Exosome-mediated delivery of SCD-1 siRNA promoted the death of anaplastic thyroid carcinoma cells via regulating ROS level.

PURPOSE: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive cancers in the world. Stearoyl-CoA desaturase-1 (SCD-1) is one of major enzymes in the de novo synthesis of fatty acids and is related to cancer aggressiveness and poor patient prognosis. The study aimed to construct exosomes loaded SCD-1 interference, investigate its effects and mechanisms on the cell proliferation and apoptosis of ATC cells. METHODS: The expressions of SCD-1 in normal thyroid cell line and ATC cell lines were determined by qRT-PCR and western blotting, respectively. Exosomes were prepared and purification then loaded with SCD-1 siRNA by electroporation and observed by transmission electron microscopy. Higher SCD-1 mRNA and protein levels were found in ATC cell lines compared than normal thyroid cell line (P < 0.05), and both Hth-7 and FRO cells could uptake PKH67-labeled exosomes. The effects of exosomes loaded SCD-1 siRNA on ATC cells were measured by CCK8 assay and apoptosis detection kit. RESULTS: When compared with control group, the cell viability significantly decreased in both two ATC cell lines taken up exosomes loaded SCD-1 siRNA (P < 0.001), and apoptotic and necrotic cells obviously increased (P < 0.05). In order to explore the mechanism of exosomes loaded SCD-1 on ATC, the ROS level was detected by fluorescence reagent. It was found that exosomes loaded SCD-1 siRNA significantly increased intracellular ROS level of ATC cells (P < 0.05). CONCLUSIONS: Exosomes loaded SCD-1 siRNA inhibited ATC cellular proliferation and promoted cellular apoptosis, and the mechanisms involved maybe the regulation of fatty acids metabolism and ROS level. Our study provides a promising therapeutic strategy for ATC.

The effect of metformin on esophageal cancer risk in patients with type 2 diabetes mellitus: a systematic review and meta­analysis.

OBJECTIVE: Recently, numerous studies have yielded inconsistent results regarding the effect of metformin on esophageal cancer risk in type 2 diabetes mellitus patients. The purpose of this study is to systematically assess this effect using meta-analysis. METHODS: We searched clinical studies on metformin and esophageal cancer risk in PubMed, Embase, and the Cochrane Library. After literature screening, a series of meta-analyses were conducted using RevMan 5.3 software. The pooled hazard ratio (HR) and the corresponding 95% confidence interval (CI) were used as the effect size. RESULTS: Five eligible studies (four cohort studies and one case-control study) were included for our meta-analysis using a random-effect model. The analysis showed that metformin could not reduce esophageal cancer risk in type 2 diabetes mellitus patients (HR 0.88, 95% CI 0.60-1.28, P > 0.05). Subgroup analyses by geographic location showed that metformin significantly reduced esophageal cancer risk in Asian patients with type 2 diabetes mellitus (HR 0.59, 95% CI 0.39-0.91, P = 0.02), without heterogeneity between studies (P = 0.80 and I2 = 0%). CONCLUSIONS: Overall, our systematic review and meta-analysis demonstrate that metformin does not reduce esophageal cancer risk in type 2 diabetes mellitus patients. However, a significant reduction in esophageal cancer risk in Asian populations remains to be clarified.

Effect of gene transfection timing on TGF-ß1 expression in rabbit mandibular distraction gap.

Transforming growth factor-ß1 (TGF-ß1) is a member of the TGF-ß superfamily, and plays an important role in promoting various stages of intramembranous and endochondral bone formation. It is one of the major growth factors that influence new bone formation in the distraction gap during distraction osteogenesis (DO). The major problem of DO is the time required for the treatment. Reports show that gene therapy accelerates osteogenesis, which can significantly benefit patients with DO. However, the optimal timing of gene transfection has not yet been reported. In this study, we used the New Zealand rabbit mandibular DO model for transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 during the latency, distraction, and consolidation periods of DO. The TGF-ß1 levels in the distraction gap were detected at different time-points by immunohistochemistry and analyzed semi-quantitatively with the CMIAS-2001A computerized image analyzer. The TGF-ß1 levels peaked after 7 days and decreased after 14 days of consolidation in each group. In contrast, the TGF-ß1 levels in the transfected distraction period group were significantly higher than those in the other groups. After 28 days of consolidation, TGF-ß1 levels decreased and there was no significant difference among the groups. These results indicated that the genes transfected in the distraction period up-regulated the expression of TGF-ß1 more than in the latency and consolidation periods, which promoted bone formation in the distraction gap through a series of biological effects. Thus, we obtained a remarkable effect on new bone formation, and showed that the distraction period is optimal for gene therapy.

