[Expression and significance of VEGF, miR-205 and target protein Ezrin and Lamin A/C in ovarian cancer].

OBJECTIVE: To analyze the expression of vascular endothelial growth factor (VEGF), miR-205, Ezrinand Lamin A/C in ovarian cancer tissues. METHODS: The expression of VEGF in the serum of epithelial ovarian cancer and that of healthy volunteers were detected by enzyme-linked immunosorbent assay; the expressions of vascular endothelial growth factor receptor 1 (VEGFR-1), VEGFR-2, Ezrin and Lamin A/C were detected by immunohistochemistry and the micro-vessel density (MVD) of CD31 was detected by immunohistochemistry in epithelial ovarian cancer, benign ovarian and normal ovarian specimens; and the expression of miR-205, Ezrin and Lamin A/C were detected by real-time PCR in epithelial ovarian cancer, benign ovarian and normal ovarian specimens. RESULTS: The expression of VEGF in the serum of epithelial ovarian cancer patients (116.10± 11.94) was significantly higher than that of healthy volunteers (40.04±4.97, P<0.05). The positive expression rates of VEGFR-1 and VEGFR-2 in the epithelial ovarian cancer specimens were 75.9% and 91.4% respectively, which were significantly higher than that in the benign ovarian and the normal ovarian specimens (P<0.05). No differences were observed in the positive expression rates of VEGFR-1 and VEGFR-2 between the benign ovarian and the normal ovarian specimens (P>0.05). The average length of MVD in the epithelial ovarian cancer specimens (7.56±0.51), was significantly higher than that in the normal ovarian specimens (1.22±0.56, P<0.05) and in the benign ovarian specimens (0.7±0.39, P<0.05). No differences were observed in the average length of MVD between the benign ovarian and the normal ovarian specimens (P>0.05). The relative expression level of miR-205 was 0.106±0.035 in the epithelial ovarian cancer specimens, which was significantly higher than that in the normal ovarian specimens (0.0007±0.0005, P<0.05); the relative expression level of miR-205 in the benign ovarian specimens was (0.0002±0.0002), higher than that in the normal ovarian specimens, but with no significance (P>0.05). The positive expression rates of Ezrin and Lamin A/C in the epithelial ovarian cancer specimens were 51.7% and 60.3%, respectively, which were significantly lower than those in the benign ovarian and the normal ovarian specimens (P<0.05). No differences were observed in the positive expression rates of Ezrin and Lamin A/C between the benign ovarian and the normal ovarian specimens (P>0.05). The relative expression levels of Ezrin and Lamin A/C mRNA in the epithelial ovarian cancer specimens were (0.026±0.003) and (0.060±0.007), respectively, which were significantly lower than those in the normal ovarian specimens (P<0.05). There was no statistical significance between the relative expression level of Ezrin and Lamin A/C mRNA in the epithelial ovarian cancer specimens and that in the benign ovarian specimens (0.029± 0.011, 0.089 ± 0.019; P>0.05) . CONCLUSION: VEGF is significantly expressed in the serum of epithelial ovarian cancer patients; and miR-205 is up-regulated in the epithelial ovarian cancer specimens. Ezrin and Lamin A/C are down-regulated in the epithelial ovarian cancer samples. VEGF, miR-205 and target protein may be associated with the invasion and metastasis of epithelial ovarian cancer.

Downregulation of Beclin 1 and impairment of autophagy in a small population of colorectal cancer.

