ILC3 cells promote the proliferation and invasion of pancreatic cancer cells through IL-22/AKT signaling.

PURPOSE: Type 3 innate lymphocytes (ILC3s) are reported to be involved in lung cancer, possibly by producing interleukin-22 (IL-22). However, whether ILC3s and their secreted IL-22 molecules contribute to the pathogenesis of pancreatic cancer (PC) remains unclear. To this end, in this study, we investigated the effects and possible mechanisms of ILC3s on PC pathogenesis. METHOD: The IL-22 and IL-2i2R levels and the ILC3s' frequency in cancer tissues from PC patients and in peripheral blood from PC patients and healthy controls were analyzed by flow cytometry, immunochemistry, or immunofluorescence. The effects of IL-22-induced AKT signaling on the proliferation, invasion, and migration of PC cells were examined by co-culturing PC cell lines with ILC3s isolated from PC tissues, with or without the addition of neutralizing IL-22 antibody, IL-22R antibody or AKT inhibitor. RESULTS: Our results showed that IL-22 and ILC3s were significantly upregulated in the PBMCs and cancer tissues of PC patients, and the IL-22R level was increased in PC cells. The increased frequency of ILC3s was positively correlated with the clinical features of PC patients. Co-culture experiments indicated that ILC3s promoted the proliferation, invasion, and migration of PC cell lines by secreting IL-22 to activate AKT signaling because IL-22/IL-22R or AKT blockage markedly counteracted such effects on PC cells. CONCLUSION: Our data demonstrated that ILC3s may promote PC pathogenesis through IL-22/IL-22R-AKT signaling, suggesting a potential intervention target for PC treatment in the future.

Development of polymorphic microsatellite markers based on expressed sequence tags in Populus cathayana (Salicaceae).

Populus cathayana occupies a large area within the northern, central, and southwestern regions of China, and is considered to be an important reforestation species in western China. In order to investigate the population genetic structure of this species, 10 polymorphic microsatellite loci were identified based on expressed sequence tags from de novo sequencing on the Illumina HiSeq 2000 platform. All microsatellite primers were tested on 48 P. cathayana individuals from four locations on the Qinghai-Tibet Plateau. The observed heterozygosity ranged from 0.000 to 1.000, and the null-allele frequency ranged from 0.000 to 0.904. These microsatellite markers may be a useful tool in genetic studies on P. cathayana and closely related species.

Constructing gene network based on biclusters of expression data.

Two genes can be co-regulated and possibly have the similar function if they are similarly expressed, which provides a theoretical basis for construction of gene regulatory networks using gene expression data. Herein, a new method of gene regulatory network was constructed based on biclusters in this paper. Given a bicluster, this paper analyzes the correlation between genes in the clusters and then constructs the gene regulatory network by selecting genes with a correlation coefficient.

MicroRNA-215 functions as a tumor suppressor and directly targets ZEB2 in human pancreatic cancer.

It has been shown that microRNA-215 (miR-215) is dysregulated in several human malignancies, and this correlates with tumor progression. However, its expression and function in pancreatic cancer is still unclear. The aim of this study was to explore the effects of miR-215 on pancreatic cancer formation and progression. Using quantitative RT-PCR, we detected miR-215 expression in pancreatic cancer cell lines and primary tumor tissues. The association of miR-215 expression with clinicopathological factors and prognosis was also analyzed. We then observed the effects of miR-215 on the biological behavior of pancreatic cancer cells. Lastly, the potential regulatory function of miR-215 on ZEB2 expression was investigated. miR-215 expression levels were significantly downregulated in pancreatic cancer samples and cell lines. Decreased miR-215 expression was significantly associated with large tumor size, advanced TNM stage, lymph node metastasis, vessel invasion, and lower overall survival. Multivariate regression analysis corroborated that downregulation of miR-215 was an independent unfavorable prognostic factor. Overexpression of miR-215 inhibited pancreatic cancer cell proliferation, invasion, and migration; promoted cell apoptosis in vitro; and suppressed tumorigenicity in vivo. Further, ZEB2 was confirmed as a direct target of miR-215 by using a luciferase reporter assay. These findings indicate that miR-215 may act as a tumor suppressor in pancreatic cancer cells, and could serve as a novel therapeutic target for miR-based therapy.

Genetic variability of genes involved in DNA repair influence treatment outcome in osteosarcoma.

