MicroRNA-577 promotes the sensitivity of chronic myeloid leukemia cells to imatinib by targeting NUP160.

OBJECTIVE: To explore the effect of microRNA-577 on the drug sensitivity of chronic myeloid leukemia (CML) and the underlying mechanism. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of microRNA-577 in peripheral blood of patients with chronic myeloid leukemia. Meanwhile, the expression of microRNA-577 was detected in CML cell line after imatinib treatment. Cell counting kit-8 (CCK-8) and flow cytometry assay were applied to verify the effect of microRNA-577 on cell proliferation and cycle. NUP160 was identified as a target gene of microRNA-577 by dual-luciferase reporter gene assay. Cell reverse test was performed to figure out whether microRNA-577 can enhance the sensitivity of CML to imatinib. RESULTS: QRT-PCR results revealed that microRNA-577 level was notably decreased in peripheral blood of patients with CML, and microRNA-577 could inhibit the proliferation and cycle of CML cells. In addition, the result of dual-luciferase reporting assay indicated that microRNA-577 had a binding relationship with NUP160, and up-regulation of microRNA-577 in CML cell lines reduced the expression of NUP160, and vice versa. Lastly, cell reverse experiments confirmed that microRNA-577 can alleviate the resistance of CML to imatinib. CONCLUSIONS: We found that microRNA-577 promotes the sensitivity of chronic myeloid leukemia cells to imatinib by down-regulating the expression of NUP160.

MiR-34a promotes myocardial infarction in rats by inhibiting the activity of SIRT1.

OBJECTIVE: To investigate the effect of micro ribonucleic acid (miR)-34a regulating silent information regulator 1 (SIRT1) on myocardial infarction (MI) rats. MATERIALS AND METHODS: A total of 30 male, 8-week-old rats were divided into three groups, including: sham group (M group), MI group and MI + miR-34a treatment group (miR group). Tissue morphology in the MI region was observed via hematoxylin-eosin (HE) staining. Myocardial apoptosis in the three groups was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Furthermore, the protein levels of SIRT1, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in myocardial cells were detected via Western blotting. RESULTS: Compared with M group, left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) increased significantly in MI group and miR group (p<0.05), while left ventricular ejection fraction (LVEF) and fractional shortening (FS) decreased obviously (p<0.05). The results of HE staining showed that the inflammatory infiltration of myocardial cells and intercellular collagen fibers significantly increased, and the neuronal damage was remarkably aggravated in MI group and miR group when compared with M group (p<0.05). Compared with MI group, myocardial necrosis, inflammatory cell infiltration and intercellular collagen fibers all increased significantly in miR group (p<0.05). Moreover, the results of TUNEL assay revealed that myocardial apoptosis rate in MI group [(21.35±3.12)%] was remarkably higher than that of M group [(9.53±1.17)%]. Meanwhile, it was significantly higher in miR group [(42.38±3.44)%)] than that of MI group, displaying statistically significant differences (p<0.05). The number of apoptotic cells increased obviously in MI group when compared with M group, while it decreased significantly in MI group when compared with miR group (p<0.05). Besides, the protein levels of SIRT1 and Bcl-2 in myocardial tissues in miR group were remarkably lower than those of M group and MI group (p<0.05). Furthermore, the protein level of Bax in miR group was higher than that of M group and MI group, and there were statistically significant differences (p<0.05). CONCLUSIONS: Overexpression of miR-34a inhibits the activity of SIRT1, thereby promoting the apoptosis of MI.

[The effect of air test and methylene blue perfusion test on detecting the quality of anastomosis during laparoscopic rectal cancer excision (Dixon)].