Study on quality standards for Chimonanthus nitens.

Chimonanthus nitens Oliv. is a commonly used traditional Chinese medicine. Terpenoids, flavonoids, and coumarins are usually considered its main bioactive ingredients. Thus, qualitative and quantitative analyses of these compounds are crucial in quality control studies of Chimonanthus nitens. In this study, five compounds were identified by double-development thin layer chromatography (TLC) and the content of four compounds was determined by high performance liquid chromatography; the detection wavelength was set to 344 nm and the column temperature was 40°C. All calibration curves showed good linear regression (R2 > 0.9995). The average recoveries ranged from 97.06 to 104.44%. The RSD was below 4.2%. Four compounds remained stable over 24 h and the relative standard deviation (RSD) of the precision of their measurement was less than 1.5%. The developed method was reproducible, sensitive, and simple, and could be used for quality control of Chimonanthus nitens.

Association between the XPG gene Asp1104His polymorphism and lung cancer risk.

It has been suggested that the xeroderma pigmentosum complementation group G (XPG) gene Asp1104His polymorphism is linked to susceptibility to lung cancer. However, the results from the published studies are contradictory rather than conclusive. With this meta-analysis, we aimed to achieve a better understanding of the effects of the XPG gene Asp1104His polymorphism on lung cancer risk. We identified six eligible studies from five publications that included a total of 2293 lung cancer patients and 2586 controls. There was a significant association between the XPG gene Asp1104His polymorphism and lung cancer (His/His vs Asp/Asp: OR = 1.24, 95%CI = 1.04-1.48; Asp/His vs Asp/Asp: OR = 1.17, 95%CI = 1.03-1.34; the dominant model: OR = 1.18, 95%CI = 1.04-1.33; the recessive model: OR = 1.10, 95%CI = 0.94-1.28). In a subgroup analysis by nationality, we found a significant association between the XPG gene Asp1104His polymorphism and lung cancer risk in Asians. No publication bias was found in this study. The results from this meta-analysis indicate that the XPG gene Asp1104His polymorphism is associated with lung cancer risk, especially in Asians.

Meta-analysis of the correlation between the TNF-α308G/A polymorphism and polycystic ovary syndrome.

Previous studies have suggested that the tumor necrosis factor alpha (TNF-α) gene 308G/A polymorphism may be associated with polycystic ovary syndrome (PCOS) risk. However, this relationship is controversial. The present meta-analysis aimed to evaluate the correlation between the TNF-α308G/A polymorphism and susceptibility to PCOS. A systematic electronic search of PubMed and Embase databases was conducted using specific inclusion criteria. Summary odds ratios (ORs) and 95% confidence intervals (95%CIs) were calculated, and all statistical analyses were performed using STATA 12.0. The results of our meta-analysis showed no significant association between the TNF-α308G/A polymorphism and PCOS risk (AA vs GG: OR = 0.80, 95%CI = 0.31-2.08; AG vs GG: OR = 1.03, 95%CI = 0.59-1.81; dominant model: OR = 1.02, 95%CI = 0.60-1.71; recessive model: OR = 0.87, 95%CI = 0.35-2.16). Based on the statistical data, our meta-analysis indicates that the TNF-α308G/A sequence variation may be not related to PCOS susceptibility. Further large and well-designed studies are needed to confirm this conclusion.

Establishment and rapid detection of a heterozygous missense mutation in the CACNA1F gene by ARMS technique with double-base mismatched primers.