We conducted a perspective study to investigate the role of ERCC1 (rs11615), ERCC2 (rs13181 and rs1799793), ERCC4 (rs1800067), and ERCC5 (rs17655) in NER pathway in the prognosis of osteosarcoma patients. In total, 146 osteosarcoma patients were recruited between 2008 and 2013. ERCC1 rs11615, ERCC2 rs13181 and rs1799793, ERCC4 rs1800067, and ERCC5 rs17655 gene polymorphisms were assessed by polymerase chain reaction-restriction fragment length polymorphism assay. By multivariate Cox proportional hazards models, we found that carriers of ERCC1 rs11615 TT genotype showed significantly favorable survival compared to wide-type CC genotype, and the adjusted OR (95%CI) was 0.24 (0.08-0.96). Moreover, we found that subjects with ERCC2 rs1799793 AA genotype were associated with decreased hazards of death in multivariate analysis (HR = 0.22, 95%CI = 0.12-0.93). In conclusion, our results suggest that ERCC1 rs11615 and ERCC2 rs1799793 may be useful genetic prognostic markers for osteosarcoma in a Chinese population.

Characterization and effects of miR-21 expression in esophageal cancer.

The aim of this study was to investigate the expression of miR-21 in esophageal cancer and the impact of miR-21 on apoptosis, invasion, and the expression of target genes in esophageal cancer cells. Fluorescence quantitative polymerase chain reaction analysis was used to detect the expression of miR-21 in human esophageal tissues, adjacent tissues, and an esophageal cancer cell line (TE-13). The antisense miR-21 oligonucleotide was generated commercially using the solid-phase chemical synthesis method. Transient transfection was used to transfect esophageal cancer cells (TE-13 antisense and TE-13 control cells). Flow cytometry and Transwell cell assays were used to detect the apoptosis and invasion of esophageal cancer cells, respectively. The western blot method was used to detect the expression of PTEN, PDCD4, and K-ras proteins. These analyses determined that mir-21 expression significantly increased in esophageal cancer tissues and in TE-13 cells, and that this phenomenon was not associated with staging or lymph node metastasis. The apoptosis rate of TE-13 control cells was lower than that of antisense TE-13 cells indicating an enhanced invasive ability. In tissues adjacent to esophageal cancer and in TE-13 antisense cells, the expression of PTEN and PDCD4 was found to be higher than that in the control group, whereas the expression of K-ras showed the opposite pattern. Together, these results suggest that miR- 21 might be involved in the development and metastasis of esophageal cancer, through interaction with its PDCD4 and K-ras target genes.

Association of miR-21 with esophageal cancer prognosis: a meta-analysis.

The present study aimed to explore the relationship between miRNA expression and survival in patients with esophageal cancer (EC) using meta-analysis. We searched PubMed, EMBASE, CNKI, Wanfang, and ISI Web of Science databases without time restrictions, and extracted relevant data, such as the name of first author, publication year, age, gender, number of case, etc. from the studies included. We calculated the pooled hazard ratios (HRs) using the RevMan 5.2 software. A total of five studies involving 504 subjects were included in the meta-analysis, with the purpose of analyzing the association of miRNA-21 expression with EC prognosis. The pooled HR of elevated versus decreased miR-21 expression in EC was 1.87 [95% confidence interval (CI): 1.37-2.55, P < 0.001], with elevated miR-21 expression being associated with poorer prognosis for patients with EC. Our results support a prognostic role for miR-21 in EC.

Analysis of Fcgrt gene polymorphism in indigenous Chinese sheep and its association with colostrum IgG concentration.

The neonatal Fc receptor (FcRn) plays an important role in regulating IgG homeostasis in the body and passive protection to the offspring. Changes in FcRn expression levels caused by genetic polymorphisms of Fcgrt, which encodes FcRn, may lead to inter-individual differences in colostrum IgG levels in sheep. In this study, we sequenced the FcRn partial heavy chain from 179 sheep from Xinjiang Province, China, and detected the differences in colostrum IgG levels and Fcgrt genotypes to identify the correlation between the Fcgrt genotype and colostrum IgG levels in 4 sheep breeds. The DNA sequencing of a 680-bp fragment of the Fcgrt gene revealed various patterns depending on the single-strand conformation in the Suffolk breed. Sequencing analysis revealed a total of 3 patterns, AA, BB, AB, in this fragment, among which the absence of AB and BB genotype acted as a marker for breed identification and characterization, while the AA genotype was shared by Suffolk and 3 other breeds. The only allele found in all 4 breeds was allele A, indicating that natural selection may be favoring the AB and BB genotypes in general and B allele in particular, as the colostrum IgG concentration was relatively higher in the Suffolk breed compared to the other 3 breeds.

