Mechanism Study Of IL-37 Inhibits Tumor Invasion And Metastasis Via Targeting IL-6/STAT3 Signaling

Objective: Observe the correlation between IL-37 expression level and HCC clinical pathological characteristics, evaluate the value of IL-37 in HCC prognosis, to investigate the effect and preliminary molecular mechanism of IL-37 in invasion and metastasis of HCC cells.Methods:The IL-37 expression in 54 pairs of HCC tissues and their non-cancerous counterparts were detected by q PCR and IHC. To determine the correlation between IL-37 expression and HCC clinical pathological characteristics and further more evaluate the value of IL-37 in HCC prognosis, spearman test and Kaplan-Meier curve were performed.Fragment of IL-37 gene was amplified by PCR, and then ligated with enzyme digested lentiviral vector to build recombinant lentivirus vector GV358-IL1F7. PCR and DNA sequencing were performed to identify the positive clones. Then the recombinant plasmid was transfected into 293 T cells and its expression of GFP was evaluated by fluorescence microscope,while its protein expression of IL-37 was detected by Western-blot.Co-transfecting recombinant vector and lentivirus packaging system into293 T cells was performed to package the lentivirus carrying IL-37. Drug screening was conducted to determine the titer of lentivirus.The m RNA and protein expression of IL-37 in HCC cell lines and normal liver cell lines were detected. The regulating functions of IL-37 in invasion and metastasis of HCC cells were detected by Transwell assay and Wound-Healing assay.The expression levels of STAT3, p STAT3, E-cadhrin, Vimentin, MMP-9in rh IL-6 pretreated HCC cells were detected using Western-blot. At last,we subcutaneously injected LV_IL1F7 or LV_NC-transfected SMMC-7721 cells into the the liver capsule of nude mice to establish the nude mice lung metastasis model of HCC, then the tumor number of Pulmonary metastatic tumors was counted.Results: Compared with the adjacent non-tumor liver tissues(ANLTs), signifcantly lower expression of IL-37 m RNA was observed in68.52%(37/54) of HCC samples; m RNA and protein expression of IL-37 was lower in the human HCC cell lines compared with normal liver cells.Consistent with the q PCR findings, IL-37 had higher expression in ANLTs and decreased expression in HCC tissues. Based on the IHC results, the HCC patients were divided into IL-37 high-expression group(n=27) and low-expression group(n=27) to investigate the association between the IL-37 expression and the clinical features in HCC patients. We observed that the IL-37 expression was significantly associated with vascular invasion(P=0.013); HCC patients with high IL-37 expression had muchlonger OS time(median survival time, 40.0 vs. 27.0 months,P=0.040) than patients with low IL-37 expression, HCC patients with high IL-37 expression had longer DFS time(median survival time, 27.0 vs. 17.0months,P= 0.006); The target gene IL-37, prepared by PCR amplification,merged with vector GV358. All of the clones were confirmed by PCR. The PCR products from positive clones were 849 bp, while from negative clones were 185 bp. The results of DNA sequencing demostrated that GV358-IL1F7 lentiviral vector was bulit successfully; After transfected293 T cells with recombinant GV358-IL1F7 vector, high intensity GFP could be observed, which forecaste that plasmid transfection was successful. Specific stripe, detected by Western-blot, showed at around30 k D molecular weight, which is the same molecular weight as target gene.Co-transfected 293 T cells with the recombinant lentiviral vector and other two kinds of helper plasmids(p Helper 1.0 and p Helper 2.0), then, cellular supernatant was collected 48 h later and the virus was concentrated. Drug screening data indicated that the final virus titer was 9E+5 TU/ml. We constructed the IL-37 stably overexpresion HCC cell lines and observed the fluorescence distribution of these cells, we found the green fluorescence in each group rose to more than 90% distribution 72 h post-transfection; By using Western-blot, we found the expression of IL-37 was dramaticaly upregulated; We performed Wound-Healing assays and Transwell assays using SMMC-7721 and Hep G2 cells, it was noted that overexpressed IL-37 significantly suppressed wound healing in the studies with both SMMC-7721(17.33%±2 vs. 55±3%,P<0.001) and Hep G2 cells(10±2% vs.64±3%,P < 0.001). Transwell assays with Matrigel further demonstrated that IL-37 markedly inhibited the invasive capacity of SMMC-7721(35±6vs. 86±11,P=0.01) and Hep G2 cells(29±9 vs. 62±8,P=0.002)as compared with control cells. On the other hand, IL-37 inhibition had the opposite effects in Wound-Healing assays, IL-37 knockdown significantly suppressed wound healing in the studies with both SMMC-7721(83.33%±3.6 vs. 53±2%,P<0.001) and Hep G2 cells(90±3%vs. 60±7%(P<0.001); Transwell assays with Matrigel demonstrated that IL-37 knockdown markedly promoted the invasive capacity of SMMC-7721(94±18 vs. 49±4, P=0.002) and Hep G2 cells(102±12 vs.39±9, P=0.002) as compared with control cells; IL-37 significantly attenuated the STAT3 activition by IL-6; Compared with control group,the expression of E-cadherin,MMP2 was significantly downregulated and the Vimentin was significantly upregulated in SMMC-7721LV_IL1F7cells, in the mean time IL-37 attenuated the change of EMT proteins induced by IL-6; In the end, we subcutaneously injected LV_IL1F7 or LV_NC-transfected SMMC-7721 cells into the the liver capsule of the nude mice to establish the nude mice lung metastasis model of HCC and we found the SMMC-7721LV_IL1F7 group had a significanty lower number of Pulmonary metastatic tumors than the SMMC-7721LV_NCgroup.Conclusion: We have determined that IL-37 is down-regulated in HCC. IL-37 potentially possesses the efficiency to suppress the invasion and metastasis of HCC, at least in part, through the inhibition of IL-6/STAT3 signaling. Therefore, IL-37 could function not only as a fundamental inhibitor of innate immunity, but also as a novel tumor suppressor in HCC. The identification of IL-37 and its crosstalk within different oncogenic pathways in HCC progression may help in a better understanding of therapeutic strategy for HCC or other cancers.