Experimental Study On The Effect Of CDK1AP1 On The Human Lung Cancer A549 Cells

Lung cancer is a malignant tumor which seriously threat human health in these years. The morbidity of lung cancer, one of the most common primary malignant carcinomas in the world, has increased annually. There is a prevalence of more than 1.5 million cases worldwide per year, with high incidence especially in China.The aging population, the progression of urban industrialization as well as the contamination and destruction of human life environment result in the rising incidence. Furthermore, to date, the therapeutic efficacy for this malignancy has not improved due to current high mortality and poor long-term survival rates.The developing knowledge of molecular biology of tumor progression has revealed changes of gene expression during lung cancer progression. And the elucidation of molecular mechanism under carcinogenesis would be of great importance in the prevention and treatment of cancers. CDK2AP1 (cyclin-dependent kinase 2-associated protein 1), also known as DOC1, ST19, DORC1, doc-1, or p12DOC-1, was first isolated from normal keratinocytes as growth inhibitory factor by Todd et al in 1995. Located on human chromosome 12q24.31, the full length of CDK2AP1 gene is 1.6 kb, and its encoded protein is 12.4 KD(115 amino acids). CDK2AP1 interacted with DNA polymerase a, regulating the phosphorylation of the large subunit p180 and negatively modulating DNA replication in S phase of cell cycle.CDK2AP1 could also arrest cell cycle at DNA synthesis phase, leading to programmed cell apoptosis. Some study confirmed that CDK2AP1 played an important role in TGF-β1-modulated growth inhibitory system. Recently, it is revealed that the defective expression of CDK2AP1 was accompanied by the abnormal TGF-β-smad pathway, which ultimately resulted in the resistance of human oral squamous cell carcinoma cells to TGF-β-induced growth inhibitory effect. CDK2AP1 could regulate the activity of CDK2, the phosphorylation of pRB and p180, and TGF-β-Smad signaling pathway so as to play its inhibitory effect on cell growth.Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, to date, no study on the function of CDK2AP1 in human lung cancer has ever been reported. In this study, overexpressing and lentiviral vectors containing CDK2AP1 cDNA and CDK2AP1 shRNA (short hairpin RNA) were constructed, and up-regulated and down-regulated CDK2AP1 expression in human lung cancer A549 cells was found, and explored the function of CDK2AP1 in lung cancer cell's development and progression.Study objectives:(1) CDK2AP1 siRNA design and pGCSIL-GFP-CDK2AP1 shRNAs construction.(2) Construction of overexpressing vector pGC-FU-CDK2AP1 cDNA.(3) To investigate the effect of CDK2AP1 on the growth in the cells in vitro and in vivo.Methods:(1) The human CDK2AP1 cDNA sequence was searched for suitable siRNA target sequences, and three CDK2AP1 siRNA sequences were designed by the Ambion siRNA Target Finder. DNA oligos containing the target sequences were chemically synthesized, annealed, and inserted into the expression vector pGCSIL-GFP by double digestion and ligation in accordance with the manufacturer's guidelines. The ligation product was then transformed into competent Escherichia coli DH5acells.As a control for CDK2AP1 siRNA, a corresponding random siRNA sequence was used. The targeting sequence of the short hairpin RNA (shRNA) was then confirmed by sequencing.(2) CDK2AP1 cDNA was obtained the CDK2AP1-mRNA of lung tissue,by the RT-PCR assay, and then cloned into pGC-FU by double digestion, and in accordance with the manufacturer's guidelines. The ligation product was transformed into competent Escherichia coli DH5a cells. The recombinant was identified by PCR and confirmed by sequencing.(3) The selection of the most effective CDK2AP1 shRNA with the Western blot assay.(4) Lentiviral particle production with the vectors pHelper 1.0 and pHelper 2.0, and the detection of knockdown efficiency of CDK2AP1 shRNA with TR-PCR and Western blot assay.(5) In vitro studies:The Lewis lung cancer cell lines were infected by the both plasmid vectors. The cycle phase distribution of the cells were analyzed with the flow cytometry. The effective function of CDK2AP1 in the infected the lung cancer cells was revealed with the MTT assay, the BrdU assay, the Cell Invasion Assay Kit (The transwell assay) and the Colony formation.(6)In vivo studies:we builded a BALB/c nude mouse tumor xenograft model. The mice were transplanted with the both infected(CDK2AP1 and si-CDK2AP1) Lewis lung cancer cells into the right flank. Then, we got the results of the volume and weight of the tumors.Statistical analysisComparison between groups was performed with the Student's t test and X2analysis using a SigmaStat statistical software package(SPSS, Chicago,IL). P<0.05 was taken as showing significance.Results:(1)The plasmids which were designed for expression of CDK2AP1 siRNA was constructed, and confirmed by restriction enzyme digest and sequence analysis. (2) The plasmids were designed for over-expression of CDK2AP1 was constructed, and confirmed by restriction enzyme digest and sequence analysis.(3) The both vectors were transfecting into the 293T cells. The most effective CDK2AP1 shRNA, the target 1, was founded with the westen blot assay.(4) The study in vitro:The cell cycle phase distribution analyzed with a flow cytometry,the MTT assay, the BrdU assay, the Cell Invasion Assay Kit (The transwell) and the Colony formation showed that the over-expression of CDK2AP1 vector can inhibit the proliferation, invasion, and promote apoptosis of human lung cancer A549 cells.(5) The study in vivo:The BALB/c nude mouse tumor xenograft model was builded. The results of the volume and weight of the the Lewis lung cance cells revealed that CDK2AP1 inhibited the growth of the cells.Conclusions1.The vectors of Lentivirus-mediated RNA interference and over-expression of CDK2AP1 were constructed successfully.2.The study revealed that CDK2AP1 could inhibit the proliferation, invasion, and promote apoptosis of human lung cancer A549 cells in both vitro and vivo.