Effect And Mechanism Of LIF In Radiation Response To Different LET Rays In Esophageal Squamous Cell Carcinoma

Background and purposeEsophageal squamous cell carcinoma(ESCC)is moderately sensitive to low linear energy transfer(LET)X-ray,and is prone to local recurrence and distant metastasis after radiotherapy,with the worse clinical prognosis.Carbon ion is high LET ray,which have better physical and radiobiological properties than low LET rays,and have better therapeutic effects for tumor which is insensitive or resistant to low LET X-ray.The leukemia inhibitory factor(LIF)is a pluripotent cytokine that activates the STAT3 signaling pathway through the leukemia suppressor receptor(LIFR)and glycoprotein(GP-130),which stimulates tumor growth,proliferation and metastasis,and is associated with chemoradiotherapy resistance.In this study,low LET X-ray and high LET carbon ion were used to irradiate ESCC cells,then analyze the effects of LIF on the biological behavior of ESCC cells irradiated by different LET rays and to explore the molecular mechanism of its action,so as to provide a theoretical basis for the treatment of ESCC with high LET carbon ion.Method1.Study on radiation biological behavior of ESCC cells: ECA109 and KYSE150 cells were irradiated by 0,1,2 and 4 Gy X-rays.Colony formation assay was used to detect the effects of different doses of X-rays on the survival of ECA109 and KYSE150 cells.CCK8 method was used to detect the effects of different doses of X-rays on the proliferation of ECA109 and KYSE150 cells at 24,48 and 72 h after irradiation.Flow cytometry was used to detect the effects of various doses of X-rays on apoptosis and cell cycle of ECA109 and KYSE150 cells at 24 and 48 h after irradiation.Scratches and Transwell experiments were used to detect the effects of 2 Gy X-rays on the migration and invasion of ECA109 cells at 48 h.The effect of 2 Gy X-ray irradiation on the expression of ?-H2 AX protein in ECA109 cells was detected by immunofluorescence confocal assay.,Western blot was used to detect the effect of 2-Gy X-ray irradiation on the expression of Bax,Bcl-2 and ? H2 AX protein in ECA109 cells.2.Screening of differential protein molecules of radiation resistance in ECA109 cells: According to the results of the previous experiment,ECA109 cells which one were cultured for 48 h after 2 Gy X-ray irradiation and the another were unirradiated were selected for proteomic detection by TMT protein quantitative technology.Combined with bioinformatics methods,the results were analyzed by difference analysis,and the extremely significant differences of LIF protein molecules were obtained.3.Clinical significance of serum LIF in patients with ESCC: ELISA was used to detect the changes of serum LIF concentration before and after radiotherapy in 60 patients with ESCC,and compared with 30 healthy controls.Then observe the relationship between serum LIF concentration and clinicopathological features and disease prognosis.4.The exploration of biological effects of LIF targeting STAT3 in esophageal squamous cell carcinoma: After down-regulating LIF molecule in ECA109 cells by si RNA technique,the biological effects of LIF in ESCC were detected by clone formation test,CCK8 test and flow cytometry,and the metastatic changes of ESCC cells were detected by Transwell chamber.RT-PCR and Western blot were used to verify the expression of LIF in ESCC and its regulation of STAT3 signal pathway.5.LIF regulates the radiation response of ESCC cells to carbon ion through STAT3 signal pathway and its mechanism: ECA109 cells were irradiated by carbon ion with 0,1,2 and 4 Gy,and the radiation effects of different doses of carbon ion were detected by colony formation test.CCK8 method was used to detect the effects of different doses of carbon ion on the proliferation of ESCC cells in different time periods.Flow cytometry was used to detect the effects of different doses of carbon ion radiation on apoptosis and cycle of ECA109 cells at 24 and 48 h.Scratches and Transwell experiments were used to detect the migration and invasion ability of ECA109 cells after carbon ion irradiation.RT-PCR was used to detect the relative expression of