BACKGROUND: Autophagy is a highly conserved mechanism for degradation and recycling of long-lived proteins and damaged organelle to maintain cell homeostasis. Deregulation of autophagy has been associated with tumorigenesis. Beclin 1 is an essential autophagy protein and its upregulation has been observed in most colorectal cancer tissues. However, there is a small population of colorectal cancers with downregulation of Beclin 1. AIM: The purpose of this study was to investigate the role autophagy plays in colorectal cancers with downregulation of Beclin 1. METHODS: LC3 protein, an autophagosome marker, was assessed by ICH and WB in colorectal cancers tissues. An anti-tumor effect of Beclin 1 was examined by introducing exogenous Beclin 1 in vitro. Colony formation assay, growth curves and mouse xenograft were analysed. RESULTS: Our results showed that LC3 was suppressed in the colorectal cancers (9.86 %) with downregulation of Beclin 1. Moreover, overexpression of Beclin 1 inhibited colorectal cancer cell growth and enhanced the rapamycin-induced antitumor effect in vitro. CONCLUSION: Downregulation of Beclin 1 and autophagy inhibition play an important role in a part of colorectal cancers. Activating autophagy or overexpression of Beclin 1 may be an effective treatment for some colorectal cancers. Detection of expression profile of Beclin 1 in colorectal cancers could be a strategy for new diagnostic and therapeutic methods.

Evaluation of potential reference genes for qRT-PCR studies in human hepatoma cell lines treated with TNF-α.

In this study, the expression of eight candidate reference genes, B2M, ACTB, GAPDH, HMBS, HPRT1, TBP, UBC, and YWHAZ, was examined to identify optimal reference genes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis in two human hepatoma cell lines, BEL-7402 and SMMC-7721, treated with tumor necrosis factor-α (TNF-α) for different time periods. The expression stability of these genes was analyzed by three independent algorithms: geNorm, NormFinder, and BestKeeper. Results showed that TBP was the most stably expressed gene in BEL-7402 and SMMC-7721 cell lines under current experimental conditions, and that the optimal set of reference genes required for accurate normalization was TBP and HMBS, based on the pairwise variation value determined with geNorm. UBC and ACTB were ranked as the least stable genes by same algorithms. Our findings provide evidence that using TBP alone or in combination with HMBS as endogenous controls could be a reliable method for normalizing qRT-PCR data in human hepatoma cell lines treated with TNF-α.

Applicability of partial characterization of cytochrome oxidase I in identification of forensically important flies (Diptera) from China and Egypt.

Precise species identification of every insect sample collected from criminal scenes play an essential role in the accurate estimation of postmortem interval. The morphological similarity poses a great challenge for forensic entomologists. DNA-based method can be used as a supplemental means of morphological method. In the present study, we demonstrate the applicability of the 304-bp cytochrome oxidase I gene fragment in molecular identification of forensically important Diptera. We analyzed 75 specimens belonging to 19 species of 3 families originating from China (Calliphoridae: Chrysomya megacephala, Chrysomya albiceps, Chrysomya rufifacies, Chrysomya nigripes, Aldrichina grahami; Lucilia bazini, Lucilia caesar, Lucilia cuprina, Lucilia sericata, Lucilia porphyrina; Muscidae: Musca autumnalis, Musca domestica, Fannia canicularis, Stomoxys calcitrans; Sarcophagidae: Sarcophaga albiceps, Sarcophaga dux, Helicophagella melanura) and Egypt (Calliphoridae: C. megacephala, C. albiceps, L. sericata; Muscidae: M. domestica, F. canicularis, S. calcitrans, Synthesiomyia nudiseta; Sarcophagidae: Sarcophaga argyrostoma). This region was amplified using polymerase chain reaction followed by direct sequencing of the amplification products. Nucleotide sequence divergences were calculated using the Kimura two-parameter distance model and a neighbor-joining phylogenetic tree generated. Intraspecific variation ranged from 0-0.8 % and interspecific variation occurred between 1-19 %. Although all examined specimens were assigned to the correct species and formed distinct monophyletic clades, the data of the phylogenetic analysis were not completely in accordance with the traditional morphological classification. As both C. nigripes and A. grahami unexpectedly joined with Muscidae and Sarcophagidae groups respectively. Moreover, both Calliphorinae and Luciliinae clades failed to represent Calliphoridae as a separate group. Therefore, although molecular methods are beneficial especially if the available evidence are immature stage or degraded fragments of insect, we suggest using this fragment only in addition to other identification methods.

Molecular characterization of the carbon dioxide receptor in the oriental latrine fly, Chrysomya megacephala (Diptera: Calliphoridae).

