MCCC2 promotes HCC development by supporting leucine oncogenic function.

BACKGROUND: The role of methylcrotonoyl-CoA carboxylase 2 (MCCC2) in the development of tumors is well-established, and the involvement of leucine in the liver is well-known. However, the role of MCCC2 and the correlation between MCCC2 and leucine in the progression of hepatocellular carcinoma (HCC) have not yet been reported. METHODS: In this study, the Gepia database was used to evaluate the prognostic value of MCCC2 in HCC. The expression and localization of MCCC2 in HCC cells were determined by western blot and immunofluorescence assays. Flow cytometry and CCK-8 and transwell assays were carried out to explore the effect of MCCC2 on cell proliferation, migration, and invasion. In addition, mass spectrometry analysis was used to predict the potential cell function of MCCC2 in HCC. RESULTS: We found that the expression of MCCC2 increased in HCC tissues and that high expression of MCCC2 could predict poor outcomes in HCC patients. Knockdown expression of MCCC2 in HCC cells could reduce cell proliferation, migration, and invasion ability in vitro and could inhibit HCC cell proliferation in vivo. Interestingly, we found that HCC cells transfected with MCCC2-sgRNA failed to respond to leucine deprivation. Meanwhile, leucine deprivation inhibited cell proliferation, migration, and invasion in HCC cells where MCCC2 was present rather than in cells where MCCC2 was absent. In addition, knockdown of MCCC2 significantly reduced the glycolysis markers, glucose consumption, lactate secretion, and acetyl-CoA level, which is a product of leucine metabolism. Furthermore, we found that MCCC2 promotes the activation of ERK. Profiling the MCCC2 binding proteins revealed that MCCC2-associated proteins are enriched in biological processes, such as protein metabolism, energy pathway, and metabolism in HCC cells. CONCLUSIONS: Our findings revealed that MCCC2 plays a critical role in the development of HCC, and the leucine metabolism pathway might be a novel target in HCC treatment.

Identification and candidate gene screening of qCIR9.1, a novel QTL associated with anther culturability in rice (Oryza sativa L.).

KEY MESSAGE: A novel QTL, qCIR9.1, that controls callus induction rate in anther culture was identified on chromosome 9 in rice, and based on RNA-seq data, Os09g0551600 was the most promising candidate gene. Anther culture, a doubled haploid (DH) technique, has become an important technology in many plant-breeding programmes. Although anther culturability is the key factor in this technique, its genetic mechanisms in rice remain poorly understood. In this study, we mapped quantitative trait loci (QTLs) responsible for anther culturability by using 192 recombinant inbred lines (RILs) derived from YZX (Oryza sativa ssp. indica) × 02428 (Oryza sativa ssp. japonica) and a high-density bin map. A total of eight QTLs for anther culturability were detected in three environments. Among these QTLs, a novel major QTL for callus induction rate (CIR) named qCIR9.1 was repeatedly mapped to a ~ 100 kb genomic interval on chromosome 9 and explained 8.39-14.14% of the phenotypic variation. Additionally, RNA sequencing (RNA-seq) was performed for the parents (YZX and 02428), low- (L-Pool) and high-CIR RILs (H-Pool) after 16 and 26 days of culture. By using the RNA of the bulked RILs for background normalization, the number of differentially expressed genes (DEGs) both between the parents and between the bulked RILs after 26 days of culture was drastically reduced to only 78. Among these DEGs, only one gene, Os09g0551600, encoding a high-mobility group (HMG) protein, was located in the candidate region of qCIR9.1. qRT-PCR analysis of Os09g0551600 showed the same results as RNA-seq, and the expression of this gene was decreased in the low-callus-induction parent (YZX) and L-Pool. Our results provide a foundational step for further cloning of qCIR9.1 and will be very useful for improving anther culturability in rice.

Nucleostemin promotes hepatocellular carcinoma by regulating the function of STAT3.

