Role of cold-inducible RNA-binding protein in acute renal injury in a mouse model of myocardial ischemia-reperfusion: relationship with NF-κB signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of cold-inducible RNA-binding protein (CIRP) in acute renal injury in a mouse model of myocardial ischemia-reperfusion (I/R) and the relationship with nuclear factor kappa B (NF-κB) signaling pathway.Methods:Twenty-four SPF-grade healthy male C57BL/6 mice, aged 6-8 weeks, with body mass index of 24-28 g, were divided into 3 groups ( n=8 each) using a random number table method: sham operation group (Sham group), myocardial I/R group (I/R group) and myocardial I/R + CIRP-derived peptide C23 group (I/R+ C23 group). The model of myocardial I/R was developed by ligation of the left anterior descending coronary artery for 30 min followed by 120-min reperfusion in anesthetized animals. CIRP-derived peptide C23 8 mg/kg was intraperitoneally injected before myocardial ischemia and reperfusion in I/R+ C23 group, while Sham group was only threaded without ligation. Blood samples were collected from the right internal carotid artery at 120 min of reperfusion for determination of the serum creatine kinase isoenzymes (CK-MB), lactic dehydrogenase (LDH), creatinine (Cr) and blood urea nitrogen (BUN) concentrations. Renal tissues were obtained for examination of the pathological changes, and the tubular injury score was assessed. The expression of NF-κB, phosphorylated NF-κB (p-NF-κB), Nod-like receptor protein 3 (NLRP3), interleukin-1beta (IL-1β) and IL-18 in renal tissues was detected by Western blot. The expression of Toll-like receptor 4 (TLR4), NLRP3, IL-1β, TNF-α and IL-6 mRNA was determined by real-time polymerase chain reaction. Results:Compared with Sham group, the levels of serum CK-MB, LDH, Cr and BUN and renal tubule injury score were significantly increased, the expression of p-NF-κB, NLRP3, IL-1β and IL-18 was up-regulated, the expression of TLR4, NLRP3, IL-1β, TNF-α and IL-6 mRNA was up-regulated ( P<0.05), and the pathological injury to renal tissues was aggravated in I/R group. Compared with I/R group, the serum CK-MB, LDH, Cr, BUN and renal tubular injury score were significantly decreased, and the expression of p-NF-κB, NLRP3, IL-1β and and IL-18 was down-regulated, the expression of TLR4, NLRP3, IL-1β, TNF-α and IL-6 mRNA was down-regulated ( P<0.05), and the pathological injury to renal tissues was alleviated in I/R+ C23 group. Conclusions:CIRP is involved in the process of acute renal injury in a mouse model of myocardial I/R, which is associated with activation of NF-κB signaling pathway and promotion of inflammatory responses.

Mechanism for regulation of trophoblast invasion through piR-3127964 via PIWIL-3/sialophorin pathway / 中华围产医学杂志

