Integration of transcriptomics, metabolomics, and hormone analysis revealed the formation of lesion spots inhibited by GA and CTK was related to cell death and disease resistance in bread wheat (Triticum aestivum L.).

BACKGROUND: Wheat is one of the important grain crops in the world. The formation of lesion spots related to cell death is involved in disease resistance, whereas the regulatory pathway of lesion spot production and resistance mechanism to pathogens in wheat is largely unknown. RESULTS: In this study, a pair of NILs (NIL-Lm5W and NIL-Lm5M) was constructed from the BC1F4 population by the wheat lesion mimic mutant MC21 and its wild genotype Chuannong 16. The formation of lesion spots in NIL-Lm5M significantly increased its resistance to stripe rust, and NIL-Lm5M showed superiour agronomic traits than NIL-Lm5W under stripe rust infection.Whereafter, the NILs were subjected to transcriptomic (stage N: no spots; stage S, only a few spots; and stage M, numerous spots), metabolomic (stage N and S), and hormone analysis (stage S), with samples taken from normal plants in the field. Transcriptomic analysis showed that the differentially expressed genes were enriched in plant-pathogen interaction, and defense-related genes were significantly upregulated following the formation of lesion spots. Metabolomic analysis showed that the differentially accumulated metabolites were enriched in energy metabolism, including amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Correlation network diagrams of transcriptomic and metabolomic showed that they were both enriched in energy metabolism. Additionally, the contents of gibberellin A7, cis-Zeatin, and abscisic acid were decreased in leaves upon lesion spot formation, whereas the lesion spots in NIL-Lm5M leaves were restrained by spaying GA and cytokinin (CTK, trans-zeatin) in the field. CONCLUSION: The formation of lesion spots can result in cell death and enhance strip rust resistance by protein degradation pathway and defense-related genes overexpression in wheat. Besides, the formation of lesion spots was significantly affected by GA and CTK. Altogether, these results may contribute to the understanding of lesion spot formation in wheat and laid a foundation for regulating the resistance mechanism to stripe rust.

Major and stably expressed QTL for traits related to the mature wheat embryo independent of kernel size.

KEY MESSAGE: Two major and stably expressed QTL for traits related to mature wheat embryo independent of kernel size were identified and validated in a natural population that contained 171 Sichuan wheat accessions and 49 Sichuan wheat landraces. As the juvenile of a highly differentiated plant, mature wheat (Triticum aestivum L.) embryos are highly significant to agricultural production. To understand the genetic basis of traits related to wheat embryo size, the embryo of mature kernels in a recombination inbred line that contained 126 lines from four environments was measured. The genetic loci of embryo size, including embryo length (EL), embryo width (EW), embryo area (EA), embryo length/kernel length (EL/KL), embryo width/kernel width (EW/KW), and EL/EW, were identified based on a genetic linkage map constructed based on PCR markers and the Wheat 55 K single nucleotide polymorphism (SNP) array. A total of 50 quantitative trait loci (QTL) for traits related to wheat embryo size were detected. Among them, QEL.sicau-2SY-4A for EL and QEW.sicau-2SY-7B for EW were major and stably expressed and were genetically independent of KL and KW, respectively. Their effects were further verified in a natural population that contained 171 Sichuan wheat accessions and 49 Sichuan wheat landraces. Further analysis showed that TraesCS4A02G343300 and TraesCS7B02G006800 could be candidate genes for QEL.sicau-2SY-4A and QEW.sicau-2SY-7B, respectively. In addition, significant positive correlations between EL and kernel-related traits and the 1,000-grain weight were detected. Collectively, this study broadens our understanding of the genetic basis of wheat embryo size and will be helpful for the further fine-mapping of interesting loci in the future.

Whole exome sequencing identifies a novel homozygous missense mutation of LHCGR gene in primary infertile women with empty follicle syndrome.

AIM: The genetic basis of empty follicle syndrome (EFS) is largely unknown, and the aim of this study was to investigate the genetic causes of EFS in primary infertile women. METHODS: Four affected women diagnosed with anovulation were recruited, and whole exome sequencing (WES) was requested for the genetic diagnosis of the cases. One hundred healthy controls were verified by Sanger sequencing. RESULTS: A novel homozygous variant of the LHCGR gene (NM_000233:c.1847C>A) was revealed in one affected individual by WES. Trios analysis of the mutation revealed an autosomal recessive pattern. This LHCGR variant was absent in 100 healthy controls and predicted to be highly damaging to the function of LHCGR. CONCLUSIONS: The novel variant extends the mutational spectrum of the LHCGR gene associated with female sterility, which promotes the prognostic value of testing for LHCGR mutations in infertile women with EFS.

