Dajiang Li

Ars2 is overexpressed in human cholangiocarcinomas and its depletion increases PTEN and PDCD4 by decreasing microRNA-21†

Due to the lack of effective diagnostic tools, most patients with cholangiocarcinoma (CCA) have no chance of surgical resection. Ars2 is a protein that was reported to be important for microRNA (miR) biogenesis, and its depletion can reduce the levels of several miRs, including miR‐21, which is overexpressed in CCAs. We hypothesized that Ars2 was also present in CCAs and could be an early diagnostic marker. In our experiments, Ars2, PTEN, PDCD4, and miR‐21 were evaluated in 18 CCAs and paired normal tissues. ShArs2, miR‐21 mimics, and Ars2 were transfected into CCA and bile duct epithelial cells either alone or together. Cell proliferation, tumorigenicity analysis and expression changes of Ars2, PTEN, PDCD4, and miR‐21 were evaluated. We found that both Ars2 and miR‐21 were overexpressed, with 95% sensitivity and 100% specificity, and an ROC of 0.995 in distinguishing between CCAs and paired normal tissues by qRT‐PCR. PTEN and PDCD4 were reversed in immunohistochemistry, but no difference was observed using qRT‐PCR. The knockdown of Ars2 in CCA cells decreased the level of miR‐21, inhibited cell proliferation and prevented tumor formation in nude mice. Ars2 knockdown also led to an increase in both PTEN and PDCD4 protein levels. Both proteins decreased when the miR‐21 mimic was con‐transfected. However, the overexpression of Ars2 alone could not get the opposite results. Based on our data, we conclude that Ars2 is overexpressed in human CCA and may be a diagnostic marker. Ars2 depletion increases PTEN and PDCD4 protein levels via the reduction of miR‐21.

Epithelial-Mesenchymal Transition Induced by Hepatitis C Virus Core Protein in Cholangiocarcinoma

BackgroundCholangiocarcinoma (CC) is associated with chronic hepatitis C virus (HCV) infection. We investigated the effect of hepatitis C virus core protein (HCVc) on epithelial-mesenchymal transition (EMT) in CC and tried to identify its target trigger.MethodsFirst, we examined expression of HCVc and epithelial and mesenchymal markers in CC tissues. Then we transient-transfected HCVc gene into a CC cell line and examined expression of lysyl oxidase-like 2 (LOXL2) and epithelial and mesenchymal markers by quantitative real-time polymerase chain reaction (PCR) and Western blotting. Finally, LOXL2 gene silencing was shown in QBC939/HCVc cells by RNA interference (RNAi), and we further examined expression of epithelial and mesenchymal markers by quantitative real-time PCR and Western blotting.ResultsThrough immunohistochemical staining, the present study showed that HCVc is significantly associated with CC invasion and metastasis. In vitro study showed that HCVc expression induces EMT in CC cell line QBC939, and a mechanism through LOXL2 pathway is suggested. Expression of HCVc was significantly correlated with greater migratory and invasive potential of CC cells.ConclusionsThese observations indicate that HCVc plays a critical role in promoting invasion and metastasis of CC cells.

MiR-605 represses PSMD10/Gankyrin and inhibits intrahepatic cholangiocarcinoma cell progression.

The aberrant expression of PSMD10 has important functions in various malignancies. This study showed that PSMD10 was highly expressed and inversely correlated with the expression of miR‐605 in intrahepatic cholangiocarcinoma (ICC) specimens. MiR‐605 directly targeted and repressed PSMD10 expression. In addition, over‐expression of miR‐605 inhibited ICC cell progression both in vitro and in vivo. This effect of miR‐605 on ICC cells was similar to that of PSMD10 knock‐down by RNAi. Moreover, restoration of PSMD10 could reverse the phenotypic alteration caused by miR‐605 in ICC cells. These results suggest a new therapeutic strategy in ICC by restoring miR‐605, which is regulated by p53.

Aberrant expression of GATA binding protein 6 correlates with poor prognosis and promotes metastasis in cholangiocarcinoma.

AIM GATA6, a zinc-finger transcription factor, functions as a tumour promoter or suppresser according to different tumour origins. We investigated the clinical significance of GATA6 and its role in invasion and metastasis in cholangiocarcinoma (CCA). METHODS Expression of GATA6 in 87 cancerous, 24 paracancerous, 32 lymph-node metastatic and 8 liver metastatic samples from 87 CCA patients undergoing surgical resection was detected by immunohistochemistry. Impact of GATA6 on invasion, metastasis and 67kDa laminin receptor expression (67LR) was evaluated in CCA cells by shRNA lentivirus or expressed-plasmid transfection. RESULTS Aberrant expression of GATA6 in CCAs was significantly associated with lymph-node metastasis. GATA6 expression was higher in lymph-node and liver metastatic tissues compared with primary cancerous tissues. Kaplan-Meier analysis showed GATA6 expression correlated with poor overall survival and early recurrence in CCAs. Cox analysis suggested GATA6 was an independent prognostic marker for overall survival and recurrence-free survival. CCA cell invasion and migration were decreased by GATA6 knockdown and enhanced by GATA6 overexpression in vitro. Knockdown of GATA6 reduced CCA cell metastasis by xenotransplantation into nude mice. 67LR, which is overexpressed in CCAs and promotes invasion and metastasis through several pathways, positively correlated with GATA6 expression in 87 CCAs. Both mRNA and protein levels of 67LR were regulated by GATA6 in CCA cells. Moreover, ChIP analysis showed GATA6 bound to 67LR gene promoter in CCA cells. CONCLUSION Aberrant expression of GATA6 correlates with poor prognosis and promotes invasion and metastasis in CCA.

