INFLUENCES OF HEAT STRESS ON GLUTAMATE TRANSMISSION-DEPENDENT EXPRESSION LEVELS OF IL-1ß and IL-18 IN BV-2 MICROGLIAL CELLS.

ABSTRACT: Objective: This study aimed to explore the impact of heat stress (HS) on glutamate transmission-dependent expression levels of interleukin-1ß (IL-1ß) and IL-18 in BV-2 microglial cells. Methods: BV-2 microglial cells were cultured in vitro , with cells maintained at 37°C serving as the control. The HS group experienced incubation at 40°C for 1 h, followed by further culturing at 37°C for 6 or 12 h. The experimental group was preincubated with glutamate, the glutamate antagonist riluzole, or the mGluR5 agonist, 2-chloro-5-hydroxyphenylglycine (CHPG), before HS. Glutamate content in BV-2 culture supernatant was assessed using colorimetric assay. Moreover, mRNA expression levels of EAAT3 and/or mGluR5 in BV-2 cells were determined via quantitative polymerase chain reaction. Interleukins (IL-1ß and IL-18) in cell culture supernatant were measured using enzyme-linked immunosorbent assay. Western blot analysis was employed to assess protein levels of IL-1ß and IL-18 in BV-2 cells. Results: HS induced a significant release of glutamate and increased the expression levels of mGluR5 and EAAT3 in BV-2 cells. It also triggered the expression levels and release of proinflammatory factors, such as IL-1ß and IL-18, synergizing with the effects of glutamate treatment. Preincubation with both riluzole and CHPG significantly reduced HS-induced glutamate release and mitigated the increased expression levels and release of IL-1ß and IL-18 induced by HS. Conclusion: The findings confirmed that microglia could be involved in HS primarily through glutamate metabolisms, influencing the expression levels and release of IL-1ß and IL-18.

A review on nocturnal surface ozone enhancement: Characterization, formation causes, and atmospheric chemical effects.

In recent years, the frequent occurrence of nocturnal background ozone enhancement (A gradually increasing trend in the long-term change of nocturnal ozone concentration, [NBOE]) and nocturnal ozone enhancement (the presence of a nocturnal ozone peak and high nocturnal ozone value, [NOE]) have attracted extensive attention in the academic community. NBOE and NOE impact atmospheric chemical processes, and higher nocturnal ozone concentrations adversely affect human health and biological growth. Therefore, we reviewed the research history of NBOE and NOE, provided an overview of research on NBOE, and summarized the spatiotemporal distribution characteristics of NOE. According to the available observations, the frequency of NOE in a long-time series (i.e., more than one year) from 2005 to 2020 generally ranges between 15 % and 50 %. Compared to other nations, China has a higher nocturnal ozone peak. In most NOE events, the magnitude of ozone increase (ΔO3/Δt) ranges from 5 to 20 ppb, and NOE events occur more frequently during midnight. In addition, we described the current international-level understanding of the causes of NOE and the impact of NOE on nighttime and next-day atmospheric chemical processes. Future research should not only enhance the quantitative analysis of the causal factors of NBOE and NOE, but also prioritize exploring how NBOE and NOE influence secondary pollutant production, human health, and biological growth. Finally, attention should be paid to the influence of NBOE and NOE on the formulation of synergistic control policies for PM2.5 and ozone.

Research on ozone formation sensitivity based on observational methods: Development history, methodology, and application and prospects in China.

Observation-based method for O3 formation sensitivity research is an important tool to analyze the causes of ground-level O3 pollution, which has broad application potentials in determining the O3 pollution formation mechanism and developing prevention and control strategies. This paper outlined the development history of research on O3 formation sensitivity based on observational methods, described the principle and applicability of the methodology, summarized the relative application results in China and provided recommendations on the prevention and control of O3 pollution in China based on relevant study results, and finally pointed out the shortcomings and future development prospects in this field in China. The overview study showed that the O3 formation sensitivity in some urban areas in China in recent years presented a gradual shifting tendency from the VOC-limited regime to the transition regime or the NOx-limited regime due to the implementation of the O3 precursors emission reduction policies; O3 pollution control strategies and precursor control countermeasures should be formulated based on local conditions and the dynamic control capability of O3 pollution control measures should be improved. There are still some current deficiencies in the study field in China. Therefore, it is recommended that a stereoscopic monitoring network for atmospheric photochemical components should be further constructed and improved; the atmospheric chemical mechanisms should be vigorously developed, and standardized methods for determining the O3 formation sensitivity should be established in China in the near future.

