The Role And Mechanism Of CHAF1B In Promoting The Proliferation Effect Of Non-small Cell Lung Cancer

Objective:Lung cancer is one of the most prevalent malignancies and its mortality rate is the first among all the malignancies,making it a major public health issue worldwide.Non-small cell lung cancer（NSCLC）accounts for 80-85%of the total number of lung cancer.In recent 20 years,with the development of the mapping of NSCLC gene mutation,the treatment of NSCLC has been transformed from empirically used cytotoxic chemotherapy to individualized therapy strategy based on different types of oncogene.The malignant proliferation of tumor cells is accompanied with active DNA reproduction,and chromatin assembly factor 1 subunit B（CHAF1B）is the subunit of histone chaperone chromatin assembly factor-1（CAF-1）and engages in the DNA replication-coupled nucleosome assembly.Studies have revealed that CHAF1B overexpression is significantly associated with poor outcomes and that CHAF1B has potential in predicting the prognosis in various types of cancers,including high-grade gliomas,prostate,tongue and breast cancer,salivary gland tumors,renal carcinoma,endometrial and cervical cancer,and melanoma.However,no study has evaluated the expression or the clinical value of CHAF1B in NSCLC.Therefore,in the present study we aimed to investigate the association of CHAF1B expression with clinicopathological features and prognosis and its function in NSCLC.Furthermore,the effects and mechanism of CHAF1B on NSCLC cell proliferation were examined.Our findings provide important insights into identifying new oncogene driver so as to give more benefits to NSCLC patients from targeted therapy.Methods:1.Archived formalin-fixed paraffin-embedded tumor tissues from 135 NSCLC patients were used for immunohistochemistry（IHC）examination.In addition,the fresh cancer and paired non-neoplastic tissues from 27 patients were collected,and real-time quantitative PCR method was applied to determine the mRNA expression of CHAF1B on transcriptional level.Based on the semi-quantitative results from IHC,the dividing value between high CHAF1B expression group and low CHAF1B expression group was identified according to the time-dependent ROC curve.The relationship between the expression level of CHAF1B and the gender,age,smoking status,pathologic types,differentiation,lymph node metastasis,vascular invasion,tumor size and clinical stages of NSCLC patients was analyzed.At least 5-year follow-up for 135 NSCLC patients was conducted by phone call,and the survival analysis of these patients was made.2.The mRNA and protein expression level of CHAF1B in lung cancer cell line 95-D,NCI-H292,A549 as well as normal bronchial epithelium cell line 16HBE was determined by real-time quantitative PCR and Western blot,and the lung cancer cell lines with high expression were screened.The expression vector of pGPU6/GFP/Neo-shRNA for CHAF1B was constructed and transfected into 95-D lung cancer cell line by liposome,then screening the positive clones by G418 to establish stably transfected cell lines.Real-time quantitative PCR and Western blot were employed to determine the mRNA and protein expression level of CHAF1B so as to clarify the gene expression efficiency.The proliferation ability effect of lung cancer cell line was determined by CCK8;the cloning effect of NSCLC was determined by colony formation experiment;the cell apoptosis and cycle effect of NSCLC was determined by flow cytometry.3.In vivo,the lung cancer cell lines transfected by pGPU6/GFP/Neo-CHAF1B-shRN-A（CHAF1B-shRNA）and pGPU6/GFP/Neo-shNC（NC-shRNA）were respectively inoculated into subaxillary region of nude mice subcutaneously,and the subcutaneous transplanted tumor model of nude mice was established.The length and width of tumor was measured periodically to calculate the tumor volume and the growth curve was drawn.The mice were sacrificed after 40 days,with the tumor dissected and weighed.The expression of CHAF1B and Ki-67 protein was determined by IHC staining technique,and mRNA level of CHAF1B was determined by quantitative PCR.4.When the archived formalin-fixed paraffin-embedded tumor tissues from 135 NSCLC patients were determined by IHC for CHAF1B,the expression of Ki-67（a classical proliferation index）was also determined and the correlation analysis between the expression of Ki-67 and CHAF1B was made.5.In order to identify the preliminary mechanism of CHAF1B in promoting NSCLC proliferation,the expression of critical genes p53,BAK,Bcl-2,and caspase-3 with a intrinsic apoptotic pathway induced by p53 was determined by real-time quantitative PCR and Western blot.Results:1.The results from IHC detection show that