LncRNA UCA1 mediates Cetuximab resistance in Colorectal Cancer via the MiR-495 and HGF/c-MET Pathways.

Background: Cetuximab is one of the most widely used monoclonal antibodies to treat patients with RAS/BRAF wild-type metastatic colorectal cancer (mCRC). Unfortunately, cetuximab resistance often occurs during targeted therapy. However, the underlying epigenetic mechanisms remain unclear. Our previous study demonstrated that the exosomal transfer of urothelial carcinoma-associated 1 (UCA1) confers cetuximab resistance to CRC cells. The goal of this study was to elucidate the detailed role of UCA1 in cetuximab resistance in CRC and the underlying molecular mechanism. Methods: In vitro and in vivo functional studies were performed to assess the role of UCA1 in cetuximab resistance in CRC cell lines and xenograft models. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to examine UCA1 localization and expression. Bioinformatics analysis was performed to predict the potential mechanism of UCA1, which was further validated by the dual-luciferase reporter assay and the RNA immunoprecipitation (RIP) assay. Cells treated with indicators were subjected to Cell Counting Kit-8 (CCK-8) and western blotting to investigate the role of hepatocyte growth factor (HGF)/c-mesenchymal-epithelial transition (c-MET) signalling in UCA1-mediated cetuximab resistance. Results: We showed that UCA1 decreased CRC cell sensitivity to cetuximab by suppressing apoptosis. Mechanistic studies revealed that UCA1 promoted cetuximab resistance by competitively binding miR-495 to facilitate HGF and c-MET expression in CRC cells. Moreover, HGF was shown to attenuate the cetuximab-induced inhibition of cell proliferation by activating the HGF/c-MET pathway in CRC cells. Conclusion: We provide the first evidence of a UCA1-miR-495-HGF/c-MET regulatory network involved in cetuximab resistance in CRC. Therefore, UCA1 has potential as a predictor and therapeutic target for cetuximab resistance.

MiR-181c sensitizes ovarian cancer cells to paclitaxel by targeting GRP78 through the PI3K/Akt pathway.

Primary cytoreductive surgery with platinum-taxane-based chemotherapy is the standard treatment for ovarian cancer (OC) patients; however, resistance to chemotherapy is a contributing factor to OC mortality. Paclitaxel (PTX), the most widely used taxane, has become the first-line drug against OC. The molecular mechanism of PTX resistance is different from that of platinum-based agents and is still not completely elucidated. Our previous study showed that glucose-regulated protein 78 (GRP78) is involved in the resistance of OC cells to PTX. However, little is known regarding endogenous inhibitors of this gene. MicroRNAs (miRNAs) play critical roles in the regulation of gene expression; therefore, we sought to identify miRNA(s) with potential to target GRP78 under the hypothesis that miRNA(s) could serve as potential therapeutic targets. Here, we show that miR-181c, predicted to target GRP78, was downregulated in PTX-resistant OC cells and tissues. MiR-181c downregulated GRP78 expression and induced apoptosis by directly targeting its 3'-untranslated region (UTR). Overexpression of miR-181c sensitized resistant OC to PTX by inhibiting the PI3K/Akt pathway in vitro and in vivo. Taken together, our findings indicate that the delivery of miR-181c can efficiently suppress GRP78 expression and GRP78-mediated PTX resistance in OC and suggest that this strategy has therapeutic potential.

The Crystal Structure and Molecular Docking of CYP76AH3 in the Tanshinone Biosynthesis Pathway / 中国生物化学与分子生物学报

Salvia miltiorrhiza is widely used in the treatment of the angina pectoris, coronary heart disease and myocardial infarction. CYP76AH3 is the key P450 enzyme, locating at the branch point of the tanshinone synthesis pathway. The crystal structural study and key amino acid analysis are of great significance for synthetic biology study on tanshinone. But it is always a challenge for scientists to carry out protein purification, crystallization and crystal structural studies on transmembrane type Ⅱ P450 enzymes. In this study, the prokaryotic expression plasmid was generated, and the high-purity target protein was purified. CYP76AH3 was successfully crystallized, and the crystal structure was solved.After docking range was determined by Cavityplus analysis, molecular docking with Discovery Studio was conducted. The docking result indicated that Gly298 and Asp294 had hydrogen bond interaction with the substrate, while Phe479, Leu367 and Leu293 had hydrophobic interaction with the substrate.In addition, the effect of mutations at the key amino acids on the protein structure stability was predicted throung point mutation simulation. This study would provide a target for protein engineering of CYP76AH3 and lay a foundation for the study of synthesis biology on tanshinones.