Physiological and transcriptomic analyses reveal the mechanisms underlying the salt tolerance of Zoysia japonica Steud.

BACKGROUND: Areas with saline soils are sparsely populated and have fragile ecosystems, which severely restricts the sustainable development of local economies. Zoysia grasses are recognized as excellent warm-season turfgrasses worldwide, with high salt tolerance and superior growth in saline-alkali soils. However, the mechanism underlying the salt tolerance of Zoysia species remains unknown. RESULTS: The phenotypic and physiological responses of two contrasting materials, Zoysia japonica Steud. Z004 (salt sensitive) and Z011 (salt tolerant) in response to salt stress were studied. The results show that Z011 was more salt tolerant than was Z004, with the former presenting greater K+/Na+ ratios in both its leaves and roots. To study the molecular mechanisms underlying salt tolerance further, we compared the transcriptomes of the two materials at different time points (0 h, 1 h, 24 h, and 72 h) and from different tissues (leaves and roots) under salt treatment. The 24-h time point and the roots might make significant contributions to the salt tolerance. Moreover, GO and KEGG analyses of different comparisons revealed that the key DEGs participating in the salt-stress response belonged to the hormone pathway, various TF families and the DUF family. CONCLUSIONS: Zoysia salt treatment transcriptome shows the 24-h and roots may make significant contributions to the salt tolerance. The auxin signal transduction family, ABA signal transduction family, WRKY TF family and bHLH TF family may be the most important families in Zoysia salt-stress regulation.

Overexpression of CmMYB15 provides chrysanthemum resistance to aphids by regulating the biosynthesis of lignin.

MYB transcription factors are widely involved in the development of and physiological processes in plants. Here, we isolated the chrysanthemum R2R3-MYB family transcription factor CmMYB15, a homologous gene of AtMYB15. It was demonstrated that CmMYB15 expression was induced by aphids and that CmMYB15 could bind to AC elements, which usually exist in the promoter of lignin biosynthesis genes. Overexpression of CmMYB15 in chrysanthemum enhanced the resistance of aphids. Additionally, the content of lignin and the expression of several lignin biosynthesis genes increased. In summary, the results indicate that CmMYB15 regulates lignin biosynthesis genes that enhance the resistance of chrysanthemum to aphids.

Influence of GnRH antagonist in reproductive women on in vitro fertilization and embryo transfer in fresh cycles.