Potential role of heat-shock proteins in giant cell tumors.

We investigated the differential expression protein profile of giant cell tumors (GCTs), which can be used to monitor the tumor's recurrence and metastasis, to provide preliminary results for further study. We also explored heat-shock protein (HSP) inhibitor that prevents tumors from recurring and migrating. A stable isotope-labeling strategy using isobaric tags for relative and absolute quantitation coupled with two-dimensional liquid chromatography tandem mass spectrometry was used to separate and identify differentially expressed proteins. A total of 467 differentially expressed proteins were identified in GCT tissues. Up to 311 proteins were upregulated, whereas 156 proteins were downregulated in GCT tissues. Three of the differentially expressed HSPs, namely HP90A, HSPB1, and HSPB2, were upregulated. The differentially expressed proteins of GCT tissues will provide a scientific foundation for tumor prognosis, and for further studies exploring HSP inhibitor to prevent tumor recurrence and migration.

Association between the EGF rs4444903 polymorphism and liver cancer susceptibility: a meta-analysis and meta-regression.

Emerging evidence suggests that a common functional polymorphism, rs4444903 (A>G), in the EGF gene might impact an individual's susceptibility to liver cancer; however, individually published results are inconclusive. This meta-analysis aimed to derive a more precise estimation of the relationship between the EGF rs4444903 polymorphism and liver cancer risk. A literature search was conducted in the PubMed, Embase, Web of Science, and CBM databases from inception through May 1st, 2013. Seven case-control studies were included with a total of 1408 liver cancer cases and 1343 healthy controls. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Our meta-analysis results indicated that the G variant of the rs4444903 polymorphism might be associated with an increased risk of liver cancer (G allele vs A allele: OR = 1.25, 95%CI = 1.01-1.56, P = 0.040; GG + AG vs AA: OR = 1.65, 95%CI = 1.27-2.15, P < 0.001; GG vs AA: OR = 1.77, 95%CI = 1.34-2.35, P < 0.001). Further subgroup analysis by ethnicity also showed significant associations between the G variant of the rs4444903 polymorphism and an increased risk of liver cancer among Asian, Caucasian, and African populations. No publication bias was detected in this meta-analysis. In conclusion, the current meta-analysis suggests that the G variant of the rs4444903 polymorphism may increase the risk of liver cancer. The EGF rs4444903 (A>G) polymorphism can be useful as a biomarker in predicting the development of liver cancer.

Effect of variation of ABCB1 and GSTP1 on osteosarcoma survival after chemotherapy.

We conducted a comprehensive study to investigate the role of genes involved in metabolic and transport pathways in response to chemotherapy and clinical outcome of osteosarcoma patients. Genotyping of seven gene polymorphisms was performed on a 384-well plate format on the Sequenom MassARRAY platform in 162 patients with osteosarcoma. We studied the correlation of the seven gene polymorphisms with response to chemotherapy and clinical outcome of patients. Individuals with the ABCB1 TT genotype had a higher probability of responding poorly to chemotherapy, indicated by an odds ratio (OR) of 2.64 (95%CI=1.04-6.83). Similarly, the genotype of GSTP1 GG was significantly associated with improved responses to chemotherapy, indicated by an OR of 3.33 (95%CI=1.26-8.99). The ABCB1 TT and GSTP1 GG genotypes were significantly associated with a shorter overall survival (OS). Our study found that two gene polymorphisms in two transporter genes and one Phase II metabolism enzymes are associated with response to chemotherapy and OS in osteosarcoma patients, suggesting the potential of the two gene polymorphisms as prognostic biomarkers for osteosarcoma.

Effects of metallothionein-3 and metallothionein-1E gene transfection on proliferation, cell cycle, and apoptosis of esophageal cancer cells.

Metallothionein (MT)-3 has cell growth inhibitory activity, and is the only currently known MT subtype with unique physiological functions. The expression levels of MT-1E, a subtype of MT-1, were positively correlated with the degree of esophageal cancer malignancy. The present study aimed to investigate the effects of MT-3 and MT-1E gene transfection on the proliferation, cell cycle, and apoptosis of esophageal cancer cells. The cationic liposome method was used to transfect the esophageal cancer strains Eca-109 and TE13. Reverse transcription-polymerase chain reaction was used to detect target gene expression, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction was applied to detect cell proliferation, and flow cytometry was used for cell cycle and apoptosis detection. Esophageal cancer cells with MT-3 and MT-1E gene transfection showed high expression of the foreign target gene and mRNA. Cells with MT-3 gene transfection showed markedly inhibited proliferation (P < 0.05), a significantly higher proportion of cells in the G0/G1 phase (P < 0.05), a significantly lower proportion of cells in the S phase (P < 0.05), and a significantly increased apoptosis rate (P < 0.05). Cells with MT-1E gene transfection did not show significant changes in proliferation, cell cycle, or apoptosis rate (P > 0.05). Therefore, the upregulation of MT-3 gene expression can inhibit esophageal cancer cell proliferation and induce apoptosis, which may be achieved by blocking the tumor cell growth cycle, whereas effects of the MT-1E gene on the proliferation of esophageal cancer cells were not evident.