5-Hydroxymethylcytosine expression is associated with poor survival in cervical squamous cell carcinoma.

OBJECTIVE: Deoxyribonucleic acid methylation is an important epigenetic modification that is frequently altered in cancer. Recent reports showed that the level of 5-hydroxymethylcytosine was altered in various types of cancers. The influence of deoxyribonucleic acid methylation in cervical squamous cell carcinoma is not fully understood. In this study, we investigated 5-hydroxymethylcytosine and ten-eleven translocation expression in cervical squamous cell carcinoma and whether they are associated with poor survival in cervical squamous cell carcinoma. METHODS: We detected the expression of 5-hydroxymethylcytosine, 5-methylcytosine and TET1/2/3 in 140 patients with cervical squamous cell carcinoma and 40 patients with normal cervical tissues by immunohistochemistry. We assessed the prognostic values of 5-hydroxymethylcytosine, 5-methylcytosine and TET2 in the clinical outcome of cervical squamous cell carcinoma. RESULTS: Expression of 5-hydroxymethylcytosine was significantly decreased in cervical squamous cell carcinoma compared with normal cervix tissues. In contrast, 5-methylcytosine expression was significantly increased in cervical squamous cell carcinoma compared with normal cervix tissues. Moreover, expression of TET2, but not TET1 and TET3, was decreased in cervical squamous cell carcinoma. Our study showed that the decreased level of 5-hydroxymethylcytosine predicts poor prognosis of cervical squamous cell carcinoma patients. The expression of 5-hydroxymethylcytosine was an independent prognostic factor for both disease-free and overall survival of cervical squamous cell carcinoma patients. CONCLUSIONS: In cervical squamous cell carcinoma, less aggressive tumor behavior was correlated with 5-hydroxymethylcytosine and TET2. Our data indicated that 5-hydroxymethylcytosine may become a prognostic marker for cervical squamous cell carcinoma and the decreased expression of TET2 may be an underlying mechanism for decreased 5-hmC in cervical squamous cell carcinoma.

Heat shock pretreatment improves stem cell repair following ischemia-reperfusion injury via autophagy.

AIM: To investigate whether heat shock pretreatment (HSP) improves mesenchymal stem cell (MSC) repair via autophagy following hepatic ischemia-reperfusion injury (HIRI). METHODS: Apoptosis of MSCs was induced by 250 mM hydrogen peroxide (H2O2) for 6 h. HSP was carried out using a 42 °C water bath for 1, 2 or 3 h. Apoptosis of MSCs was analyzed by flow cytometry, and Western blot was used to detect Bcl-2, Bax and cytochrome C expression. Autophagy of MSCs was analyzed by flow cytometry and transmission electron microscopy, and the expression of beclin I and LC3-II was detected by Western blot. MSCs were labeled in vivo with the fluorescent dye, CM-Dil, and subsequently transplanted into the portal veins of rats that had undergone HIRI. Liver levels of proliferating cell nuclear antigen (PCNA) were quantified by fluorescent microscopy. Serum aminotransferase activity and the extent of HIRI were also assessed at each time point. RESULTS: HSP for 2 h reduced apoptosis of MSCs induced by H2O2 as seen by a decrease in apoptotic rate, a decrease in Bax and cytochrome C expression and an increase in Bcl-2 expression (P < 0.001). In addition, HSP for 2 h induced autophagy of MSCs exposed to H2O2 as shown by an increase in acidic vesicular organelle-positive cells, beclin 1 and LC3-II expression, and autophagosome formation (P < 0.05). Treatment with 3-methyladenine attenuated HSP-induced autophagy and abolished the protective effects of HSP on the apoptosis of MSCs. Rapamycin failed to have additional effects on either autophagy or apoptosis compared with HSP alone. The phosphorylation of p38MAPK was significantly elevated and the phosphorylation of mTOR was downregulated in heat shock pretreated MSCs. Treatment with the p38MAPK inhibitor, SB203580, reduced HSP-induced autophagy in MSCs. In vivo studies showed that the transplantation of HSP-MSCs resulted in lower serum aminotransferase levels, lower Suzuki scores, improved histopathology and an increase in PCNA-positive cells (P < 0.05). CONCLUSION: HSP effectively induces autophagy following exposure to H2O2 via the p38MAPK/mTOR pathway, which leads to enhanced MSC survival and improved MSC repair following HIRI in rats.