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor in the liver and the second leading cause of cancer-related death worldwide. The collaborative function between Nucleostemin (NS) and STAT3 has been reported but not well studied in HCC. Here, we found a significant correlation between NS expression and STAT3 phosphorylation, not only in HCC cancers but also in HCC tissues. Patients with high expression of both NS and p-STAT3 show a very poor survival rate. High expression of both NS and p-STAT3 is also associated with tumor size and microvascular invasion. Knocking down the expression of NS greatly reduces the phosphorylation of STAT3. Conversely, overexpression of NS significantly promotes STAT3 phosphorylation. NS and p-STAT3 are located in the nucleus and physiologically interact with each other. Furthermore, NS greatly enhances cell migration and invasion by promoting the epithelial-mesenchymal transition (EMT). NS also supports cell proliferation and colony formation. The importance of NS in HCC was further demonstrated by evaluating tumor formation in vivo. Therefore, we demonstrate a critical collaborative function between NS and STAT3 in HCC, providing an invaluable insight into the mechanism of HCC. The concomitant expression of NS and p-STAT3 might be a potential prognostic indicator and therapeutic target in patients with HCC.

RelB promotes liver fibrosis via inducing the release of injury-associated inflammatory cytokines.

Liver fibrosis is a serious chronic disease that developed by a coordinated interplay of many cell types, but the underlying signal transduction in individual cell type remains to be characterized. Nuclear factor-κB (NF-κB) is a widely accepted central player in the development of hepatic fibrosis. However, the precise role of each member of NF-κB in different cell type is unclear. Here, we generated a mouse model (RelbΔhep ) with hepatocyte-specific deletion of RelB, a member of NF-κB family. RelbΔhep mice born normally and appear normal without obvious abnormality. However, in the CCl4-induced liver fibrosis, RelbΔhep mice developed less severe disease compared with wide-type (WT) mice. The denaturation and necrosis of hepatocytes as well as the formation of false lobules in RelbΔhep mice were significantly reduced compared with WT mice. The production of α-SMA and the level of collagen I and Collagen III were greatly reduced in RelbΔhep mice comparing with WT mice. Furthermore, in patients with liver fibrosis, RelB is up-regulated along with the stage of diseases. Consistently, CCl4 treatment could up-regulate the expression of RelB as well as inflammatory cytokines such as IL-6 and TGF-ß1 in hepatoma cell as well as in WT mice. Knockdown the expression of RelB in hepatoma cells greatly reduced the expression of CCl4-induced inflammatory cytokines. In summary, we provide the genetic evidence to demonstrate the critical and hepatocellular role of RelB in liver fibrosis. RelB is an important transcription factor to drive the expression of inflammatory cytokines in the initiation phase of injury.

Coupling function of cyclin-dependent kinase 2 and Septin2 in the promotion of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common and aggressive malignant tumor with a poorly defined molecular mechanism. Cyclin-dependent kinase 2 (CDK2) and Septin2 (SEPT2) are 2 known oncogenic molecules but the mechanism of functional interactions remains unclear. Here, we interestingly found that CDK2 and SEPT2 show very similar dynamic expression during the cell cycle. Both CDK2 and SEPT2 show the highest protein levels in the G2/M phase, resulting in CDK2 interacting with SEPT2 and stabilizing SEPT2 in HCC. In a panel of 8 pairs of fresh HCC tissues and corresponding adjacent tissues, both western blot and immunohistochemistry (IHC) assays demonstrate that CDK2 expression is highly correlated with SEPT2. HCC with high expression of both CDK2 and SEPT2 are more likely to relapse. This observation is further demonstrated by a large panel of 100 HCC patients. In this large panel, high expression of both CDK2 and SEPT2 significantly correlates with tumor differentiation and microvascular invasion, which is an independent prognostic factor in HCC patients. In summary, our results reveal a cooperative function between CDK2 and SEPT2. HCC with high expression of CDK2 and SEPT2 might be more aggressive and respond poorly to current therapy.