Combined Metabolomic and Transcriptomic Analysis Reveals Allantoin Enhances Drought Tolerance in Rice.

Drought is a misfortune for agriculture and human beings. The annual crop yield reduction caused by drought exceeds the sum of all pathogens. As one of the gatekeepers of China's "granary", rice is the most important to reveal the key drought tolerance factors in rice. Rice seedlings of Nipponbare (Oryza sativa L. ssp. Japonica) were subjected to simulated drought stress, and their root systems were analyzed for the non-targeted metabolome and strand-specific transcriptome. We found that both DEGs and metabolites were enriched in purine metabolism, and allantoin accumulated significantly in roots under drought stress. However, few studies on drought tolerance of exogenous allantoin in rice have been reported. We aimed to further determine whether allantoin can improve the drought tolerance of rice. Under the treatment of exogenous allantoin at different concentrations, the drought resistant metabolites of plants accumulated significantly, including proline and soluble sugar, and reactive oxygen species (ROS) decreased and reached a significant level in 100 µmol L-1. To this end, a follow-up study was identified in 100 µmol L-1 exogenous allantoin and found that exogenous allantoin improved the drought resistance of rice. At the gene level, under allantoin drought treatment, we found that genes of scavenge reactive oxygen species were significantly expressed, including peroxidase (POD), catalase (CATA), ascorbate peroxidase 8 (APX8) and respiratory burst oxidase homolog protein F (RbohF). This indicates that plants treated by allantoin have better ability to scavenge reactive oxygen species to resist drought. Alternative splicing analysis revealed a total of 427 differentially expressed alternative splicing events across 320 genes. The analysis of splicing factors showed that gene alternative splicing could be divided into many different subgroups and play a regulatory role in many aspects. Through further analysis, we restated the key genes and enzymes in the allantoin synthesis and catabolism pathway, and found that the expression of synthetase and hydrolase showed a downward trend. The pathway of uric acid to allantoin is completed by uric acid oxidase (UOX). To find out the key transcription factors that regulate the expression of this gene, we identified two highly related transcription factors OsERF059 and ONAC007 through correlation analysis. They may be the key for allantoin to enhance the drought resistance of rice.

EMT-related long non-coding RNA in hepatocellular carcinoma: A study with TCGA database.

Accumulating evidence suggest that dysregulated expression of long non-coding RNA (lncRNA) plays a critical role in human tumorigenesis. However, little is known about the lncRNA implicated in the epithelial-to-mesenchymal transition (EMT) process. In this study, we performed data mining in The Cancer Genome Atlas (TCGA) hepatocellular carcinoma (HCC) data set and identified the a spectrum of differentially expressed lncRNAs implicated the EMT process of HCC, and functionally validated their roles in LM3 cells. Especially, lncRNA WDFY3-AS2-, LINC00472-, MIAT-, and MEG3-associated genes were significantly enriched in EMT-linked pathways. Loss-of-function study showed that genetic silencing of WDFY3-AS3, MIAT, and MEG3, but not LINC00472, resulted in reduced N-cadherin expression, cell migration, and cell invasion. Collectively, our results identify several lncRNAs that regulate the EMT process of HCC, which provides critical information for HCC tumorigenesis and potential therapeutic targets.

Nicotinamide Mononucleotide, an NAD+ Precursor, Rescues Age-Associated Susceptibility to AKI in a Sirtuin 1-Dependent Manner.

