Objective To investigate whether KAP-1 could induce CSC-self renewal in pancreatic cancer cell lines.Methods KAP-1 expression was examined in 14 cases of pancreatic cancer with immunohistochemistry.KAP-1 shRNA amplified by PCR was inserted into pGC-LV in vector,and then identified by restriction endonuclease digestion and nucleotide sequencing.The lentiviral vector pGC-shRNA-KAP-1 was co-transfected with pHelper 1.0 and pHelper 2.0 packaging plasmids into HEK 293T cells,and the lentivirus was collected and virus titer was measured.The expressions of KAP-1 and vimentin were detected by Western blot and RT-qPCR when human pancreatic cancer cell line Panc-1 was infected by the lentivirus.Sphere forming assay was conducted to assess the capacity of CSC or CSC-like cell self-renewal in this study.Results The KAP-1 expression level in cancerous tissues on immunohistochemistry was significantly higher than in the corresponding normal tissues (P=0.002).After infected by lentivirus,the expressions of KAP-1 and vimentin were knocked down,which could be detected by Wesren blot and RT-qPCR.Compared to Panc-1-GFP (NC) cells,the outcomes suggested that knocking down the expression of KAP-1 could decrease the formation of pancreatospheres,which further suggested the capacity of CSC-self-renewal in primary and secondary pancreatospheres of Panc-1-shRNA-KAP-1 and NC cells.Conclusions KAP-1 expression in pancreatic cancer tissues has been identified.The lentiviral vector for shRNAs targeting KAP-1 was constructed successfully.The formation of pancreatospheres decreased by knocking down the KAP 1 gene.KAP-1 is involved in the regulation of CSC phenotype.The mechanism is probably related to the upregulated expression of the EMT marker vimentin.

Objective To investigate the effects of KAP-1 in promoting the epithelial-mesenchymal transition (EMT) of pancreatic cancer cell line Capan-2.Methods The lentiviral vector of LV-plenti-GFP-KAP-1 was constructed.Capan-2 cells were divided into the experimental group (cells transfected by lentiviral vector of LVplenti-GFP-KAP-1),negative control group (cells transfected by empty vector) and blank control group 1 (cells cultured in 1640 medium plus 10％ fetal calf serum).Capan-2 cells in the experimental group were subdivided into the miR-100-5p inhibitor transfection group (cells transfected with miR-100-5p inhibitor),empty vector control group (cells transfected with microRNAs inhibitor),blank control group 2 (cells cultured in 1640 medium plus 10％ fetal calf serum).The lentivirus was identified by double endonuclease restriction and sequencing,and the virus titer was detected.The morphological changes of the cells were observed after transfecting lentiviral vector of LV-plenti-GFP-KAP-1 to the Capan-2 cells.The expressions of KAP-1,genes of EMT proteins and mRNA of miR-100-5p were detected by real-time quantitative polymerase chain reaction.The protein expressions of KAP-1,EMT proteins in all the groups were detected by Western blot.The measurement data were presented by mean ± standard deviation,and were analyzed using the analysis of variance.Results The lentiviral vector of LV-plentiGFP-KAP-1 was successfully constructed,and the virus titer was 2 × 108 TU/ml.Compared with the control group,the mesenchymal transition of the Capan-2 cells was detected in the experimental group after transfecting the Capan-2 cells with lentiviral vector of LV-plenti-GFP-KAP-1 for 48 hours.The relative mRNA expressions of KAP-1,N-cadherin,vimentin,E-cadherin,miR-100-5p were 1.77 ± 0.83,2.62 ± 0.71,2.50 ± 0.21,7.20 ± 1.17 and 1.81 ±0.40 in the experimental group,5.03 ±0.29,5.07 ±1.53,3.83 ±0.57,7.83 ±0.78,7.01 ± 0.96 in the negative control group,5.13 ± 1.14,5.81 ± 1.49,4.92 ± 0.90,3.07 ± 0.36,6.87 ± 0.35 in the blank control group 1,with significant difference among the 3 groups (F =5.99,7.62,7.88,6.62,4.64,P ＜0.05).The relative mRNA expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 1.56 ± 0.42,4.89 ± 0.61,5.20 ± 0.38,with significant difference among the 3 groups (F =5.14,P ＜ 0.05).The relative mRNA expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.10 ± 1.37,3.44 ± 0.94,3.08 ±1.16,with no significant difference among the 3 groups (F =0.49,P ＞ 0.05).The results of western blot showed that the relative protein expressions of KAP-1,N-cadherin,vimentin,E-cadherin were 2.77 ± 1.99,1.31 ±0.38,4.25 ± 0.63,0.62 ± 0.06 in the experimental group,0.83 ± 0.46,0.41 ± 0.37,1.03 ± 0.33,1.17 ± 0.45 in the negative control group,0.71 ± 0.26,0.08 ± 0.04,1.37 ± 0.92,3.04 ± 0.65 in the blank control group 1,with significant difference among the 3 groups (F =5.54,4.68,3.19,8.18,P ＜ 0.05).The relative protein expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 2.27 ±0.71,0.56 ±0.43,0.61 ±0.39,with significant difference among the 3 groups (F =4.81,P ＜0.05).The relative protein expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.19 ± 0.55,3.93 ± 0.06,3.61 ± 0.73,with no significant difference among the 3 groups (F =0.04,P ＞ 0.05).Conclusion KAP-1 promotes the EMT of Capan-2 cells by specifically down-regulating the miR-100-5p expression.

