The Effect Of IL-27 On Proliferation,Invasion And Migration Of Non-small-cell Lung Cancer Cell Line H1299 By MiR-935 In Vitro

Background and Objective: IL-27 is a heterodimer cytokine composed of IL-27p28 chain and EBI3.It is a member of the IL-6 /IL-12 cytokine superfamily and is secreted mainly by monocytes,macrophages,dendritic cells and microglia.IL-27 R is a heterodimer composed of gp130 and IL-27 R.IL-27 binds to the gp130/IL-27 R complex and conducts signal transduction by activating the JAK/STAT signaling pathway,mainly involving the phosphorylation of STAT1 and STAT3.IL-27 has been reported as a tumor suppressor and plays a significant role in a variety of cancers,such as inhibiting tumor growth,invasion,metastasis,and promoting cell death.Micro RNAs(mi RNAs)are small non-coding RNAs with a length of about 20-25 nucleotides,which play a key role in a variety of biological processes and have a dual role in cancers from different sources.Therefore,this study first explored the effect of IL-27 on the proliferation,invasion and migration of H1299 cells in non-small cell lung cancer in vitro,and then discussed the relationship between mi R-935 and IL-27,so as to explore the effect of mi R-935 on the proliferation,invasion and metastasis of H1299 cells by regulating IL-27.Methods: Tumor tissues and adjacent normal lung tissues of patients with NSCLC were collected,and m RNA expression levels in tissues were detected by real-time quantitative PCR.The effect of IL-27 on the proliferation of H1299 cells was detected by CCK8 assay.In addition,the effect of IL-27 on the proliferation of H1299 cells was evaluated by colony formation assay.Transwell invasion test was used to evaluate the effect of IL-27 expression on the invasion ability of H1299 cells.The wound healing assay was used to simulate the process of cell migration in vivo,so as to clarify the effect of IL-27 on the migration ability of H1299 cells.We determined the relationship between mi R-935 and IL-27 by detecting the expression of mi R-935 in NSCLC tissues.H1299 cells were transfected with mi R-935 or treated with IL-27 for cell survival test,and CCK8 test,wound healing assay and Transwell invasion test were used to evaluate the effect of mi R-935 on the proliferation,invasion and migration of H1299 cells through IL-27.Results: The overall mean expression level of IL-27 m RNA in 76 pairs of non-small cell lung cancer tissue samples was significantly lower than that in adjacent normal tissues(p<0.01).IL-27 m RNA and IL-27 expression levels of 6 non-small cell lung cancer cell lines(HCC827,SPAC1,H1299,H1650,A549,H520)were significantly lower than those of normal lung epithelial cell lines(BEAS-2B).Theeffect of IL-27 on the proliferation,invasion and migration of H1299 cells in vitro suggested that the absorbance of the IL-27 group was significantly lower at day 1,day3 and day 5 than that of the control group(P<0.01),reflecting the reduced proliferation of H1299 cells.The colony formation rate in the IL-27 group was significantly lower than that in the control group(P<0.01).In H1299 cells,the relative invasion rate of the control group was 100%,while the IL-27 group was 38.8%,the invasion rate of IL-27 group was significantly lower than that of the control group(P<0.01).Wound healing assay suggested that the wound width of IL-27 group was significantly larger than that of the control group in H1299 cells.The expression of mi R-935 in NSCLC tissues was significantly higher than that in adjacent normal tissues,and mi R-935 was negatively correlated with IL-27 level in NSCLC.We predicted the binding site of mi R-935 to the target gene IL-27 3'-UTR through Pic Tar and Targetscan.The overexpression of mi R-935 significantly reduced the luciferase activity of wild-type IL-27 3'-UTR in H1299 cells,but had no significant effect on the luciferase activity of mutant IL-27 3'UTR.We transfected H1299 cells with mi R-935 or treated with IL-27 for cell proliferation,invasion and migration.The results indicated that the overexpression of mi R-935 could promote the proliferation of H1299 cells,and the addition of IL-27 on this basis could inhibit the proliferation of H1299 cells.The wound healing assay was used to simulate the process of cell migration in vivo.The results showed that mi R-935 reversed the migration inhibition induced by IL-27 in H1299 cells.The Transwell invasion assay was evaluated and showed that mi R-935 reversed the invasion inhibition induced by IL-27 in H1299 cells.Conclusion: IL-27 can significantly inhibit the proliferation,invasion and migration of H1299 cells in vitro.The expression of mi R-935 in non-small cell lung cancer tissues is significantly higher than that in adjacent normal tissues,and is negatively associated with the level of IL-27,suggesting that mi R-935 can negatively regulate the expression of IL-27 in non-small cell lung cancer.mi R-935 promotes the proliferation,invasion and metastasis of H1299 cells by negatively regulating IL-27.The data in this study demonstrated that mi R-935,as a tumor-related mi RNAs,can regulate the expression of IL-27 in non-small cell lung cancer.Therefore,inhibition of mi R-935 expression may be a therapeutic target for future non-small cell lung cancer.