The rapid growth of an aging population creates challenges regarding age-related diseases, including AKI, for which both the prevalence and death rate increase with age. The molecular mechanism by which the aged kidney becomes more susceptible to acute injury has not been completely elucidated. In this study, we found that, compared with the kidneys of 3-month-old mice, the kidneys of 20-month-old mice expressed reduced levels of the renal protective molecule sirtuin 1 (SIRT1) and its cofactor NAD+ Supplementation with nicotinamide mononucleotide (NMN), an NAD+ precursor, restored renal SIRT1 activity and NAD+ content in 20-month-old mice and further increased both in 3-month-old mice. Moreover, supplementation with NMN significantly protected mice in both age groups from cisplatin-induced AKI. SIRT1 deficiency blunted the protective effect of NMN, and microarray data revealed that c-Jun N-terminal kinase (JNK) signaling activation associated with renal injury in SIRT1 heterozygotes. In vitro, SIRT1 attenuated the stress response by modulating the JNK signaling pathway, probably via the deacetylation of a JNK phosphatase, DUSP16. Taken together, our findings reveal SIRT1 as a crucial mediator in the renal aging process. Furthermore, manipulation of SIRT1 activity by NMN seems to be a potential pharmaceutical intervention for AKI that could contribute to the precise treatment of aged patients with AKI.

Effect of photoperiod change on chronobiology of cercarial emergence of Schistosoma japonicum derived from hilly and marshy regions of China.

The chronobiology of cercarial emergence appeared to be a genetically controlled behavior, adapted to definitive host species, for schistosome. However, a few physiological and ecological factors, for example the change of photoperiod, were reported to affect the rhythmic emergence of cercariae. Therefore, the effect of photoperiod change on cercarial emergence of two Schistosoma japonicum isolates, the hilly and the marshland, was investigated. Four shedding experiments each under a different photoperiod were conducted. Under a natural photoperiod, two distinct shedding modes, one from the hilly region and one from the marshland, were observed. Under a reversed photoperiod, the regular pattern (i.e. under a natural photoperiod) of S. japonicum cercarial emergence was reversed for the marshland isolate and disappeared for the hilly isolate. With an input of a 2 h darkness from 7am to 9am, the cercarial emergence peak were delayed for the two isolates; whereas with an input of a 2 h darkness from 5pm to 7pm, neither effect on the cercarial emergence rhythm was observed. The total cercariae emerged for both parasite isolates varied with a different photoperiod. The results indicate that the change of photoperiod could affect the chronobiology of S japonicum cercarial emergence.

[Cloning, expression and antigenic analysis of merozoite surface protein MSPDBL2-DBL2 domain from Plasmodium falciparum].

OBJECTIVE: To clone and express the DBL domain of Plasmodium falciparum merozoite surface protein MSPDBL2(DBL2), and investigate its antigenicity. METHODS: The DBL2 fragment was amplified by PCR and cloned into pET28a vector. The recombinant pET28a-DBL2 plasmid was transformed into E. coli BL21 (DE3) and protein expression was induced by IPTG. The expressed product was purified by Ni-NTA affinity chromatography, and analyzed by SDS-PAGE and Western blotting. RESULTS: DBL2 gene fragment of Plasmodium falciparum merozoite surface protein MSPDBL2 (950 bp) was obtained by PCR. The recombinant pET28a-DBL2 plasmid was identified by PCR, double enzyme digestion, and DNA sequencing. The recombinant DBL2 protein was expressed in an inclusion body form with Mr 340,000 after being induced with IPTG. Moreover, the purified recombinant DBL2 protein was recognized by sera from patients with falciparum malaria. CONCLUSION: The recombinant pET28a-DBL2 plasmid has been constructed. The purified rDBL2 protein shows adequate antigenicity.

Efficient induction and rapid identification of haploid grains in tetraploid wheat by editing genes TtMTL and pyramiding anthocyanin markers.

Doubled haploid (DH) technology provides an effective way to generate homozygous genetic and breeding materials over a short period of time. We produced three types of homozygous TtMTL gene-edited mutants (mtl-a, mtl-b, and mtl-ab) by CRISPR/Cas9 in durum wheat. PCR restriction enzymes and sequencing confirmed that the editing efficiency was up to 53.5%. The seed-setting rates of the three types of mutants ranged from 20% to 60%. Abnormal grain phenotypes of kernel, embryo, and both embryo and endosperm abortions were observed in the progenies of the mutants. The average frequency of embryo-less grains was 25.3%. Chromosome counting, guard cell length, and flow cytometry confirmed that the haploid induction rate was in the range of 3%-21% in the cross- and self-pollinated progenies of the mtl mutants (mtl-a and mtl-ab). Furthermore, we co-transformed two vectors, pCRISPR/Cas9-MTL and pBD68-(ZmR + ZmC1), into durum wheat, to pyramide Ttmtl-edited mutations and embryo-specifically expressed anthocyanin markers, and developed a homozygous durum haploid inducer with purple embryo (DHIPE). Using DHIPE as the male parent to be crossed with the wild-type Kronos, the grains with white embryos were identified as haploid, while the grains with purple embryos were diploid. These findings will promote the breeding of new tetraploid wheat varieties.