Involvement of GRP78 in the Resistance of Ovarian Carcinoma Cells to Paclitaxel.

BACKGROUND: Glucose regulated protein 78 (GRP78) is a type of molecular chaperone. It is a possible candidate protein that contributes to development of drug resistance. We first examined the involvement of GRP78 in chemotherapy-resistance in human ovarian cancer cell. MATERIALS AND METHODS: The expression of GRP78 mRNA and protein were examined by RT-PCR and western blotting, respectively, in human ovarian cancer cells line (HO-8910). Sensitivity of HO-8910 to paclitaxel was determined with methyl thiazolyl tetrazolium (MTT). Suppression of GRP78 expression was performed using specific small-interfering RNA (siRNA) in HO-8910 cells, and cell apoptosis was assessed by flow cytometry. Statistical analysis was performed using the SPSS 15.0 statistical package. RESULTS: HO-8910 cells, with high basal levels of GRP78, exhibited low sensitivity to paclitaxel. The mRNA and protein levels of GRP78 were dramatically decreased at 24h, 48h and 72h after transfection and the sensitivity to paclitaxel was increased when the GRP78 gene was disturbed by specific siRNA transfection. CONCLUSIONS: The results suggested that high GRP78 expression might be one of the molecular mechanisms causing resistance to paclitaxel, and therefore siRNA of GRP78 may be useful in tumor-specific gene therapy for ovarian cancer.

Prognostic values of 5-hmC, 5-mC and TET2 in epithelial ovarian cancer.

PURPOSE: DNA methylation is an important epigenetic modification that is frequently altered in cancer. Recent reports showed that the level of 5-hydroxymethylcytosine (5-hmC) was altered in various types of cancers. The influence of DNA methylation in epithelial ovarian cancer (EOC) is not fully understood. Therefore, the aim of the present study was to investigate factors involved in DNA demethylation in EOC compared with normal ovarian tissues. METHODS: We examined the expression of 5-hmC, 5-mC, and TET2 by immunohistochemistry in 130 cases of EOC and 40 cases of normal ovarian tissues. We assessed the prognostic values of 5-hmC, 5-mC, and TET2 in clinical outcome of EOC. RESULTS: We discovered a significant decrease in 5-hmC and TET2 expression in EOC compared with normal ovarian tissues. In contrast, there was a significant increase in 5-mC expression in EOC compared with normal ovarian tissues. The expression of 5-hmC, 5-mC, and TET2 correlated with pathologic stage, tumor grading, lymph node metastasis, and vascular thrombosis. Furthermore, decreased level of 5-hmC predicts poor prognosis of EOC patients. The expression of 5-hmC was an independent prognostic factor for overall survival of EOC patients. CONCLUSIONS: The data suggest that loss of 5-hmC is an epigenetic event of EOC, and the expression of 5-hmC could serve as a prognostic factor for EOC.

Notch signaling: a novel regulating differentiation mechanism of human umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells in vitro.

BACKGROUND: Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) could be induced to differentiate into insulin producing cells (IPCs) in vitro, which have good application potential in the cell replacement treatment of type-1 diabetes. However, the mechanisms regulating this differentiation have remained largely unknown. Notch signaling is critical in cell differentiation. This study investigated whether Notch signaling could regulate the IPCs differentiation of human UCB-MSCs. METHODS: Using an interfering Notch signaling protocol in vitro, we studied the role of Notch signaling in differentiation of human UCB-MSCs into IPCs. In a control group the induction took place without interfering Notch signaling. RESULTS: Human UCB-MSCs expressed the genes of Notch receptors (Notch 1 and Notch 2) and ligands (Jagged 1 and Deltalike 1). Human UCB-MSCs with over-expressing Notch signaling in differentiation resulted in the down-regulation of insulin gene level, proinsulin protein expression, and insulin-positive cells percentage compared with the control group. These results showed that over-expressing Notch signaling inhibited IPCs differentiation. Conversely, when Notch signaling was attenuated by receptor inhibitor, the induced cells increased on average by 3.06-fold (n = 4, P < 0.001) in insulin gene level, 2.60-fold (n = 3, P < 0.02) in proinsulin protein expression, and 1.62-fold (n = 6, P < 0.001) in the rate of IPCs compared with the control group. Notch signaling inhibition significantly promoted IPCs differentiation with about 40% of human UCB-MSCs that converted to IPCs, but these IPCs were not responsive to glucose challenge very well both in vitro and in vivo. Hence, further research has to be carried out in the future. CONCLUSIONS: Notch signaling may be an important mechanism regulating IPCs differentiation of human UCB-MSCs in vitro and Notch signaling inhibition may be an efficient way to increase the number of IPCs, which may resolve the shortage of islet of cell replacement treatment of type-1 diabetes.