The blowfly Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) cannot only act as a mechanical vector of various pathogens, but also infest man and animals causing human health problems and economic losses in the livestock and fish industries. As in other insects, olfaction of this species plays an important role in host location and is presumably mediated via transmembrane receptor signaling pathways. Here, we isolate and characterize CmegGr1 and CmegGr2, two new members of the chemosensory receptor gene family from C. megacephala. The open reading frames of CmegGr1 and CmegGr2 cDNA clones encode 453 and 486 amino acid residues, respectively. These two deduced proteins display high amino acid conservation with previously identified carbon dioxide (CO2) receptors, such as Drosophila melanogaster Gr21a/Gr63a and Anopheles gambiae s.s. Gr22/Gr24. Further sequence analysis showed that both proteins are consistent with their corresponding orthologs in the membrane topology prediction with some ambiguities in the location of N terminus and the number of transmembrane domains. The transcripts of CmegGr1 and CmegGr2 were detected in the major chemosensory organs including the antennae and proboscises with maxillary palps attached. These results suggest that CmegGr1 and CmegGr2 are likely to be the primary receptors for CO2 detection in C. megacephala. Knowledge of the molecular identity of the blowfly olfactory CO2 receptors may aid in the development of novel control strategies designed to take advantage of this unique and critical olfactory pathway.

Identification of forensically important Sarcophagidae (Diptera) based on partial mitochondrial cytochrome oxidase I and II genes.

Entomological evidence is of great importance in forensic cases for postmortem interval calculation. The use of Sarcophagidae (Diptera) for postmortem interval estimation is limited because morphological determination is often hampered because of similar characteristics in the larval, pupal, and even adult stage. To make the species identification more accurate and reliable, DNA-based identification is considered. In this study, we assessed the use of partial mitochondrial cytochrome oxidase I and II genes for discrimination of forensically important Sarcophagidae from Egypt and China [Sarcophaga argyrostoma (Robineau-Desvoidy), Sarcophaga dux (Thomson), Sarcophaga albiceps (Meigen), and Wohlfahrtia nuba (Wiedemann)]. This region was amplified using polymerase chain reaction followed by direct sequencing of the amplification products and using restriction enzymes HinfI and MfeI. Nucleotide sequence divergences were calculated using the Kimura 2-parameter distance model, and a neighbor-joining phylogenetic tree was generated. All examined specimens were assigned to the correct species. Combinations of the restriction enzymes HinfI and MfeI provide different restriction fragment length polymorphism profiles even among 3 sympatric species that belong to the Sarcophaga genus. Therefore, this study demonstrates that the studied partial mitochondrial cytochrome oxidase I and II genes were found to be instrumental for the molecular identification of these forensically important flesh fly species.

[Changes in expression of cyclooxygenase-2 in the spinal dorsal horn after intrathecal p38MAPK inhibitor SB203580 on neuropathic pain in rats].

OBJECTIVE: To investigate the changes of cyclooxygenase-2 (COX-2) expression in the spinal cord dorsal horn after intrathecal a specific p38MAPK inhibitor-SB203580 on neuropathic pain in rats induced by chronic constrictive injury (CCI) to the sciatic nerve. METHODS: Twenty-four male SD rats after intrathecal catheter placement were randomly divided into 4 groups: a sham group with sham surgery, the neuropathic pain model of a NS group, a DMSO group and an SB group were established by CCI to sciatic nerve. NS or DMSO or SB203580 was injected IT NS or 2%DMSO or SB203580 twice a day for 5 consecutive days starting at 6th day when the model of chronic constrictive injury was established. Mechanical stimuli were measured before the surgery and on 1st, 3rd, 5th, 7th, 9th, and 11th day after the surgery. Then all rats were sacrificed and the lumbar segment of spinal cord was removed to determine the COX-2 expression in the dorsal horn by immunocytochemistry. RESULTS: Day 1 to 11 after the surgery, the threshold to mechanical on the surgery side was significantly lower in the NS group and the DMSO group than in the sham group. Day 7 to 11 after the sugery, the threshold to mechanical on the surgery side was significantly lower in the SB group than in the NS group and the DMSO group. The expression of spinal COX-2 was higher in the NS group and the DMSO group than in the sham group, but lower in the SB group than in NS group and the DMSO group. CONCLUSION: Intrathecal administration of SB203580 has significant analgesic effect in the CCI rat model. Expression of COX-2 is significantly reduced when p38MAPK is inhibited by intrathecal SB203580, and p38MAPK stimulation is essential for COX-2 expression.