Status and determinants of health behavior knowledge among the elderly in China: a community-based cross-sectional study.

BACKGROUND: Limited studies are available on health behavior knowledge among the elderly and the interaction between the elderly and their children living with them. Using a survey of the elderly in the community and their children living with them, we explored the characteristics that determine health behavior knowledge. We also identified the influencing factors affecting such knowledge. The relationship between the health behavior knowledge of the elderly and that of their children was also investigated. METHODS: A total of 1207 elderly people were selected by multistage probability sampling (number of children of the elderly = 201). We used a self-designed behavior knowledge questionnaire (38 questions) to survey the participants. After the 38 questions were answered, the total score was determined. The group with adequate health behavior knowledge (score ≥ 24) and that with inadequate knowledge (score < 24) were distinguished by logistic regression to explain the influencing factors affecting health knowledge. RESULTS: On the basis of the responses to 38 questions in the survey, approximately 46.7% of elderly people were identified as having a good knowledge of health. Knowledge of the proper amount of certain foods and liquids as well as that of psychological disorder among the elderly recorded a low percentage at < 60%. Factors related to health behavior knowledge among the elderly were as follows educational attainment, past occupation, and location of residence. Participants who finished high school or higher had 6, 4, and 3 times greater odds of possessing adequate health knowledge than those who attained below primary school, primary school, and junior high school levels, respectively. Those with experience as administrative and technical personnel, workers, migrant workers, and farmers had 2.5, 2.3, 3.9, and 2.1 times greater odds of possessing adequate health knowledge, respectively, than those who were unemployed. Respondents living in the city had 3.7 times greater odds of possessing adequate health knowledge than those living in the countryside. In the stem family, the health knowledge of the elderly was significantly lower than that of their children (P < 0.001). However, the influence of their children's knowledge upper their elder's was relatively weak. CONCLUSION: The need to improve health behavior knowledge among the Chinese elderly remains high. Medical personnel in the community should provide health education related to proper diet and alcohol intake, as well as psychological health, particularly for those elderly who only reached primary school and below, used to be unemployed, and are living in rural areas. Children of stem families should be guided to enhance their health education and contribute to the health knowledge of their elderly parents.

Combined metabolomic and transcriptomic analysis reveals key components of OsCIPK17 overexpression improves drought tolerance in rice.

Oryza Sativa is one of the most important food crops in China, which is easily affected by drought during its growth and development. As a member of the calcium signaling pathway, CBL-interacting protein kinase (CIPK) plays an important role in plant growth and development as well as environmental stress. However, there is no report on the function and mechanism of OsCIPK17 in rice drought resistance. We combined transcriptional and metabonomic analysis to clarify the specific mechanism of OsCIPK17 in response to rice drought tolerance. The results showed that OsCIPK17 improved drought resistance of rice by regulating deep roots under drought stress; Response to drought by regulating the energy metabolism pathway and controlling the accumulation of citric acid in the tricarboxylic acid (TCA) cycle; Our exogenous experiments also proved that OsCIPK17 responds to citric acid, and this process involves the auxin metabolism pathway; Exogenous citric acid can improve the drought resistance of overexpression plants. Our research reveals that OsCIPK17 positively regulates rice drought resistance and participates in the accumulation of citric acid in the TCA cycle, providing new insights for rice drought resistance.

SGK1 mediates hypotonic challenge-induced proliferation in basilar artery smooth muscle cells via promoting CREB signaling pathway.