Highly Sensitive Aptasensor for Trace Arsenic(III) Detection Using DNAzyme as the Biocatalytic Amplifier.

A highly sensitive fluorescence biosensing system was designed for the detection of trace amounts of arsenic(III) (As3+) based on target-triggered successive signal amplification strategy. The specific recognition between the target As3+ and the aptamer sequence results in the release of the blocking DNA to trigger the subsequent signal amplification steps. Exonuclease III (Exo III)-mediated DNA recycling digest process is introduced into the sensing system to generate numerous Mg2+-dependent DNAzymes. After magnetic separation, the active DNAzyme with multiple turnovers could catalyze the continuous cleavage of the fluorophore-quencher-functionalized substrate strands, thus yielding a significantly amplified fluorescence signal for target detection. Due to the synergetic signal amplification of Exo III and DNAzyme, the fluorescent biosensor exhibits ultrasensitivity for As3+ monitoring, with a detection limit of 2 pM. Our established biosensor also displays excellent selectivity toward the target As3+ and has been successfully applied to the determination of As3+ in water samples with satisfactory accuracy. This sensing platform can be developed as a universal approach for the fast, sensitive, and accurate detection of aptamer-binding molecules.

An ultrasensitive and simple fluorescence biosensor for detection of the Kras wild type by using the three-way DNA junction-driven catalyzed hairpin assembly strategy.

In this work, a label-free fluorescence biosensor was proposed for simple detection of the Kras wild type by using the three way DNA junction-driven catalyzed hairpin assembly strategy. In this system, a three-way DNA junction probe (JP) and two hairpin probes (H1 and H2) were designed. In the presence of the Kras wild type, an autocatalytic DNA machine can be activated. This leads to the generation of numerous free G-rich sequences, which can associate with a fluorescent dye N-methylmesoporphyrin IX (NMM) to yield an amplified fluorescence signal for the target detection. This sensing platform showed a high sensitivity towards the Kras wild type with a detection limit as low as 2.7 fM without any labelling, immobilization, or washing steps. The designed sensing system also exhibits an excellent selectivity for the Kras wild type compared with other interference DNA sequences. Furthermore, the presented biosensor is robust and has been successfully applied for the detection of the Kras wild type in a real biological sample with satisfactory results, suggesting that this method is promising for simple and early clinical diagnosis of genetic diseases. Thanks to its simplicity, cost-effectiveness, and ultrasensitivity, our proposed sensing strategy provides a universal platform for the detection of other genetic diseases by substituting the target-recognition element.

A cascade toehold-mediated strand displacement strategy for label-free and sensitive non-enzymatic recycling amplification detection of the HIV-1 gene.

In this work, a label-free fluorescence biosensor for simple detection of the HIV-1 gene was proposed by using toehold-mediated strand displacement reactions (TMSDRs) combined with a non-enzymatic target recycling amplification strategy. In this system, two TMSDRs were used. In the presence of the HIV-1 gene, an autocatalytic DNA machine can be activated. This leads to the generation of numerous free G-rich sequences, which can associate with a fluorescent dye N-methylmesoporphyrin IX (NMM) to yield an amplified fluorescence signal for the target detection. This sensing platform showed a high sensitivity towards the HIV-1 gene with a detection limit as low as 1.9 pM without any labelling, immobilization, or washing steps. The designed sensing system also exhibits an excellent selectivity for the HIV-1 gene compared with other interference DNA sequences. Furthermore, the presented biosensor is robust and has been successfully applied for the detection of the HIV-1 gene in a real biological sample with satisfactory results, suggesting that this method is promising for simple and early clinical diagnosis of HIV infection. Thanks to its simplicity, cost-effectiveness and ultrasensitivity, our proposed sensing strategy provides a universal platform for the detection of other genes by substituting the target-recognition element.