Identification of forensically important arthropods on exposed remains during summer season in northeastern Egypt.

OBJECTIVE: To document the arthropod succession pattern and to identify forensically important species in northeastern Egypt (32° 15' E and 30° 36' N) for the first time. METHODS: Carcasses were exposed in an open area for 60 days during summer season. Ambient daily temperature (maximum and minimum) and relative humidity (RH) were recorded and existing keys were used for identification of different species. RESULTS: During the period of study, the mean of maximum and minimum temperatures were 34.85 °C and 29.2 °C respectively, while the mean of RH was 53.5%. Four stages of decomposition were observed: fresh, bloat, decay and dry. The most abundant orders were found to be Diptera, Coleoptera and Hymenoptera. Arthropods were collected belonging to 4 families of Diptera: Muscidae, Fanniidae, Calliphoridae and Sarcophagidae. While there were 2 families of Coleoptera: Dermestidae and Histeridae. Monomorium species was the only Hymenoptera family in this study. CONCLUSION: The present work provided a basis for further studies dealing with insect colonization of carcasses in different seasons and locations in Egypt.

TGF-ß1 alters microRNA profile in human gastric cancer cells.

OBJECTIVE: MicroRNAs (miRNAs) are important regulators that play a key role in tumorigenesis and tumor progression. Transforming growth factor-ß1 (TGF-ß1) is involved in invasion and metastasis in many tumors. In this study, we investigated the microRNAs (miRNA) profiles altered by TGF-ß1 in gastric cancer (GC) cells. METHODS: We detected the expression profiles of miRNA by miRNA microarray and quantitative real-time polymerase chain reaction. Migration and invasion, wound-healing assay, prediction of miRNA targets, Western blot and qRT-PCR analysis were carried out to determine the role of one selected miRNA, namely miR-193b, in affecting the biological behaviors of GC BGC823 cells. RESULTS: Among 847 human miRNAs in the microarray, three miRNAs (miR-27a, miR-29b-1 and miR-194) were up-regulated and three (miR-574-3p, miR-193b and miR-130b) were down-regulated in BGC823 cells treated with TGF-ß1 compared with control. miR-193b suppressed the invasion and metastasis of GC cells in vivo and in vitro, and down-regulated urokinase-type plasminogen activator (uPA) protein in GC cells. CONCLUSIONS: TGF-ß1 altered miRNA expression profile in BGC823 cells. Among the altered miRNAs, TGF-ß1 induced the down-regulation of miR-193b, which inhibited cell invasion and metastasis in vivo and in vitro, and down-regulated uPA protein in GC cells.

[Corrigendum] CXCR1 promotes malignant behavior of gastric cancer cells in vitro and in vivo in AKT and ERK1/2 phosphorylation.

Subsequently to the publication of the above article, the authors contacted the Editorial Office to explain that they had inadvertently included data from the same original source in the first row of data panels in Fig. 4B on p. 2191 (showing the results of cell migration assay experiments) to represent two differently performed experiments. Specifically, these images (second and third data panels) containing partially overlapping data corresponded to the 'Vacant­BGC823' in the empty plasmid transfection group and the background 'BGC823 cell' groups, respectively. However, the authors had retained their original data, which they presented to the office for our inspection, and were able to reassemble the data correctly in the figure. The revised version of Fig. 4, showing the replacement data for the 'Vacant­BGC823' and 'BGC823' Migration panels in Fig. 4B, is shown on the next page. The authors are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish a Corrigendum, and all the authors agree with its publication. Furthermore, the authors apologize to the readership for any inconvenience caused. [International Journal of Oncology 48: 2184­2196, 2016; DOI: 10.3892/ijo.2016.3428].