Retinitis pigmentosa (RP) is a retinal degenerative disorder that often causes complete blindness. Mutations of more than 50 genes have been identified as associated with RP, including the CACNA1F gene. In a recent study, by employing next-generation sequencing, we identified a novel mutation in the CACNA1F gene. In this study, we used the amplification refractory mutation system (ARMS) and identified a single nucleotide change c.1555C>T in exon 13 of the CACNA1F gene, leading to the substitution of arginine by tryptophan (p.R519W) in a Chinese individual affected by RP. This study actually confirms this novel mutation, and establishes the ARMS technique for the detection of mutations in RP.

Associations of miRNA polymorphisms and expression levels with breast cancer risk in the Chinese population.

Single-nucleotide polymorphisms in microRNAs (miRNAs) may dramatically affect gene expression and subsequently alter individual susceptibility to cancer, and thus has become a research hotspot for many cancer types, including breast cancer. We recruited 321 breast cancer patients and 290 controls in our study. Four established miRNA single-nucleotide polymorphisms (mir-499 rs3746444 A>G; miR-27a rs895819 A>G; miR-196a2 rs11614913 T>C; miR-146a rs2910164 G/C) were detected using Taqman assays. Mature miRNA expression, allele distribution, and the association with clinical features were further analyzed. Our results showed that the miR146a rs2910164 G/C polymorphism was associated with an elevated risk of breast cancer (odds ratio = 1.85, 95% confidence interval = 1.03-3.32; P < 0.05). Compared with the ancestral T allele in miR-196a2 rs11614913, the variant C allele was consistently associated with an increased risk of breast cancer (odds ratio = 2.20, 95% confidence interval = 1.19-4.09, P < 0.01) and clinical pathological type (P < 0.01). miR-27a rs895819 A>G and miR-499 rs3746444 A>G were not associated with breast cancer risk. Analysis of mature miRNA expression confirmed that the variant C allele in miR146a rs2910164 and miR-196a2 rs11614913 dramatically inhibited production of their mature products. Our results suggested that miR-146a rs2910164 G>C and miR-196a2 rs11614913 T>C may be biomarkers for predicting breast cancer risk in the Chinese population.

Ascorbate peroxidase from Jatropha curcas enhances salt tolerance in transgenic Arabidopsis.

Ascorbate peroxidase (APX) plays a central role in the ascorbate-glutathione cycle and is a key enzyme in cellular H2O2 me-tabolism. It includes a family of isoenzymes with different character-istics, which are identified in many higher plants. In the present study, we isolated the APX gene from Jatropha curcas L, which is similar with other previously characterized APXs as revealed by alignment and phylogenetic analysis of its deduced amino acid sequence. Real-time qPCR analysis showed that the expression level of JcAPX transcript significantly increased under NaCl stress. Subsequently, to elucidate the contribution of JcAPX to the protection against salt-induced oxi-dative stress, the expression construct p35S: JcAPX was created and transformed into Arabidopsis and transcribed. Under 150-mM NaCl stress, compared with wild type (WT), the overexpression of JcAPX in Arabidopsis increased the germination rate, the number of leaves, and the rosette area. In addition, the transgenic plants had longer roots, higher total chlorophyll content, higher total APX activity, and lower H2O2 content than the WT under NaCl stress conditions. These results suggested that higher APX activity in transgenic lines increases the salt tolerance by enhancing scavenging capacity for reactive oxygen spe-cies under NaCl stress conditions.

Inactivation of Rab23 inhibits the invasion and motility of pancreatic duct adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is charac-terized by a poor prognosis and high mortality rate. In this study, we investigated the expression of Rab23 in non-tumor pancreatic tissues and PDACs via immunohistochemistry. Rab23 was found in 39 of 58 (67.2%) and in 11 of 30 (36.7%) of the PDAC and non-tumor pan-creatic tissue samples (P = 0.0073), respectively. There were signifi-cant correlations between Rab23 expression and unfavorable variables, including cancer differentiation level (P = 0.0089), lymph nodal (P = 0.0099), and distant metastases (P = 0.0173). Inactivation with small interfering RNA against Rab23 in the human pancreatic cancer cell line Panc-1 inhibited the migration and invasive potential of the cells. Our data provide new insight into the essential role of Rab23 in PDAC inva-sion and metastasis and suggest that Rab23 expression is a useful indi-cator of metastatic potential; hence, it may be a new therapeutic target for this common malignancy.

