Effect Of ShRNA-mediated Silencing Of AFAP1L1 Gene On The Proliferation Of Lung Adenocarcinoma Cell In Vitro And In Vivo

Objective1.To investigate the clinical significance of actin filament-associated protein 1-like 1(AFAP1L1)expression in non-small cell lung cancer(NSCLC).2.To explore the effect of shRNA silencing AFAP1L1 on the proliferation,cell cycle,apoptosis and invasion of lung adenocarcinoma A549 cells,and preliminary explore the molecular mechanism of AFAP1L1 involved in regulating the progression of lung cancer.3.By constructing a lentiviral expression vector to mediate the shRNA to silence the AFAP1L1 gene and observing its inhibitory effect on the growth of subcutaneously implanted tumors of lung cancer in nude mice,it may provides an experimental basis for gene therapy of lung cancer.Methods1.The Max Vision immunohistochemical technique was used to detect the expression level of AFAP1L1 protein in 84 cancer tissues and 69 normal tissues respectively,and to analyze the difference of AFAP1L1 protein expression in cancer tissues and normal tissues and its relationship with clinical pathological characteristics and pathological stage of patients.2.The shRNA interference technique was used to knock down the AFAP1L1 gene in lung adenocarcinoma cell line A549 to suppress its intracellular transcription and translation level.Subsequently,cell proliferation was detected using Celigo image cytometry,cell cycle was evaluated by flow cytometry,and Annexin V(anchored protein)was used as a marker to determine the level of apoptosis.Transwell experiments were used to investigate the ability of AFAP1L1 to knock down the invasion of lung adenocarcinoma cells.Further,the Path Scan intracellular signal transduction array was used to analyze the downstream cancer signaling proteins of AFAP1L1 knockdown A549 cells.3.Construction of shRNA lentiviral expression vector of recombinant AFAP1L1 gene.The effect of AFAP1L1 knockdown on the proliferation and invasion of lung adenocarcinoma cells was verified in vivo by nude mice tumorigenesis experiments.Results1.We detected 62 cases of AFAP1L1 in 84 NSCLC cancer tissues(73.8%),while 16cases(23.2%)were detected in 69 normal tissues by Max Vision immunohistochemistry.AFAP1L1 is highly expressed in NSCLC cancer tissues and low or no expression in normal tissues(?~2=38.84 and P<0.005),with statistically significant differences.2.Further analysis revealed that the expression of AFAP1L1 is related to the pathological stage of lung cancer.The expression of AFAP1L1 was detected in 53.8%and 65.6%of tissue specimens of stage I and II NSCLC patients,respectively,while the expression rate of AFAP1L1 in stage III+IV NSCLC patients was 88.5%.There were statistical differences(?2=5.596 and P<0.05).3.The expression level of AFAP1L1 is related to lymph node metastasis,pleural invasion and vascular invasion of lung cancer.The positive rate of AFAP1L1 expression in specimens with lymph node metastasis was 85.1%,and the positive rate of AFAP1L1expression in specimens without lymph node metastasis was 62.2%.The results of the comparison between the groups were?~2=4.653 and P<0.05,there was a statistical difference.The positive rates of the group with pleural invasion and the group without pleural invasion were 82.5%and 61.4%respectively,the difference was statistically significant(?~2=4.416,P<0.05).The positive rates of vascular infiltration group and non-vascular infiltration group were 82.1%and 57.1%,respectively,with statistical difference(?~2=4.811,P<0.05).In addition,the expression level of AFAP1L1 protein was not statistically different from the pathological type,gender,and age of the patients(P>0.05).4.We successfully constructed AFAP1L1-shRNA lentiviral expression vector.The study found that knocking down the AFAP1L1 gene of lung adenocarcinoma A549 cells significantly inhibited its proliferation and invasion ability;in the cell cycle study,it was found that knocking down the AFAP1L1 gene promoted the cell cycle to G1 and G2/M phase transition,G1 and G2/M phase cell ratio Increase;compared with negative control shRNA transfected cells,apoptosis increased in AFAP1L1-shRNA transfected cells.Transwell measurement results showed that compared with the blank control group and the plasmid control group,the number of cells that interfered with the membrane penetration of the experimental group was significantly reduced,and the difference was statistically significant(P<0.05).Using Path Scan intracellular signal transduction array analysis,we found that the down-regulation of AFAP1L1 significantly activated P38and caspase 3,and inhibited PRAS40 activation.5.The analysis of the growth of the transplanted tumors using the in vivo imaging technique through nude mice tumorigenesis experiments showed that the blank control group and the experimental group had tumor volumes of 619.80±278.97mm~3 and289.50±47.07mm~3,respectively,with significant differences.The tumor weights of the blank control group and the experimental group were 0.966±0.185g and 0.583±0.057g,respectively.The tumor weight of the experimental group was significantly lower than that of the blank control group.In addition,by in vivo imaging of the experimental group and the blank control group,the results showed that the fluorescent expression of the experimental group was reduced compared with the blank control group(P<0.05).Conclusions:1.The expression level of AFAP1L1 protein in NSCLC cancer tissues is significantly higher than that in normal tissues,and it is closely related to the pathological stage of NSCLC,pleural invasion,lymph node metastasis,and vascular infiltration.2.AFAP1L1 accelerates the cycle of lung adenocarcinoma cells,inhibits lung cancer cell apoptosis,and promotes lung cancer cell invasion.3.As an efficient gene editing technology,RNA interference can silence the expression of target genes in lung cancer cells.Construction of a lentiviral vector(shRNA-AFAP1L1-LVs)stably expressing the target gene RNA interference and subsequent experiments found that the specific inhibition of the expression of AFAP1L1 gene in human lung cancer cell lines in vivo and in vitro can inhibit human lung adenocarcinoma cell A549 The ability of growth,proliferation and invasion.Using in vivo imaging technology,the effect of silencing the AFAP1L1 gene on the biological behavior of lung adenocarcinoma cells was verified.To lay the foundation for further research on the malignant behavior of lung cancer cells,and to provide more evidence to reveal the molecular mechanism of lung cancer development,AFAP1L1 has the potential to become a target for lung cancer gene therapy.