| Background and ObjectiveIntrahepatic cholangiocarcinoma (ICC) is a rare primary malignant liver cancer. It is definited that cholangiocarcinoma arises from the secondly ductular epithelium of the biliary tree, so it also be called peripheral cholangiocarcinoma. It has been reported that ICC accounts for only about 10%-15% of primary liver cancers. However, in recent years the incidence of ICC has increased worldwide. ICC is considered to be a highly fetal carcinoma because of present late, early invasion, widespread metastasis, and the lack of an effective therapy. Despite improved diagnostic and operative techniques, the prognosis of ICC remains poor. In contrast to HCC, early lymphatic spread tends to occur in ICC. Moreover, lymph node metastasis is an important factor to prognosis of ICC.PRL-3 phosphatase belongs to a small class of tyrosine phosphatases, which also include PRL-1 and PRL-2. The genes encoding PRL-1, PRL-2, and PRL-3 are located on chromosomes 6q12, 1p35, and 8q24.3, respectively. All the three members are proteins of about 20-kD and share 76% to 87% amino acid sequence identity. These proteins are prenylated in vitro and in vivo and this posttranslational modification is important for their intracellular distribution. More evidences suggested that PRL phosphatases, especially PRL-3, might play causal role in growth regulation, proliferation, promoting cell motility, invasion and metastasis. PRL-3 is a newly identified metastasis-related gene. The original report about PRL-3 showed it was consistently over-expressed in all metastases of colorectal cancers, suggesting that PRL-3 was associated with colorectal cancer metastasis. The expression of PRL-3 was also up-regulated in the metastases of gastric cancer and ovarian cancer, and was correlated with cancer progression and metastasis. Many evidences suggested that an excess of PRL-3 was a key alteration for the acquisition of metastatic properties of the tumor cells. Overexpression of PRL-3 could enhance cell motility and invasive ability in vitro, and promote metastasis in mouse model systems. Oppositely, down-regulation of PRL-3 expression by interfering RNA could attenuate the ability of motility and invasion in vitro and suppress metastasis in nude mice. Furthermore, PRL-3 is expected to be a valuable prognostic indicator for tumor patients. Studies have revealed that patients with high PRL-3 expression had shorter survival time in several cancers including gastric cancer and colorectal cancer. Collectively, these observations demonstrated that PRL-3 played a causative role in tumor metastasis, and was a potential marker for clinical evaluation of tumor aggressiveness and prognosis.However, up to now, the PRL-3 expression level in ICC and its role in metastasis of intrahepatic cholangiocarcinoma are still unclear. In this study, we aimed to detect the expression of PRL-3 in ICC by immunohistochemistry assay and investigate the relationship between PRL-3 and clinicopathologic features including prognosis. PRL-3 siRNA was synthesized and transfected into HCCC-9810 intrahepatic cholangiocarcinoma cell line, and the effect of silencing of PRL-3 expression on migration and invasion of HCCC-9810 cell was evaluated. Furthermore, the role and mechanism of PRL-3 in the process of metastasis was also investigated.Methods1. The expression of PRL-3 in ICC:The expression of PRL-3 in 102 intrahepatic cholangiocarcinoma,102 correspoding non-cancerous tissues, and 62 matched lymphatic metastasis tissues was detected by immunohistochemistry (S-P) and the relationship between PRL-3 and clinicopathologic parameters was analyzed. Survival analysis was employed to evaluate the impact of high PRL-3 expression on the prognosis of patients with ICC.2. Synthesis of PRL-3 siRNA and transfection:Human intrahepatic cholangiocarcinoma cell line, HCCC-9810, were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% antibiotics in an atmosphere of 37℃in 5% CO2. The expression of PRL-3 was measured in HCCC-9810. Specific PRL-3 siRNA was synthesized by chemical method in vitro. Specific PRL-3 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 selectivity of PRL-3 knockdown.3. Effect and role of PRL-3 on metastasis of ICC:Motllity 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. In order to understand the probalble mechanism of PRL-3 involved in the process of metastasis, the expression of MMP-7 