The Role of MAPK-ERK Pathway in 67-kDa Laminin Receptor-Induced FasL Expression in Human Cholangiocarcinoma Cells

Background and AimsCancer cells are thought to possess immune evasion properties due to FasL overexpression in many types of human tumors. In the present study, we set out to investigate the role of MAPK-ERK pathway in 67-kDa laminin receptor induced FasL expression and FasL-mediated apoptosis in human cholangiocarcinoma cells.MethodsThe expression of FasL and its promoter activity in cultured cholangiocarcinoma cells were examined after treatment with laminin or transfection with plasmids containing siRNA targeted to 67-kDa laminin receptor. The effects of MAPK-ERK cascade inhibitor and c-Myc inhibition by siRNA on 67-kDa laminin receptor-induced FasL expression were determined. Apoptosis assay was performed to analyze the apoptosis of lymphocytes cocultured with cholangiocarcinoma cells treated with or without MAPK-ERK cascade inhibitor.ResultsOur results revealed that the specific MAPK-ERK cascade inhibitor, PD98059, significantly attenuated phosphorylation of c-Myc on Ser-62 and FasL upregulation in QBC-939 cells and these cells showed decreased cytotoxicity against Fas-sensitive Jurkat T cells. A luciferase reporter assay revealed that FasL promoter activity was significantly reduced in cells treated with PD98059 or transfected with c-Myc siRNA.ConclusionsBased on these results, we conclude that 67LR induces FasL expression and cytotoxicity against Fas-sensitive Jurkat T cells in human cholangiocarcinoma cells through the phosphorylation of c-Myc on Ser-62 and the subsequent activation of the FasL promoter through the ERK pathway.

Surgical treatment of congenital biliary duct cyst

BackgroundIt is acknowledged that total cyst excision is a safe and ideal surgical treatment for congenital biliary duct cyst, compared to simple internal drainage. The aim of this study was to determine the optimal operation occasion and the effect of laparoscopy on congenital biliary duct cyst based upon total cyst excision.MethodsFrom January 2002 to January 2011, 217 patients were admitted to Southwest Hospital for congenital biliary duct cyst. To determine the optimal surgery occasion, we divided these subjects into three groups, the infant group (age ≤ 3 years), the immaturity group (3 < age ≤ 18 years), and the maturity group (age > 18 years), and then evaluated the feasibility, risk and long-term outcome after surgery in the three groups. To analyze the effect of laparoscopic technique on congenital biliary duct cyst, we divided the patients into the laparoscopy and the open surgery groups.ResultsAmong the three groups, the morbidity from cholangiolithiasis before surgical treatment had obvious discrepancy (p < 0.05) (lowest in the infant group), and intraoperative blood loss also had apparent diversity (p < 0.05). Furthermore, long-term outcomes (secondary cholangiolithiasis, stoma stenosis and cholangiocarcinoma) showed no significant difference between different groups (p > 0.05).Similarly, no significant discrepancy was observed in the morbidity from postoperative complications or long-term postoperative complications (p > 0.05) between the laparoscopic and the open surgery groups.ConclusionsWe conclude that total cyst excision should be performed as early as possible. The optimal treatment occasion is the infant period, and laparoscopic resection may be a new safe and feasible minimally invasive surgery for this disease.

Role of cholangiocyte bile Acid transporters in large bile duct injury after rat liver transplantation.

Background. The pathogenesis of nonanastomotic strictures with a patent hepatic artery remains to be investigated. This study focuses on the role of cholangiocyte bile acid transporters in bile duct injury after liver transplantation. Methods. Sprague-Dawley rats were divided into three groups (n=20 for each): the sham-operated group (Sham), the transplant group with 1-hr donor liver cold preservation (CP-1h), and the transplant group with 12-hr donor liver cold preservation (CP-12h). Bile was collected for biochemical analysis. The histopathologic evaluation of bile duct injury was performed and the cholangiocyte bile acid transporters apical sodium-dependent bile acid transporter (ASBT), ileal lipid binding protein (ILBP), and Ost&agr;/Ost&bgr; were investigated. Results. The immunohistochemical assay suggested that ASBT and ILBP were expressed exclusively on large bile duct epithelial cells, whereas Ost&agr; and Ost&bgr; were expressed on both small and large bile ducts. Western blot and quantitative polymerase chain reaction analysis showed that the expression levels of these transporters dramatically decreased after transplantation. It took seven to 14 days for ILBP, Ost&agr;, and Ost&bgr; to recover, whereas ASBT recovered within 3 days and even reached a peak above the normal level seven days after operation. In the CP-12h group, the ratios of the ASBT/ILBP, ASBT/Ost&agr; and ASBT/Ost&bgr; expression levels were correlated with the injury severity scores of large but not small bile ducts. Conclusions. The results suggest that the unparallel alteration of cholangiocyte bile acid transporters may play a potential role in large bile duct injury after liver transplantation with prolonged donor liver preservation.

