The Effect And Molecular Mechanisms Of Period2 Gene On The Proliferation And Apoptosis Of A549 Lung Cancer Cell

Objective:To investigate the effect of the circadian clock gene Period2 (Per2) on proliferation and apoptosis of human lung cancer A549 cells and further study the related mechanisms to identify the biological functions of Per2 gene in tumor cells.Methods:1. Experiments in vitro:The effect of Per2 gene on cloning, proliferation and apoptosis of A549 cells was detected by the plate colony-forming assay,MTS assay and flow cytometry.The effect of Per2 gene on the apoptosis morphology of A549 lung cancer cells was observed by Hoechst33258 staining. And the expression levels of proliferation and apoptosis-related genes were detected by Western blot and RT-qPCR.2. Animal experiments in vivo:The effect of Per2 on tumor formation rate and the growth of tumor of A549 cells was investigated through the experiments of nude mice, and the cell apoptosis of tumor transplanted subcutaneous ly in nude mice was further detected.The Per2 and Ki-67 expression levels in tumor transplanted subcutaneously were detected by immunohistochemistry.Results:1.The clone forming assay and MTS assay showed that, Per2 gene exhibited an apparent inhibition on the plate colony formation ability and cell proliferation activity of A549 cells, having statistically significant difference (P <0.05).2. The flow cytometry showed that, compared with the control group, the percent of cells in G1 phase with Per2 overexpression increased significantly, and the percent of cells in S phase decreased; the apoptosis rate of A549 cells with Per2 gene overexpression was higher than that of the control group, (23.27 ±3.37)% and (14.30±4.57%), respectively.3. Data of qRT-PCR and Western blot showed that compared with the control group, the mRNA and protein expression of genes in A549 cells with Per2 gene overexpression increased, such as P53, P21, Bax, Caspase8, while the expression of c-Myc, Bcl-2 genes decreased.4. Animal experiments showed that, the tumor formation rate of nude mice in the two groups was 100%, but in the Per2 overexpression group, the growth rate of the tumors transplanted subcutaneously was significantly slower than the control group, and the tumor weight was lighter than the control group, exhibiting statistically significant difference (P<0.05). Calculated according to the formula, the tumor inhibition rate of Per2 gene on A549 cells in nude mice was 36.77%.5.The apoptosis assay of tumors transplanted subcutaneously showed that, the apoptosis rates in the Per2 overexpression group and control group were (9.45±3.57)‰, (3.66±1.27)‰, respectively, with statistically significant difference (P<0.05); Immunohistochemistry showed that, the IRS score in the Per2 overexpression group and control group were (6.38±2.504 VS 3.12±2.232), respectively, with statistically significant difference (P<0.05). and compared with the control group, the percentage of Ki-67 positive cells in the Per2 overexpression group was higher, were(40.62±12.082 VS 71.25±6.409)%, with statistically significant difference (P<0.05).Conclusions:The overexpression of Per2 gene not only had apparent inhibition on the growth of tumor cells and the growth of tumors transplanted in nude mice,but also could induce the apoptosis of tumor cells. The underlying mechanism is possibly associated with up-regulation of expression of P53, P21, Bax, Caspase8,and the down-regulation of expression of c-Myc, Bcl-2, Ki-67; the specific mechanism is to be further studied.