MicroRNA-193a-3p participates in the progression of rheumatoid arthritis by regulating proliferation and apoptosis of MH7A cells through targeting IGFBP5.

OBJECTIVE: This study aims to explore the regulatory effect of microRNA-193a-3p on rheumatoid arthritis (RA) and its underlying mechanism. PATIENTS AND METHODS: Expression level of microRNA-193a-3p in synovial tissues extracted from 30 RA patients and healthy controls was detected by quantitative Real-time polymerase chain reaction (qRT-PCR). MH7A cells were subjected to TNF-α induction for constructing the in vitro RA model. After transfection of microRNA-193a-3p inhibitor in MH7A cells, proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Enzyme-linked immunosorbent assay (ELISA) was conducted to determine levels of interleukin 6 (IL-6) and IL-8 in MH7A cells. Subsequently, the dual-luciferase reporter gene assay was carried out to verify the binding condition between microRNA-193a-3p and IGFBP5. Rescue experiments were conducted to evaluate the proliferation and apoptosis of MH7A cells with knockdown of microRNA-193a-3p and IGFBP5. RESULTS: MicroRNA-193a-3p was highly expressed in synovial tissues of RA patients and TNF-α-induced MH7A cells than those of controls. TNF-α induction significantly increased the proliferative rate of MH7A cells, reaching the peak at 96 h. After knockdown of microRNA-193a-3p, the promoted proliferation by TNF-α induction was significantly inhibited. In addition, TNF-α induction significantly inhibited the apoptosis of MH7A cells. After inhibition of microRNA-193a-3p expression, the inhibited apoptosis by TNF-α induction remarkably increased. TNF-α induction upregulated levels of IL-6 and IL-8 in MH7A cells, which were remarkably reduced after the microRNA-193a-3p knockdown. Dual-luciferase reporter gene assay confirmed that IGFBP5 could bind to microRNA-193a-3p, and its expression was negatively regulated by microRNA-193a-3p. The regulatory effects of microRNA-193a-3p on proliferation and apoptosis of MH7A cells were reversed by IGFBP5 knockdown. CONCLUSIONS: MicroRNA-193a-3p is highly expressed in the synovial tissues and cells of rheumatoid arthritis. MicroRNA-193a-3p participates in the process of rheumatoid arthritis by regulating the proliferation, apoptosis and inflammatory response of MH7A cells through targeting IGFBP5.

Short Communication: Genetic linkage map of Cucurbita maxima with molecular and morphological markers.

Cucurbita maxima is one of the most widely cultivated vegetables in China and exhibits distinct morphological characteristics. In this study, genetic linkage analysis with 57 simple-sequence repeats, 21 amplified fragment length polymorphisms, 3 random-amplified polymorphic DNA, and one morphological marker revealed 20 genetic linkage groups of C. maxima covering a genetic distance of 991.5 cM with an average of 12.1 cM between adjacent markers. Genetic linkage analysis identified the simple-sequence repeat marker 'PU078072' 5.9 cM away from the locus 'Rc', which controls rind color. The genetic map in the present study will be useful for better mapping, tagging, and cloning of quantitative trait loci/gene(s) affecting economically important traits and for breeding new varieties of C. maxima through marker-assisted selection.

Sequence variations in the FAD2 gene in seeded pumpkins.

Seeded pumpkins are important economic crops; the seeds contain various unsaturated fatty acids, such as oleic acid and linoleic acid, which are crucial for human and animal nutrition. The fatty acid desaturase-2 (FAD2) gene encodes delta-12 desaturase, which converts oleic acid to linoleic acid. However, little is known about sequence variations in FAD2 in seeded pumpkins. Twenty-seven FAD2 clones from 27 accessions of Cucurbita moschata, Cucurbita maxima, Cucurbita pepo, and Cucurbita ficifolia were obtained (totally 1152 bp; a single gene without introns). More than 90% nucleotide identities were detected among the 27 FAD2 clones. Nucleotide substitution, rather than nucleotide insertion and deletion, led to sequence polymorphism in the 27 FAD2 clones. Furthermore, the 27 FAD2 selected clones all encoded the FAD2 enzyme (delta-12 desaturase) with amino acid sequence identities from 91.7 to 100% for 384 amino acids. The same main-function domain between 47 and 329 amino acids was identified. The four species clustered separately based on differences in the sequences that were identified using the unweighted pair group method with arithmetic mean. Geographic origin and species were found to be closely related to sequence variation in FAD2.

First Report of Phytophthora Blight of Lily Caused by Phytophthora nicotianae in China.