Application of Joint Detection of AFP, CA19-9, CA125 and CEA in Identification and Diagnosis of Cholangiocarcinoma

OBJECTIVE To explore the application of joint detection of serum AFP, CA19-9, CA125 and CEA in identification and diagnosis of cholangiocarcinoma (CC). MATERIALS AND METHODS The levels of serum AFP, CA19-9, CA125 and CEA of both 30 patients with CC and 30 patients with hepatocellular carcinoma (HCC) were assessed. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic effects of single and joint detection of those 4 kinds of tumor markers for CC. RESULTS The levels of serum CA19-9, CA125 and CEA in CC patients were higher than that in HCC patients,whereas that of serum AFP was significantly lower s. The area under ROC curve of single detection of serum AFP, CA19-9, CA125 and CEA were 0.05, 0.86, 0.84 and 0.83, with the optimal cutoff values of 15.4 ng/ml, 125.1 U/ml, 95.7 U/ml and 25.9 ng/ml, correspondingly, and the percentage correct single diagnosis was <79%. With joint detection, the diagnostic effect of combined AFP, CA19-9, CA125 and CEA was the highest, with an area under the ROC curve of 0.94 (95%CI 0.88~0.99). CONCLUSIONS Single detection of serum CA19-9, CA125 and EA is not meaningful. The sensitivity, specificity, the rate of correct diagnosis and the area under ROC curve of joint detection of AFP, CA19-9, CA125 and CEA are highest, indicating that the joint detection of these 4 tumor markers is of great importance in the diagnosis of CC.

Downregulation of mucins in graft bile ducts after liver transplantation in rats.

Background. Nonanastomotic biliary strictures represent a serious complication after orthotopic liver transplantation (OLT). This study investigates the potential role of mucins in bile duct injury after OLT. Methods. Sprague-Dawley rats were divided into four groups: normal group (Normal, n=5), sham-operated group (Sham, n=20), OLT group with 1 hr donor cold ischemic time (n=20), and OLT group with 12 hr donor cold ischemic time (OLTn=20). Expression of mucins and GATA factors in bile ducts was examined by real-time polymerase chain reaction, immunohistochemistry, and immunoblotting. Bile was collected for biochemical analysis, and the histological changes associated with bile duct injury were evaluated. Results. In normal bile ducts, Muc1, Muc2, Muc3A, Muc4, and Muc6 mRNA were expressed, whereas Muc5AC mRNA was undetectable. The expression of Muc1, Muc3A, and Muc4 but not Muc2 and Muc6 at mRNA level in graft bile ducts decreased remarkably early after OLT. The decreased expression of Muc1 and Muc4 was further confirmed at protein level by immunohistochemistry and immunoblotting. Downregulation of Muc1 and Muc3A expression by prolonged cold ischemic time was significantly associated with the injury severity scores of large but not small bile ducts. Among six GATA factors, GATA3, GATA4, and GATA6 mRNA were expressed in normal bile ducts. GATA4 and GATA6 mRNA levels decreased significantly after OLT. Conclusion. Downregulation of Muc1 and Muc3A expression by prolonged cold ischemic time may play a potential role in large bile duct injury early after OLT.

A polysaccharide from Pinellia ternata inhibits cell proliferation and metastasis in human cholangiocarcinoma cells by targeting of Cdc42 and 67 kDa Laminin Receptor (LR)

In this study, we isolated and purified a polysaccharide (PTPA) from the tubers of Pinellia ternate. We aimed to evaluate the cytotoxic effects of PTPA on human cholangiocarcinoma (CCA) cell lines and to identify the underlying molecular mechanism. PTPA at the dose from 25 to 200μg/mL showed significant inhibitory effect on the proliferation of four cancer cell lines (SNU-245, CL-6, Sk-ChA-1 and MZ-ChA-1), among which Sk-ChA-1 was a most sensitive cell line to PTPA treatment via induction of apoptosis. Interestingly, RNA interference of Sk-ChA-1 cells with 67LR or Cdc42-targeted shRNAs resulted a similar potency in decreasing cell viability and causing apoptotic death. Moreover, PTPA (100μg/mL) or 67LR or Cdc42 special shRNAs increased the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2, induced the activation of caspase-9 and caspase-3, but not caspsase-8, and inhibited the expression of 67LR or Cdc42 protein in Sk-ChA-1 cells. Taken together, the inhibitory effect of PTPA on the cell growth of Sk-ChA-1 cells was at least in part mediated via the activation of the intrinsic mitochondrial apoptotic pathway and the downregulation of 67LR or Cdc42 protein expression. Thus, PTPA may be developed as a promising candidate for chemopreventive agent in the prevention and treatment of human CCA.