The EZH2/HAS3 Regulates Radiation-induced Lung Cancer Cells Migration And Invasion Effects And Mechanisms

Research background:Lung cancer is the most common cancer worldwide.Radiation therapy is one of the main means of tumor treatment,which has a vital position and function.Hyaluronic acid（HA）,as an important biomacromolecule,is one of the main components of the extracellular matrix and is involved in many physiological and pathological processes,such as cell proliferation,cell migration,and cell apoptosis.Recent studies have shown that HA not only plays an important role in normal biological processes but also plays an important role in the occurrence and development of diseases,such as a tumor,inflammation,and cardiovascular diseases.Hyaluronate synthase（HAS）is responsible for HA synthesis.Studies have found that the expression level of HAS increases significantly in a variety of tumor cells.These changes in HA are closely related to the biological characteristics of tumor cell proliferation,invasion,and metastasis.The HAS family includes HAS1,HAS2,and HAS3,among which HAS1 and HAS2 mainly synthesize high molecular weight HA and HAS3 synthesize low molecular weight HA.It has been reported that low molecular weight HA can promote cell migration and invasion.EZH2（Enhancer of zest homolog 2）is a histone methyltransferase that is one of the core members of Polycomb’s recombinant protein Complex 2（PRC2）.The downstream gene expression could be inhibited by H3K27me3.Studies have shown that EZH2 is highly expressed in a variety of tumor tissues and is closely related to the growth,differentiation,and metastasis of tumor cells.Recent studies have shown that EZH2 is involved in hyaluronic acid biosynthesis and tumor development.Therefore,this study aims to explore the effects and mechanisms of EZH2/HAS3 in regulating radiation-induced migration and invasion of lung cancer cells,providing new ideas and new basis for radiation therapy of lung cancer.Research purpose:The cell migration,invasion ability,and cytoskeleton changes of A549 cells and H1299 cells after HAS3 knockdown were observed under radiation conditions.Verify the regulatory relationship between EZH2 and HAS3.Research methods:1.The migration ability of A549 cells and H1299 cells before and after radiation was observed by scratch test;Under radiation conditions（A549 cells irradiated 10Gy,H1299 cells irradiated 6Gy）,the migration ability of A549 cells and H1299 cells was observed after HAS3 knockdown;The migration ability of H1299 cells after overexpression of EZH2 was observed.2.Transwell experiment was used to observe the changes in the cell invasion ability of A549 cells and H1299 cells before and after radiation;The invasion ability of A549 cells and H1299 cells was observed after HAS3 knockdown;The invasion ability of H1299 cells after EZH2 overexpression was observed.3.Ghost-pen cyclic peptide cytoskeleton staining was used to observe the cytoskeleton changes of A549 cells and H1299 cells before and after radiation;The cytoskeletal changes of A549 cells and H1299 cells after HAS3 knockdown were observed.4.The m RNA and protein expression levels of HAS3 in A549 cells and H1299cells were detected by transient transfection assay;The m RNA and protein expression levels of EZH2 in H1299 cells were detected by transient EZH2 overexpression plasmid.5.Lentivirus transfection experiment was used to construct the A549-EZH2^LOWcell model.6.The expression level of HAS3 m RNA in A549 cells and H1299 cells was detected by RT-q PCR.m RNA expression levels of EZH2 and HAS3 were detected after overexpression of EZH2 in H1299 cells.7.Western Blot assay was used to detect the expression levels of HAS3,EZH2,and HAS family proteins in A549 and H1299 cells before and after radiation;The expression level of HAS3 protein in A549 cells and H1299 cells were detected after transient si HAS3 transfer;The expression level of EZH2 protein was detected after overexpression of EZH2 in H1299 cells;H3K27me3 protein expression was detected in the A549-EZH2^LOWcell model.8.The EZH2-mediated H3K27me3 enrichment in the HAS3 promoter region was detected by CHIP-PCR assay.Research result:1.Migration,invasion,and cytoskeletal changes of lung cancer cells under radiation conditions（1）The changes in lung cancer cell migration abilityScratch test results showed that the migration ability of A549 cells in the irradiation group was significantly improved compared with that in the sham group,and the results were statistically significant at 24h and 48h after 10Gy irradiation（P<0.01）;The cell migration ability of the H1299 cell irradiation group was also significantly improved compared with the sham irradiation group,and the results were statistically significant 24h after 6Gy irradiation（P<0.05）.（2）The change in invasive ability of lung cancer cellsThe results of the cell Transwell experiment showed that the number of cells in the subcompartment increased significantly in the A549 cell irradiation group compared with the sham irradiation group,and the result was statistically significant24h after 10Gy irradiation（P<0.05）;The number of cells in the subcompartment was also significantly increased in the H1299 cell irradiation group compared with the sham irradiation group,and the results were statistically significant at 24h and 48h after 6Gy irradiation（P<0.01,P<0.05）.（3）Cytoskeletal changes in lung cancerGhost pen cyclic peptide staining showed that the antennae of A549 cells in the irradiation group were longer and larger than those in the sham group;The microfilament structure of H1299 cells in the irradiation group was significantly increased compared with that in the sham irradiation group.2.Changes in HAS3 expression in lung cancer cells under radiationThe expression level of HAS3 protein in A549 and H1299 cells was significantly increased after radiation.3.Cell migration,invasion ability,and cytoskeletal changes of lung cancer cells after HAS3 knockdown under radiation condition（1）Detection of m RNA and protein levels in lung cancer cells after HAS3knockdownA549 cells and H1299 cells were transferred to si HAS3 instantaneously,and m RNA expression levels were detected 24h later.RT-q PCR results showed that the expression level of HAS3 m RNA in the experimental group was significantly decreased compared with the control group（P<0.05）;The total protein was extracted48 hours later,and the Western Blot results showed that the expression level of HAS3protein in the two lung cancer cells was significantly decreased compared with the control group.