Studies Of CHAF1A Gene Function And Its Molecular Mechanism In Non-small Cell Lung Cancer (NSCLC)

Objectives1.To determine the expression of CHAF1 A in non-small cell lung cancer(NSCLC).We examined the expression levels of CHAF1 A in NSCLC tissues and corresponding normal lung tissues.The relations of clinicopathological parameters and CHAF1 A were studied.2.We examined the expression of CHAF1 A genes in human lung cancer cell line A549,H1299 and H1975.The si RNA interference technology was applied for lentivirus packaging,infecting cells,decreasing CHAF1 A gene and interfering CHAF1 A gene expression.The cell growth,proliferation and apoptosis,were observed.3.By detecting the cell cycle to prove that CHAF1 A gene knockdown could interfere with the cell cycle of lung cancer cells.4.To detect changes of the downstream target genes expression in non-small cell lung cancer cells after CHAF1 A gene knocking down,to explore the predictive biomarkers of non-small cell lung cancer growth,proliferation and metastasis potential,and to provide new ideas for the diagnosis and treatment of lung cancer.Methods1.Samples were collected from patients in cardiothoracic surgery department in the second affiliated hospital of Shanxi Medical University from December 2010 to January 2016.All of the cases were tested by immunohistochemistry and real-time PCR to clarify CHAF1 A expression in non-small cell lung cancer.The relationship of clinicopathological parameters and CHAF1 A were studied.2.Real-time PCR was applied to detect efficiency of CHAF1A-si RNA-lentivirusin infection lung cancer cell line H1299 cells.3.MTT and cell clone formation test were used to study the proliferation of CHAF1 A knockdown H1299 lung cancer cells.4.Flow cytometry was applied to study the cell cycle change and cell apoptosis of CHAF1 A knockdown H1299 cells.5.Western blot was used to detect the downstream target genes expression level,including p21 and p27 protein,cell cycle dependent kinase 2(CDK2),Skp2 protein,cell cycle cyclin D1 and other related expression protein level in CHAF1 A knockdown H1299 cells.Results1.Immunohistochemical results showed CHAF1 A mainly expressed in the cytoplasm and cell membrane in non-small cell lung cancer cells.Compared with normal lung tissue,CHAF1 A express level was remarkably up-regulated in NSCLC.It was showed CHAF1 A express level had no relevance with gender,age,but had significant relevance with smoking status,tumor size,and tumor metastasis of NSCLC patients.The CHAF1 A gene expresses in human lung cancer cell line A549,H1299,H1688,and H1975.2.H1299 cells were infected with lentivirus-si RNA-CHAF1 A and lentivirus-si RNA-control respectively.The results showed more than 80% of the cell emitted green fluorescence in both groups after 72 hours transfection.After lentivirus infection,CHAF1 A gene expression in H1299 cells at m RNA level was significantly inhibited(P < 0.05).The knockdown efficiency reached 84.7% as demonstrated in the results of real-time PCR.3.Results from Cellomics count(5 days)showed significant decrease of cell counting in CHAF1 A knockdown group compared with control(162 ±10 vs.523 ± 8 cells/field,P <0.05).Clone formation experiment proved that CHAF1 A knockdown significantly inhibited H1299 cells clone formation(21 ± 9 vs.84 ± 12 colonies/dish,P < 0.05)compared with control.MTT results showed that CHAF1 A knockdown H1299 cell growth ability decreased significantly(P < 0.05).4.Flow cytometry results showed CHAF1 A knockdown H1299 cell apoptosis was promoted(P < 0.05)compared with control.5.Compared with control,CHAF1 A knockdown H1299 cells in G1 and G2/M phases significantly reduced(P < 0.05),while cells in S phase markedly increased(P < 0.05).These results suggested inhibition of the S to G2/M phase transition in CHAF1 A knockdown H1299 cell and arrest of the cell in S phase.6.Western blot analysis showed significant change of CHAF1 A downstream protein level,in which cyclin D1,CDK2 and Skp2 expression increased,while p21 and p27 protein level decreased significantly.Conclusions 1.Compared to normal tissue,CHAF1 A m RNA and protein expression in non-small cell lung cancer tissues is up-regulated.The CHAF1 A gene expresses in human lung cancer cell line A549,H1299,H1688,and H1975.2.The over-expression of CHAF1 A closely related to clinical pathology in non-small cell lung cancer patients.CHAF1 A may be an important indicator of the prognosis of non-small cell lung cancer.3.CHAF1 A m RNA expression level can be significantly and effectively suppressed using si RNA interfering technique and lentivirus packaging and infection.4 H1299 cell proliferation and clone formation are inhibited significantly after CHAF1 A gene knockdown.5.H1299 CHAF1 A knockdown induces cell cycle arrest in S phase,resulting in inhibition of cell proliferation and growth.Additionally,H1299 cells apoptosis significantly increased after CHAF1 A knockdown.6.CHAF1 A knockdown in H1299 cell,markedly downregulates cyclin D1,CDK2,Skp2 expression,and upregulates p21 and p27 expression,resulting in DNA synthesis inhibition,the tumor cycle arrested in S phase and tumor growth suppressed.These findings provide strong evidence that CHAF1 A gene is closely related to the occurrence and development non-small cell lung cancer.This study suggest that CHAF1 A is a potential target for non-small cell lung cancer prevention and which provides a new clue for the pathogenesis and treatment of NSCLC.