Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro / 中国血吸虫病防治杂志

Objective To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412 (rSjCP1412) on proliferation, cell cycle, apoptosis and activation of human hepatic stellate cells LX-2 in vitro, and explore the underlying mechanisms. Methods The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression, and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis. Yeast RNA was digested using 12.5, 25.0, 50.0 µg rSjCP1412 proteins at 37 °C for 2, 3, 4 h, and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of rSjCP1412 protein. The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay, and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining, while the percentage of LX-2 cells at G0/G1, S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining. The type I collagen, type III collagen and α-smooth muscle actin (α-SMA) mRNA expression was quantified using quantitative florescent real-time PCR (qPCR) assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of rSjCP1412 protein for 48 h, while soluble egg antigen (SEA) served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments. The expression of collagen I, α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein, transforming growth factor-β1 (TGF-β1) alone or in combination, to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells. Results The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared, which had a RNase activity. Compared with the normal group, the survival rates of LX-2 cells significantly decreased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein and SEA for 48 h (F = 22.417 and 20.448, both P values < 0.05). The apoptotic rates of LX-2 cells significantly increased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h (F = 11.350, P < 0.05), and treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h resulted in arrest of LX-2 cells in G0/G1 phase (F = 20.710, P < 0.05). Treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h caused a significant reduction in relative expression levels of collagen I (F = 11.340, P < 0.05), collagen III (F = 456.600, P < 0.05) and α-SMA mRNA (F = 23.100, P < 0.05) in LX-2 cells, and both rSjCP1412 protein and SEA treatment caused a significant reduction in collagen I (F = 1 302.000, P < 0.05), α-SMA (F = 49.750, P < 0.05) and Smad4 protein expression (F = 52.420, P < 0.05) in LX-2 cells. In addition, rSjCP1412 protein treatment inhibited collagen I (F = 66.290, P < 0.05), α-SMA (F = 31.300, P < 0.05) and Smad4 protein expression (F = 27.010, P < 0.05) in LX-2 cells activated by TGF-β1. Conclusion rSjCP1412 protein may induce apoptosis of LX-2 cells and inhibit proliferation, cell cycle and activation of LX-2 cells through down-regulating Smad4 signaling molecules.

Hsa-miRNA-143-3p Reverses Multidrug Resistance of Triple-Negative Breast Cancer by Inhibiting the Expression of Its Target Protein Cytokine-Induced Apoptosis Inhibitor 1 In Vivo / 한국유방암학회지

PURPOSE: Multidrug resistance (MDR) remains a major obstacle in the treatment of triple-negative breast cancer (TNBC) with conventional chemotherapeutic agents. A previous study demonstrated that hsa-miRNA-143-3p plays a vital role in drug resistance of TNBC. Downregulation of hsa-miRNA-143-3p upregulated the expression of its target protein cytokine-induced apoptosis inhibitor 1 (CIAPIN1) in order to activate MDR, while upregulation of hsa-miRNA-143-3p effectively enhances the sensitivity of drug-resistant TNBC cells to chemotherapeutics. The present study aimed to further verify these findings in vivo. METHODS: We established a hypodermic tumor nude mice model using paclitaxel-resistant TNBC cells. We expressed ectopic hsa-miRNA-143-3p under the control of a breast cancer-specific human mammaglobin promoter that guided the efficient expression of exogenous hsa-miRNA-143-3p only in breast cancer cells. Thereafter, we overexpressed hsa-miRNA-143-3p in xenografts using a recombinant virus system and quantified the expression of hsa-miRNA-143-3p, CIAPIN1 protein, and proteins encoded by related functional genes by western blot. RESULTS: We successfully completed the prospective exploration of the intravenous virus injection pattern from extensive expression to targeted expression. The overexpression of hsa-miRNA-143-3p significantly alleviated chemoresistance of TNBC by inhibiting viability. In addition, we observed that the expression of CIAPIN1 as a hsa-miRNA-143-3p target protein was remarkably decreased. CONCLUSION: We partly illustrated the mechanism underlying the hsa-miRNA-143-3p/CIAPIN1 drug resistance pathway. HsamiRNA-143-3p as a tumor suppressive microRNA may be a novel target to effectively reverse MDR of TNBC in vivo.