Objective: To investigate the feasibility and safety of air test (AT) and methylene blue perfusion test (MBPT) to detect the quality of the anastomosis in laparoscopic rectal cancer excision (Dixon), and compare the two approaches. Methods: AT is performed by filling the pelvis with saline solution and insufflating the rectum with air through a size 22 G balloon catheter (Foley). MBPT is carried out by surrounding clean sponges around anastomosis and injecting methylene blue solution into the rectum as like as AT. The balloon catheter connected manometer,ensuring the pressure in rectum can reach 40 cmH(2)O during AT and MBPT. The presence of air bubbles and overt blue-stained spillage indicated anastomotic leaks which are were resolved during surgery. All 28 patients undergoing laparoscopic rectal excision received both AT and MBPT intraoperatively in a randomized fashion. The integrity of the anastomosis, postoperative vital signs, blood examination, drainage and postoperative imaging were analyzed. Results: All 28 patients received both tests successfully with no adverse event. MBPT Level 1 was detected in 15 cases, level 2 in 8 cases, level 3 in 5 cases. No MBPT level 4 was observed. AT level 1 was detected in 22 cases, level 2 in 5 cases, level 3 in 1 cases. No AT level 4 was founded. Three cases were diagnosed with postoperative anastomotic leakage (3/28, 10.71%), of which 2 cases were Grade B [definition and grading proposed by the international study group of rectal cancer (ISREC) in 2010]. One case was Grade C. The positive rate of MBPT was superior to AT (the McNemar testing, P<0.01). Conclusions: The two intraoperative tests are both technically feasible and safe. Compared to AT, MBPT has the advantage of localizing the leak site with a higher positive accuracy, and represents a promising standardized approach for intraoperative test of the anastomosis quality. Intraoperative repair is absolutely helpful for the level 3 and 4 intraoperative tests.

Clinical significance of serum EGFR gene mutation and serum tumor markers in predicting tyrosine kinase inhibitor efficacy in lung adenocarcinoma.

OBJECTIVE: To study the clinical significance of serum epidermal growth factor receptor (EGFR) gene mutation and serum tumor markers in the prediction of tyrosine kinase inhibitor (TKI) efficacy in patients with lung adenocarcinoma. METHODS: Ninety patients with pathologically diagnosed lung adenocarcinoma were enrolled. Further, 51 out of 90 patients received the EGFR-TKI therapy, oral gefitinib. The correlations among serum EGFR gene mutations in exons 18-21, serum tumor markers such as carcinoembryonic antigen (CEA), carbohydrate antigen 24-2 (CA24-2), carbohydrate antigen 125, carbohydrate antigen 15-3 as well as carbohydrate antigen 19-9 (CA19-9) levels, and EGFR-TKI efficacy were determined. RESULTS: There was a high consistency of EGFR gene mutation rate between serum and tissue samples. The serum EGFR gene mutation rate in female patients or non-smokers was significantly higher than that in male patients or smokers, respectively. Serum CA19-9, CA24-2, and CEA levels were significantly correlated with serum EGFR mutation. After receiving gefitinib, the progression-free survivals (PFSs) of patients with high serum CEA level, high serum CA19-9 level, or serum EGFR gene mutation were significantly higher than those of normal patients, respectively. The PFSs were significantly prolonged in patients with EGFR gene mutation and high serum CEA level or patients with EGFR gene mutation and high serum CA19-9 level compared with those in patients with one abnormal biomarker and normal patients. CONCLUSION: Combined detection of EGFR gene mutations as well as CA19-9 and CEA levels in peripheral blood can predict the efficacy of EGFR-TKI in the treatment of patients with lung adenocarcinoma.

Prolyl isomerase PIN1 regulates the stability, transcriptional activity and oncogenic potential of BRD4.

BRD4 has emerged as an important factor in tumorigenesis by promoting the transcription of genes involved in cancer development. However, how BRD4 is regulated in cancer cells remains largely unknown. Here, we report that the stability and functions of BRD4 are positively regulated by prolyl isomerase PIN1 in gastric cancer cells. PIN1 directly binds to phosphorylated threonine (T) 204 of BRD4 as revealed by peptide binding and crystallographic studies and enhances BRD4's stability by inhibiting its ubiquitination. PIN1 also catalyses the isomerization of proline 205 of BRD4 and induces its conformational change, which promotes its interaction with CDK9 and increases BRD4's transcriptional activity. Substitution of BRD4 with PIN1-binding-defective BRD4-T204A mutant in gastric cancer cells reduces BRD4's stability, attenuates BRD4-mediated gene expression by impairing its interaction with CDK9 and suppresses gastric cancer cell proliferation, migration and invasion, and tumor formation. Our results identify BRD4 as a new target of PIN1 and suggest that interfering with their interaction could be a potential therapeutic approach for cancer treatment.