Lily (Lilium spp.) is an economically important cut flower in China. In August 2009, 30 to 40% of plants of lily cv. Siberia in a greenhouse for cut flower production in Yunnan, China were severely diseased. Infected plants developed water-soaked lesions and soft rot on the base of stems and leaves near the soil surface. As the disease progressed, stems bent and plants collapsed. Soft rot symptoms were observed on some bulbs and roots of severely diseased plants. Small, diseased tissue fragments (approximately 3 mm) were surface disinfected with 0.5% NaOCl and then plated to Phytophthora selective medium (10% V8 juice agar) (4). Inoculated dishes were incubated at 25°C in the dark. After 5 days, white colonies with abundant aerial mycelia developed from all plated tissue samples. The fungus had aseptate hyphae. Sporangia were papillate, both caducous and noncaducous, and the shape ranged from ovoid to spherical. The dimensions of sporangia were 30 to 62 × 21 to 46 µm. On the basis of morphological features, isolates were identified as Phytophthora nicotianae Breda de Haan. The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS1/ITS4 and sequenced. BLAST analysis of the 835-bp fragment showed a 99% homology with the sequence of P. nicotianae AY833527. The nucleotide sequence has been assigned GenBank No. GU299778. PCR amplification of genomic DNAs using the P. nicotianae-specific primer pair ITS3-PNIC1 generated a 455-bp sequence (3). The result further confirmed the identity of P. nicotianae. Pathogenicity tests were conducted in the greenhouse on lily cv. Siberia grown in pots. Ten 3-month-old plantlets were inoculated by watering the wounded stem bases and soil surface with 30 ml of zoospore suspensions (105 spores per ml). Five uninoculated plantlets were used as controls. All plantlets were covered with plastic bags and incubated at room temperature (22 to 26°C) for 48 h. Inoculated plants developed initial symptoms of slight chlorosis and wilting of lower leaves. Within a 3-week period, all plants died due to soft rot of stem bases and leaves. The pathogen was reisolated from inoculated plants but not from control plants that were symptomless. P. nicotianae has been reported as the causal agent of Phytophthora blight on lily in Korea, Japan, and Hungary (1,2). To our knowledge, this is the first report of Phytophthora blight of lily in China. References: (1) J. Bakonyi et al. Plant Pathol. 50:795, 2001. (2) H. J. Jee and W. G. Kim. Plant Pathol. J. 14:452, 1998. (3) P. W. Tooley et al. Appl. Environ. Microbiol. 63:1467, 1997. (4) X. B. Zheng. Phytophthora and Its Research Technology. Beijing. China Agriculture Press, Beijing, 1997.

First Report of Southern Blight Caused by Sclerotium rolfsii on Lily in China.

Lily (Lilium spp.) is an economically important cut flower cultivated in China. The soilborne fungus, Sclerotium rolfsii, is a major pathogen on many plants. During July 2005, severe basal stem rot and bulb rot symptoms were observed on an oriental lily cultivar (Sorbonne) in some commercial fields in northern Kunming (China). Disease incidence ranged from 20 to 30% across fields. Leaves of infected plants were chlorotic initially. As the disease progressed, stems and bulbs rotted and plants wilted. In the presence of abundant moisture, a white mycelium occurred on infected tissues. White or light-to-dark brown sclerotia (1 to 3 mm in diameter) developed from mycelium. Fungal isolates from infected bulbs grown on potato dextrose agar (PDA) produced white mycelia and 1- to 2-mm diameter dark brown sclerotia. Sclerotia were nearly round with smooth surfaces and distributed over the entire colony. Isolates were identified as S. rolfsii on the basis of mycelial characteristics and color, size, and distribution of sclerotia. Pathogenicity was tested in the greenhouse on oriental lily cv. Sorbonne grown in pots (1 plant per pot, five replicates). Inoculum that consisted of 1 g per pot of wheat kernels infested with mycelium and sclerotia was placed at the base of each inoculated plant. Five noninoculated plants served as controls. The inoculation trial was repeated once. After inoculation, all plants were covered with a polyethylene bag for 72 h and kept at temperatures ranging between 25 and 27°C. Inoculated plants developed symptoms of leaf yellowing within 12 days, soon followed by the appearance of white mycelium and sclerotia, and then eventually wilted. Control plants remained symptomless. S. rolfsii was reisolated from inoculated plants. To our knowledge, this is the first report of southern blight caused by S. rolfsii on lily in China. Infection of lily bulbs by S. rolfsii may cause losses in production fields in China, and the presence of infected bulbs may also interfere with bulb shipment.