An A/G polymorphism rs3746444 in miR-499 is associated with increased cancer risk: a meta-analysis.

An A/G polymorphism (rs3746444) has been identified in the miR-499 gene that can change the conformation of the secondary gene structure and thereby directly affect binding to target mRNAs and the microRNA (miRNA) maturation process, thus altering protein expression and potentially contributing to cancer susceptibility. Numerous studies investigating the association between the rs3746444 polymorphism and cancers have been published; however, results are inconsistent and inconclusive. To clarify the relationship between the miR-499 rs3746444 polymorphism and cancer, we conducted a comprehensive meta-analysis on 14 case-control studies comprising 7189 cases and 8577 controls. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by using dominant, recessive, and co-dominant genetic models. A publication bias test and subgroup analysis were also performed. Results showed that the G allele was associated with a significantly increased cancer risk compared to the A allele (OR = 1.09; 95%CI = 1.00-1.18). Similarly, moderately elevated risks were also observed in overall analyses in the dominant model (OR = 1.13; 95%CI = 1.01-1.26). Moreover, significantly increased risks were observed in Asian populations (G allele vs A allele: OR = 1.18; 95%CI = 1.01-1.37; GG vs AA: OR = 1.36; 95%CI = 1.07-1.73; dominant model: OR = 1.19; 95%CI = 1.00-1.41; recessive model: OR = 1.31; 95%CI = 1.03-1.66), but not in European populations. These findings indicate that the miR-499 rs3746444 polymorphism is associated with an increased cancer risk.

Expressions of Src homology 2 domain-containing phosphatase and its clinical significance in laryngeal carcinoma.

We investigated the expression of Src homology 2 domain-containing phosphatase (SHP-2) in laryngeal carcinoma and its clinical significance. Expression of SHP-2 was detected by immunohistochemical staining in normal mucosal tissues and various grades of laryngeal carcinoma. We looked for possible correlations between expression of SHP-2 in laryngeal carcinoma and clinical staging and lymph node metastasis. Immunochemical staining results revealed that the SHP-2 expression was significantly higher (88.24%) in laryngeal carcinoma than in normal mucosal tissue (25%). Additionally, the expression of SHP-2 was significantly correlated with lymph node metastasis, but not with clinical stage and gender of patients with laryngeal carcinoma. Therefore, SHP-2 may be useful as a prognostic marker for laryngeal carcinoma and as a therapeutic target in laryngeal carcinoma treatment.

Polymorphisms of three new microsatellite sites of the dystrophin gene.

To look for novel microsatellites in the dystrophin gene for the diagnosis of Duchenne muscular dystrophy, candidate microsatellite sites in the dystrophin gene were analyzed with the SSRHunter software and were also genotyped. Among the 15 candidate microsatellite sites, three novel microsatellite sites in the 60th, 30th, and 2nd intron were found to have a high degree of polymorphism. We submitted these three new loci to the European Molecular Biology Laboratory, under accession Nos. FN547040, FN547041 and FN557526, which were called DXSDMD-in60, DXSDMD-in30 and DXSDMD-in2, respectively. In these three loci, we found 9, 6 and 11 alleles, respectively, in the 205 individuals. In addition, we also detected 20, 19 and 20 genotypes for the three loci in female samples, with a polymorphism information content of more than 0.600. In conclusion, the three microsatellite sites in the intron region of the dystrophin gene have a high degree of polymorphism, and they can be used in population genetics, as well as to provide a theoretical basis for genetic diagnosis and elucidation of molecular mechanisms in Duchenne muscular dystrophy.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100 percent; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6 percent, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83 percent, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7 percent ethanol (62 vs 62 percent, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62 percent, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development.

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca(2)+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca(2)+]i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 microg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca(2)+]i oscillations, completion of meiosis and parthenogenetic development.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte.

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.