The Expression Of Calcium-Binding Protein S100A4 In The Cholangiocarcinoma And Its Role In The Metastasis Of Cholangiocarcinoma

Background&aimCholangiocarcinoma arises from the ductal epithelium of the bile duct tree. The incidence of cholangiocarcinoma is increasing in recent years. Cholangiocarcinoma is classified anatomically into intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC). Complete resection is the only way to cure the disease at present. Moreover, because cholangiocarcinoma is difficult to diagnose at an early stage, most patients have unresectable disease at clinical presentation, and prognosis is very poor. Few patients are candidates for curative resection and the recurrence rates are high even after curative resection. Chemotherapy and radiation therapy have not been clearly shown to be effective to the cholangiocarcinoma. Identifying effective prognostic biomarkers which influence the invasion and metastasis of ICC, and investigating the molecular mechanisms underlying the progression of ICC are important for the diagnosis and therapy of ICC.S100A4 belongs to the S100 protein family, which is confirmed to be related to the invasion and metastasis of many carcinomas. It was reported that S100A4 is expressed in different types of digestive tumor, including colorectal, esophageal, pancreatic, and gastric cancers. The overexpression of S100A4 was correlated with the lymph node metastasis, vasular invasion, differentiation of tumors and TNM stage and could be an independent prognostic factor. S100A4 is involved in the regulation of a wide range of intracellular and extracellular biological effects, including cell proliferation, extracellular matrix remodeling, cell motility, cell detachment, and angiogenesis.To our knowledge, there are few report to detect the expression of S100A4 in the intrahepatic and extrahepatic cholangiocarcinoma. Matrix metalloproteinases (MMPs), which belong to a family of zinc-dependent proteinases, play a key role in the degradation of extracellular matrices. Several experimental strategies have Proved substantial connection between S100A4 and certain members of the MMP family. S100A4 may promote the progression of human tumor through transcriptional activation of MMPs. In this study, we detected the expression of S100A4, MMP-9 expression in ICC and ECC by immunohistochemistry and investigated the relationship between S100A4, MMP-9 and clinicopathologic features. S100A4 siRNA was transfected into HCCC-9810 intrahepatic cholangiocarcinoma cell line, and the effect and potential mechanism of interfering of S100A4 expression on proliferation and invasion of HCCC-9810 cell were evaluated. The expression of different MMPs were also be determined.Methods1. The expression of S100A4 in cholangiocarcinoma:Formalin-fixed and paraffin-embedded 76 cholangiocarcinoma tissues and corresponding non-cancerous tissues which included 44 ICC and 32 ECC were retrieved from the Department of Pathology, Shandong Provincial Hospital. The expression of S100A4 and MMP-9 in cholangiocarcinoma was detected by immunochemistry. The relationship between the expression of S100A4 and MMP-9 and the clinicopathologic factors was evaluated.2. The expression of S100A4 in intrahepatic cholangiocarcinoma cell line HCCC9810:The expression of S100A4 in the HCCC-9810 was detected by immuncytochemistry, RT-PCR and western blot.3. Synthesis of S100A4 siRNA and transfection:S100A4 siRNA was synthesized by chemical method in vitro. Three S100A4 siRNA or negative control siRNA was transfected into HCCC-9810 cells with the Lipofectamine 2000 reagent according to the manufacturers' instruction. Western blot and RT-PCR were performed after transfection to assess the effection of S100A4 silence and choose the most effective S100A4 siRNA.4. Role of S100A4 on proliferation and invasion of ICC:The cell proliferation was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay as described in the manufacture's manual. Motility and migrating velocity of cells was tested by scratch wound healing assay. The ability of cells to invade through a Matrigel-coated filter was measured in transwell chambers. To investigate the possible mechanism of S100A4 involved in the procession of invasion and metastasis, the expression of MMP-2, MMP-7, MMP-9,MMP-13 in HCCC-9810 after S100A4 siRNA transfection was tested by Real-time RT-PCR and western blot.Results1. The expression of S100A4 