Effect of miR-133 on apoptosis of trophoblasts in human placenta tissues via Rho/ROCK signaling pathway.

OBJECTIVE: The aim of this study was to explore the role of micro ribonucleic acid (miR)-133 in the apoptosis of human placental trophoblasts through the Ras homolog gene family (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway. PATIENTS AND METHODS: The plasma samples were collected from 30 patients with pre-eclampsia (PE) undergoing treatment and 30 healthy subjects (control group) who received physical examination in our hospital. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was utilized to measure the expression of miR-133 in PE patients and healthy people. Meanwhile, blood pressure, urine protein content, liver function, and kidney function were detected in patients of both groups as well. Subsequently, the placental trophoblasts were extracted and transfected with inhibitors and miRNA mimics to suppress and overexpress miR-133, respectively. The transfection efficiency was determined by RT-PCR. The levels of interleukin-6 (IL-6), IL-1, and tumor necrosis factor-alpha (TNF-α) were measured in both groups. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was performed to determine the apoptosis of trophoblasts. Next, the RT-PCR and Western blotting were carried out to detect the expressions of the Rho/ROCK pathway. Furthermore, the influence of miR-133 on the apoptosis of trophoblasts in human placenta tissues through Rho/ROCK was comprehensively observed. RESULTS: In vivo experiments demonstrated that the urinary protein content, miR-133 level, systolic blood pressure, diastolic blood pressure, and liver function and renal function indexes were significantly elevated in pre-eclampsia (PE) patients in comparison with normal subjects (p<0.05). After transfection of mimics and inhibitors, the expression of miR-133 was remarkably up- and down-regulated, respectively. The content of the inflammatory factors in miR-133 mimics group was overtly higher than the other two groups. The TUNEL staining results showed that the number of apoptotic cells significantly increased and decreased in the miR-133 mimics group and miR-133 inhibitors group, respectively. Subsequent experiments indicated that the expressions of apoptosis gene Caspase3, pathway gene, and protein ROCKI were notably up-regulated in miR-133 mimics group. However, they were evidently down-regulated in miR-133 inhibitors group than in the control group. In addition, a consistent trend was observed in the protein expression level. CONCLUSIONS: MiR-133 participates in the development and progression of PE through the Rho/ROCK signaling pathway, which may affect the apoptosis of trophoblasts in the placenta tissues.

Circ-ZNF609 promotes carcinogenesis of gastric cancer cells by inhibiting miRNA-145-5p expression.

OBJECTIVE: To elucidate the expression pattern and biological function of circular RNA ZNF609 (circ-ZNF609) in gastric cancer (GC). PATIENTS AND METHODS: Circ-ZNF609 expression in GC tissues and adjacent normal tissues (ANT) was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The regulatory effect of circ-ZNF609 on growth and metastasis of GC cells was evaluated through the Cell Counting Kit-8 (CCK-8), colony formation and transwell invasion assay, respectively. GC cell apoptosis influenced by circ-ZNF609 was examined by flow cytometry. The binding between circ-ZNF609 and miRNA-145-5p was verified by the Dual-Luciferase reporter gene assay. Finally, a series of rescue experiments were conducted to explore the mechanism of the circ-ZNF609/miRNA-145-5p axis in regulating GC progression. RESULTS: QRT-PCR data revealed a higher level of circ-ZNF609 in GC tissues relative to ANT. Identically, circ-ZNF609 was highly expressed in GC cell lines relative to controls. The knockdown of circ-ZNF609 in BGC823 and MGC803 cells suppressed proliferative and invasive abilities. MiRNA-145-5p was predicted to be the target gene of circ-ZNF609 by bioinformatics, and further verified by the Dual-Luciferase reporter gene assay. Rescue experiments showed that miRNA-145-5p knockdown partially reversed the regulatory effect of circ-ZNF609 on growth and metastasis of GC cells. CONCLUSIONS: Circ-ZNF609 promotes proliferative and invasive abilities of gastric cancer cells by inhibiting miRNA-145-5p expression as a ceRNA, thus accelerating gastric cancer progression.

Endoplasmic reticulum stress in bone marrow-derived cells prevents acute cardiac inflammation and injury in response to angiotensin II.