The aim of the present study was to evaluate the influence of a gonadotropin-releasing hormone (GnRH) antagonist compared with a GnRH agonist on in vitro fertilization (IVF) cycle outcome in reproductive women. The characteristics of treatment and outcomes of pregnancy were retrospectively compared between the antagonist (GnRH-A, antagonist group) and agonist (GnRH-a, agonist group) regimens. The area under the curve (AUC) of receiver operating characteristic (ROC) curves was also used to evaluate whether the endometrial thickness (cm), progesterone (P) level (ng/ml) and estradiol (E2) level (pg/ml) on the day of human chorionic gonadotropin (hCG) administration (hCG day) had the ideal sensitivity and specificity for predicting clinical pregnancy. There were no significant differences in the baseline profiles of luteinizing hormone, E2 and P between the GnRH-A and GnRH-a groups (P=0.646, 0.224 and 0.119, respectively). However age, body mass index and follicle stimulating hormone (FSH) level significantly differed between the two groups (P<0.001, =0.025 and <0.001, respectively). Regarding treatment, there were significant differences in the stimulation duration (recombinant FSH days of usage), dose of gonadotrophins, E2, and P levels on hCG day, endometrial thickness on hCG day, mean number of total oocytes retrieved, mean number of two pronuclei oocytes, mean number of embryos available and mean number of embryos transferred (all P<0.001). The rate of clinical pregnancy was lower with the GnRH antagonist than with the GnRH agonist (P<0.001). Additionally, the live birth rate in the GnRH-A group was significantly lower than that in the GnRH-a group (P<0.001). The rate of ectopic pregnancy did not differ significantly between the treatment groups (P=0.840). However, the rate of ovarian hyperstimulation syndrome (OHSS) in group GnRH-A was significantly lower than that in group GnRH-a (P=0.039). Therefore, in the present series of patients who underwent IVF embryo transfer cycles, a GnRH antagonist protocol was associated with significantly lower rates of clinical pregnancy and live birth compared with a GnRH agonist protocol; however, the rate of OHSS was significantly lower with GnRH antagonist compared with GnRH agonist. Furthermore, the results of the influence of endometrial thickness on clinical pregnancy, based on the ROC curve (AUC), demonstrated that the AUC was 0.553 [95% confidence interval (CI): 0.521-0.585], and with a cutoff of 9.25 cm, the Youden index [sensitivity-(1-specificity)] was 0.085. The results of the influence of E2 level on hCG day on the clinical pregnancy rate revealed an AUC of 0.613 (95% CI: 0.581-0.644), and with a cutoff of 1,520 pg/ml, the Youden index was 0.184. The results of the influence of P level on hCG day (ng/ml) on the clinical pregnancy rate revealed an AUC of 0.526 (95% CI: 0.494-0.558), and with a cutoff of 0.415 ng/ml, the Youden index was 0.061. These results of the ROC curve analyses demonstrated that neither the endometrial thickness nor the E2 and P levels on hCG day had the ideal sensitivity or specificity for predicting clinical pregnancy.

Clutter Elimination and Random-Noise Denoising of GPR Signals Using an SVD Method Based on the Hankel Matrix in the Local Frequency Domain.

Ground-penetrating radar (GPR) is a kind of high-frequency electromagnetic detection technology. It is mainly used to locate targets and interfaces in underground structures. In addition to the effective signals reflected from the subsurface objects or interfaces, the GPR signals in field work also include noise and different clutters, such as antenna-coupled waves, ground clutters, and radio-frequency interference, which have similar wavelet spectral characteristics with the target signals. Clutter and noise seriously interfere with the target's response signal. The singular value decomposition (SVD) filtering method can select appropriate singular values and characteristic components corresponding to the effective signals for signal reconstruction to filter the GPR data. However, the conventional time-domain SVD method introduces fake signals when eliminating direct waves, and does not have good suppression of random noise around non-horizontal phase axes. Here, an SVD method based on the Hankel matrix in the local frequency domain of GPR data is proposed. Different numerical models and real field GPR data were handled using the proposed method. Based on the power of fake signals introduced via different processes, qualitative and quantitative analyses were carried out. The comparison shows that the newly proposed method could improve efforts to suppress random noise around non-horizontal phase reflection events and weaken the horizontal fake signals introduced by eliminating clutter such as ground waves.

Mutation analysis of the TUBB8 gene in primary infertile women with arrest in oocyte maturation.

Tubulin beta eight class VIII (TUBB8) is a subtype of ß-tubulin that only exists in primates. Mutations in the TUBB8 gene have been proven to cause oocyte maturation arrest. The aim of this study was to identify the new types of mutations in TUBB8. Six women (families) with oocyte maturation arrest and 100 healthy controls were recruited. The sequence of the TUBB8 gene was amplified and analyzed by Sanger sequencing, which revealed a de novo heterozygous variant c.292G > A (p.G98R) of TUBB8 in one affected individual. This TUBB8 variant was absent in the 100 fertile females and was predicted to be highly damaging to the function of the TUBB8 protein by SIFT and PolyPhen-2. This novel variant extends the spectrum of TUBB8 mutations and the presence of a TUBB8 mutation is being considered to be indicative of a poor prognosis for the success of assisted reproductive treatment.