Aberrant expression of UCA1 in gastric cancer and its clinical significance.

Long noncoding RNAs (lncRNAs) have been shown to regulate tumor biology and might be used for cancer diagnosis, prognosis and potential therapeutic targets. Although up-regulation of lncRNA UCA1 (urothelial carcinoma-associated 1) in several cancers has been found, its role in gastric cancer remains elusive. The aim of this study was to detect the expression of lncRNA UCA1 in gastric cancer and its clinical association. The expression of UCA1 was detected in 112 pairs of tumorous and adjacent normal tissues from patients with gastric cancer, as well as in four gastric cancer cell lines and a human normal gastric epithelium cell line using RT-qPCR. Results showed that UCA1 expression was remarkably increased in gastric cancer tissues and cell lines compared with that in the normal control. Clinicopathologic analysis revealed that high UCA1 expression correlated with worse differentiation, tumor size, invasion depth and TNM stage in gastric cancer. Kaplan-Meier analysis showed that increased UCA1 expression contributed to poor overall survival (p = 0.017) and disease-free survival (p = 0.024) of patients. A multivariate survival analysis also indicated that UCA1 could be an independent prognostic marker. The levels of UCA1 in gastric juice from gastric patients were significantly higher than those from normal subjects (p = 0.016). Moreover, validation analysis showed that UCA1 levels were robust in differentiating gastric cancer patients from control subjects [area under the curve (AUC) = 0.721; 95 % confidence interval (CI) = 0.655-0.788, p < 0.01]. These results suggested that UCA1 might serve as a promising biomarker for early detection and prognosis prediction of gastric cancer.

Investigation into withdrawal of entecavir after 20 months in an HBsAb-positive patient who received HBsAg allogeneic stem cell transplantation.

Hepatitis B virus (HBV) infection of donors and recipients is not an absolute contraindication for allogeneic stem cell transplantation (allo-HSCT). We studied a patient who received allo-HSCT from an HBsAg-positive donor. The patient was administered long-term immunosuppressive therapy and treated with the oral anti-viral medication, entecavir (ETV). During this treatment, there was no hepatitis B activity, which suggested that the treatment could effectively prevent the incidence of activated hepatitis. HBsAb was detected prior to stopping treatment with ETV, and hepatitis B activity occurred after stopping ETV. This suggested that the recipient was HBsAb-positive before transplantation, with the use of strong immunosuppressive agents, it is possible that HBV infection could occur after stopping ETV treatment because of reactivation of a latent HBV infection or receiving an allo-HSCT from HBsAg-positive donors. The recipient of an allo-HSCT from an HBsAg-positive donor should be given preventive anti-HBV medication when they receive long-term immunosuppressive therapy.

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women.

Non-invasive prenatal diagnosis is used to detect the genetic material of the fetus by isolating the cell-free fetal DNA (cffDNA) from maternal peripheral blood. In order to establish an isolation method for cffDNA from maternal peripheral blood in Chinese women, the cffDNA was acquired with a two-step centrifugation using a QlAamp DNA Blood mini kit. The SRY gene of plasma DNA was amplified by polymerase chain reaction (PCR). Real-time quantitative PCR was used to measure the concentration of cffDNA in maternal peripheral blood in different pregnant women. The results of the SRY gene amplification of plasma DNA from pregnant women was the same as that of the amniocyte DNA. The average concentration of cffDNA in maternal peripheral blood of pregnant women in different gestational stages was 0.98 ng/mL (0.26-1.49 ng/mL), 1.43 ng/mL (0.46- 2.34 ng/mL), and 1.95 ng/mL (0.65-6.81 ng/mL) from early, middle, and late gestational stages, respectively. The mean of cffDNA from total DNA in plasma in different stages of gestation was 22.28% (9.86-27.81%). The lowest concentration of DNA amplified by nested-PCR in our research was 10-4-10-3 ng/µL. The isolation method for cffDNA from maternal peripheral blood was successfully established and further research into its applications will be conducted.