Hypotonic stimulus enlarges cell volume and increased cell proliferation with the exact mechanisms unknown. Glucocorticoid-induced kinase-1 (SGK1) is a serine/threonine kinase that can be regulated by osmotic pressure. We have revealed that SGK1 was activated by hypotonic solution-induced lowering of intracellular Cl- concentration. Therefore, we further examined whether SGK1 mediated hypotonic solution-induced proliferation and the internal mechanisms in basilar smooth muscle cells (BASMCs). In the present study, BrdU incorporation assay, flow cytometry, western blotting were performed to evaluate cell viability, cell cycle transition, and the expression of cell cycle regulators and other related proteins. We found that silence of SGK1 largely blunted hypotonic challenge-induced increase in cell viability and cell cycle transition from G0/G1 phase to S phase, whereas overexpression of SGK1 showed the opposite effects. The effect of SGK1 on proliferation was related to the upregulation of cyclin D1 and cyclin E1, and the downregulation of p27 and p21, which is mediated by the interaction between SGK1 and cAMP responsive element-binding protein (CREB). Moreover, we overexpressed ClC-3 Cl- channel to further verify the role of SGK1 in low Cl- environment-induced proliferation. The results revealed that overexpression of ClC-3 further enhanced hypotonic solution-induced cell viability, cell cycle transition, and CREB activation, which were alleviated or potentiated by silencing or overexpression of SGK1. In summary, this study provides compelling evidences that SGK1, as a Cl--sensitive kinase, is a critical link between low osmotic pressure and proliferation in BASMCs, and shed a new light on the treatment of proliferation-associated cardiovascular diseases.

Spike Density Quantitative Trait Loci Detection and Analysis in Tetraploid and Hexaploid Wheat Recombinant Inbred Line Populations.

Spike density (SD) is an agronomically important character in wheat. In addition, an optimized spike structure is a key basis for high yields. Identification of quantitative trait loci (QTL) for SD has provided a genetic basis for constructing ideal spike morphologies in wheat. In this study, two recombinant inbred line (RIL) populations (tetraploid RIL AM and hexaploid RIL 20828/SY95-71 (2SY)) previously genotyped using the wheat55K SNP array were used to identify SD QTL. A total of 18 QTL were detected, and three were major and one was stably expressed (QSd.sau-2SY-7A.2, QSd.sau-AM-5A.2, QSd.sau-AM-7B, and QSd.sau-2SY-2D). They can explain up to 23.14, 19.97, 12.00, and 9.44% of phenotypic variation, respectively. QTL × environment and epistatic interactions for SD were further analyzed. In addition, pyramiding analysis further revealed that there were additive effects between QSd.sau-2SY-2D and QSd.sau-2SY-7A.2 in 2SY, and QSd.sau-AM-5A.2 and QSd.sau-AM-7B in AM. Pearson's correlation between SD and other agronomic traits, and effects of major or stable QTL on yield related traits indicated SD significantly impacted spike length (SL), spikelet number per spike (SNS) and kernel length (KL). Several genes related to spike development within the physical intervals of major or stable QTL were predicted and discussed. Collectively, our research identified QTL with potential applications for modern wheat breeding and broadening the genetic basis of SD.

Calcium-sensing receptor antagonist NPS-2143 suppresses proliferation and invasion of gastric cancer cells.

NPS-2143 is a calcium-sensing receptor (CaSR) antagonist that has been demonstrated to possess anticancer activity. To date, the effects of NPS-2143 on gastric cancer (GC) cell growth, motility, and apoptosis have not been investigated. In the present study, we firstly investigated the expression of CaSR in GC tissues using immunohistochemistry and western blotting. Then Cell Counting Kit-8 and colony formation assays were conducted to explore the effect of the NPS-2143 on the proliferation of GC cell line AGS. Transwell invasion and migration assays were performed to test the effect of NPS-2143 on AGS cell motility. We determined the percentage of apoptotic cells by flow cytometry and explored the changes of apoptosis-related protein by western blotting. Furthermore, we constructed a CaSR knockdown AGS cell line to determine whether NPS-2143 acted via inhibition of CaSR. We found that the protein expression level of CaSR was higher in GC tissues compared with the paired adjacent normal tissues. In addition, NPS-2143 treatment caused an inhibitory effect on the proliferation, invasion, and migration of AGS cells and a promoting effect on AGS apoptosis. The expression of Bcl-2 was decreased while the levels of Bax and active caspase 3 were enhanced in AGS cells after NPS-2143 treatment. Mechanistically, NPS-2143 lead to a significant decrease in the expression of phosphorylated (p)-AKT, phosphorylated mechanistic target of rapamycin (p-mTOR), p70, and cyclin D1. Knockdown of CaSR also suppressed cell proliferation, invasion, and migration and promoted cell apoptosis. No significant difference was observed between CaSR-silenced AGS cells with and without NPS-2143 treatment. These results confirmed that NPS-2143 has an inhibitory influence on AGS cell growth via inhibiting CaSR, which then suppresses the PI3K/Akt signaling pathway.

