The Effects And Methylation Status Of SOCS1 And SOCS3 Gene Promoter In EML4-ALK Positive H2228 Lung Cancer Cells

Background and Objective:The EML4-ALK fusion gene consist of echinoderm microtubule-associated-protein-like 4(EML4)and anaplastic lymphoma kinase(ALK),that is a newly discovered driver gene of nonsmall-cell lung cancer and exhibits special clinical and pathological features.The JAK-STAT signaling pathway,an important downstream signaling pathway of EML4-ALK,is aberrantly sustained and activated in EML4-ALK-positive lung cancer cells fusion gene,but the underlying reason remains unknown.The suppressor of cytokine signaling(SOCS)is a negative regulatory factor that mainly inhibits the proliferation,differentiation,and induction of apoptotic cells by inhibiting the JAK-STAT signaling pathway.The aberrant methylation of the SOCS gene leads to inactivation of tumors and abnormal activation of the JAK2-STAT signaling pathway.The purpose of this study was to investigate the effects of SOCS1 and SOCS3 on the EML4-ALK fusion gene positive lung cancer cells and the methylation status of SOCS1 and SOCS3 promoter regions.Methods:Methylation specific PCR was used to detect the methylation status of SOCS1 and SOCS3 promoter in EML4-ALK positive lung cancer cells and lung cancer tissues,and the results were verified by H2228 sequencing.DNA methyltransferase inhibitor 5 '-Aza-d C was used to treat H2228 cells,and the expression levels of SOCS1 and SOCS3 were detected by real-time PCR and Western blot.Use ALK inhibitor TAE684 and ALK-Si RNA treat H2228 cells to inhibite the expression of ALK,and the expression of p-JAK2,p-STAT3,p-STAT6,SOCS1 and SOCS3 were detected by Western blot.After overexpression of ALK in EML4-ALK negative HEK293 cells,the activation of JAK-STAT signaling pathway and the expression of SOCS1 and SOCS3 were detected by Western blot.PEGFp-SOCS1 and p EGFp-SOCS3 plasmids were transfected into H2228 cells,and the expression levels of SOCS1 and SOCS3 were detected by RT-PCR and Western-Blot.The transfection efficiency was tested,and the cell proliferation and cell cycle were detected by EDU,CCK8 and flow cytometry.Results:MSP and DNA sequencing assay results indicated the presence of SOCS1 promoter methylation in H2228 cells as well as in all of seven EML4-ALK-positive lung cancer tissues,and the presence of SOCS3 promoter methylation in H2228 cells as well as in three cases of seven EML4-ALK-positive lung cancer tissues.The expression level of SOCS1 and SOCS3 significantly increased in H2228 cells after 5?-Aza-d C treatment.After HEK-293 cells overexpressing ALK 48 hours,the JAK-STAT signaling pathway is activated,the expression level of ALK and p-ALK protein was significantly increased,and the expression of p-JAK2,p-STAT3 and p-STAT6 increased with the increase of ALK and p-ALK expression.H2228 cells were treated with ALK-Si-RNA and ALK inhibitor TAE-684 respectively,the expression of ALK and p-ALK protein was significantly decreased,and the expression of p-JAK2,p-STAT3 and p-STAT6 were decreased,and the JAK-STAT signaling pathway was inhibited.H2228 cells were transfected with p EGFp-SOCS1 and p EGFp-SOCS3,at the transcriptional level,the expression of SOCS1 and SOCS3 in H2228 cells was 10.3 times higher and 93.4 times higher than that of the control group respectively.The results of Western blot also showed that the expression of SOCS1 in H2228 cells transfected with SOCS1 was significantly increased,and the expression of SOCS3 in H2228 cells transfected with SOCS3 was significantly increased.With the increase of the expression of SOCS1 and SOCS3,we found that the expression of p-ALK,p-JAK2,p-STAT3 and p-STAT6 were decreased in the two groups,which indicated that the JAK-STAT signaling pathway was inhibited.The results of EDU showed that the proliferation ability of H2228 cells was significantly lower than that of transfected H2228 cells after transfection with SOCS1 and SOCS3.The statistical results showed that the cell viability of the control group was 56.25%,the cell viability of p EGFp-SOCS1 group was 36.45%(P<0.05),and the p EGFp-SOCS3 group was 38.72%(P<0.05).When H2228 cells were overexpressed in SOCS1 and SOCS3,the inhibition rates of cell metabolic activity were 48.49%(P<0.05)and 58.33%(P<0.05)respectively compared with the control group,and the results of CCK-8 were consistent with EDU.FCM cell cycle analysis showed the number of control group and experimental group G1 and S phase cells were basically no difference,but the number of the two experimental groups of G2 cells were significantly higher than the control group,the statistical results show that the two experimental group P values were less than 0.05.Conclusion:Aberrant methylation of SOCS1 and SOCS3 promoter regions exist in EML4-ALK(+)H2228 cells and some lung cancer tissues.After overexpression of ALK in HEK293 cells,the JAK-STAT signaling pathway was activated,and the JAK-STAT signaling pathway was inhibited after ALK expression was inhibited in H2228 cells.The results showed that JAK-STAT pathway might be the downstream signaling pathway of ALK.After SOCS1 and SOCS3 were overexpressed in H2228 cells,the JAK-STAT pathway was inhibited,and the metabolic activity and proliferation ability of the cells were inhibited too.Flow cytometry analysis showed that the number of G2 cells in the two experimental groups was significantly higher than that in the control group.We conclude that SOCS1 and SOCS3 inhibit cell metabolize and cell proliferation by inhibiting JAK-STAT signaling pathway,and the effect on cell cycle is mainly to block of G2/M phase.The aberrant methylation of SOCS1 and SOCS3 promoter in H2228 cells reduced the expression of SOCS1 and SOCS3,and the inhibition of JAK-STAT signaling pathway disappeared,resulting in the unlimited proliferation of tumor cells.