A secretory function of human insulin-producing cells in vivo.

BACKGROUND: Mesenchymal stem cells derived from human umbilical cord blood (UCB-MSCs) have good research and application prospects in the treatment of diabetes. We once induced UCB-MSCs to differentiate into insulin-producing cells (IPCs) in vitro, but we did not know the functions of these cells in vivo. The aim of this study was to assess the functional effects of IPCs on insulin secretion and their role in the treatment of diabetes in vivo. METHODS: UCB-MSCs were induced to IPCs by an inducing protocol with extracellular matrix gel. BALB/C nude mice were made hyperglycemic by intraperitoneal injection of streptozotocin. The diabetic mice were transplanted with 1X10(7) IPCs under the renal capsule or with phosphate-buffered saline as a control. After transplantation, the grafts were analyzed by immunocytochemistry for the expression of human insulin; the serum human insulin levels were measured; and blood glucose and body weight status were monitored. RESULTS: Immunofluorescence showed that numerous IPCs under the kidney capsule were insulin-positive. On day 14 after transplantation, the serum human insulin level of the treatment group (n=9) averaged 0.44+/-0.12 mU/L, which was higher than that of the control group (n=9) that did not express insulin (t=10.842, P<0.05). The diabetic mice remained hyperglycemic and kept losing body weight after IPC transplantation, and there was no significant difference in the control group. CONCLUSION: IPCs differentiated from UCB-MSCs generate human insulin in diabetic mice, but more research is needed to make further use of them to regulate hyperglycemia and body weight in vivo.

Assessment of duodenal circular drainage in treatment of superior mesenteric artery syndrome.

AIM: To assess the clinical value of duodenal circular drainage for superior mesenteric artery syndrome (SMAS). METHODS: Forty-seven cases of SMAS were treated with duodenal circular drainage from 1959 to 2001. Clinical data were analyzed retrospectively. RESULTS: In this group, good effects were achieved in 39 cases treated with duodenal circular drainage after 2-15 years of follow-up. The other eight cases were first treated with anterior repositioning of the duodenum (two cases), duodenojejunostomy (five cases), subtotal gastrectomy and billroth II gastrojejunostomy (one case), but vomiting was not relieved until duodenal circular drainage was performed again. A follow-up study of 8-10 years revealed satisfactory results in these eight patients. CONCLUSION: In SMAS, if the reversed peristalsis is strong and continuous, and vomiting occurs frequently, the symptom can not be relieved even if the obstruction of duodenum is removed surgically. The key treatment is the relief of reversed peristalsis. The duodenal circular drainage can resolve the drainage direction of duodenal content, thus relieving the symptom of vomiting.

Diagnosis and surgical treatment of hepatic hilar cholangiocarcinoma.

BACKGROUND: Hepatic hilar cholangiocarcinoma can be diagnosed early with the progress in diagnostic imaging, and thus the rate of resection of the tumor has increased markedly. To assess the effectiveness of resection, we reviewed 185 cases of hepatic hilar cholangiocarcinoma diagnosed and treated at our hospital. METHODS: The clinical data of 185 patients with hepatic hilar cholangiocarcinoma who had been treated surgically from 1972 to 2006 were retrospectively analyzed. RESULTS: The records of the 185 patients were divided into first stage (1972-1986) or second stage (1987-2006) according to the incidence of the tumor and its resection rate. Primary symptoms included upper abdominal discomfort or pain, anorexia, tiredness, weight loss and progressive jaundice. Ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), and magnetic resonance cholangiopancreatography (MRCP) were first line methods for atraumatic diagnosis. If the patients displayed intrahepatic bile duct dilatation or were diagnosed as suffering from extrahepatic obstructive jaundice, percutaneous transhepatic cholangiography (PTC), MRCP or endoscopic retrograde cholangiopancreatography (ERCP) should be used. In this series, 87 patients underwent resection of the tumor (47.0%). Of the 87 patients, 43 received radical resection and 44 palliative resection. Fifteen patients underwent resection in the first stage and 72 in the second stage. A total of 74 patients were followed up after the resection. The median survival time of the radical resection group was 37 months and that of the palliative resection group was 17 months (P<0.001). The other 62 patients receiving no resection died within 1.5 years. CONCLUSIONS: Once patients are diagnosed with hepatic hilar cholangiocarcinoma, they should undergo exploratory laparotomy. Resection is the most effective method for the treatment of hepatic hilar cholangiocarcinoma.

