Enhancement Of Migratory Ability Of Hepatocellular Carcinoma Cells By Interleukin-11

Background and Objection:Hepatocellular carcinoma(HCC)is the third most common cause of cancer-related death globally,accounting for 80-90%of primary liver cancers.China alone accounts for more than 50%of the world’s cases.The number of HCC patients has been increasing.Although with the constant improvement of the level of diagnosis and treatment,the curative effect of hepatocellular carcinoma patients increasingly significant,but the survival rate was not significantly improved,its main reason is that many HCC patients have been diagnosed with metastasis at beginning.However,the mechanism of invasion and metastasis of hepatocellular carcinoma is not fully understood.A considerable body of evidence suggests that the inflammatory cells and cytokines in the tumor microenvironment are major factors that determine the behavior of malignant cells.Interleukin-11(IL-11)is a member of the IL-6 family of cytokines.It signals via the IL11 receptor(R)a and gp130 receptor complex to activate the Janus tyrosine kinases(JAK).This,in turn,can induce the activation of three independent downstream signaling pathways:the signal transducer and activator of transcription(STAT)pathway,the Ras-mitogen activated protein kinase(MAPK)pathway;and the phosphotidylinositol-3 kinase(PI-3K)pathway.IL-11 was originally cloned as a mediator of plasmacytoma cell proliferation and was later found to exhibit a wide variety of biological effects in neural cells as well as in the hematopoietic and immune systems.More recently,IL-11 has been linked to the development of gastric cancer,colorectal cancer and breast cancer.IL-11 is expressed in the majority of hepatocellular carcinomas and has been associated with an aggressive phenotype and poor prognosis in hepatocellula adenocarcinoma.It reported that IL-11 is independent prognostic factors in HCC patients.However,whether and by what mechanism(s)IL-11 might contribute to tumor progression are still not clear.Epithelial-mesenchymal transition(EMT)is known to be a central mechanism responsible for invasiveness and metastasis of various cancers is a critical process for tumor invasion and metastasis.It is characterized by loss of cell polarity and cell adhesion,repression of E-cadherin expression and increased cell motility.At the same time,the traits of epithelial cells such as long-lasting morphological and molecular changes are altered to mesenchymal characteristics,such as fibroblastic morphology,increased vimentin expression,and enhanced migration and invasion capacities,thus causing cancer cell metastasis.However,the involvement of EMT and its reverse process,mesenchymal-epithelial transition(MET)in the late events of tumor cell metastasis,such as distant colonization,are still hotly debated.In the present study,Interleukin-11(IL-11)and IL11 receptor(R)a were examined in HCC patient samples and the relationships with clinicopathologic features.Moreover,the effects of IL-11 on HCC cell EMT,proliferation and migration and involvement of relevant signaling pathways were investigated.Methods:1.Clinical pathological specimens of hepatocellular carcinoma and normal hepatic tissues were collected for immunohistochemistry test(IHC).The expression of IL-11、IL-11RA were analysed according to clinical pathological data.2.The cell culture of SMMC7721 cells.They were cultured in DMEM supplemented with 10%FBS,100 U/ml penicillin,100μg/ml streptomycin at 37°C in a 5%C02 humidified atmosphere.3.The effect of recombinant human IL-11(rhIL-11)on prolification and migratory ability of SMMC7721 cells was detected by CCK-8 assay,wound scratch assay and transwell migration assay,respectively.4.The expression of EMT-related genes(including E-cadherin and Vimentin)of SMMC7721 cells and the effects of rhIL-11 on them were detected by Quantitative real-time PCR(QRT-PCR).Total RNA extraction,RNA quantitation and purity testing,cDNA synthesis and PCR reaction were done according to the specifications.We got the relevant data and curves after the amplification,and then got the result according to the relative quantification 2-ΔΔ Ct method.5.The protein level of EMT-related proteins(including E-cadherin and Vimentin)of SMMC7721 cells and the effects of rhIL-11 on them were detected by Western Blot.The steps comprise total protein extraction,protein concentration