Ultrasensitive and simple fluorescence biosensor for detection of the mecA gene of Staphylococcus aureus by using an exonuclease III-assisted cascade signal amplification strategy.

In this work, a label-free fluorescence biosensor for ultrasensitive and simple detection of the mecA gene of Staphylococcus aureus was proposed by using an exonuclease III (Exo III)-assisted cascade signal amplification strategy. The 3' end-extruding hairpin probe (HP) acted as the target recognition element and the caged G-quadruplex was used as the signal reporter. Without the mecA gene, the HP probe cannot be digested by Exo III, as the G-rich sequences are blocked in the stem of the HP probe. In the presence of the mecA gene, the hybridization of the mecA gene with the 3' end-extruding HP probe triggers the digestion reaction of Exo III, liberating the mecA gene and the mecA gene analogue. Both the released mecA gene and the mecA gene analogue can hybridize with other HP probes and activate another round of the cleavage reaction. Consequently, the released free G-quadruplex is "lit up" by N-methylmesoporphyrin IX (NMM), displaying a dramatically enhanced fluorescence intensity. This sensing platform showed a high sensitivity towards the mecA gene with a detection limit as low as 2.4 fM without any labelling, immobilization, or washing steps. The designed sensing system also exhibits excellent selectivity for the mecA gene in the presence of other interfering DNA sequences. Furthermore, the presented biosensor is robust and has been successfully applied for the detection of the mecA gene in a real food sample with satisfactory results. Owing to its simplicity, cost-effectiveness and ultrasensitivity, our proposed sensing strategy provides a promising platform for the detection of other genes by substituting the target-recognition element.

Fine Mapping of the Affecting Tillering and Plant Height Gene CHA-1 in Rice.

The plant architecture of rice is an important factor affecting yield. Strigolactones (SLs) are newly discovered carotenoid-derived plant hormones that play an important role in rice plant architecture. In this study, a high-tillering dwarf mutant, CHA-1, was identified by spatial mutagenesis. CHA-1 was located in the region of 31.52-31.55 MB on chromosome 1 by map-based cloning. Compared with the wild-type THZ, the CHA-1 mutant showed that ACCAC replaced TGGT in the coding region of the candidate gene LOC_Os01g54810, leading to premature termination of expression. Genetic complementation experiments proved that LOC_Os01g54810 was CHA-1, which encodes a putative member of Class III lipase. Expression analysis showed that CHA-1 was constitutively expressed in various organs of rice. Compared with those in THZ, the expression levels of the D17 and D10 genes were significantly downregulated in the CHA-1 mutant. In addition, the concentrations of ent-2'-epi-5-deoxystrigol (epi-5DS) in the root exudates of the CHA-1 mutant was significantly reduced compared with that of THZ, and exogenous application of GR24 inhibited the tillering of the CHA-1 mutant. These results suggest that CHA-1 influences rice architecture by affecting SL biosynthesis.

Trimetazidine inhibits liver fibrosis and hepatic stellate cell proliferation and blocks transforming growth factor-ß (TGFß)/Smad signaling in vitro and in vivo.

Trimetazidine (TMZ) has been used extensively to treat coronary artery disease and to reduce fibrosis. Liver fibrosis is a reversible process. However, the impacts of TMZ on liver fibrosis triggered by CCl4 and on hepatic stellate cells in liver fibrosis remain to be elaborated. In the current study, the liver fibrosis models were constructed by using CCl4-induced mice and TGF-ß-induced hepatic stellate cells. The involvement of TMZ in liver fibrosis was subsequently investigated. In the CCl4-induced hepatic fibrosis mouse model, it was shown that the expression levels of alanine aminotransferase and aspartate aminotransferase were reduced after TMZ treatment; the expression levels of the extracellular matrix proteins colla1 and α-SMA were down-regulated; furthermore, the expression levels of TGFß/Smad signaling proteins were inhibited. In TGF-ß-induced hepatic stellate cells, compared to the TGF-ß-induced group, cell proliferation and migration were inhibited after TMZ treatment; meanwhile, extracellular matrix protein and TGFß/Smad signaling protein expression levels followed the same trend as in the hepatic fibrosis model. In conclusion, TMZ could block the TGFß/Smad signaling in liver fibrosis model, with inhibiting liver fibrosis and hepatic stellate cell proliferation. This may broaden the application sphere of TMZ in liver fibrosis therapy.