LIF,STAT3,MMP2 and E-cadherin m RNA,Western blot was used to detect the expression of LIF,STAT3,MMP2 and E-cadherin protein after carbon ion irradiation.Result1.The biological behavior of ECA109 and KYSE150 cells was significantly affected by different doses of X rays.Compared with the 0 Gy group,the proliferation of ECA109 cells at 48 h after 2 Gy X rays was not significantly inhibited(p > 0.05),while the proliferation of KYSE150 cells was significantly inhibited(p< 0.05).ECA109 cells showed cell cycle arrest at 48 h after 4 Gy irradiation(p < 0.05),while KYSE150 cells showed cell cycle arrest at 48 h after 2 and 4 Gy irradiation(p < 0.05).Apoptosis of ECA109 cells was significantly increased 48 h after irradiation with 4 Gy(p < 0.05),while apoptosis of KYSE150 cells was significantly increased 48 h after irradiation with 2 and 4 Gy(p < 0.05).The effect of 2 Gy X ray on the invasion and migration of ECA109 cells did not change significantly(p > 0.05).There was significant difference in ?-H2 AX focus of ECA109 cells in 2 Gy group(p < 0.05),but there was no significant difference in the expression of Bax,Bcl-2 and STAT3 protein(p > 0.05).2.In this study,proteomic analysis was performed on ECA109 cells at 48 h after 2 Gy X-ray irradiation.399 differentially expressed proteins were obtained(215 down-regulated and 184 up-regulated),of which the expression of LIF was significantly different(p = 0.0003).Enrichment analysis of GO and KEGG pathway showed that LIF was involved in STAT3 signal pathway.3.The serum LIF concentrations of ESCC patients before and after radiotherapy were 0.5224 ± 0.1983?g/ml and 0.3861 ± 0.1506?g/ml,respectively,which were significantly higher than those of healthy controls(0.3233 ± 0.0985?g/ml)(p<0.05).The serum LIF concentration of ESCC was correlated with tumor T stage,lymph node stage,clinical stage and histological grade(p<0.05).The serum LIF concentration was increased in 13 patients after radiotherapy,and 47 patients was decreased than that before radiotherapy.The local recurrence rates during the follow-up period(36 months)were 69.2%(9/13)in the increased LIF group and 34.0%(16/47)in the decreased group,respectively(p= 0.023).The distant metastasis rates in the two groups were 53.8% and 23.4%,respectively(p= 0.034).The 1-,2-and 3-survival rates of the two groups were 76.9%,46.2%,30.7% and 85.1%,66.0%,46.8%,respectively(p= 0.048).4.After knocking down the LIF in ECA109 cells,the proliferation ability of ECA109 cells decreased,the apoptosis rate increased,and the migration and invasion ability of ECA109 cells were inhibited,which was significantly different from that of the control group(p<0.05).Down-regulation of LIF could reduce the expression of STAT3 in ECA109 cells.(p < 0.05).5.The results of ECA109 cells irradiated by different doses of carbon ion showed that compared with 0 Gy group,2 and 4 Gy carbon ion rays could induce apoptosis of ECA109 cells,block the cell cycle in G2 ? M phase,and inhibit the proliferation,migration and invasion of ECA109 cells(p<0.05).The results of transcription and translation showed that 2 and 4 Gy carbon ion could significantly down-regulate the expression of LIF,p-STAT3,STAT3 and MMP2 and up-regulate the expression of E-cadherin in ECA109 cells(p<0.05).Conclusion1.2 Gy X-ray had no significant inhibitory effect on the proliferation and metastasis of esophageal squamous cell carcinoma ECA109 cells.The expression of ?H2AX was significantly up-regulated after irradiation,while the expression of STAT3 was not significant.2.The expression of LIF in esophageal squamous cell carcinoma ECA109 cells was down-regulated after 2 Gy X-ray irradiation,and the concentration of serum LIF in ESCC patients was correlated with local recurrence,distant metastasis and prognosis of diseases.3.The low expression of LIF regulates the biological effects of esophageal squamous cell carcinoma ECA109 cells by targeting STAT3 molecules and mediating STAT3 signal pathway.4.Carbon ions induce the expression of relevant molecules in the STAT3 signaling pathway by down-regulating LIF in esophageal squamous cell carcinoma ECA109 cells,thus inhibiting the proliferation and metastasis of ESCC cells.