[Clinical analysis of primary small intestinal neoplasms in 305 cases].

OBJECTIVE: To summarive the experience in diagnosis and treatment of primary small intestinal neoplasm. METHODS: The data of 305 patients with pathologically confirmed primary small intestinal tumor collected from 6 hospitals around the Songhua River during the past 33 years were analyzed retrospectively. RESULTS: There were 42 benign and 263 malignant tumors in this series with a ratio of 1: 6.26. The 263 malignant tumors in this series consisted of 135 adenocarcinomas, 57 malignant stromal tumors, 37 malignant lymphomas, 20 carcinoids, and etc. Chronic occult bleeding, gradual of body weight loss and mild abdominal pain (three obscurities) were the common clinical features and alerting massage of intestinal tumor. Correct preoperative diagnostic rate was only 57.0% (174/305) due to difficulty in early diagnosis, which was 67.2% (92/137) in the duodenal tumors, and 51.9% (82/168) in the jejunoileal tumors. All of the 42 benign tumors were resected completely. For the 263 patients with malignant tumors, radical dissection was performed in 153, palliative resection in 34, and gut by-pass or biopsy in 76. The median survival of the patients who underwent radical resection of their malignant tumors was 92 months, which was significantly higher than that of the other groups. CONCLUSION: Early diagnosis of primary small intestinal tumors is difficult and with a preoperative misdiagnosis rate of 43.0%. Total intestinal barium swallowing, endoscopy and superior mesenteric arteriography are three critical examinations for diagnosis and location. Early surgical resection is crucial in improving the prognosis. The primary small intestinal tumor should be resected as early as possible if no distant metastasis is detected.

Clinical analysis of patients with iatrogenic bile duct injury.

BACKGROUND: The main cause of iatrogenic bile duct injury is misidentification of the common bile duct as the cystic duct. In this article, we summarize the experience in the treatment of 112 patients with iatrogenic bile duct injury. METHOD: Clinical data of these patients treated at 10 hospitals of Songhua river area, Heilongjiang province, China from January 1978 to January 2005 were analyzed retrospectively. RESULTS: In 55.4% patients (62/112), iatrogenic bile duct injury was due to misidentification of the anatomy of Calot's triangle before cholecystectomy. Their diagnosis was based on clinical features, celiac puncture and imaging examination in which ultrasonography was most sensitive, giving a diagnostic rate of 97.5%. Six types of injury were identified according to their locations, and type III damage was commonly seen (92/112). The curative rate in this group was 95.5% (107/112). Eighty-seven patients (77.7%) underwent Roux-en-Y choledochojejunostomy with a cure rate of 94.3% (82/87). CONCLUSION: The prevention of iatrogenic bile duct injury lies in identifying the topography of extrahepatic bile ducts. Roux-en-Y choledochojejunostomy is usually the treatment of choice.

[Analysis of clinical manifestations and surgical treatment of Crohn disease: a report of 82 cases].

OBJECTIVE: To elucidate the clinical types of Crohn disease and evaluate its surgical treatment. METHODS: Clinical data of 82 cases with Crohn disease were retrospectively analyzed from June 1972 to June 2003. RESULTS: Among 82 cases with Crohn disease,38 cases were diagnosed before operation,and 44 cases(53.7% ) were misdiagnosed. Main clinical manifestations included abdominal pain(96.3% ),diarrhea(89.0% ) and abdominal mass(28.0% ),other clinical manifestations included fistulation,intestinal hemorrhage and extra- intestinal manifestations such as ulcerative stomatitis,mycotic stomatitis. Patients received different surgical procedures as following: partial enterectomy in 57 cases,hemicolectomy and colostomy in 4 patients,partial ileectomy and ileostomy in 2,ileocolic bypass procedure in 3 patients,partial enterectomy and colectomy and anastomosis in 3 patients because of internal fistula,repair of ileal perforation in 2,lysis of adhesion in 6,drainage of intraperitoneal abscess and ostomy in 3,radical operation in 2 due to colon cancerization. Seventy- three cases(89.0% ) were cured by operation,postoperative complications occurred in 9 patients and 2 cases died. CONCLUSION: It is the key point to achieve successful operation that the corresponding operative modes respectively for varied types of Crohn disease should be adopted.