MicroRNA-152 Acts as a Tumor Suppressor MicroRNA by Inhibiting Krüppel-Like Factor 5 in Human Cervical Cancer.

Aberrant expression of microRNA-152 (miR-152) is frequently observed in human cancers including ovarian cancer, breast cancer, prostate cancer, and gastric cancer. However, its expression and functional role in cervical cancer (CC) are poorly understood. Also, the association between miR-152 and Krüppel-like factor 5 (KLF5) expression in CC remains unclear. In this study, analyzing the expression of miR-152 by quantitative real-time PCR (qRT-PCR) revealed it was sharply reduced in CC tissues and cell lines. In addition, the negative correlation of miR-152 expression and KLF5 expression was observed. The dual-luciferase reporter assay validated that KLF5 was a target of miR-152. In vitro functional assays revealed that miR-152 could inhibit cell proliferation and cell cycle progression through regulating the expression of KLF5. Taken together, our study suggested that miR-152 functions as a tumor suppressor in CC, and the miR-152/KLF5 axis may provide novel therapeutic targets for CC treatment.

Schisandrin A reverses doxorubicin-resistant human breast cancer cell line by the inhibition of P65 and Stat3 phosphorylation.

BACKGROUND: Multidrug resistance (MDR) in breast cancer therapy occurs frequently. Thus, anti-MDR agents from natural products or synthetic compounds were tested extensively. We have also explored the reverse effect and mechanism of Schisandrin A (Sch A), a natural product, on MCF-7 breast cancer doxorubicin (DOX)-resistant subline MCF-7/DOX. METHODS: MTT assay was performed to measure the viability of MCF-7 cells to assess the reverse effect of Sch A. Western blot analysis was used to study the protein levels. Laser scanning confocal microscopy was performed to detect the intercellular DOX and Rhodamine 123 accumulation. The qRT-PCR was used to analysis the target gene expression. Dual-luciferase reporter assay was performed to test the transcriptional activity of P-glycoprotein (P-gp). RESULTS: Sch A, at the concentration of 20 µM, showed selective reverse effect (better than the positive control, verapamil at 5 µM) on MCF-7/DOX cell line but not on BEL-7402/DOX, Hep G2/DOX, and K-562/DOX cells. In addition, Sch A enhanced DOX-induced cleavage of Caspase-9 and PARP levels by increasing intracellular DOX accumulation and inhibiting P-gp function. Furthermore, Sch A selectively suppressed P-gp at gene and protein levels in MCF-7/DOX cells which express high level of MDR1 but not MRP1, MRP3, or BCRP. Besides, Sch A showed inhibitory effect on P-gp transcriptional activity. Sch A significantly reduced p-IκB-α (Ser32) and p-Stat3 (Tyr705) levels which mediate P-gp expression. In addition, Stat3 knockdown enhanced the reverse effect of siP65. The combined effect of siStat3 and siP65 was better than Sch A single treatment in MCF-7/DOX cells. CONCLUSION: Sch A specifically reverses P-gp-mediated DOX resistance in MCF-7/DOX cells by blocking P-gp, NF-κB, and Stat3 signaling. Inhibition of P65 and Stat3 shows potent anti-MDR effect on MCF-7/DOX cells.

The mechanisms involved in miR-9 regulated apoptosis in cervical cancer by targeting FOXO3.

As a seriously global health problem, cervical cancer is a great risk to women which threatens their lives. Approximately 30% patients who received definitive treatment may fail to recover from this disease. Accordingly, there is an imperatively need to explore alternative therapeutic approaches for this disease. Several studies have revealed that miR-9 was a critical regulator during cervical cancer growth. Here, we reported that the miR-9 was overexpressed in cervical tumor tissue and exerted a promoting effect on human cervical cancer cell (SiHa) growth. Both in vitro and in vivo experiments confirmed that miR-9 could stimulate the proliferation and migration of SiHa cells. In contrast, inhibition of miR-9 induced apoptosis in SiHa cells. In addition, dual luciferase reporter system assay verified that there was a strong target relationship between miR-9 and FOXO3. Result of western blot assay showed that the inhibition of miR-9 increased the expression of FOXO3. Moreover, miR-9 regulated FOXO3 downstream proteins Bax, Bcl-2 and p-Akt expressions, which suggesting that miR-9 was involved in the SiHa cells apoptosis. In conclusion, our results suggest that the inhibition of miR-9 could induce apoptosis in cervical cancer by targeting FOXO3 and presented a potential molecular target for the treatment of cervical cancer patients.

