| Objective:To observe the correlation between the expression of Interleukin enhancer binding factor2(ILF2)and the degree of infiltration of CD8+T cells in esophageal squamous cell carcinoma,and to explore the effect of ILF2 expression on radiotherapy sensitivity of esophageal squamous cell carcinoma.Methods:1.The expression of ILF2 and IL-2 and infiltration of CD8+T cells in cancer tissues of ESCC patients were detected by immunohistochemistry.2.The survival of ESCC patients was followed up.DFS and OS were used as parameters to observe the effect of ILF2 expression on radiotherapy efficacy of ESCC patients.3.The ILF2-sh RNA lentivirus plasmid was used to infect human esophageal squamous cell carcinoma cell line ECA109,and the ILF2 overexpressed plasmid was transfected into human esophageal squamous cell carcinoma cell line KYSE180.The m RNA expression level of ILF2 in two different cells was detected by RT-PCR assay.Western blot assay was used to detect the expression level of ILF2 protein in two different cells.4.ECA109 cells were infected with ILF2-sh RNA lentivirus plasmid and KYSE180 cells were irradiated after transfection with ILF2 overexpressed plasmid.Four groups were set up: group NC-109(ECA109 cells transfected with empty plasmid),group KD-109(ECA109 cells transfected with ILF2 small interference group),group NC-180(KYSE180 cells transfected with empty plasmid group),group OE-180(KYSE180 cells transfected with ILF2 overexpression plasmid group).The effect of ILF2 expression on radiotherapy sensitivity of ESCC cells was detected by CCK-8 assay and clonal formation assay.Result:1.Immunohistochemical results showed that the expression level of ILF2 in ESCC tissues was significantly up-regulated compared with that in para-carcinoma tissues,and the difference was statistically significant(χ2=4.881,P=0.027).Then,according to the expression of ILF2 in ESCC tissues,the ILF2 was divided into high expression group and low expression group.The correlation between the expression degree of ILF2 and IL-2,and the infiltration degree of ILF2 and CD8+T cells in the two groups was compared.The results showed that the expression degree of IL-2 was positively correlated with the expression degree of ILF2 in ESCC tissues,and the difference was statistically significant(P=0.039).The infiltration degree of CD8+T cells was positively correlated with the expression degree of ILF2,and the difference between the two groups was statistically significant(P=0.001).The correlation between IL-2 expression and the degree of infiltration of CD8+T cells was further compared.The results showed that the degree of infiltration of CD8+T cells was positively correlated with the degree of IL-2 expression in ESCC tissues,and the difference was statistically significant(P=0.008).2.Through the follow-up of the survival of ESCC patients with DFS and OS as parameters,the statistics of 1-year,2-year and 3-year DFS rates and 1-year,2-year and3-year OS rates of ESCC-assisted radiotherapy showed that the 1-year,2-year and3-year DFS rates of ESCC patients with high ILF2 expression were 88.4%,69.0% and57.0%,respectively.Its m DFS have not been reached;The 1-year,2-year,3-year DFS rate of ESCC patients with low expression of ILF2 was 60.0%,33.0%,20.0%,and the m DFS rate was 20 months.The rate of DFS in patients with high expression was significantly higher than that in patients with low expression,and the difference between the two groups was statistically significant(P=0.009).The 1-year,2-year and3-year OS rates in ESCC patients with high expression of ILF2 were 96.15%,92.31%and 76.92%,respectively,and the m OS was not yet reached.The 1-year,2-year and3-year OS rates of ESCC patients with low expression of ILF2 were 86.67%,66.67%and 46.67%,respectively,and their m OS was 30 months.The OS rate of ESCC patients with high expression of ILF2 was significantly higher than that of patients with low expression,and the difference between the two groups was statistically significant(P=0.043).Log-Rank test results showed that the OS rate and DFS rate of ESCC patients with high expression of ILF2 were higher than those of the group with low expression of ILF2,suggesting that ESCC patients with high expression of ILF2 had better radiotherapy efficacy.3.The m RNA level of ILF2 decreased significantly after infection with ILF2-sh RNA lentivirus plasmid.The ILF2 m RNA level of KYSE180 cells was significantly increased after transfection with ILF2 overexpression plasmid.4.After infection with ILF2-sh RNA lentivirus plasmid,the expression of ILF2 protein decreased and ILF2 was successfully knocked down in ECA109 cells.After transfection of ILF2 overexpression plasmid into KYSE180 cells,the expression of ILF2 protein increased,suggesting that ILF2 was overexpressed in KYSE180 cells.5.Cells infected with ILF2-sh RNA lentivirus were exposed to 0,2,4,8,10 Gy radiation,respectively,and CCK8 assay was performed 48 hours later to observe cell proliferation.The results of CCK-8 experiment showed that radiation could inhibit cell proliferation in both KD-109 group and NC-109 group,and was positively correlated with dose,but the cell damage caused by radiation was more obvious in KD group(at 0,2,4,8Gy,the difference between the two groups was statistically significant,P<0.001;However,there was no statistical significance between the two groups in the 10 Gy radiation dose group,P>0.05),and the cell growth rate in the KD-109 group was significantly decreased compared with that in the NC-109 group.After transfection of KYSE180 cells with ILF2 overexpressed plasmid,radiation caused obvious death in OE-180 group and NC-180 group,which was positively correlated with dose(when irradiated 0,2,4,8,10 Gy,the difference between the two groups was statistically significant,P<0.001),and the cell growth rate in OE-180 group was higher than that in NC-180 group.6.To further examine the effect of ILF2 knockdown/overexpression on cell proliferation of ESCC cells after irradiation,ECA109 cells were infected with ILF2-shr NA lentvirus and KYSE180 cells were transfected with ILF2 overexpression plasmid.After receiving 0,2,4,8,10 Gy radiation,respectively,cell survival rate SF was calculated in the four groups after receiving different radiation(at 2,4,8Gy,there was statistical significance between NC-109 and KD-109 and NC-180 and OE-180 groups,P<0.05;at 10 Gy,NC-109 and KD-109 and NC-180 and OE-180 showed no statistically significant difference between the two groups,P>0.05).The results showed that after ILF2 knockdown,the radiosensitization ratio was greater than 1,so it was believed that ILF2 could inhibit the sensitivity of ESCC cells to radiotherapy.Conclusion:1.In ESCC tissues,the expression degree of IL-2 was positively correlated with the expression degree of ILF2,and the infiltration degree of CD8+T cells was positively correlated with the expression degree of ILF2 and IL-2.2.Compared with patients with low expression of ILF2,ESCC patients with high expression of ILF2 had better radiotherapy efficacy and better OS and DFS.3.ILF2 can promote the proliferation of ESCC cells.4.ILF2 inhibited the radiotherapy sensitivity of ESCC cells. |
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