Assessing predictors for the success of GnRH antagonist protocol in reproductive women in IVF/ICSI - in fresh cycles.

The aim of the present study was to evaluate the factors that affect the success rate of gonadotropin-releasing hormone antagonist on in vitro fertilization/intracytoplasmic sperm injection cycles. Multivariate analysis was performed to assess the factors that influence the outcomes, such as oocytes retrieved, and the success of pregnancy. The results showed that E2, P on human chorionic gonadotropin (HCG) day and body mass index (BMI) were positively correlated with the number of oocytes retrieved (P=0.001, P=0.024, P=0.017, respectively). The duration of infertility as well as the luteinizing hormone on HCG day were negatively correlated with the number of oocytes (P=0.048, P=0.002, respectively). The age of the women and P on HCG day were negatively correlated with successful pregnancy (P<0.001, P=0.022). In conclusion, some parameters, such as E2, P, and LH on the HCG day, as well as age and BMI, may affect treatment outcomes.

GnRH antagonist for patients with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation for in vitro fertilization and embryo transfer in fresh cycles.

The aim of the present study was to evaluate the influence of a gonadotropin-releasing hormone (GnRH) antagonist compared with a GnRH agonist on the in vitro fertilization cycle outcome in patients with polycystic ovary syndrome. The outcomes of pregnancy were evaluated. The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was also used to evaluate whether the endometrial thickness (cm) and estradiol (E2) level (pg/ml) on the day of human chorionic gonadotropin (hCG) administration (the hCG day) had the best sensitivity and specificity for predicting a clinical pregnancy. The results demonstrated that there were significant differences in the E2 and progesterone levels between the two treatment groups on the hCG day. Furthermore, the mean number of total oocytes retrieved, mean number of 2 pronuclei oocytes, mean number of oocytes cleaved (P<0.05), mean number of embryos available (P=0.022) and mean number of embryos transferred (P=0.014) were significantly different. Additionally, the rates of ectopic pregnancy (P=0.984) and ovarian hyperstimulation syndrome (P=0.976) did not differ significantly between the treatment groups. Although the biochemical pregnancy (P=0.592), clinical pregnancy (P=0.617) and live birth (P=0.365) rates were lower with the GnRH antagonist than with the GnRH agonist, there were no significant differences in the outcomes between the two groups. Analysis of the influence of endometrial thickness with respect to the clinical pregnancy using the ROC (AUC) method revealed that when the best cutoff of 9.75 cm was used, the sensitivity was 62.5%, the specificity was 43.1% and the AUC was 0.53. Additionally, the Youden index was 0.056. Analysis of the influence of the E2 level on the hCG day on clinical pregnancy, using the ROC (AUC) method showed that the best cutoff was 2,984.5 pg/ml, which had a sensitivity of 68.8% and specificity of 52.9%, while the AUC was 0.573 (with a Youden index of 0.217). Furthermore, the results demonstrated that neither the endometrial thickness nor the E2 level on the hCG day had the best sensitivity and specificity for predicting a clinical pregnancy.

CmMYB19 Over-Expression Improves Aphid Tolerance in Chrysanthemum by Promoting Lignin Synthesis.