Targeted Identification of Rice Grain-Associated Gene Allelic Variation Through Mutation Induction, Targeted Sequencing, and Whole Genome Sequencing Combined with a Mixed-Samples Strategy.

BACKGROUND: The mining of new allelic variation and the induction of new genetic variability are the basis for improving breeding efficiency. RESULTS: In this study, in total, 3872 heavy ion-irradiated M2 generation rice seeds and individual leaves were collected. The grain length was between 8 and 10.22 mm. The grain width was between 1.54 and 2.87 mm. The results showed that there was extensive variation in granulotype. The allelic variation in GS3 and GW5 was detected in 484 mixed samples (8:1) using targeted sequencing technology, and 12 mixed samples containing potential mutations and 15 SNPs were obtained; combined with Sanger sequencing and phenotype data, 13 key mutants and their corresponding SNPs were obtained; protein structural and functional analysis of key mutants screened out 6 allelic variants leading to altered grain shape, as well as the corresponding mutants, including long-grain mutants GS3-2 and GS3-7, short-grain mutants GS3-3 and GS3-5, wide-grain mutant GW5-1 and narrow-grain mutant GW5-4; whole genome sequencing identified new grain length gene allelic variants GS3-G1, GS3-G2 and GS3-G3. CONCLUSION: Based on the above studies, we found 6 granulotype mutants and 9 granulotype-related allelic variants, which provided new functional gene loci and a material basis for molecular breeding and genotype mutation and phenotype analysis. We propose a method for targeted identification of allelic variation in rice grain type genes by combining targeted sequencing of mixed samples and whole genome sequencing. The method has the characteristics of low detection cost, short detection period, and flexible detection of traits and genes.

Enhanced targeting of 3D pancreatic cancer spheroids by aptamer-conjugated polymeric micelles with deep tumor penetration.

Pancreatic cancer is a high degree malignant tumor which makes its diagnosis and treatment highly critical. The effect of conventional chemotherapy for pancreatic cancer is quite poor due to the low accumulation of the chemotherapeutic drugs at the tumor site. Therefore, enhancing the targeting efficiency and accumulation of the drug carrier at tumor site with subsequent release of drug within the effective time period is one of the key factors for successful targeted chemotherapy of pancreatic cancer. Our previous studies have demonstrated that aptamer can be a valid targeting moiety to guide the chemotherapeutic drug doxorubicin (DOX) to accumulate at the tumor tissue. Herein, the present study aims to further investigate the targeting efficiency as well as therapeutic efficacy of the drug delivery system comprised of aptamer-modified polymeric nano drug carrier encapsulated with DOX (DOX@PCL-b-PEO-Aptamer micelles). The in vitro cytotoxicity studies and laser confocal microscopy indicated that DOX@PCL-b-PEO-Aptamer micelles exhibited enhanced targeting and cytotoxic efficacy towards human pancreatic cancer cells (Panc-1 cells) as compared to free DOX and DOX-loaded PCL-b-PEO-NH2 micelles (DOX@PCL-b-PEO-NH2 micelles). Furthermore, the aptamer-decorated drug delivery system exhibited better tumor penetration into the three-dimensional (3D) spheroid of Panc-1 cells with successful release of DOX as compared to the drug delivery system without aptamer modification. Overall, this study suggests that the aptamer-modified polymeric micelles could be effectively employed for the targeted delivery of anticancer drug to treat pancreatic cancer in near future.

MicroRNA-92b-3p promotes the progression of liver fibrosis by targeting CREB3L2 through the JAK/STAT signaling pathway.