[Effects of microRNA-379-5p on proliferation, migration and invasion of hepatocellular carcinoma cell line].

OBJECTIVE: To investigate the effects of microRNA-379-5p (miR-379-5p) on proliferation, migration and invasion of hepatocellular carcinoma (HCC) cells. METHODS: Human HCC cell line HepG2 was infected with lentivirus carrying miR-379-5p (miR-379-5p group) or lentivirus carrying negative control sequences (negative control group). The untreated HepG2 cells represented blank control group. Cell proliferation was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide (MTT) assays. Cell migration and invasion were assessed by Transwell assays. The mRNA and protein expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 were analyzed by real-time quantitative polymerase chain reaction and Western blot, respectively. RESULTS: Compared with negative control group and blank control group, cell migration and invasion was significantly inhibited in miR-379-5p group (P<0.05). However, there was no significant difference in cell proliferation among the three groups (P>0.05). Furthermore, the mRNA and protein levels of MMP-2 and MMP-9 in miR-379-5p group were significantly lower than that in negative control group and blank control group (P<0.05). CONCLUSION: miR-379-5p can suppress migration and invasion of HCC cell lines, which may be achieved by inhibiting MMP-2 and MMP-9 expressions.

Effects of minimally invasive percutaneous and trans-spatium intermuscular short-segment pedicle instrumentation on thoracolumbar mono-segmental vertebral fractures without neurological compromise.

OBJECTIVE: To compare the outcomes of minimally invasive percutaneous short-segment pedicle instrumentation (SSPI) with that of trans-spatium intermuscular SSPI on thoracolumbar mono-segmental vertebral fracture without neurological compromise. METHODS: A total of 39 patients with thoracolumbar mono-segmental vertebral fracture without neurological deficit receiving treatment between January 2009 and July 2011 were enrolled. Percutaneous SSPI was performed for 18 patients (the percutaneous group), and trans-spatium intermuscular SSPI was performed for 21 patients (the trans-spatium intermuscular group). Peroperative indices, intraoperative radiation exposure time, postoperative and follow-up lumbodorsal pain, function scores, and radiological data were compared. RESULTS: The percutaneous group had significantly less intraoperative blood loss and less severe postoperative pains, but suffered significantly longer fluoroscopy time and higher hospitalization costs compared with the trans-spatium intermuscular group. No significant difference was observed in operating time. All patients were followed up for 17.3 ± 9.2 months (ranging from 5 to 35 months). No significant differences were observed between the two groups in terms of postoperative relative vertebral height (RVH) and regional kyphotic angle (RKA), as well as last follow-up RVH, RKA, lumbodorsal pain, and Oswestry disability index. CONCLUSION: Percutaneous SSPI has the virtues of less intraoperative blood loss and less severe pains in the treatment of thoracolumbar mono-segmental vertebral fracture without neurological deficit. When compared with trans-spatium intermuscular SSPI, it results in longer intraoperative radiation exposure time and a higher surgery cost. To us, percutaneous SSPI has no advantage over trans-spatium intermuscular SSPI in therapeutic outcomes. LEVEL OF EVIDENCE: Level IV. Retrospective study.

Survival of conditionally immortalized hepatocytes in the spleen of syngeneic rats.