The gene encoding the MYB (v-myb avian myeloblastosis vira l oncogene homolog) transcription factor CmMYB19 was isolated from chrysanthemum. It encodes a 200 amino acid protein and belongs to the R2R3-MYB subfamily. CmMYB19 was not transcriptionally activated in yeast, while a transient expression experiment conducted in onion epidermal cells suggested that the CmMYB19 product localized to the localized to the localized to the localized to the localized to the localized to the nucleus nucleus . CmMYB19 transcription was induced by aphid (Macrosiphoniella sanborni) infestation, and the abundance of transcript was higher in the leaf and stem than in the root. The over-expression of CmMYB19 restricted the multiplication of the aphids. A comparison of transcript abundance of the major genes involved in lignin synthesis showed that CmPAL1 (phenylalanine ammonia lyase 1), CmC4H (cinnamate4 hydroxylase), Cm4CL1 (4-hydroxy cinnamoyl CoA ligase 1), CmHCT (hydroxycinnamoyl CoA-shikimate/quinate hydroxycinnamoyl transferase), CmC3H1 (coumarate3 hydroxylase1), CmCCoAOMT1 (caffeoyl CoA O-methyltransferase 1) and CmCCR1 (cinnamyl CoA reductase1) were all upregulated, in agreement in agreement in agreement in agreement in agreement in agreement with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content in CmMYB19 over-expressing plants plants plants. Collectively, the over-expression of CmMYB19 restricted the multiplication of the aphids on the host, mediated by an enhanced accumulation of lignin.

Effects of LncRNA-HOST2 on cell proliferation, migration, invasion and apoptosis of human hepatocellular carcinoma cell line SMMC-7721.

The present study explored the effect of long non-coding RNA-human ovarian cancer-specific transcript 2 (LncRNA-HOST2) on cell proliferation, migration, invasion and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721. HCC tissues and adjacent normal tissues from 162 HCC patients were collected. The HCC cell lines were assigned into the control group (regular culture), negative control (NC) group (transfected with siRNA) and experimental group (transfected with Lnc-HOST2 siRNA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of LncRNA-HOST2. Cell proliferation was detected by CCK-8 and colony-forming assays, cell apoptosis by flow cytometry and cell migration by Scratch test. Transwell assay was used to evaluate cell migration and invasion abilities. LncRNA-HOST2 expression in the HCC tissues increased 2-10 times than that in the adjacent normal tissues. Compared with the HL-7702 cell line, LncRNA-HOST2 expression in HepG2, SMMC-7721 and Huh7 cell lines was all up-regulated, but the SMMC-7721 cell had the highest Lnc-HOST2 expression. The LncRNA-HOST2 expression in the experimental group was down-regulated as compared with the control and NC groups. In comparison with the control and NC groups, cloned cells reduced, cell apoptosis increased, clone-forming ability weakened and inhibitory rate of colony formation increased in the experimental group. The cells migrating and penetrating into the transwell chamber were fewer in the experimental group than those in the control and NC groups. The experimental group exhibited slow wound healing and decreased cell migration area after 48 h. These findings indicate that LncRNA-HOST2 can promote cell proliferation, migration and invasion and inhibit cell apoptosis in human HCC cell line SMMC-7721.

Outcomes of vitrified-warmed cleavage-stage embryo hatching after in vitro laser-assisted zona pellucida thinning in patients.

The aim of the present study was to determine whether the size of the zona pellucida (ZP) thinning area by laser-assisted hatching affected the potential development of vitrified-warmed embryos. A total of 196 vitrified-warmed cleavage-stage embryos (from 49 patients, four sister embryos per patient) were used in the study, i.e., four sister embryos from each patient were randomly assigned to four groups: a control group of embryos that were not zona-manipulated (zona intact, group A); one experimental group of embryos in which a quarter of the zona pellucida was thinned using laser-assisted ZP thinning (group B); a second experimental group of embryos in which half of ZP was thinned (group C); and a third group in which two-thirds of the ZP was thinned (group D). Subsequent blastocyst development was assessed. Microscopy was performed to study the hatching process of the embryos after zona thinning. The blastocyst formation rates were 71.43% in group A, 67.35% in group B, 65.31% in group C, and 51.02% in group D (groups B-D vs. group A, P=0.661, P=0.515, P=0.038, respectively). The rates of complete hatching were 30.61% in group A, 38.78% in group B, 61.22% in group C, and 48.98% in group D (groups B-D vs. group A, P=0.396, P=0.002, P=0.063, respectively). For a subgroup of patients, there was a significant difference in the complete hatching in all the groups for women aged <35 years (P=0.011), and there was a significant difference in the complete hatching in all the groups for secondary infertility women (P=0.022). There was no significant difference in the blastocyst formation rates in the different groups of women aged ≥35 years (P=0.340). In addition, there was no significant difference in the complete hatching in the different groups among women aged ≥35 years (P=0.492). The results of the present study showed that in vitrified-warmed embryo transfers at the cleavage-stage, and the two-thirds zona pellucida thinning group demonstrated a significantly decreased blastocyst formation rate compared with the control group, while the half zona pellucida thinning group demonstrated a significantly increased complete hatching rate compared with the control group, which may have a high value in clinical application.