Cholangiocarcinoma is a bile duct adenocarcinoma derived from the bile duct epithelium. Current treatment strategies for cholangiocarcinoma include surgery, chemotherapy, and radiotherapy. Surgical treatment is the first choice for all subtypes of cholangiocarcinoma. However, cholangiocarcinoma has the characteristics of high malignancy, easy recurrence after surgery, and low 5-year survival rates. Recent studies have found that many traditional Chinese medicines exhibit excellent anti-tumor effects in the treatment of various cancers, including cholangiocarcinoma. These medicines have advantages, such as low prices and abundant reserves, and are considered as an effective and safe complementary and alternative therapy for the treatment of cholangiocarcinoma. This article aims to review the effects of traditional Chinese medicine on cholangiocarcinoma from different aspects and levels in recent years. Results will provide new ideas for the prevention and treatment of cholangiocarcinoma.

Objective To evaluate the MUC1 promoter's role in driving gene expression in pancreatic cancer and its therapeutic significance.Methods Two plasmids were made.The plasmid pEGFP-MUC1N1 contained MUC1 promoter fragment connected to the pEGFP-N1 vector with the EGFG reporter gene.The pShuttle-MUC1-EGFP plasmid contained MUC1 promoter fragment and EGFP reporter gene connected to pShuttle plasmid.Lipofectamine 2000 was used to transfect the two plasmids into cells of MUC1-positive human pancreatic cell line Panc-1 and MUC1-negative human cervical carcinoma Hela.Fluorescence microscopy and flow cytometry compared the specificity and activity of the MUC1 promoter and CMV promoter.Results Reporter gene EGFP-positive cells 48 hours after transfection with pEGFP-MUC1-N1 and pShuttleMUC1-EGFP plasmid were 69.6％ and 63.6％ respectively,in Panc-1 cells,and 4.2％ and 3.7％ respectively,in Hela cells.Conclusions MUC1 promoter can drive reporter gene activity in MUC1-positive tumor cells targeting functional expression.There is potentially a use of targeted therapy in pancreatic cancer at the genetic level.

Objective To investigate the therapeutic effects of endoscopic retrograde cholangiopancreatography (ERCP) and laparoscopic bile duct exploration lithotomy (LBDEL) in treatment of intra/extra-hepatic duct stones. Methods There were 110 patients whose intrahepatic stones located in Ⅰ , Ⅱ hepatic duct and 378 patients whose stones only located in the common bile duct. These patients respectively underwent LBDE combined with choledochoscope laser lithotripsy or ERCP combined with endoscopic sphincterotomy (EST) and endoscopic nasobiliary drainage (ENBD) to remove the stones. Common bile ducts were performed primary suture or T tube placement in the LBDEL cases. The evaluation was carried out for perioperative complications and postoperative recovery of the surgical methods. Results The residual stone rate was 31.82% in 110 cases. The rate was higher in ERCP group (51.06%) than that in LBDEL group (17.46%) (P < 0.05). Postoperative recovery was better in LBDEL group than that in ERCP group. The residual stone rate was 8.20% in 378 cases. The rate was lower in ERCP group (3.68%) than that in LBDEL group(11.63%) (P < 0.05). Between the two groups, there had no statistical significance in postoperative recovery. The incidences of bile leakage and pulmonary infection were higher in LBDEL group than in ERCP group. The incidences of abdominal cavity infection, acute pancreatitis, digestive tract perforation and gastrointestinal bleeding were higher in ERCP group than that in LBDEL group. 2 of the 378 patients occurred death were happened digestive tract perforation which were induced during ERCP procedure. Conclusion LBDEL and ERCP demonstrated the same therapeutic effects in the treatment of common bile duct stones. However, ERCP has no large advantages in the treatment of hepatolithiasis, and shows higher complication rates. LBDEL has a significant curative effect for intra-and extrahepatic bile duct calculi and can maintain the integrity of Oddi sphincter. This technology is easy to spread to the basic-level hospital to benefit the majority of patients.