in cholangiocarcinoma:The expression of S100A4 and MMP-9 in 76 cholangiocarcinoma tissues was detected by immunochemistry. S100A4 and MMP-9 were positively expressed in 18(40.9%) and 23(52.3%) in ICC patients, and 17(53.1%) and 19(59.4%) in ECC patients. The positive correlation between S100A4 and MMP-9 expression was statistically significant in the cholangiocarcinoma(P=0.027). S100A4 positive expression was significantly correlated with lymph node metastasis in the ICC patients. S100A4 expression is also inclined to be correlated with lymph node metastasis in the ECC patients(P=0.060).2. The expression of S100A4 in intrahepatic cholangiocarcinoma cell line HCCC9810:Immuncytochemistry, RT-PCR and western blot showed that S100A4 was high expressed in the HCCC-9810 cell line.3. Transfection of S100A4 siRNA:Three S100A4 targeting siRNA (siRNA 1,2,3) and negative control siRNA were transfected into HCCC-9810 cells with siRNA dilivering lipofectamine 2000. The efficiency of S100A4 siRNA sequence against the expression of S100A4 was detected by Real-time RT-PCR and western blot. Real-time RT-PCR showed that the relative mRNA levels in S100A4siRNA-1,2, 3 group was 56.5%,21.3% and 13.1%, compared to the negative control group. All the three siRNA significantly reduced the S100A4 mRNA(P<0.05), and the S100A4 siRNA-3 showed strongest inhibition. Western blot showed that the protein level of S100A4 was significantly down-regulation in the S100A4 siRNA-2 and S100A4 siRNA-3 group, with strongest down-regulation in the S100A4siRNA-3 group (P<0.05). Real-time RT-PCR and western blot suggested that S100A4 siRNA-3 showed strongest inhibition of the expression of S100A4 compared with S100A4 siRNA-1,2. S100A4 siRNA-3 was choosed for the following experiments to investigate the role of S100A4 in the invasion of HCCC9810 cell lines.4. Role of S100A4 on proliferation and invasion of ICC:1) S100A4 siRNA sequence could not alter the HCCC-9810 cell proliferation. A MTT assay revealed that treatment with S100A4 siRNA did not change the number of viable cells, compared with the control groups. (P>0.05) 2) S100A4 siRNA sequence could reduce the migration of HCCC-9810:Scratch wound healing results showed that The wound distance 24h after scratching in negative control cells NC siRNA cells, and S100A4 siRNA cells were respectively (77.91±8.41)%, (60.90±8.13)%,(56.24±7.34)%, and (61.63±7.49)%, (23.10±8.41)%, (20.50±5.04)% at 48 h after scratching. The migrating distances of S100A4 siRNA cells was significantly shorter than those of NC siRNA cells and negative control cells.3) S100A4 siRNA sequence could reduce the invasiveness of HCCC-9810:Transwell assays applying Matrigel-coated filters revealed that the number of cells penetrated through the basement membrane was (33.8±8.41) /HP in S100A4 siRNA group, (67.6±13.27)/HP in NC siRNA group and (61.3±13.20)/HP in negative control group. The number of cells that penetrated into the lower chamber was significantly smaller in the S100A4siRNA group(P<0.05).4) S100A4 siRNA sequence down-regulated the expression of MMP9:Real-time RT-PCR suggested that the expression of MMP-9 was significant lower in S100A4 siRNA group than in the control groups (P<0.05). There is no significant difference in the expression of MMP-2,MMP-7,MMP-13 between S100A4 siRNA group and the control groups. Western blot results further confirmed that the expression of MMP-9 was down regulated in the S100A4 siRNA group.Conclusion1. Both S100A4 and MMP-9 are overexpressed in the intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma. The positive correlation between S100A4 and MMP-9 expression was statistically significant.2. The overexpression of S100A4 is correlated with lymph node metastasis and vascular invasion in intrahepatic cholangiocarcinoma, and is also inclined to be correlated with lymph node metastasis in the ECC patients which indicated that S100A4 may play an important role in the invasion of cholangiocarcinoma.3. Transfection of S100A4 siRNA could inhibit the expression of S100A4 in the intrahepatic cholangiocarcinoma cell line HCCC-9810 sufficiently, and reduce invasiveness and MMP-9 expression of HCCC-9810. S100A4 may promote the metastasis and invasion of cholangiocarcinoma by up-regulating the MMP-9 expression.4. S100A4 may play a key role in the invasiveness and metastasis of cholangiocarcinoma, and could be a potential therapeutic target for cancer therapy.