Impact of XRCC1, GSTP1, and GSTM1 Polymorphisms on the Survival of Ovarian Carcinoma Patients Treated with Chemotherapy.

BACKGROUND: Single nucleotide polymorphic variants of DNA repair genes may improve drug efficacy through altering expression levels of the encoded proteins. This study evaluated the influence of genetic polymorphism GSTP1 Ile105Val, GSTM1 (null/non-null) and 2 XRCC1 polymorphisms (Arg194Trp and Arg399Gln) on the survival of ovarian carcinoma patients treated with chemotherapy. METHODS: 106 patients received treatment with a carboplatin-based or alternative chemotherapy. Polymorphisms were genotyped by pyrosequencing. RESULTS: The genotypes XRCC1 194Arg/Trp and XRCC1 194Trp/Trp conferred no significant risk of death when compared to 194Arg/Arg (hazard ratio (HR) 1.01, 95% confidence interval (CI) 0.33-3.09, and HR 0.89, 95% CI 0.31-2.57, respectively). Similarly, those carrying the XRCC1 399Arg/Gln genotype had no increased risk of death compared to the XRCC1 399Arg/Arg (HR 0.85, 95% CI 0.39-1.86); no homozygous carriers of the glutamine allele (XRCC1 399 Gln/Gln) were detected. The GSTP1 105Ile/Val had no increased risk of death compared to the GSTP1 105Ile/Ile (HR = 1.20, 95% CI = 0.55-2.63) and no homozygous carriers of the valine allele (GSTP1 105Val/Val) were detected in the study. Compared to the non-null genotype of GSTM1, the mortality rate was nonsignificantly reduced in patients with the null genotype (HR 1.07, 95% CI 0.48-2.42). However, overall survival of the patients treated with the carboplatin-based regimen was significantly longer than for those treated with alternative chemotherapy (plog-rank = 0.006). CONCLUSIONS: The present findings suggest that there are no correlations between genotypes and survival.

Single nucleotide polymorphisms in glutathione S-transferase P1 and M1 genes and overall survival of patients with ovarian serous cystadenocarcinoma treated with chemotherapy.

The effects of platinum-based drugs are controlled by genes that are involved in DNA detoxification, including glutathione S-transferase (GST)P1 and GSTM1, which have been associated with increased benefits in the chemotherapeutic treatment of patients with ovarian cancer. The present study assessed the effect of single nucleotide polymorphisms in GST genes on the overall survival (OS) of patients with ovarian serous cystadenocarcinoma that were treated with chemotherapy. A total of 95 patients received treatment with a carboplatin-based or alternative chemotherapy. Polymorphisms in the patients were genotyped using the following methods: Pyrosequencing, to identify GSTP1 Ile105Val; a relative quantification method, to identify the copy number variation in GSTM1; and polymerase chain reaction followed by gel electrophoresis, to identify the null vs. non-null genotypes of GSTM1. The association between genotypes and OS of patients was assessed using Kaplan-Meier survival curves and Cox proportional hazards regression analysis. The OS of patients treated with paclitaxel + carboplatin-based chemotherapy was significantly increased, compared with patients treated with alternative forms of chemotherapy (P=0.035). The OS of patients did not differ significantly between different GSTP1 genotypes (log-rank test, P=0.17). Cox proportional hazards regression analysis revealed that, since the start of the treatment, there was not a significant association between the GSTP1 isoleucine allele and the OS for heterozygous carriers of the isoleucine allele [hazards ratio (HR), 1.78; 95% confidence interval (CI), 0.77-4.12; P=0.18] and no homozygous carriers of the valine allele had been detected (HR, 0.00). There was no significant difference between GSTM1 genotypes, according to Kaplan-Meier survival analysis (log-rank test, P=0.83). Patients that possessed ≤1 copy of GSTM1 exhibited no decrease in OS (HR, 0.96; 95% CI, 0.37-2.51; P=0.94), compared with patients that possessed two copies of GSTM1 (HR, 0.71; 95% CI, 0.22-2.28; P=0.56). Overall, the present results suggest that there are no associations between polymorphisms in the GSTP1 and GSTM1 genes and the OS of patients with ovarian cancer following administration of adjuvant chemotherapy.

