| ObjectiveE-cadherin (E-cad, CDH1) is preferentially expressed in human and animal epithelial cells, As a cell adhesion molecule, it is very essential for cell-cell and cell-matrix interactions and has been shown to maintain normal epithelial morphology. E-cadherin is generally considered to be a tumor suppressor in recent years. Downregulation of E-cad can promote EMT (Epithelial to Mesenchymal Transition),which makes cells organized loosely and acquisition of migratory and invasive properties. Clinical studies have shown E-cad is downregulated in many tumors such as gastric cancer, colon cancer, especially in hepatocellular carcinoma. E-cad is associated with poor prognosis and postoperative recurrence. But the mechanism of E-cad downregulation is not very clear yet.Inflammation, especially chronic inflammation has been advanced as a major risk factor to tumor development, while it induces the inflammatory cells to promote tumor growth, progression, metastasis.It is well established that Tumor-associated macrophages (TAM) within the microenvironment may contribute to tumor growth and spread in hepatocellular carcinoma.There is evidence that the abundance of TAM is correlated with poor prognosis in hepatocellular carcinoma.To make it clear by which mechanisms TAM influences tumour cells invasion, we analyzed the relationship between E-cad expression and TAM infiltration in HCC tissues and co-cultured the human hepatocellular cancer cell lines BEL-7402,SMMC-7721with macrophages from THP-1. we found that the downregulation of E-cad in tumor hepatocellular cancer cells was correlated with TAM infiltration,co-incubation enhanced invasiveness of HCC cells. Besides, NF-κB Signaling pathway may play a key role in promoting HCC cells invasion.Methods1Detection of E-cadherin expression and TAM by Immunohistoche-mistry in HCC patients(1) Selection of HCC clinical specimensIn this study,we collected HCC clinical specimens which from Qilu hospital of shandong university. All of the patients were not treated before receiving surgical. Clinicopathologic information,including gender, age, tumor size and pathological differentiation,were obtained from the patients’medical records.(2) Immunohistochemistry for E-cadherin and CD68Immunohistochemistry for E-cadherin was performed on a4-mm-thick section and used a mono-clonal mouse anti-E-cad antibady(1:100dilution); For CD68used a mono-clonal mouse anti-CD68antibady(1:100dilution). Both used a rabbit anti-mouse IgG antibody conjugated to HRP as the secondary antibody, high pressure cooking retrieval in the EDTA buffer(pH9.0), the endogenous peroxidase was blocked by incubation with3%H2O2. Incubation with the primary antibodies in a humidity chamber at4℃overnight, initial dyeing with DAB and counterstained with hematoxylin. For both antibodys, negative controls were performed through omitting two primary antibodies.(3) Analysis of the correlation between E-cadherin expression and TAM infiltration numberBoth of E-cadherin and CD68expression were evaluated with a digital image system. Five or six images were selected each patient and captured at a magnification of200×,then saved as TIFF files.The positive staining of E-cadherin and CD68were analyzed by software Image-Pro Plus version6.0. TAM were identified as the CD68positive staining.The cell numbers were counted manually on the consecutive section. The numbers of TAM in each patient were determined by averaging the number of TAM in3high-power fields where the most CD68staining was observed and there were not necrosis areas.The data was analysis with software GraphPad Prism5.2Detection of E-cadherin and other EMT associated genes expression of HCC cells co-cultured with macrophages drived from THP-1cells(1) Co-culture of HCC cells with macrophages from PMA-treated THP-1cellsHuman HCC cell lines BEL-7402,SMMC-7721and human monocytic cell line THP-1were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science;7402,7721and THP-1were respectively maintained in1640,EMEM,DMEM supplemented with10%heat-inactivated fetal bovine serum, cultured at37℃with5%CO2.THP-1cells were treated with phorbol-12-myristate-13-acetate (PMA) at320nM in order to make sure that THP-1cells diverentiate into macrophages. Before co-culture, macrophages were washed with PBS and were co-cultured with7402,7721by a six-well transwell apparatus with0.4-um pore size.(2) Detection of genes expression by RT-PCRTotal RNA of HCC cellswere extracted. Expression of E-cad,Snail,Slug,Twist,MMP2,MMP9were detected by RT-PCR.(3) Western BlottingDetection of Expression of E-cad of HCC cells co-cultured with macrophages.3Detection of E-cad expression of HCC cells co-cultured with macrophages drived from PBMCs(1) Isolation and differentiation of human peripheral blood mononuclear cells We selected24healthy volunteers and isolated peripheral blood mononuclear cells(PBMCs) by Ficoll Paque density centrifugation in a sterile environment. The cells were seeded into six-well platesand and macrophages were acquired by a plastic adsorptive process and treated with PMA.(2) HCC cells co-cultured with macrophages from PBMCs7402,7721co-cultured with macrophages from PBMCs by Transwsll apparatus.