Molecular characterization and expression pattern of an odorant receptor from the myiasis-causing blowfly, Lucilia sericata (Diptera: Calliphoridae).

The blowfly Lucilia sericata (Diptera: Calliphoridae) is a facultative ectoparasite that causes myiasis in both man and animals, leading to serious human health problems and economic losses in the livestock industry. Like other insects, olfaction of this species plays an important role in host location and is presumably mediated by a seven transmembrane receptor family. Here, we isolate and characterize LserOR1, which is the first candidate member of the odorant receptor gene family from L. sericata. LserOR1 displayed high amino acid conservation with previously identified Or83b orthologs from different insect species. The transcripts of LserOR1 were detected in the major olfactory organs including the antennae and maxillary palps, as well as in traditionally non-olfactory tissues such as the legs and female ovipositors. In developmental studies, a quantitative real-time PCR assay was developed and validated for determining the relative expression levels of LserOR1 during several stages. In contrast to its extremely high expression in the adult stage, LserOR1 expression was at the lowest level during the egg stage, and then increased to a peak through the first two larval stages before declining in the third-instar stage. These results suggest that a broadly expressed LserOR1 receptor is likely to be essential for olfactory sensory processes throughout the lifetime of L. sericata. The present study provides the information that may aid in the development of novel blowfly repellents using olfactory proteins as molecular targets.

[Cytotoxicity of a new biomedical titanium alloy Ti-25Nb-10Ta-1Zr-0.2Fe].

OBJECTIVE: To evaluate the cytotoxicity of a new type of titanium alloy Ti-25Nb-10Ta-1Zr-0.2Fe by studying the induced proliferation of L929 cells in contrast with other titania widely used in clinical practice. METHODS: The cell line was treated with extracting liquid containing different concentrations of titanium alloys. The number and morphology of cells was observed under an inverted phase contrast microscope. MTT was used to measure the relative growth rate (RGR) and judge the cytotoxicity grade. Flow cytometry was used to observe cell cycle progression. RESULTS: The RGR of TNTZ group cells at the 3 time points was (93.7±0.8), (100.6±0.4), and (106.4±0.3); the cytotoxicity grade was 1, 0 and 0 after treating for 1, 3 and 5 days; with influence on neither the cell morphology nor the cell cycle. The flow cytometry showed that the sequence of S phase cells was Ti>TNTZ>TC4>blank control >TC4ELI, with no significant difference (P>0.05). None of the 4 materials inhibited the cell proliferation. CONCLUSION: The cell morphology and proliferation are not affected by TNTZ. The new titaniu alloys shows good cyto-compatibility. The cytotoxicity is grade 0, meeting the clinical application standard.

The expression of ADAM23 and its correlation with promoter methylation in non-small-cell lung carcinoma.

ADAM23, a member of a disintegrin and metalloprotease (ADAM) family, has been reported to be expressed in several types of tumours. The exact role of ADAM23 and the possible mechanisms in which it is involved in non-small-cell lung carcinoma (NSCLC) remains unclear. Therefore, this study was designed to explore the expression of ADAM23 and its correlation with promoter methylation in NSCLC. Immunohistochemistry and RT-PCR together with Western blotting methods were used to analyse the expression of ADAM23 in 52 cancer tissue samples and eight benign pulmonary lesions as well as four cell lines. The methylated status of ADAM23 gene was determined with methylation-specific PCR (MSP). The results of immunohistochemistry showed that the expression of ADAM23 protein was lower in NSCLC than that in corresponding normal tissues and benign pulmonary lesions (38.5%vs. 86.5% and 87.5%, P < 0.05), and decreased as NSCLC progressed. Meanwhile, methylation of ADAM23 gene was observed in 21 of 52 NSCLC tissues (40.4%), much higher than that of adjacent normal tissues (7.6%) and benign pulmonary lesions (0/8). In the cancer tissues of ADAM23-negative samples, the rate of ADAM23 gene methylation was 50.3% (17/32). ADAM23 expression and its promoter methylation were negatively associated (r = -0.328, P = 0.017). Moreover, weak expression of ADAM23 in methylated cancer cells increased after treatment with 5-aza-2'-deoxycytidine (5-Aza-2'-dC), confirming that methylation was responsible for the gene downregulation. Our results demonstrate that the expression level of ADAM23 is likely to be involved in the progression of NSCLC and its downregulation is probably correlated with promoter methylation. These findings may provide potential diagnostic and prognostic information about NSCLC.