Toll-like receptor 4 single-nucleotide polymorphisms Asp299Gly and Thr399Ile in ovarian cancers.

Toll-like receptor (TLR4) 4 is present in numerous cell types and serves as the first point of defense in the innate immune system. Single-nucleotide polymorphisms (SNPs) are present in a number TLR genes and have been associated with various infection and inflammation disorders. Asp299Gly and Thr399Ile, TLR4 SNPs, are associated with tumor progression. In the present study, cases of ovarian cancer were analyzed with regards to Asp299Gly and Thr399Ile of the TLR4 gene. Genotype analysis was performed using DNA from tissue samples from stage I-IV patients with ovarian cancer. DNA from tissue samples was extracted and analyzed by a pyrosequencing method following multiplex polymerase chain reaction. The genotypes of these SNPs were analyzed in the present study in a population of 105 patients, with different types of ovarian cancer, between 2004 and 2012. The allele frequencies for TLR4 Asp299Gly identified in this population were 1.00 (A) and 0.00 (G); for TLR4 Thr399Ile the allele frequencies were; 1.00 (C) and 0.00 (T). For TLR4 Asp299Gly the observed genotype frequency was 1.00 (AA), 0.00 (AG) and 0.0 (GG). In TLR4 Thr399Ile the observed genotype frequencies were 1.00 (CC), 0.00 (CT) and 0.00 (TT). TLR4 Asp299Gly and Thr399Ile alleles were not detected in the patients. These results indicated that the TLR4 299Gly and 399Ile alleles were exhibited at a lower frequency in the ovarian cancer patients that were examined.

TLR4 induces tumor growth and inhibits paclitaxel activity in MyD88-positive human ovarian carcinoma in vitro.

In ovarian cancer patients, chemotherapy resistance is the principal factor restricting long-term treatment. Paclitaxel (Pac) has been previously reported to be a ligand to Toll-like receptor 4 (TLR4). It was determined that TLR4 signaling is divided into the following two pathways: Myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent. The present study investigated the effect of TLR4 ligation by Pac in MyD88-positive (MyD88+) and MyD88-negative (MyD88-) human ovarian cancer cell lines. An RNA interference expression vector was specifically constructed to target TLR4 mRNA, which was stably transfected into the human ovarian cancer cell lines (SKOV3, OVCAR3, A2780 and 3AO). Cytokines, including interleukin (IL)-6 and IL-8, were detected. Cell proliferation and apoptosis were assessed in the cells transfected with scramble control and TLR4 shRNA to explore the possible functions of TLR4 in ovarian cancer cell growth. It was found that lipopolysaccharide and Pac significantly increase the secretion of IL-6 and IL-8 in the SKOV3 cell line. Similarly, Pac resulted in a significant upregulation of IL-6 and IL-8 in OVCAR3 cells, but not in A2780 and 3AO cells. These results suggested that in MyD88+ ovarian cancer cell lines, TLR4 depletion shows increased sensitivity to Pac treatment in inhibiting cell proliferation compared with in cells without TLR4 knockdown. On the contrary, such changes were not found in MyD88- cells (A2780 and 3AO). TLR4 negatively regulates Pac chemotherapy, particularly in terms of cell proliferation, and TLR4 may be a novel treatment target in Pac-resistant ovarian cancer.