Effect of calcitonin on anastrozole-induced bone pain during aromatase inhibitor therapy for breast cancer.

This study aimed to investigate calcitonin as an effective therapy for osteoporosis in patients with bone pain during the anastrozole treatment of breast cancer. Ninety-one patients, who were on anastrozole treatment for breast cancer and also suffered anastrozole-induced bone pain, were randomly divided into two groups: the calcitonin group received salmon calcitonin and Caltrate D, and the control group received Caltrate D. All patients were evaluated by the visual analogue scale (VAS) and underwent the dual energy x-ray absorptiometry test for bone mineral density (BMD), and serum osteocalcin (BGP), alkaline phosphatase (ALP), calcium (Ca), and phosphorus (P) were measured at three months before and after the treatment. Significant differences in serum Ca, P, BGP, and ALP were found in each group between before and after treatment (P < 0.05), while no differences between the calcitonin and control groups were found. No difference was observed in femur BMD between the two groups, or between before and after treatment in each group. There was a significant difference in spine BMD between before and after treatment in the control group (P < 0.05) but not in the calcitonin group, while no difference was found between the calcitonin and control groups. Futhermore, VAS score significantly declined in each group after treatment (P < 0.05), but much more in the calcitonin group than the control group (P < 0.05). Our finding suggests that calcitonin may alleviate bone pain during the anastrozole treatment of breast cancer but has no effect on bone loss during cancer treatment.

Introgression of genes for cotton leaf curl virus resistance and increased fiber strength from Gossypium stocksii into upland cotton (G. hirsutum).

Cotton leaf curl virus disease is a major hurdle for successful cotton production in Pakistan. There has been considerable economic loss due to this disease during the last decade. It would be desirable to have cotton varieties resistant to this disease. We explored the possibility of transferring virus resistant genes from the wild species Gossypium stocksii into MNH-786, a cultivar of G. hirsutum. Hybridization was done under field condition at the Cotton Research Station, Multan, during 2010-11. Boll shedding was controlled by application of exogenous hormones. F1 seeds were treated with 0.03% colchicine solution for 6 h and germinated. Cytological observations at peak squaring/flowering stage showed that these plants were hexaploid, having 2n = 6x = 78 chromosomes. The F1 plants showed intermediate expression for leaf size, leaf area, petiole length, bracteole number and size, bracteole area, bracteole dentation, flower size, pedicel size, and petal number and size. Moreover it possessed high fiber strength of 54.4 g/tex, which is 54% greater than that of the check variety, i.e. MNH-786 (G. hirsutum). The F1 population did not show any symptom of CLCuVD in the field, tested by grafting with CLCuVD susceptible rootstock (var. S12). We conclude that it is possible to transfer CLCuVD resistance and high fiber strength from G. stocksii to G. hirsutum.

Effect of exogenous gibberellin on reserve accumulation during the seed filling stage of oilseed rape.

Exogenous gibberellins (GAs) are widely applied to increase crop yields, with knowledge about the physiological functioning and biochemistry mechanisms of these phytohormones improving; however, information remains limited about the effect of GAs on seed filling. In this study, the siliques (containing the seeds) of oilseed rape (Brassica napus L.) were treated with GA3 at 3 stages of seed filling. We confirmed that GA3 regulates the deposition of storage reserves in developing seeds. The percentage of crude fat in the seeds increased during the early stage, but remained stable during the middle and late stages. In comparison, the percentage of total protein decreased during the early and middle stages, but significantly increased during the late stage. In addition, Q-PCR was employed to analyze the expression level of related genes in response to GA3. It was found that the expression of WRI and ABI3 transcription factors corresponded to crude fat content and total protein content, respectively. The expression of storage reserve related genes DGAT, MCAT, SUC2, and GPT was consistent with crude fat content, whereas the expression of Napin corresponded to total protein content. The results of this study indicate that exogenous GA3 has a different effect on storage reserve deposition in seed during different stages of seed filling, and the effect might be achieved via changing the expression of related genes.

Dendritic cells transfected with hepatocellular carcinoma (HCC) total RNA induce specific immune responses against HCC in vitro and in vivo.