[Retracted] MicroRNA-204 inhibits proliferation, migration, invasion and epithelial-mesenchymal transition in osteosarcoma cells via targeting Sirtuin 1.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the cell Transwell assay data in the article (featured in Figs. 3B and 6B) were strikingly similar to data that appearing in different form in another article by different authors at different research institutions, which had already been published elsewhere at the time of the present article's submission. Owing to the fact that the contentious data in the above article had already appeared in different form in another article prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. The authors did not reply to indicate whether or not they agreed with the retraction of the paper. The Editor apologizes to the readership for any inconvenience caused. [the original article was published on Oncology Reports 34: 399­406, 2015; DOI: 10.3892/or.2015.3986].

Expression and functions of the repressor element 1 (RE-1)-silencing transcription factor (REST) in breast cancer.

The repressor element 1 (RE-1)-silencing transcription factor (REST), also known as the neuron-restrictive silencer factor (NRSF) or repressor binding to the X2 box (XBR), REST/NRSF/XBR, is originally discovered as a transcriptional repressor of a large number of primarily terminal neuronal differentiation genes in non-neuronal cells and neural stem cells (NSCs). Recently, the tumor-suppressor function of REST is finally proved. However, the expression profile and function of REST in breast cancer are not very clear. In this study, the expression of REST was detected in breast cancer tissue by immunohistochemistry. The results showed that REST expression was significantly lower in breast cancer samples compared to normal and benign breast samples (P < 0.05). Furthermore, the shRNA approach was used to investigate the function of REST in human breast cancer cells. Knocking down REST expression by shRNA in the human breast cancer MCF-7 cells resulted in an increase in cell proliferation, suppression in apoptosis, and reduced sensitivity to anticancer drug with a concurrent significantly up-regulated expression of Bcl-2. These data implied a significant role of REST in breast cancer. The reduced expression of REST might contribute to the breast cancer pathogenesis.

Reduced stratifin expression can serve as an independent prognostic factor for poor survival in patients with esophageal squamous cell carcinoma.

UNLABELLED: Stratifin plays an important role in cancer biology by interfering with intracellular signalling pathways and cell-cycle checkpoints. Decreased expression of stratifin gene has been reported to be a poor prognostic indicator in a variety of human malignant tumors. AIM: To clarify the role and prognostic significance of stratifin in esophageal squamous cell carcinoma (ESCC). METHODS: The alteration of stratifin messenger RNA (mRNA) and protein was analyzed by reverse-transcription and quantitative real-time polymerase chain reaction (QRT-PCR) and Western blotting in 20 paired ESCC and nonneoplastic esophageal mucosa tissues, respectively. Then, immunohistochemistry (IHC) was used to evaluate expression of stratifin in tissues of 148 ESCC patients (including the former 20 pairs of tissues) and correlate it with clinicopathological parameters and prognosis of ESCC patients. RESULTS: The stratifin level of mRNA and protein was markedly downregulated in ESCC tissue compared with in corresponding nonneoplastic esophageal epithelium (P<0.05). Similarly, the positive rate of stratifin protein expression was lower in the esophageal cancer than in paired nonneoplastic esophageal epithelium as detected by IHC (P=0.007). Statistically, the downregulation of stratifin expression was correlated with tumor infiltration depth (P=0.003), lymph node metastasis (P=0.008), distant metastasis (P=0.013), and lymphovascular invasion (P=0.007) of ESCC. Furthermore, the reduced stratifin expression was associated with shorter 5-year survival rate of ESCC patients after curative surgery (P<0.0001). On the basis of univariate and multivariate Cox regression analysis, we found that reduced stratifin expression, T4 stage, lymph node metastasis, and distant metastasis were independent risk factors for worse prognosis in ESCC patients. CONCLUSION: The present report indicates that stratifin could be a useful indicator for prognosis of this disease, as well as a potential target for more effective therapy.