Liver fibrosis is a common feature of almost all chronic liver diseases, which eventually leads to cirrhosis and even hepatocellular carcinoma (HCC). The current study showed that miR-92b plays an important role in the progression of HCC but its role in liver fibrosis is still unclear. Here we aimed to explore the role and underlying molecular mechanism of miR-92b-3p in the activated hepatic stellate cells (HSCs) and the pathological process of hepatic fibrosis. We found that miR-92b-3p was highly expressed both in fibrotic liver tissues from patients and model mice and in activated LX-2 cells stimulated with TGF-ß1. However, the expression of miR-92b-3p was downregulated in inactivated LX-2 cells treated with adipogenic differentiation mixture (MDI). In addition, we found that miR-92b-3p mimic could promote the activation, proliferation, and migration of LX-2 and HSC-T6 cells, while miR-92b-3p inhibitor could reverse this process. From the TargetScan databases, we found that CREB3L2 is a potential target of miR-92b-3p and the luciferase assay revealed the suppressed CREB3L2 expression by miR-92b-3p. Mechanistically, we found that miR-92b-3p promotes the activation of HSCs and thereby the progression of liver fibrosis by activating JAK/STAT pathway via targeting CREB3L2, providing a new target for the diagnosis and treatment of liver fibrosis.

Autocatalytic DNA circuit for Hg2+ detection with high sensitivity and selectivity based on exonuclease III and G-quadruplex DNAzyme.

Utilizing G-quadruplex as the signal report probe, an ultrasensitive and label-free autocatalytic DNA circuit for Hg2+ detection on the basis of exonuclease III (Exo III)-assisted cascade signal amplification has been proposed. In the absence of Hg2+, the hairpin A and the DNA1 cannot hybridize due to the thymine-thymine (T-T) mismatches. Therefore, hairpin probes with the 3'-protruding terminus can be resistant to Exo III digestion, preventing the G-rich sequence to be released. In the presence of Hg2+, the combination of the DNA1 with the 3' end-extruding hairpin A via T-Hg2+-T coordination chemistry triggers the digestion reaction of Exo III, leading to the release of the DNA1 and the sequence with domains c, d, and e. Both of the DNA1 and the sequence with domains c, d, and e can combine with other hairpin probes and activate another round of the cleavage reaction. The produced G-rich sequence can form G-quadruplex structure by binding with N-Methyl mesoporphyrin IX (NMM). The biosensor exhibits excellent selectivity and high sensitivity for Hg2+. The linear range of this biosensor is from 10 fM to 100 nM, and the linear equation can be expressed as: F610 = 1.3 × 105 Lg C + 7.40 × 104 (R2 = 0.998), in which F610 is the fluorescence intensity at 610 nm, C represents the Hg2+ concentrations, and Lg is the logarithm of 10. The detection limit is 10 fM. The biosensor is robust and can be applied to the detection of Hg2+ in water samples. By substituting the target-recognition elements, this sensing system can also be used for the detection of other metal ions.

Investigating the Mechanisms of AquaporinZ Reconstitution through Polymeric Vesicle Composition for a Biomimetic Membrane.

Aquaporin-Z (AqpZ) are water channel proteins with excellent water permeability and solute rejection properties. AqpZ can be reconstituted into vesicles utilizing cell-like bilayer membranes assembled from amphiphilic block copolymers, for the preparation of high-performance biomimetic membranes. However, only a few copolymers have been found suitable to act as the membrane matrix for protein reconstitution. Hence, this work analyzes the mechanism of protein reconstitution based on a composition-reconstitution relationship. The vesicle formation and AqpZ reconstitution processes in various amphiphilic block copolymers were investigated in terms of size, morphology, stability, polymeric bilayer membrane rigidity, and thermal behavior. Overall, this study contributes to the understanding of the composition-reconstitution relationship of biomimetic membranes based on AqpZ-reconstituted polymeric vesicles.

