IL-1RA Inhibits Iymph Node Metastasis Via MMP9 And VEGF-C In ESCC

Background: Esophageal cancer has a high incidence in China,of which,95% of the pathological types are esophageal squamous cell carcinoma(ESCC).ESCC in China have the characteristics of high incidence,high mortality and low survival rate.In recent years,the treatment of ESCC has become increasingly diverse with the improvement and breakthrough of surgical treatment,radiotherapy and chemotherapy,targeted therapy and immunotherapy,but the survival prognosis has not been significantly improved.Therefore,exploring more diagnostic and therapeutic targets is still Extremely demanded for ESCC.We found that the expression of Interleukin 1 receptor antagonist(IL-1RA)protein in ESCC was decreased in the D-2 mass spectrometry analysis,but its role and mechanism in ESCC is still unclear.Methods: 1.The differentially expressed genes in 5 pairs of ESCC tissues and corresponding adjacent tissues were detect by whole transcriptome high-throughput sequencing,and conduct bioinformatics analysis based on the differential genes screened.2.RT-qPCR and Western Blotting were used to detect the expression of IL-1RA in 8 pairs of ESCC and its corresponding adjacent tissues,and immunohistochemistry was used to detect the expression of IL-1RA in 76 pairs of ESCC and its adjacent tissues.The clinical significance of IL-1RA was analyzed combined with clinical data and survival rates.3.Lentiviral overexpression technology Was used to construct a stable transgenic cell line of ESCC with IL-1RA overexpression,which was verified by Western Blotting and RT-qPCR.4.CCK8 cells,plate clone formation experiment and nude mouse subcutaneous tumor formation experiment were used to detect the effect of IL-1RA overexpression on the proliferation and the metastasis ability was detected by cell scratch experiment,Trans-well migration experiment and nude mouse tail vein injection Experimental;lymphatic vessel looping,lymphatic vessel migration experiments and animal experiments to detect the effect of IL-1RA on tumor lymphangiogenesis.5.The effect of IL-1RA over-expression on the transcription of ESCC genes was detected by whole transcriptome sequencing,and the specific menchanism was predicted via GO function analysis and KEGG signal pathway analysis.6.The effect of IL-1RA in PI3K/NF-?B signaling pathway related proteins(PI3K,p-PI3 K,NF-?B,p-NF-?B),matrix metalloproteinase family(MMP2,MMP9)by RT-qPCR and Western Blotting methods And MMP13);detect the effect of IL-1RA on the expression and secretion of lymphangiogenesis regulatory factors(VEGF-A/B/C/D,HIF-1? and COX-2)through ELISA experiments;7.The role of MMP9 in EMT progression caused by IL-1RA was verified via rounds Supplementary experiments;8.The therapeutic effect of Anakinra on ESCC was evaluated via Anakinra administration experiment In nude mice.Results: 1.Whole transcriptome sequencing analysis and biological database data indicated that IL-1RA expression was decreased in ESCC.2.The results of clinical verification showed that the decrease expression of IL-1RA is related to poor clinical staging,positive lymph node metastasis and poor prognosis of ESCC.3.In vitro cell experiment and in vivo nude mouse function experiment found that overexpression of IL-1RA can promoted the proliferation and migration ability of ESCC.4.Overexpression of IL-1RA can inhibit the progression of EMT of by inhibiting the expression of MMP9 in ESCC.5.Overexpression of IL-1RA can inhibit the expression and secretion of EVGF-C by inhibiting the transcription of VEGF-C gene via PI3K/NF-?B pathway,and inhibit tumor-induced lymphangiogenesis.6.The interleukin 1 receptor antagonist drug Anakinra can inhibit ESCC lymphangiogenesis in nude mice,thereby inhibiting the proliferation and metastasis of ESCC.Conclusion: 1.The expression of IL-1RA is decreased in ESCC,and its decreased is closely related to poor clinical staging,positive lymph node metastasis and poor prognosis of ESCC.2.IL-1RA can inhibit the lymph node metastasis of ESCC by regulating the EMT process mediated by MMP9 and the lymphangiogenesis regulated by PI3K/NF-?B/VEGF-C.3.Anakinra can inhibit the proliferation and metastasis of ESCC,and may be an effective drug for the treatment of ESCC.