Inflammation plays an important role in hypertensive cardiac injury. The endoplasmic reticulum (ER) stress pathway is involved in the inflammatory response. However, the role of ER stress in elevated angiotensin II (Ang II)-induced cardiac injury remains unclear. In this study, we investigated the role of ER stress in Ang II-induced hypertensive cardiac injury. Transcriptome analysis and quantitative real-time PCR showed that Ang II infusion in mice increased ER stress-related genes expression in the heart. C/EBP homologous protein (CHOP) deficiency, a key mediator of ER stress, increased infiltration of inflammatory cells, especially neutrophils, the production of inflammatory cytokines, chemokines in Ang II-infused mouse hearts. CHOP deficiency increased Ang II-induced cardiac fibrotic injury: (1) Masson trichrome staining showed increased fibrotic areas, (2) immunohistochemistry staining showed increased expression of α-smooth muscle actin, transforming growth factor ß1 and (3) quantitative real-time PCR showed increased expression of collagen in CHOP-deficient mouse heart. Bone marrow transplantation experiments indicated that CHOP deficiency in bone marrow cells was responsible for Ang II-induced cardiac fibrotic injury. Moreover, TUNEL staining and flow cytometry revealed that CHOP deficiency decreased neutrophil apoptosis in response to Ang II. Taken together, our study demonstrated that hypertension induced ER stress after Ang II infusion. ER stress in bone marrow-derived cells protected acute cardiac inflammation and injury in response to Ang II.

First Report of Greenhouse Tomato Wilt Caused by Plectosphaerella cucumerina in China.

Tomato (Lycopersicon esculentum Mill.) is one of the most important vegetable crops in China. In May 2011, root rot and plant wilt were observed on tomato plants (variety Jinguan No. 5 and Meina) in 26 commercial greenhouses in Huludao city, Liaoning Province, China. Disease incidence was 30 to 95%. At beginning of fruit set, symptoms were chlorosis of lower leaves and lack of turgidity in young leaves. Severely affected plants were wilted and stunted as fruit approached maturity. Primary and secondary roots became necrotic with few fine feeder roots. Symptomatic roots were collected and cut into small pieces, disinfested in 2% sodium hypochlorite for 2 min, rinsed with sterile water, and placed on potato dextrose agar. After incubation at 25°C for 5 days, 20 axenic cultures were obtained from single conidia. Colonies were buff or salmon pink, moist, and had appressed, slimy mycelium. Aerial mycelium was sparse with simple or branched conidiophores. Conidia were 4.0 to 8.9 × 2.0 to 4.0 (average 6.9 × 2.8) µm, aggregated in slimy heads, hyaline ellipsoidal and ovoid, smooth, and 0 to 1 septate. Conidia were borne on phialides that were 6.3 to 24.3 × 1.4 to 3.3 (average 15.9 × 2.2) µm. These characteristics are typical of Plectosphaerella cucumerina (Lindf.) W. Gams (1) (anamorph: Fusarium tabacinum) (3). The internal transcribed spacer (ITS) region of the 20 isolates was amplified using the primers ITS1/ITS4 and sequenced. Identical sequences were obtained from all 20 isolates, and the sequence of isolate HLDT15 was submitted to GenBank (Accession No. KC894931). BLAST analysis of the sequence showed 100% similarity to P. cucumerina (AB469880). Pathogenicity tests were conducted with tomato variety Jinguan No. 5. Six 12-liter pots were filled with sterilized potting mix (equal parts sand, peat, and soil) and 200 ml conidial suspension (1 × 105 conidia ml-1). The conidial suspension of the isolate of P. cucumerina was prepared from 7-day-old cultures grown in potato dextrose broth on a shaker (120 rpm) at 25 ± 1°C. Six control pots were filled with potting mix and 200 ml of sterilized potato dextrose broth. Each pot was sown with six surface-sterilized (2% sodium hypochlorite for 2 min) tomato seeds. All the pots were kept in a greenhouse at 23 to 28°C. Three to four weeks after seedling emergence, all inoculated plants were dwarfed and lower leaves were chlorotic. Roots were necrotic and produced fewer fibrous roots. All characteristics were similar to original observations on the host of origin. Control plants remained asymptomatic. The same results were obtained when pathogenicity tests were repeated twice. P. cucumerina was reisolated from inoculated plants and matched the morphological and molecular characteristics of the original isolates. P. cucumerina was reported as a pathogen of tomato in Italy (2) and Australia (4). To our knowledge, this is the first report of P. cucumerina causing tomato wilt in China. So far, we have observed the disease on tomatoes in commercial greenhouses of Pulandian and Panjin city, Liaoning Province, China. The spread of this disease may pose a threat to tomato production in China. References: (1) A. Carlucci et al. Persoonia 28:34, 2012. (2) A. Matta et al. Riv. Patol. Veg. 14:119, 1978. (3) M. E. Palm et al. Mycologia, 87:397, 1995. (4) I. G. Pascoe et al. Mycol. Soc. 83:343, 1984.