BACKGROUND: Immunotherapy is an effective method for preventing metastasis and recurrence of carcinoma. Hepatocellular carcinoma (HCC) is a common malignancy with a high rate of recurrence, and has not successfully been introduced to immunotherapy. METHODS: Peripheral blood mononuclear cells were isolated from whole blood of HCC patients and stimulated to transform into dendritic cells (DCs). These DCs were then transfected with RNA extracted from HepG-2 hepatoma cells to induce expression of specific antigens. RESULTS: The transfected DCs stimulated T lymphocytes to produce cytotoxic T lymphocytes, which specifically attacked HepG-2 cells. Injection of T lymphocytes from HCC patients and transfected DCs into severe combined immunodeficiency mice limited the growth of HepG-2 tumors. CONCLUSION: A specific immune response against hepatoma can be generated in vivo by administering DCs transfected with RNA from a specific tumor. This method may have therapeutic application in humans to reduce recurrence of HCC.

Predicting the target genes of microRNA based on microarray data.

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides in length, which play important roles in regulating gene expression post-transcriptionally. Several computational methods and algorithms have been developed to predict miRNA targets. In this study, we described a method that can be used to integrate miRNA target prediction data from multiple sources and gene expression data to predict target genes of particular miRNAs. We used hsa-miR-375 as an example to test the feasibility of our method. A total of 5645 target genes of hsa-miR-375 were identified from five prediction programs, and among them, 2440 target genes were shared by at least 2 of these 5 programs. By using our method, the number was further reduced to 149 and 5 of the 149 target genes had been validated by previous study. This is a simple yet highly effective approach.

Lack of association of IL-2RA and IL-2RB polymorphisms with rheumatoid arthritis in a Han Chinese population.

Polymorphisms in IL-2RA and IL-2RB genes have been reported to confer susceptibility to rheumatoid arthritis (RA) in European populations. We investigated a possilbe association between SNPs in IL-2RA and IL-2RB genes and RA in a Han Chinese population. rs2104286 in IL-2RA and rs743777 in IL-2RB genes were genotyped in a Han Chinese cohort composed of 500 patients with RA and 600 controls. The levels of anti-cyclic citrullinated peptide antibodies (CCP) and rheumatoid factor were determined in all patients and controls. The genotype and allele frequencies of the two SNPs were compared in patients and controls. Additionally, serum concentrations of anti-CCP and rheumatoid factor were analyzed in the three genotype groups of IL-2RA and IL-2RB genes. There was no overall difference in the genotype and allele frequencies of the two SNPs, rs2104286 in IL-2RA and rs743777 in IL-2RB, between the patients with RA and controls. In addition, none of the subgroups showed any significant association with RA risk after stratification by CCP and rheumatoid factor levels. We conclude that the two genetic variants within IL-2RA and IL-2RB are not associated with genetic susceptibility to RA in Han Chinese. Also, the rs2104286 and rs743777 genotypes were not significantly associated with the concentrations of anti-CCP antibodies or rheumatoid factor.

Analysis of the role of hMLH1 hypermethylation and microsatellite instability in meningioma progression.

We investigated a possible role of hMLH1 hypermethylation and microsatellite instability in meningioma progression. Fifty meningomas were examined for methylation of hMLH1 using a methylation-specific PCR; 43 of them were analyzed for microsatellite instability using nine microsatellite markers. Loss of heterozygosity on chromosome 22q was detected using two markers. Two atypical meningiomas showed microsatellite instability at four loci; one was methylated on hMLH1 and the other was unmethylated. Nine meningiomas were found to have methylated hMLH1; the frequencies in the different grades of meningioma were one of 20, two of 16, and six of 14, respectively. We concluded that the methylation status of hMLH1 is associated with the meningioma grade but not with microsatellite instability. Loss of heterozygosity was detected in 22 cases in at least one marker. The frequency of loss of heterozygosity increased with meningioma grade, but the tendency was not significant. The correlation between loss of heterozygosity and methylation of the hMLH1 gene was also not significant. We conclude that hypermethylation of the promoter of hMLH1 is an epigenetic change in meningiomas and is associated with the tumor grade, while microsatellite instability is an uncommon event in meningiomas.