（2）Changes in cell migration ability of lung cancer cells after HAS3knockdown under radiation conditionIn A549 cells,the migration ability of the experimental group was significantly decreased compared with the control group,and the results at 24h and 48h were statistically significant（P<0.01）;Under the condition of 10Gy irradiation,the migration ability of the experimental group was significantly decreased compared with the control group,and the result of 24h was statistically significant（P<0.05）.In H1299 cells,the migration ability of the experimental group was significantly decreased compared with the control group,and the result of 24h was statistically significant（P<0.05）;Under the condition of 6Gy irradiation,the migration ability of the experimental group was significantly decreased compared with the control group,and the result of 24h was statistically significant（P<0.05）.（3）Changes in cell invasion ability of lung cancer cells after HAS3 knockdown under radiation conditionIn A549 cells,the number of cells in the lower chamber of the experimental group was significantly reduced compared with the control group,and the results at24h and 48h were statistically significant（P<0.01,P<0.05）;Under the condition of10Gy irradiation,the number of cells in the lower chamber of the experimental group was significantly reduced compared with the control group,and the result of 24h was statistically significant（P<0.05）.Similarly,in H1299 cells,the number of cells in the lower chamber of the experimental group was significantly reduced compared with the control group,and the result was statistically significant at 48h（P<0.05）;Under the condition of 6Gy irradiation,the number of cells in the lower chamber of the experimental group was significantly reduced compared with the control group,and the result of 48h was statistically significant（P<0.05）.（4）Changes in the cytoskeleton of lung cancer cells after HAS3 knockdown under radiationCompared with the control group,the morphology of A549 cells in the experimental group was deformed from a normal shuttle to an ellipse or circle,and the cell microfilament structure was significantly reduced.Compared with the control group,the cell morphology of the experimental group also changed from normal spindle shape to oval or circular shape under the condition of 10Gy irradiation.Similarly,H1299 cells in the experimental group showed a significant decrease in cell size and microfilament structure compared with the control group.The microfilament structure of the experimental group was also significantly reduced compared with the control group under the irradiation condition of 6Gy.4.Radiation-induced changes of EZH2 and HAS family in lung cancer cellsA549 cells irradiated by 10Gy and H1299 cells irradiated by 6Gy showed significantly lower expression levels of EZH2 protein and significantly higher expression levels of HAS family proteins than those in the sham group.5.Changes of HAS family after overexpression of EZH2 and regulation of HAS3 by EZH2（1）Changes of HAS family after EZH2 overexpression in H1299 cellsThe expression level of EZH2 protein in H1299 cells was significantly lower than that in SK-MES-1 and A549 cells.H1299 cells transferred EZH2 overexpression plasmid,and the m RNA expression level was detected 24h later.RT-q PCR results showed that the m RNA expression level of EZH2 in group 1299-EZH2^OEwas significantly increased compared with group H1299-Vector（P<0.05）;Total proteins were extracted 48 hours later,and Western Blot results showed that the expression level of EZH2 protein was significantly higher than that of the H1299-Vector group.After EZH2 was overexpressed in H1299 cells,the protein expression levels of HAS1and HAS2 were not significantly changed,while the m RNA expression level of HAS3 was significantly decreased（P<0.01）;The expression level of HAS3 protein was significantly decreased.（2）Validation of the regulatory relationship between EZH2 and HAS3In the A549-EZH2^LOWcell model,H3K27me3 protein expression was significantly lower than that in the A549-Vector group.The results of biogenic analysis predicted the existence of three H3K27me3 enrichment sites in the HAS3promoter region,named PP1,PP2,and PP3,respectively.The concentration difference of H3K27me3 in the HAS3 promoter region of A549-Vector cells and A549-EZH2^LOWcells was detected by Ch IP-PCR assay.The experimental results showed that H3K27me3 was enriched in the PP2 region,which was statistically significant compared with A549-Vector（P<0.05）.6.Changes in migration and invasion ability of H1299 cells after EZH2overexpressionThe migration experiment results after overexpression of EZH2 in H1299 cells showed that the migration ability of the H1299-EZH2^OEgroup was significantly decreased compared with the H1299-Vector group,and the result of 24h was statistically significant（P<0.01）;After overexpression of EZH2 in H1299 cells,Transwell experiment results showed that the number of H1299 cells in the lower chamber of the H1299-EZH2^OEgroup was significantly reduced compared with that in the H1299-Vector group.It indicated that the cell invasion ability was significantly decreased,and the result was statistically significant at 24h（P<0.05）.Conclusion:1.HAS3,as one of the hyaluronic acid synthases,can promote the migration and invasion of A549 and H1299 lung cancer cells;2.As an important component protein of PRC2,EZH2 can negatively regulate HAS3 expression in a H3K27Me3-dependent manner;3.Radiation can enhance the migration and invasion ability of A549 and H1299lung cancer cells by affecting the expression of EZH2 and HAS3.