BACKGROUND: Hepatocyte transplantation has been shown to be effective in the treatment of liver failure; however, the shortage of donor organs limits its clinical application. Several reports have suggested that conditionally immortalized hepatocytes (CIH) could be an alternative to primary hepatocytes. However, CIH are known to undergo apoptosis in vitro at a non-permissive temperature, which is similar to body temperature. METHODS: To investigate the duration of survival and in vivo apoptosis of CIH in the syngeneic host, the L2A2 cells (a kind of CIH) that were established from hepatocytes of a Lewis rat with a gene for a temperature-sensitive Simian Virus 40 (SV40) large T antigen were transplanted into the spleen. Cells were isolated from the spleen that was removed periodically up to 6 months, and used to detect the presence of the L2A2 cells among them with the selective culture for CIH and T-antigen PCR. In situ apoptosis of L2A2 cells was also examined. In order to improve the survival of transplanted L2A2 cells in the host, a group of rats were partially hepatectomized 1 day before transplantation was performed. RESULTS: The L2A2 cells secreted albumin at a rate of 1.17 +/- 0.18 microg/24 h per 10(6) cells in vitro. After transplantation, L2A2 cell colonies and PCR amplification bands appeared up to 14 and 7 days, respectively, but this duration was not prolonged by a partial hepatectomy. The spleen showed a large number of hepatocytes that were in the process of dying on the 5th day, and only a number of ghost hepatocytes were present on the 7th day of transplantation. No tumors were found during the 6-month observation period. CONCLUSIONS: Conditionally immortalized hepatocytes can survive in the spleen for a limited period, in spite of the growth stimulation, most likely because they undergo apoptosis in vivo as well as in vitro at a non-permissive temperature. These data suggest that the use of these cells in hepatocyte transplantation be limited to temporary hepatic support.

Autoimmune hepatitis in a patient with myasthenia gravis and thymoma--a report on the first case in Korea.

Myasthenia gravis is an autoimmune disease that results from an antibody-mediated reaction and occurs with thymoma in 15% of patients. It is very rarely associated with autoimmune hepatitis. Four cases of myasthenia gravis with autoimmune hepatitis have been reported in the world. We recently experienced a case of 30-year-old man with myasthenia gravis associated with thymoma and autoimmune hepatitis. This condition is the first case that has not been reported previously in Korea. We report this rare condition along with a brief review of the literature.

Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage.

The rat hepatocytes were immortalized using a temperature-sensitive mutant of SV40 large T antigen (tsT) to develop as a possible substitute for primary hepatocytes. Four rat hepatocyte lines that have been developed and maintained more than passage 50, were characterized for their cellular morphology, T antigen and p53 expression, chromosomes, liver-specific differentiation, telomerase activity and anchorage independent growth. All of four cell lines showed a typical epithelial cell morphology, but the population-doubling time became short with passage: 18 to 60%. T antigen expression was increased with passage about 3 to 65 times at permissive temperature but decreased significantly at non-permissive temperature. The expression level of p53 unchanged during passages was also decreased at non-permissive temperature. The distribution of chromosome number changed somewhat with passage. The production levels of albumin and urea in four cell lines were 2.4 to 13.0% and 7.5 to 19.9% of those produced in primary hepatocytes, respectively and were decreased to an undetectable level with passage. Telomerase activity was increased 10 fold following immortalization of cells, but anchorage independent growth of cells did not develop. These results indicate that conditionally immortalized hepatocytes become dedifferentiated with in vitro passage, which may be caused by marked chromosomal damages that occur with compulsive and continuous replications by the increment of T antigen content with passage and its sequential inhibition of p53 function.

Mutational abrogation of the PTEN/MMAC1 gene in gastrointestinal polyps in patients with Cowden disease.

BACKGROUND & AIMS: To understand the molecular etiology of Cowden disease-associated gastrointestinal polyps, we analyzed the mutational status of PTEN/MMAC1, a recently identified Cowden disease gene located at 10q23, in gastric hamartomas, colonic adenoma, and juvenile polyps of 3 patients with Cowden disease. METHODS: Messenger RNA expression, gene deletion, and sequence alteration of PTEN/MMAC1 were evaluated by quantitative polymerease chain reaction (PCR), PCR-single-strand conformation polymorphism, and sequencing analysis. RESULTS: Germline missense mutation at codon 289 (AAA to GAA, Lys to Glu) and deletion of the wild-type allele were detected in the polyps of 2 patients with Cowden disease in the same family. Germline allelic deletion and transcriptional silencing of the remaining allele, probably caused by abnormal methylation, were also observed in a gastric hamartoma of 1 patient. CONCLUSIONS: The germline mutation and alteration of the remaining allele observed in this study strongly support that PTEN/MMAC1 functions as a tumor suppressor in Cowden disease. This study is the first to show that the mutational abrogation of PTEN/MMAC1 plays a causal role in the genesis of gastrointestinal polyps in Cowden disease, providing molecular genetic evidence that colonic adenoma, juvenile polyp, and gastric hamartoma could be included in the manifestations of Cowden disease.