[Effect of ß-catenin gene silencing by shRNA on biologic characteristics of human esophageal carcinoma cells].

OBJECTIVE: To study the effects of short hairpin RNA (shRNA) mediated gene silencing of ß-catenin on the biological characteristics of esophageal carcinoma cells, and to provide theoretical and experimental evidence for the gene therapy of esophageal carcinoma through target inhibition of ß-catenin gene. METHODS: Single strand DNA was synthesized according to the hairpin RNA sequence, and then subcloned into eukaryotic expression vector pGenesil-3 to construct a shRNA-expression pDNAs driven by human U6 promoter of ß-catenin (pGen-3-CTNNB1). One additional construct of random siRNA (pGen-3-con) without homologous to any human genes was constructed in a similar fashion as control.Positive clones were identified and verified by restriction cleavage and DNA sequencing analyses. pGen-3-CTNNB1 and pGen-3-con were then transfected into esophageal carcinoma cell line Eca-109 with liposome, respectively. Positive colonies were selected with G418. Expression of ß-catenin protein and mRNA in the transfected and nontransfected Eca-109 cells were examined by Western blotting, immunofluorescence and RT-PCR, respectively. Xenograft tumor model was used to compare the tumorigenesis of three different cells.Expressions of ß-catenin in all tumor tissues were examined by immunohistochemistry staining. The invasive abilities of three different cells were examined with transwell invasion filter and Matrigel. RESULTS: ß-catenin expression levels were found markedly decreased in Eca-109 cells transfected with pGen-3-CTNNB1. In vivo, transfection with ß-catenin shRNA greatly impeded the tumor growth, pGen-3-con (1.18 ± 0.13) g, Eca-109 (1.38 ± 0.21) g, pGen-3-CTNNB1 (0.42 ± 0.09) g, P < 0.05. Immunohistochemistry staining showed a significantly decreased expression of ß-catenin in ß-catenin shRNA transfected cells than in random shRNA transfected and nontransfected cells (P < 0.05). The infiltration abilities of esophageal carcinoma cells were significantly suppressed, pGen-3-con (81 ± 5)/HPF, Eca-109 (77 ± 6)/HPF, pGen-3-CTNNB1 (41 ± 4)/HPF, P < 0.01; along with significantly decreased migration abilities, pGen-3-con (73 ± 5)/HPF, Eca-109 (69 ± 5)/HPF, pGen-3-CTNNB1 (38 ± 4)/HPF (P < 0.05). CONCLUSIONS: There are abnormal expression of ß-catenin and activation of Wnt signaling pathway in human esophageal carcinoma cell line Eca-109. RNA interference targeting ß-catenin gene suppresses the growth of xenograft tumorigenesis in nude mouse and the invasiveness and metastatic capability of esophageal carcinoma cells.

[Forensic analysis of 74 tumor related medical malpractice cases].

OBJECTIVE: To analyze the causes of medical malpractice in patients with tumor, to determine the medical responsibility, and to recommend the related preventions. METHODS: Seventy four medical malpractice cases, which were involved in tumor and collected from 2000 to 2009 in medicolegal expertise center of west China, were analyzed retrospectively. RESULTS: The medical malpractice cases in the patients with tumor showed an increasing tendency in recent years. The main causes are missed diagnosis, misdiagnosis, improper chemotherapy and neglect of complications. The causes of medical malpractice were different in the different levels of medical services. The occurrence of medical malpractice in surgery and OB-GYN showed more frequent than the others. CONCLUSION: Forensic pathology autopsy is important to resolve medical malpractice of tumor patients by finding out the cause of death and clarifying the medical responsibility. The occurrence of medical malpractice could be reduced by the clinical doctors through improving serve consciousness, obtaining the patients' trust, improving the medical treatment, following related laws and rules, fulfiling duty of medical careness.