Detection of novel mutations related to maturity-onset diabetes of the young / 基础医学与临床

Objective To find novel genes related to maturity-onset diabetes of the young(MODY)or novel muta-tions of known MODY related genes and provide the basis for MODY diagnosis. Methods Taking the major clinical features of MODY as screening criteria,we selected four patients from the Endocrine Department of Peking Union Medical College Hospital and prepared their genomic DNA sample for whole exome sequencing.PCR and Sanger se-quencing are used to validate the sequencing results. Results Two novel mutations of the GCK gene and HNF4α gene, c.1348G>T(p.Ala450Thr)and c.758G>A(p.Arg253Gln)were found in two patients. Conclusions These two patients are both MODY patients and this is the first time the novel mutations were found.

Autologous bone marrow stem cell transplantation for treatment terminal liver diseases / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of autologous bone marrow-derived stem cell (BMSCs) transplantation in the treatment of liver failure and decompensated hepatic cirrhosis.</p><p><b>METHODS</b>Bone marrow was harvested (65-95 ml) from 24 patients in the transplantation group. The BMSCs were isolated and infused into liver or spleen of patients via hepatic or splenic artery. At different time points after the transplantation, the patients' liver function and prothrombin time (PT) were evaluated, and the survival rate and symptoms of the patients were recorded.</p><p><b>RESULTS</b>All the serum biochemical indexes remained stable 2 weeks after the transplantation, and at 4 weeks after transplantation, albumin level increased significantly in comparison with the preoperative level (P<0.05). At 12 weeks, the albumin level further increased (P<0.01) along with Pre-ALB (P<0.01), while total bilirubin, tolal bile acid, PT and fibrinogen were all significantly lowered (P<0.05), and globulin, ALT, and AST remained unchanged (P>0.05). One week after the transplantation, improved appetite was observed in 22 cases (91.67%), and 21 cases (87.5%) showed better physical strength; at 2 weeks, hepatic face improved in 15 cases (62.5%), and spider telangiectasia was significantly reduced in one case; at 12 weeks, the survival rate of the patients was 62.5%, and 9 died or gave up treatment due to chronic liver failure complicated by spontaneous bacterial peritonitis, hepatorenal syndrome, or DIC. No complications associated with the transplantation occurred in these patients.</p><p><b>CONCLUSION</b>BMSC transplantation can significantly improve the liver function of patients with terminal liver disease with good safety and effectiveness.</p>

A novel mutation in the myocilin gene identified in a Chinese primary open angle glaucoma family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the possible myocilin molecular genetic defect underlying POAG in China and to identify the pathogenic mutation causing the disease.</p><p><b>METHODS</b>The majority of 1 branch of a large Chinese POAG family were personally examined by two senior ophthalmologists. The diagnoses were made by both doctors according to the signs of elevated intraocular pressure, glaucomatous optic neuropathy and glaucomatous visual field defect. All coding sequences of the myocilin gene plus the flanking sites were amplified by polymerase chain reaction (PCR) using genomic DNA from all examined family members followed by sequencing of the PCR products. One hundred normal control subjects were screened by single strand confirmational polymorphism analysis for the mutation.</p><p><b>RESULTS</b>This Chinese pedigree exhibited autosomal dominant mode of inheritance. The onset age ranged from 26 to 59 years. A novel disease-causing missense mutation T455K in the third exon of the myocilin gene was identified in all affected family members, all glaucoma suspects and 4 individuals who have not shown apparently signs of glaucoma. None of the subjects without the mutation had glaucoma. Affected individuals with the T455K mutation showed variable onset between 26 and 59 years of age. Filtering surgery was performed on all of 7 affected family members. The T455K mutation in myocilin gene was not found in the normal controls. A previously reported polymorphism IVS2+35(A to G)was detected in 4 individuals.</p><p><b>CONCLUSION</b>The novel myocilin sequence alteration T455K that is highly associated with the development of glaucoma and locates in a very conserved residue is proven to be a disease-causing missence mutation. All affected individuals and all POAG suspects in this family are identified to have this mutation. The mutation in this family is associated with a phenotype characterized by mix-onset open angle glaucoma and associated with a high penetrance. It is important for the mutation screening and periodical checkups of presymptomatic individuals belonging to the family of a POAG patient with T455K mutation.</p>