MMP-2 and MMP-9 gene polymorphisms associated with cervical cancer risk.

AIM: To inquire into whether there is any relationship between MMP-2 rs2285053 and MMP-9 rs3918242 polymorphisms and the onset risk of cervical cancer (CC) and invasion and metastasis. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was utilized to examine the frequency and distribution of MMP-2 rs2285053 and MMP-9 rs3918242 polymorphisms in 150 CC patients and 120 healthy individuals who were matched therewith in age and gender, and the CC risk and invasion and metastasis condition of individuals carrying different genotypes of the two single nucleotide polymorphisms (SNPs) were compared. The data were processed by SPSS 18.0 software. RESULTS: The differences of genotype distribution frequencies of the two SNPs between the two groups were not apparent (P > 0.05), and no significant relevance was found between the two SNPs and the lymphatic metastasis of CC. The CC risk for those carrying CC/TT (rs2285053) and CT/TT (rs3918242), CC (rs2285053) and CC (rs3918242), and CC (rs2285053) and CT/TT (rs3918242) respectively was 0.135, 1.138, and 1.182 times as much as that for those carrying CT/TT (rs2285053) and CC (rs3918242) (95% CI=0.028-0.660; 95% CI=0.615-2.103; 95% CI=0.558-2.502), reflecting that the two SNPs had interactions there between. CONCLUSIONS: MMP-2 rs2285053 and MMP-9 rs3918242 polymorphisms may have correlations with CC susceptibility.

Effects of celastrol on enhancing apoptosis of ovarian cancer cells via the downregulation of microRNA­21 and the suppression of the PI3K/Akt­NF­κB signaling pathway in an in vitro model of ovarian carcinoma.

Celastrol has previously been used to treat rheumatoid arthritis, bruises, back pain and additional diseases. At present, efficacy studies predominantly focus on the anti­inflammatory, antioxidative and antitumor effects of celastrol. However, the effect of celastrol on ovarian cancer cells is not fully elucidated. In the present study, the effects of celastrol were investigated in ovarian cancer cells and the mechanisms involved were explored. In OVCAR3 cells, celastrol was observed to suppress cellular proliferation, induce apoptosis and increase caspase­9 and ­3 activity in a dose­ and time­dependent manner. The expression levels of microRNA­21 (miRNA­21) were reduced, in addition to a reduction in the levels of phosphoinositide 3­kinase (PI3K)/p­Akt­NF (NF)­κB following treatment with celastrol. Notably, reduced expression of miRNA­21 replicated the effect of celastrol on OVCAR3 cells and inhibited the PI3K/p­Akt­NF­κB signaling pathway in an in vitro model of ovarian carcinoma. To the best of our knowledge this is the first study to indicate that celastrol may represent a potential agent for the treatment of human ovarian carcinoma, via the induction of apoptosis through the downregulation of miRNA­21 and the PI3K/Akt­NF­κB signaling pathway in an in vitro model of ovarian carcinoma.

Metabolic behavior prediction of pazopanib by cytochrome P450 (CYP) 3A4 by molecular docking.

Metabolism-mediated drug adverse effects (e.g., drug-drug interaction, bioactivation, etc.) strongly limit the utilization of clinical drugs. The present study aims to predict the metabolic capability of cytochrome P450 (CYP) 3A4 toward pazopanib which is an excellent drug exhibiting therapeutic role toward various cancers especially for ovarian cancer. Pazopanib can be well docked into the activity cavity of CYP3A4, and the interaction structure in pazopanib was methyl group located besides nitrogen in the five-membered ring. The distance between the hydrogen atom in methyl group and active center is 3.64 Å. The interaction amino acid is Glu374. Furthermore, both pazopanib and ketoconazole were docked into the activity cavity of CYP3A4 to compare their binding potential. The distance between ketoconazole and activity center (2.10 Å) is closer than the distance between pazopanib and activity center of CYP3A4, indicating the easy influence of CYP3A4 inhibitor toward the metabolism of pazopanib. All these data were helpful for the clinical application of pazopanib, and R&D of other tinib drug candidates as new anti-tumor drugs.