CHAF1B expression was significantly unregulated in lung cancer tissues but weakly or not expressed in normal lung tissues.The results from semi-quantitative analysis show that the integral from cancer tissues was 5.04±0.23 and the one from normal tissues was 0.69±0.07（n=135,P<0.0001）.The results from real-time quantitative analysis show that the transcription level of mRNA for CHAR1B in NSCLC is 8.5-fold higher than that of adjacent normal tissues,this difference being particularly significant in squamous cell carcinoma.The dividing value between high expression group and low expression group was determined as 4.83 according to ROC curve.The correlation analysis between the CHAF1B expression level of patients and the clinical pathogenic indicators was conducted,and it was found that the expression level of CHAF1B is correlated with gender（P=0.008）,pathological type（P<0.001）,smoking（P<0.001）,differentiation degree（P=0.007）,clinical stage（P=0.002）,and tumor size（P<0.001）,but not correlated with lymph node metastasis（P=0.085）,pulse tube invasion（P=0.719）,and age（P=0.406）.The database for clinical materials,recurrent-metastasis time,and survival time was created,and the Kaplan-Meier survival curve was drawn.The survival rate difference between the high and the low expression group of CHAF1B was compared via log-rank method,and it was found that low differentiation and medium differentiation(^c2=5.811,P=0.016);having and having no lymph node metastasis(^c2=21.944,P<0.001);different clinical phase(^c2=26.521,P<0.001);different tumor size(^c2=8.553,P=0.014);different survival rate between high expression and low expression of CHAF1B（c²=16.716,P<0.001）.With the employment of COX proportional hazards regression models,it was found that different clinical phase,whether the lymph node metastasis occurred,and the expression level of CHAF1B were the independent risk factors affecting the survival of NSCLC patients,in which the mortality risk of patients with high CHAF1B expression is2.44-fold higher than those with low CHAF1B expression.2.Real-time quantitative PCR was employed to determine the expression level of normal bronchial epithelial cells HBE and of NSCLC A549,95-D,and NCI-H292.The results shown the expression of CHAF1B in 95-D cells is significantly increased,so95-D cells were chosen as subsequent biological functional study.The recombinant expression vector pGPU6/GFP/Neo-CHAF1B-shRNA from successfully-built target CHAF1B gene was confirmed to effectively suppress the expression of CHAF1B.The suppression ratio of pGPU6/GFP/Neo-CHAF1B-HOMO-550 to CHAF1B was 70%,so liposome was used to transfect it to 95-D lung cancer cell line and the positive clones was screened by G418 to establish stably transfected cell lines.In vitro cytology functional experiment demonstrated that,for CHAF1B-shRNA,when compared with those of control group,its proliferation ability decreased,colony formation reduced,.apoptotic cells increased,and S phase distribution in cell cycle decreased.3.The mice-transplanted tumor experiment shown that,for CHAF1B-shRNA,when compared withNC-shRNA,the growth rate of subcutaneous implanted tumors decreased,the tumor weight lightened,and the tumor volume shrank;meanwhile,the expression level of CHAF1B for both mRNA transcription and protein was reduced.4.IHC staining was employed to determine CHAF1B and Ki-67,and the semi-quantitative results shown that there was a notable positive correlation between the expression of CHAF1B and that of Ki-67（r=0.858,P<0.001）.5.The mechanism of CHAF1B in promoting proliferation was preliminarily investigated.It was found that for CHAF1B-shRNA group,when compared with NC-shRNA group,its p53 expression increased;pro-apoptotic protein BAK rose significantly;the expression of pro-apoptotic protein Bcl-2 decrease significantly;the ratio of BAK/Bcl-2increased;the level of caspase-3 increased.Conclusions:1.The expression of CHAF1B in NSCLC is high.The NSCLC patients with high CHAF1B expression have shorter disease-free survival time and higher mortality rate;CHAF1B is an independent predictor for the poor prognosis of NSCLC patients.2.The expression vector of pGPU6/GFP/Neo-CHAF1B-shRNA targeted CHAF1B gene is successful constructed,and it can effectively inhibit the expression of CHAF1B.3.Both in vivo and in vitro experiment demonstrate interfering CHAF1B expression could inhibit the malignant proliferation of NSCLC,induce the stagnation of cell cycle,and boost apoptosis.4.CHAF1B is positively correlated with classic proliferation index Ki-67 and could be used as proliferation marker.5.CHAF1B probably boost apoptosis via p53-induced intrinsic apoptotic pathway and inhibit tumor growth.