Genome-wide association study reveals novel genetic loci contributing to cold tolerance at the germination stage in indica rice.

Low temperature at the germination stage is one of the major abiotic stresses limiting rice (Oryza sativa L.) production, especially in regions where rice seeds are sown directly. However, few relevant genetic loci and genes have been identified. In this study, we report the phenotypic analysis of low temperature germination (LTG) in 200 indica rice varieties and a genome-wide association study (GWAS) of LTG in this collection using 161,657 high-quality SNPs, which were identified via genotyping-by-sequencing (GBS) of all the rice varieties. A total of 159 genetic loci were detected, and they were evenly distributed on all 12 chromosomes. Among them, 51 loci were detected more than twice; in particular, 23 loci were detected repeatedly in both the wet and dry seasons, and 569 genes were predicted in the 200-kb genomic region harbouring these 23 loci. Furthermore, 14,742 differentially expressed genes (DEGs) were identified using RNA sequencing. By integrating GWAS and RNA sequencing, 179 candidate DEGs were obtained. Sequence variation in the region of loci 95 was analyzed using 20 varieties with extreme phenotype. The polymorphisms of three DEGs (Os07g0585500, Os07g0585700, Os07g0585900) were associated with their phenotypes. Haplotype analysis of the three genes demonstrated that almost all the varieties with the same haplotype as japonica Nipponbare on the three DEGs showed high LTG ability. These findings provide valuable information for understanding the genetic control of LTG and performing molecular breeding with marker-assisted selection in indica rice.

Quantitative Trait Locus Analysis of Seed Germination and Early Seedling Growth in Rice.

Seed germination and early seedling growth are important agricultural traits for developing populations of both irrigated and directly seeded rice (DSR). To investigate the genetic mechanisms underlying seed germination and early seedling growth in rice, 275 recombinant inbred lines (RILs) were genotyped in this study via the genotyping-by-sequencing (GBS) approach to construct a high-density linkage bin map based on the parent-independent genotyping method. Quantitative trait loci (QTLs) for 12 traits related to seed germination and early seedling growth were analyzed. Totally, 22 additive loci were detected, after analysis of the interaction between additive QTLs and environments, five stable additive loci were obtained. Among them, loci 4, 5, 12 and 14 exhibited clear pleiotropic effects that were associated with multiple traits. Analysis of the effects of the five additive stable loci showed that a single locus increased the corresponding phenotypic value. Ten of the 275 RILs pyramided the excellent alleles of the five stable genetic loci. Most phenotypic values of the ten RILs were greater than the average values. Four RILs (G260, G342, G371, and G401) with more excellent phenotypic values were subsequently selected; these RILs could serve as donor parents of favorable alleles in the breeding process. Due to the existence of pleiotropy, the use of these genetic loci for pyramid breeding can further increase the efficiency to reach breeding goals. In addition, these five stable loci have an average physical interval of only 170 kb, we also further identified five promising candidate genes by qRT-PCR, which provides us with a basis for future cloning of these genes. Overall, this work will help broaden our understanding of the genetic control of seed germination and early seedling growth, and this study provides both a good theoretical basis and a new genetic resource for the breeding of direct-seeded rice.

Development of a core SNP arrays based on the KASP method for molecular breeding of rice.