measure,electrophoresis,transferred to a membrane,immune response,exposure and development.6.The protein level of EMT-related proteins(including E-cadherin and Vimentin)of SMMC7721 cells and the effects of rhIL-11 on them were also detected by immunofluorescence(IF).7.Statistical Analysis.All statistical tests were performed using SPSS software version 13.0 and all data were expressed as means ± SEM.Differences between two groups were analyzed using the Student’s t test;and differences among groups were analyzed using one-way ANOVA if variance was homogeneous and then we chose Bonferroni method to make multiple comparisons among groups,if not,Welch method was needed to make differences among groups and we chose Dunnett T3 method to make multiple comparisons among groups.A p value of less than 0.05 was considered statistically significant.All experiments were conducted at least three times.Results:1.Localization of IL-11 was primarily in the cytoplasm of tumor cells.And localization of IL-11RA was primarily in the membrance and cytoplasm of tumor cels.Of the 57 patients,the expression of IL-11 in intratumoral was stasitical higher than that in peritumorla.And there is nothing to do with age,gender,tumor size,serum AFP and liver cirrhosis.But the expression of IL-11 was sharply related to histological differentiation,lymph node metastasis and potal vein tumor thrombosis.Similarly,the expression of IL-11 RA in intratumoral was significantly higher than that in peritumoral.The expression of IL-11RA has no relationship with age,gender,tumor size,serum AFP,liver cirrhosis and metastasis.But there is significantly related to histological differentiation and potal vein tumor thrombosis.2.Compared with untreated SMMC7721 cells,there is no significant correlation between IL-11 and cell proliferation viability.Howere,we demonstrated that IL-11 increased migratory ability of SMMC7721 cells.3.The mRNA expression of related genes(E-caherin and Vimentin)of SMMC7721 cells were detected by QRT-PCR The expression of E-caherin mRNA decreased with the treatment of rhIL-11,but the expression of Vimentin mRNA increased.The alteration of expression of E-caherin and Vimentin mRNA was stastistic different with the control group.4.The protein level of E-caherin and Vimentin of SMMC7721 cells and the effects of thIL-11 on them were detected by Western Blot.Same as the gene expression,the protein level of E-caherin decreased while the protein level of Vimentin increased.5.The expression of E-cadherin and Vimentin were also detected by immunofluorescence.This showed the same results of previous studies(QRT-PCR and Western Blot).6.The influence of rhIL-11 on the phosphorylation level of STAT3,AKT and ERKI/2 was detected by Western Blot.The ptrotein level of p-STAT3 and p-AKT in SMMC7721 cells treated with trIL11 were significantly higher than that in control group,while there is no alteration of ptrotein level of p-ERK1/2.7.SMMC7721 cells co-incubated with IL-lland LY294002(PI3K inhibitor)reduced cell invasion and migration conpared to cells treated with IL-11 alone.Similarly,cells co-incubated with IL-11and S31-201(STAT3 inhibitor)reduced cell migration compared to cells treated with IL-11 alone.Conclusion:In the present study,we found that intratumoral expression of IL-11 and IL-11RA was significantly higher than that in perintratumoral,suggested that IL-11 may play an important role in the process of HCC.We investigated the effect of IL-11 in hepatocellular carcinoma cell function-proliferation and migration.Then we observed that IL-11 can enhance migration ability in SMMC7721 cells while has no influence on cell growth.Moreover,we demonstrated that IL-11 may induce EMT,for it can reduce the expression of E-cadherin and increase the expression of Vimentin The ptrotein level of p-STAT3 and p-AKT in SMMC7721 cells treated with rhIL11 were significantly higher than that in control group,and PI3K inhibitor and STAT3 inhibitor can suppress the effect of IL-11 on migration in SMMC7721 cells,respectively.In summary,these findings suggested that IL-11 could play an important role in HCC progression by induction of EMT via,at least partially,PI3K-Akt and JAK-STAT3 pathway,which can provide novel theory and practice to explain the pathogenesis of invasion and metastasis for HCC,and IL-11 may serve as a valuable prognostic biomarker and potential therapeutic target.