Objective To investigate the expression of microRNA-1290 in pancreatic cancer and its role in invasion and metastasis of pancreatic cancer.Methods The expression of microRNA-1290 in pancreatic cancer tissue microarray and pancreatic cancer cell lines (AsPC-1,BxPC-3,Capan-2,Panc-1,and MIA PaCa-2) were detected by immunohistochemistry and QT-PCR.The pancreatic cancer cell lines Panc-1 and MIA PaCa-2 in logarithmic growth phase were treated with microRNA-1290 inhibitor,and the invasion and metastasis ability of pancreatic cancer cells were detected by Transwell and wound healing asssay.Western Blot was used to detect the expression of invasion and metastasis-associated proteins cyclooxygenase 2 (COX-2) and matrix metalloproteinase 2(MMP-2) in pancreatic cancer cell lines.Results (1) The expression of microRNA-1290 in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues and adjacent tissues (P ＜ 0.05).(2) Compared with pancreatic normal epithelial cells (HPDE),the expression of microRNA-1290 was significantly higher in different pancreatic cancer cell lines (P ＜ 0.05).The expression level of MicroRNA-1290 in Panc-1 and MIAPaCa-2 pancreatic cancer cells was significantly higher than that in other pancreatic cancer cell lines (P ＜ 0.05).(3) The number of invasive and metastatic cells was significantly decreased after treatment with microRNA-1290 inhibitor (P ＜0.05).(4) The expression of MMP-2 and COX-2 were decreased in Panc-1 and MIAPaCa-2 pancreatic cancer cells treated with MicroRNA-1290 inhibitor.Conclusion The expression of MMP-2 and COX-2 may be involved in the invasion and metastasis of pancreatic cancer cell by regulating the expression of microRNA-1290 in pancreatic cancer.

ObjectiveTo investigate the effect of ankyrin-repeat domain-containing protein 22 （ANKRD22） on the proliferation， invasion， and migration of human hepatoma cells and its molecular mechanism. MethodsThe TCGA database was used to analyze the expression level of ANKRD22 in normal liver tissue and hepatocellular carcinoma tissue and its association with prognosis. Western Blot and qRT-PCR were used to measure the expression of ANKRD22 in human normal liver cells （L-02） and human hepatoma cells （Huh7， HepG2， MHCC-97H， SK-HEP-1， and SMMC-7721）； CCK-8 assay， EdU， wound healing assay， and Transwell assay were used to observe the effect of ANKRD22 on the proliferation， invasion， and migration of hepatoma cells； Western Blot was used to investigate the association of ANKRD22 with cyclins and EMT-related proteins； KEGG and ssGSEA analyses were performed to investigate the mechanism of action of ANKRD22 in hepatoma cells， and related experiments were conducted for validation. The independent-samples t-test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsIn the TCGA database， the expression level of ANKRD22 in hepatoma tissue was significantly higher than that in normal liver tissue （t=5.083， P<0.05）， and the patients with a high expression level of ANKRD22 had longer overall survival and disease-related survival than those with a low expression level of ANKRD22 （P<0.05）. The expression level of ANKRD22 in various human hepatoma cell lines was higher than that in human normal liver cells （all P<0.05）. Cell proliferation assay showed that the ANKRD22 overexpression group had significantly higher EdU positive rate and proliferation rate than the Vector group （t=19.60 and 6.72， both P<0.001）， and compared with the si-NC group， the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower EdU positive rate and proliferation rate （all P<0.001）. Compared with the Vector group， the overexpression group had significantly higher expression levels of Cyclin E1， Cyclin D1， CDK7， and CDK4 （t=3.54， 4.95， 6.34， and 5.19， all P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）. The overexpression group had a significantly lower expression level of P27 than the Vector group （t=6.12， P<0.001）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group （both P<0.001）. Invasion and migration experiments showed that compared with the Vector group， the ANKRD22 overexpression group had significantly higher migration rate and number of crossings through the membrane （migration group and invasion group） （t=5.01， 25.60， and 3.67， all P<0.05）， and compared with the si-NC group， thesi-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower migration rate and number of crossings through the membrane （migration group and invasion group） （all P<0.01）. The overexpression group had significantly higher expression levels of N-cadherin， Vimentin， and Snail than the Vector group （t=12.13， 8.85， and 13.97， all P<0.001）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）； the overexpression group had a significantly lower expression level of E-cadherin than the Vector group （t=4.98， P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group （both P<0.001）. The KEGG enrichment analysis and the ssGSEA analysis showed that ANKRD22 was associated with the PI3K/AKT/mTOR signaling pathway in hepatocellular carcinoma， and the overexpression group had significantly higher expression levels of p-AKT/AKT， p-PI3K/PI3K， and p-mTOR/mTOR than the Vector group （t=12.21， 3.43， and 9.75， all P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）. ConclusionANKRD22 is highly expressed in hepatoma cells and can promote the proliferation， invasion， and migration of hepatoma cells and the activation of the PI3K/AKT/mTOR signaling pathway.