(3) Detection of genes expression by RT-PCRHCC cells were collected after co-culture6h,24h,total RNA were extracted. Expression of E-cadherin,Snail,SlugvTwist,MMP2,MMP9were detected by RT-PCR for both of HCC cell lines.4Detection of morphological changes of HCC cells co-cultured with macrophages(1) Co-culture of HCC cells with macrophages from PMA-treated THP-1cells(2) Detection of morphological changes of HCC cells co-cultured with macrophagesThe morphology of7402and7721were evaluated with the digital image system after co-culture with THP-1differentiated macrophages.5Migration of HCC cells co-cultured with macrophages7402and7721cell lines were grown in a six-well plate with growth medium. Straight wounds were created using a sterile lOul pipette tip, washed with PBS and made record the next day. After co-culture with THP1-differentiated macrophages made record again.6Expression of genes of HCC after blocking NF-κB signal pathways(1) Blocking of NF-κB signal pathways in HCC cells in co-culture system7402and7721cell lines were treated with pyrollidine dithiocarbamate (PDTC) at10ng/ml for1h to inhibit of NF-κB pathway. Before co-culture,both cell lines were washed with PBS.(2) Detection of E-cad expression and genes expression associated with EMT of HCC cellsExpression of E-cad,Snail,Slug,Twist,MMP2,MMP9were detected.7Detection of HCC cell lines’migration after blocking of NF-κB signal pathways(1) Blocking of NF-κB signal pathways in HCC cells in co-culture system(2) Migration of HCC cells by the wound healing assayResults1Down-expression of E-cadherin was associated with TAM in hepatocellular carcinoma tissues(1) Analysis of E-cadherin expression and TAM in HCC tissue samplesSix images of representative fields were selected each patient and captured, three images of six were slected respectively to high and low expression of each sample, and we analysised eighty-three images altogether. The number of TAM in each sample was determined by averaging the number of TAM in3high-power fields where the most CD68staining was observed and there were not necrosis areas.(2) The down-expression of E-cad was associated with TAM numberTAM number of E-cad high-expressed group is small, the average is58; while TAM number of group E-cadherin low-expressed is larger, average is172.There were significantly differences(P<0.0001). The results clearly revealed that there is a significant correlation between down-expression of E-cadherin and TAM infiltration in hepatocellular carcinoma.2Expression of E-cad in HCC cells was down-regulated after co-culture with macrophages from THP-1cells.We observed that E-cad of HCC cell lines co-cultured with macrophages were low expression in mRNA level in significantly after6h and24h with P values＜0.001.Meanwhile,we also observed that E-cad of the two types of HCC cell lines was low-expressed markedly after co-cultured48h in protein level. 3EMT associated genes in HCC cells were up-regulated after co-culture with macrophagesIn7402cells co-cultured,slug and twist those were suppressors of E-cadherin, were up-regulated in mRNA level,MMP9was were up-regulated too.In7721cells,MMP9was highly expressed.These results show that macrophages inhibited the expression of E-cad via raising the expression of slug and twist,and promoted the origin of EMT by combining with the high-expression of MMP9.4Expression of E-cad and other genes in HCC cells were regulated after co-culture with macrophages from PBMCs.HHC cell lines co-cultured with macrophages from peripheral blood mononuclear cells(PBMCs),then detection of E-cad and other genes in HCC cells were as results of2and3.It indicated that both of macrophages from THP-1and PBMCs can down-regulated E-cad and induced process of EMT.5Macrophages cells activation alters the morphology of hepatocellular carcinoma cellsHepatocellular carcinoma cells arranged in loose,cell gap became wide rather than forming aggregated after co-culturewith macrophages.7402cells had an elongated spindle shape and7721had filamentous tails at cell poles. It indicated HHC cells co-cultured showed a striking morphological change:they lossed of epithelial morphology, became dissociated from others, and acquired mesenchymal phenotypes, a process of EMT.6The migration ability of HCC cells co-cultured with macrophages from THP-1cellsFor wound-healing assay,the migration number of7402is larger.The average migration after wounding of in co-cultured group was863um,and it was530um in control group, P valued<0.001,the differences between two groups were obvious.While the average of migration cells in co-cultured group was283um,and it was only112um in control group in7721,P valued<0.001,the differences were significant.These results indicated that the migration ability of two types of cell lines was significantly enhanced after co-cultured with macrophages from THP-1cells.7Alteration of E-cad and other genes expression was inhibited by blocking NF-κ B signal pathway in HCC cellAfter Blocking NF-κ B signal pathway in HCC cells with PDTC, the decreased expression of E-cad induced by macrophages was inhibited in both mRNA and protein levels.9The increase of migration ability disappeared after blocking NF-K B signal pathway in HCC cellsBlocking NF-κ B signal pathway in HCC cells.It found that the average of migration cells in control group,co-cultured group and PDTC co-cul group were as follows112,283,83.The cell migration of the third group decreased, significantly lower than the second group.These results indicates that the NF-κ B signal pathway of HCC cells play a vital role in the effect of macrophages promoting EMT.Conclusion1The downexpression of E-cadherin was associated with TAM in HCC.2Macrophages induced down-regulation of E-cadherin in HCC cells and promote its process of EMT.3Macrophages promote EMT of HCC through NF-κ B signal pathway. |
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