Validation of quantitative fluorescent-PCR for rapid prenatal diagnosis of common aneuploidies in the Chinese population.

Quantitative fluorescent polymerase chain reaction (QF-PCR) is an accurate and reliable method for rapid detection of aneuploidy; however, it is not routinely used in China. We aimed to validate QF-PCR as a means for prenatal common aneuploidy screening and to analyze the heterozygosities of short tandem repeat (STR) markers in the Chinese population. The sequences of 19 STR markers in chromosomes 21, 18, 13, X, and Y were designed; three kinds of fluoresceins were used to label the primers, and the QF-PCR detecting conditions were explored and optimized. The results of analysis of 210 prenatal samples by multiplex QF-PCR were compared with karyotyping analysis. All cases were successfully tested by QF-PCR and conventional cytogenetic analysis. QF-PCR results were consistent with the results of cytogenetic analyses, with the exception of two cases. The sensitivity and specificity of QF-PCR to diagnose common aneuploidies were 94.74 and 100%, respectively. The heterozygosities of most of the markers were lower than reported for Western populations, but relatively similar to those of other Asian populations. We conclude that QF-PCR is able to detect the common aneuploidies for prenatal diagnosis with high detection efficacy; therefore it is suitable for rapid prenatal diagnosis and for large-scale testing in laboratories. However, we need to add new STR markers or to find alternative STR markers with high heterozygosity in order to make this technique useful for routine diagnosis.

Nexin-1 inhibits the activity of human brain trypsin.

Trypsin and other trypsin-like serine proteases have been shown to play important roles in neural development, plasticity and neurodegeneration. Their activity is modulated by serine protease inhibitors, serpins. However, for human brain trypsin, trypsin-4, no brain-derived inhibitors have been described. Here, we report that nexin-1 inhibits trypsin-4, and forms stable complexes only with this trypsin-isoenzyme. This result suggests that nexin-1 could modulate trypsin activity in brain where both nexin-1 and trypsin-4 are expressed.

Functional reconstruction of maxilla with pedicled buccal fat pad flap, prefabricated titanium mesh and autologous bone grafts.

The aim of this study was to evaluate the use of pedicled buccal fat pad flap (PBFPF), prefabricated titanium mesh and autologous bone graft in maxillary reconstruction. Seventeen patients with a unilateral class I-III maxillary defect were involved. Preoperatively, a solid model was manufactured based on virtual maxillectomy and reconstruction of the abnormal maxilla. Intraoperatively, PBFPF was applied to repair the soft-tissue defect, serving as nasal lining and the receiving bed for bone grafts. Titanium mesh was prefabricated on the solid model and then, together with bone grafts from iliac crest, fixed to residual bones to reconstruct the hard-tissue defect. Postoperative aesthetic appearance and function were followed up. No exposure of titanium mesh, leakage or oronasal regurgitation occurred. Of the patients with a class I or II defect 91% (10/11) and of those with a class III defect 50% (3/6) gained a good appearance. Fifteen patients were articulate. Eleven patients received dental rehabilitation and had a normal diet. PBFPF with prefabricated titanium mesh and autologous bone grafts is a reliable option for reconstruction of unilateral maxillary defects of class I and II, but this method alone should be used cautiously in defects of class III and beyond.

Clusterin plays an important role in hepatocellular carcinoma metastasis.

To identify genes associated with tumor metastasis in hepatocellular carcinoma (HCC), gene expression profiles between a pair of primary HCC (H2-P) and their matched metastatic HCC (H2-M) were compared. Overexpression of clusterin (CLU) was found in H2-M cells. To determine the roles CLU played in HCC metastasis, CLU was transfected into H2-P cells. Overexpression of CLU in H2-P cells increased cell migration by twofold in vitro and formation of metastatic tumor nodules in liver by eightfold in vivo. To evaluate the correlation of CLU expression with HCC metastasis, the expression levels of CLU in HCCs were investigated using a tissue microarray (TMA) containing 104 pairs of primary HCCs and their matched metastases. The frequency of CLU overexpression increased significantly in metastatic HCCs (59.1%) compared with that in primary tumors (32.6%, P<0.001). To gain additional insight into the function of CLU, the expression profile of H2P-CLU was compared with vector-transfected H2-P cells by cDNA microarray. A total of 35 upregulated and 14 downregulated genes were detected in H2P-CLU. One of the upregulated genes known as YKL-40, which is implicated in matrix-remodeling and metastasis, was further studied using TMA. A significant correlation (P<0.001) between the expression levels of YKL-40 and CLU was observed, implying that the CLU-YKL-40 pathway may play an important role in HCC metastasis.