BACKGROUND: The development and utilization of genetic markers play a pivotal role in marker-assisted breeding of rice cultivars during pyramiding of valuable genes. Among molecular markers, SNPs have become the most promising due to their wide distribution within genomes and suitability for high -throughput automated genotyping. Although metadata of SNPs have been identified via next generation sequencing in rice, a large gap between the development of SNP markers and the application in breeding still exists. To promote the application of SNP markers based on the KASP (Kompetitive Allele-Specific PCR) method in rice breeding, a set of core SNP arrays was built via the screening of SNP databases and literature resources based on the KASP method. RESULTS: Five hundred and ninety six SNPs classified into eight subsets including quality control, indica-indica variation, highly polymorphic, functional genes, key genes targeting sites, gene cloned region, important trait associated and gap filling sites were chosen to design KASP primers and 565 out of them were successfully designed, and the assay design success rate was 94.8%. Finally, 467 out of the 565 successfully-designed SNPs can display diversity at the loci were used to develop a set of core SNP arrays. To evaluate the application value of the core SNP markers in rice breeding, 481 rice germplasms were genotyped with three functional KASP markers designed from the sequences of GBSSI, SSIIa, and Badh2 from the core SNP arrays for estimation of their grain quality performance. Eighteen rice lines, including Xiangwanxian 13, Basmati 370, Ruanhua A, and PR 33319-9-1-1-5-3-5-4-1, harbor all three favorable alleles. The core KASP arrays were also used for rice germplasm assessment, genetic diversity and population evaluation. Four hundred and eighty-one rice germplasms were divided into 3 groups: POP1, POP2 and POP3. POP1 and POP2 were indica rice subgroups consisting of 263 and 186 rice germplasms, respectively. POP3 was a japonica rice subgroup consisting of 32 rice germplasms. The average FST value for the three subgroups was 0.3501; the FST value of POP1 and POP3 was the largest (0.5482), while that of POP1 and POP2 was the smallest (0.0721). The results showed that the genetic distance between the japonica and indica rice subspecies was large, indicating that the core SNP markers were effective at discriminating the population structure of the germplasms. Finally, the core KASP arrays were used for association analysis with milled grain traits. A total of 31 KASP markers were significantly associated (P < 0.01) with ML and the LWR. Among the 31 markers, 13 were developed based on cloned genes or on identified loci related to yield traits. Notably, several KASP markers associated with grain quality were also found to be associated with brown planthopper resistance or green leafhopper resistance simultaneously. CONCLUSIONS: The core KASP arrays developed in our study were efficient and versatile for rice germplasm assessment, genetic diversity and population evaluation and are valuable for promoting SNP molecular breeding in rice. Our study demonstrated that useful assays combined with molecular breeding can be exploited for important economic trait improvement in rice breeding.

High expression of Anxa2 and Stat3 promote progression of hepatocellular carcinoma and predict poor prognosis.

AIM: To elucidate whether the interaction between Anxa2 and Stat3 could promote the progression of hepatocellular carcinoma (HCC) and that high co-expression of Anxa2 and Stat3 could predict poor prognosis in HCC patients. METHODS: We investigated Anxa2 and Stat3 expression using Western blot analysis in 4 HCC and adjacent nontumor tissues and using immunohistochemistry in 100 patients' paraffin sections. Then we assessed the expression of Stat3, Anxa2 and co-expression of Stat3 and Anxa2 with relevant clinical pathological parameters and their prognostic value in HCC patients. The recurrence and overall survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models. RESULTS: The incidence of high Stat3 expression in HCC tissues (35%) was significantly higher than that in non-HCC tissues (8%) (P < 0.001). The same result was observed in Anxa2 (P < 0.001). Also, the overexpression of Stat3 or Anxa2 showed a significant relationship with the recurrence of the 100 HCC patients (P = 0.012; P = 0.003). Additionally, tumor size >3 cm in diameter, multiple tumor number, and the presence of microvascular tumor thrombus were also significantly associated with recurrence in 100 patients. Then, all enrolled patients were divided into four groups according to IHC score of Stat3 and Anxa2, and the results indicated a significant difference in recurrence time between the subgroups (P < 0.001). What's more, co-highexpression of Stat3 and Anxa2 was related to the presence of microvascular tumor thrombus (P = 0.003) and poor tumor differentiation (P < 0.001), but not relevant with other clinical features (All P > 0.05). CONCLUSION: The expression of Stat3, Anxa2, or co-high-expression of the two proteins was associated with HCC recurrence and survival.