[Comparison of mtDNA extraction from different parts of sarcosaphagous insects].

OBJECTIVE: To explore mitochondrial DNA (mtDNA) extraction effects of different parts from sarcosaphagous insects using improved cetyltriethylammnonium bromide (CTAB) method. METHODS: Thirteen Lucilia sericata (Meigen) and 13 Nicrophorus fossor (Erichson) were collected from the corpses of rabbits placed on the outdoor lawn in Huhehot district. Four parts (head, chest muscle, legs and wings) of insect were collected, and the mtDNA of all samples were extracted using CTAB method. The purity and concentration were tested using protein and nucleic acid spectrophotometry. The integrity of the extracted mtDNA and PCR products were checked by agarose gel electrophoresis. The PCR products were sequenced and the obtained sequences were imputed into GenBank for comparison. RESULTS: mtDNA were successfully extracted from 10 head samples, 6 legs samples, 4 wing samples and 13 chest muscle samples of the Lucilia sericata (Meigen). Also, mtDNA were successfully extracted from 5 head samples, 8 legs samples, 3 wing samples and 13 chest muscle samples of the Nicrophorus fossor (Erichson). CONCLUSION: mtDNA can be obtained from chest muscle and other parts of sarcosaphagous insects using the improved CTAB method.

[Sequencing of mitochondrial DNA cytochrome oxidase subunit I gene in sarcosaphagous flies from 14 provinces in China].

OBJECTIVE: To detect the 278 bp region of gene of the cytochrome oxidase subunit I (COI) in mitochondral DNA (mtDNA) of sarcosaphagous flies, identify the species of sarcosaphagous flies, and provide reference for forensic application. METHODS: Samples were collected in Baotou and Chifeng of Inner Mongolia, Tianjin, Nanning, Fuzhou, Linyi of Shandong, Shijiazhuang, Yinchuan, Lanzhou, Huairou of Beijing, Xinxiang and Nanyang of Henan, Datong of Shanxi, Wuhu of Anhui, Quzhou of Zhejiang, Changsha, Zhuzhou and Yongzhou of Hunan. A total of 38 flies were randomly collected from rabbits, dogs and pigs which were set outdoors, then the flies' mitochondrial DNA (mtDNA) were extracted by the improved small insects DNA homogenate method. Amplification was conducted by Perkin-Elmer 9600 thermal cycler, then vertical non-denaturing 7% polyacrylamide gelectrophoresis. PCR products were purified using the nucleic acid purification kit. Sequences of both strands were obtained by direct sequence of the double-stranded PCR product using one of the PCR primers and the ABI PRISM big dye terminator cycle sequencing dit. Sequence reactions were electrophorsed on ABI Model 3730 DNA Sequencers. A UPGMA tree was contrasted using the maximum composite likelihood method in MEGA4. RESULTS: The 38 sarcosaphagous flies belonged to 3 families(Muscidae, Calliphoridae, and Sarcophagidae), 10 genuses (Musca Linnaeus, Hydrotaea Robineau-Desvoidy, Aldrichina Townsend, Hemipyrellia Townsend, Achoetandrus Bezzi, Protophormia Townsend, Chrysomya Robineau-Desvoidy, Lucilia Robineau-Desvoidy, Helicophagella Enderlein, and Boettcherisca Rohdendorf), and 12 species [Musca domestica (Linnaeus), Hydrotaea (Ophyra) capensis (Wiedemann), Lucilia caesar (Linnaeus), Lucilia illustris (Meigen), Aldrichina graham (Aldrich), Hemipyrellia ligurriens, Achoetandrus (Chrysomya) rufifacies (Macquary), Protophormia terraenovae (Robineau-Desvoidy), Chrysomya megacephala (Fabricius), Lucilia sericata (Meigen), Helicophagella melanura (Meigen), and Boettcherisca peregrine (Robineau-Desvoidy)]. CONCLUSION: The genus of the sarcosaphagous flies can be identified by 278 bp gene sequence analysis of CO I in mtDNA. This method is rapid, convenient and precise.