a Cyclin D1 siRNA plasmid vector to simultaneously down-regulate Cyclin D1. Expressions of Cyclin D1,MMP-2 mRNA and protein were examined by Real time RT-PCR and Western blot, and RhoA,Rac1 were examined by immunofluorescence stain in A549-untreated, A549-hCCND13 and A549-scrambled siRNA cells respectively. The cell cycle was examined by flow cytometry. Ability of cell proliferation and invasion was compared with MTT proliferation assay, soft agar clone formation assay,and Boyden chamber model. Primary tumor growth and experimental metastasis were examined using back subcutaneouly tumor xenograft models and abdominal cavity tumor xenografts metastasis models planted with A549-untreated, A549-hCCND13 and A549-scrambled siRNA cells in nude mice.Results1. The overexpression of Cyclin D1 was 74.1%(43/58). Cyclin D1 expression was closely related to tumor size,grade of cell differentiation,lymph node metastasis and clinic stage. There was positive correlation between NF-?Bp65 and Cyclin D1, and negative correlation between Cyclin D1 and P27(P<0.05).2. The siRNA expression vectors were successfully constructed.hCCND13 was most effective which down-regu1ated the mRNA and protein of Cyclin D1 at 48h after transfection(P<0.01).The inhibition rate of Cyclin D1 expression was 61%(mRNA) and 52%(protein).3. Vector hCCND13 can simultaneously down-regulate Cyclin D1 expression in stably transfected A549 cells. The inhibition rate of Cyclin D1 expression was 72%(mRNA) and 65%(protein).The down-regulate of Cyclin D1 expression resulted in decrease of MMP-2,RhoA,Rac1 protein levels. And the rate of G1 phage cells was increased from 52.3% to 71.7%.The transfected cells exhibited a marked decrease in the rate of cell growth,in contrast to the original lines(P<0.01).The inhibition rate of cell growth was 40% examined by MTT and 53% examined by soft agar clone formation assay. As shown by boyden chamber assay, invasive capacity was significantly decreased in the infection, compare with untreated and scrambled siRNA group in vitro(P<0.01). Back subcutaneouly tumor xenograft models and abdominal cavity tumor xenograft metastasis models were successfully constructed. The time of tumor formation in hCCND13 group(12.1±0.8 day)was more than in untreated group(7.3±0.9day).Cyclin D1 siRNA infection caused the growth of tumors slowly in vivo. The inhibition rate of tumor growth was 50% in hCCND13 group.And the Cyclin D1 siRNA infection inhibited lung and lymph node metastasis in the metastatic models(P<0.05).The expression of Cyclin D1,MMP-2,RhoA and Rac1 protein exhibited a marked decrease in hCCND13 group, in contrast to other groups.Conclusions1. Cyclin D1 and relation genes were related to the oncogenesis and progression of NSCLC.Overexpression of Cyclin D1 can promote the proliferation, invasion and metastasis of lung carcinoma.Decreasing the overexpression of Cyclin D1 may inhibit the proliferation, invasion and metastasis of lung carcinoma.2. The siRNA expression vectors targeting Cyclin D1 were successfully constructed. The expression of Cyclin D1 gene was inhibited effectively in A549 cells transfected by hCCND13 ,Which laid a basis for its application in the experimental treatment of lung cancer.3. These data suggest that Cyclin D1 siRNA shows its antitumor activity against both tumor growth and metastasis. It is the reason of inhibition tumor growth that Cyclin D1 siRNA can inhibit the cell cycle progression.And the mechanism of inhibition tumor metastasis was related with the decrease of MMP-2,RhoA and Rac1 expression after Cyclin D1 decreased by Cyclin D1 siRNA.