| About 90% of hepatocellular carcinoma(HCC)develops from chronic liver fibrosis.Before developing into HCC,the microenvironment of fibrotic liver is characterized by inflammation and fibrosis and called premalignant microenvironment(PME).The inflammation of PME is mainly caused by the liver injury induced infiltration of numerous immune cells.Among these infiltrated immune cells,monocytes differentiate into M1 type macrophages and secrete pro-inflammatory factors such as TGF-?,IL-1?,TNF-? and IL-6,leading to the expansion of inflammation.Hepatic stellate cells(HSCs),an important effector cells of intrahepatic inflammation,are activated by TGF-? and other cytokines,and then transdifferentiate into myofibroblast-like cells which can secrete a variety of collagen,non-collagen glycoproteins,and proteoglycans to reshape the extracellular matrix(ECM),ultimately leading to the formation of liver fibrosis.In recent years,it has been reported that PME usually coexists with tumor microenvironment(TME)in HCC.The ECM in PME contains a large number of multifunctional cytokines,which interact with tumor cells in TME and promote tumor proliferation and metastasis.As the main source cells of ECM in PME,activated HSCs are considered to be a key factor driving the development of HCC.However,the specific regulatory mechanism is still unclear.Therefore,this paper focuses on exploring how activated HSCs regulate the progress of HCC.After co-culturing of HCC cells with activated HSCs,the transcriptome sequencing results of HCC cells showed that the expression levels of a large number of genes changed significantly.GSEA of those up-regulated genes showed that they were mainly related to functions such as metastasis(EMT)and proliferation(E2F target).Furthermore,HCC cells and activated HSCs were mixed(10:90)and implanted subcutaneously in nude mice.Consistent with RNA-seq results,it was found that the subcutaneous tumor proliferated rapidly and a large number of HCC cells metastasized to the liver.Activated HSCs are characterized by the secretion of a variety of cytokines and extracellular matrix.Therefore,we speculated that the above phenomenon was caused by the paracrine pathway of HSCs.Generally,the paracrine pathway functions by activation of phosphorylation signal transduction in target cells.Therefore,HCC cells were incubated with condition medium(CM)derived from activated HSCs for 10 minutes,followed by high-throughput protein phosphorylation mass spectrometry experiments.The results showed that CM stimulation caused significant activation of JAK-STAT,MAPK and PI3K-Akt signaling pathways that promote tumor proliferation and metastasis in HCC cells,and this result was further confirmed by Western Blotting experiments.These data suggested it is the paracrine pathway through which the activated HSCs promote the proliferation and metastasis of HCC cells.In order to explore the key paracrine signaling molecules secreted by activated HSCs which promote proliferation and metastasis,and meanwhile exclude the autocrine effect of HCC cells,we used RNA-seq to analyze the transcriptional changes of HSCs before and after activation,and analyzed the protein mass spectra of CM from HCC cells.and activated HSCs respectively.It was found that IL-11 was highly expressed in activated HSCs and was specifically secreted by HSCs,while barely by HCC cells.Thus,IL-11 was speculated to be the key paracrine signaling molecule.It was further confirmed by Western Blotting experiment that IL-11 stimulation caused significant activation of JAK-STAT,MAPK and PI3K-Akt signaling pathways in HCC cells.After knocking down the expression of IL-11 RA using RNA interference technology,the activation of signal pathways caused by CM stimulation was significantly inhibited in HCC cells.Taken together,these results all suggested that HSCs promoted the proliferation and metastasis of HCC cells by paracrine molecules IL-11.To investigate the effect of IL-11 on HCC cells in vivo,HCC cells were endowed the ability to secret IL-11 through lentivirus infection and implanted subcutaneously in nude mice.We were surprised to find that the subcutaneous tumors of nude mice in this group grew abnormally fast,and after tumors growing to a certain size,the nude mice showed weight loss and abdominal bulge.Moreover,we found that a large number of metastatic HCC cells in the liver of nude mice in this group,however,HCC cells that could not secrete IL-11 did not undergo liver metastasis.In the process of investigating the role of IL-11 in promoting the metastasis of HCC cells,we found that IL-11 could not only induce the increase of vascular permeability of mice in vivo,but also was a non-classical neutrophil chemokines and could induce neutrophils to strongly release NETs.NETs contains a large number of proteases that can cleave the basement membrane around the tumor,resulting in the destruction of the basement membrane structure and distant metastasis of tumor cells.In the tissues of clinical HCC patients,we found that there was a large amount of IL-11 around the cancer nest,and the neutrophil infiltration was obvious.Meanwhile,we also found that some neutrophils released a large amount of NETs.These results suggest that activated HSCs can promote the proliferation of hepatoma cells by secreting IL-11 specifically,and also induce the increase of vascular leakage and neutrophil infiltration.The infiltrated neutrophils could destroy the basement membrane structure and promote the distant metastasis of HCC cells by releasing NETs under the action of IL-11.These functions of IL-11 contribute to the rapid progress of HCC and reduce the survival rate of HCC patients greatly.Therefore,IL-11 specifically secreted by HSCs may be a noval target for HCC precise therapy. |
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