and MMP-9 in HCCC-9810 which was treated with PRL-3 siRNA was tested by Western blot.Results1. The expression of PRL-3 in ICC:PRL-3 expression was negative or low in adjacent non-cancerous intrahepatic bile ducts. But in cholangiocarcinoma tissues and lymph node metastases (LNM), the rate of positive PRL-3 expression was 47.1%(48/102) and 80.6%(50/62) respectively. The PRL-3 expression in primary ICC and its LNM was significantly higher than that in normal intrahepatic bile ducts (P＜0.05). In 62 primary tumor lesions, the proportion of high PRL-3 expression was 61.3%(38/62). We also assessed the PRL-3 expression of 40 primary cancer lesions without LNM, and the result showed that 25% of them was with positive PRL-3 expressions. Statistic analysis showed that expression of PRL-3 was more frequently detected in the primary lesion with LNM than that without LNM (P＜0.001). Expression of PRL-3 in primary tumors was significantly associated with progression and metastasis of tumors, such as TNM (P＜0.001), T stage (P<0.001), vascular invasion (P=0.002), and LNM (P＜0.001) and high serum CA-199 level (P＜0.05). The Kaplan-Meier survival curve showed that patients with high level of PRL-3 in primary tumor tended to have shorter survival time than those with low level of PRL-3. In a multivariate analysis, the result showed that PRL-3 expression was an independent prognostic marker of overall survival (RR 1.886, P= 0.024).2. Transfection of siRNA:Both RT-PCR and western blot results demonstrated HCCC-9810 cell expressed high level PRL-3, and therefore we can employ HCCC-9810 cell line for the following RNA interference studies. The PRL-3 siRNA and negative control siRNA were transfected into HCCC-9810 cells by lipofectamine 2000. RT-PCR and western blot were used to identify an efficient and specific PRL-3 siRNA sequence against the expression of PRL-3. The mRNA expression levels of PRL-3 in parental HCCC-9810, negaive-transfected control, lipo2000 control (only lipo2000 in cells) and PRL-3 siRNA-1,-2,-3 transfecting cells were respectively 0.596±0.106,0.581±0.045,0.651±0.055,0.183±0.085,0.197±0.058 and 0.396±0.086; the protein expression levels of PRL-3 in each group were:0.653±0.077,0.589±0.049,0.611±0.066,0.071±0.018,0.059±0.024 and 0.280±0.101. These results suggested all the three of PRL-3 siRNA could inhibit endogenous PRL-3 expression in HCCC-9810, especially PRL-3 siRNA-2. PRL-3 siRNA-2 was choosed for the following experiments. Expression of PRL-3 either in mRNA level or in protein level was significantly lower in the group transfected into PRL-3 siRNA compared to the group negative control siRNA and the blank one (P＜0.05). There is no siginificant difference between negative control group and parental HCCC-9810 cells (P>0.05).3. Effect and role of PRL-3 on metastasis of ICC:Scratch wound healing results showed:the ratio of wound distance 24h after scratching and original wound distance in PRL-3 siRNA cells, negative siRNA cells and parental HCCC-9810 cells, were respectively (62.12±6.28)%, (23.88±2.55)%and (21.20±6.07)%. The migrating distances of PRL-3 siRNA transfecting cells was significantly shorter than those of negative siRNA cells and parental HCCC-9810 cells (P<0. 05). In invasion assay, the number of cells that had migrated into the lower chamber were 19.40±2.30/HP in PRL-3 siRNA group,64.00±2.73/HP in negative control group and 67.20±3.11/HP in parental HCCC-9810 group. The difference was significant between group transfected with PRL-3 siRNA and the other two groups (P＜0.05). Western blot result suggested the expression of MMP-7 and MMP-9 was significant lower in group transfected with PRL-3 siRNA than negative congtrol and the blank one (P<0.05). There is no siginificant difference between negative control group and parental HCCC-9810 cells (P>0.05).Conclusion1. High PRL-3 expression was deteced in ICC. Moreover, PRL-3 expression in LNM is higher than primary lesion. PRL-3 plays an important role in progression and metastasis of ICC, and overexpression of PRL-3 is considered to be an independent prognostic factor for overall survival in patients with ICC.2. PRL-3 specific siRNA can inhibit the expression of PRL-3 in HCCC-9810 cells and weaken the invasion and migration potency of HCCC-9810 cells. PRL-3 maybe facilitate the invasion and migration of tumor cells by the activation of MMPs. |
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