Objective:To investigate the molecular mechanism for regulation of trophoblast invasion by piR-3127964, which is differentially expressed in placental tissues of preeclamptic and healthy pregnant women.Methods:Placenta samples of healthy (control group, n=12) and preeclamptic pregnant (PE group, n=10) women who delivered by caesarean section and chorionic villi specimens of patients undergoing artificial abortion were collected in the Department of Obstetrics of the First Affiliated Hospital of Chongqing Medical University during November 2020 to August 2021. Total RNA was extracted from placenta samples and sequenced and the expression of piR-3127964 in different tissues was determined by real-time quantitative-polymerase chain reaction (qRT-PCR). The expressions of PIWI proteins including PIWIL-1, PIWIL-2 and PIWIL-3 in different tissues were detected by Western blot. The expressions of two candidate targets, guanine nucleotide-binding protein-like 3-like (GNL3L) mRNA and sialophorin (SPN) mRNA were evaluated by qRT-PCR after exogenous treating HTR-8/SVneo cells with mimics, inhibitor or negative control of piR-3127964, respectively. qRT-PCR was also used to detect the relative expression of GNL3L and SPN at mRNA level in placentas of all women. The interactions between GNL3L/SPN and piR-3127964 were analyzed by double luciferase reporter gene detection. The localization of piR-3127964 and SPN in chorionic villi was detected by fluorescence in situ hybridization and immunofluorescence. Transwell assay was performed to analyze the influence of piR-3127964 on the invasion of HTR-8/SVneo cells and the possible mechanism. Independent sample t-test, analysis of variance, and LSD post test were used for analysis Results:(1) Enrichment pathways of candidate targets predicted by differentially expressed piR-3127964 were associated with cell motility. There were statistically significant differences in piR-3127964 expression in villi, healthy and preeclamptic placentas (2.950±0.853 vs 1.036±0.303 vs 0.254±0.155, F=27.35, P<0.05), and piR-3127964 was predominantly expressed in extravillous cytotrophoblasts (EVTs). (2) The expression of PIWIL-3 protein in placentas of preeclamptic patients was significantly lower than that in healthy placentas and villi (0.810±0.400 vs 3.175±0.429 and 6.843±1.379, F=49.36, P<0.05). (3) Compared with the control group, exogenous piR-3127964 mimics (piR-mimics) and inhibitors (piR-inhibitor) significantly affected the expression of SPN mRNA (0.971±0.045 vs 0.732±0.010, F=6.50; 1.076±0.073 vs 1.293±0.092, F=7.58; both P<0.05), while the expression of GNL3L mRNA had no significant correlation with piR-3127964 level. (4) The luciferase activity of wild-type SPN (SPN-WT) plasmids was significantly affected by piR-mimics (1.010±0.049 vs 0.645±0.047, t=9.34, P<0.05) and piR-inhibitor (1.035±0.058 vs 1.397±0.015, t=－10.60, P<0.05). (5) SPN mRNA was significantly upregulated in placentas of preeclamptic patients than in healthy placentas (2.097±0.239 vs 1.305±0.290, t=－4.22, P<0.05), but no significant difference in the expression of GNL3L mRNA was observed. Immunofluorescence experiment showed that SPN was expressed in EVTs. (6) The invasive potential of HTR8/SVneo cells treated with piR-inhibitor was significantly inhibited, but this effect could be reversed by SPN knockdown (160.714±53.860 vs 371.667±103.061 and 344.333±120.267, F=9.76, both P<0.05). Conclusions:piR-3127964 expression is abnormally downregulated in placentas of preeclamptic patients, resulting in inhibition of trophoblasts invasion through upregulation of SPN expression, which may be related to the pathogenesis of preeclampsia.

A case of combined oxidative phosphorylation deficiency type 10 with tachypnea as first manifestation / 中华围产医学杂志

A term infant born small for gestational age presented with tachypnea as the first symptom one week after birth and had recurrent lactic acidosis. Whole-exome sequencing revealed compound heterozygous variants of c.1640C>T(p.A547V) and c.1274delG(p.G425Efs*23) in MTO1 gene, based on which the patient was diagnosed as combined oxidative phosphorylation deficiency type 10. The patient developed Klebsiella pneumoniae sepsis and died at 41 days of age. Combined oxidative phosphorylation deficiency type 10 is a type of mitochondrial disease inherited in an autosomal recessive manner. Patients with the onset of symptoms in the neonatal period are likely to have a poor prognosis and there is no effective treatment at present. The heterozygous variants of c.1640C>T and c.1274delG detected in this case are de novo variants, which expand the spectrum of variants in MTO1 gene.

Effect of long non-coding RNA LNC 01309 on proliferation and migration abilities of human hepatoma cells and its mechanism of action / 临床肝胆病杂志

Objective To investigate the effect of long non-coding RNA (lncRNA) LNC01309 on the proliferation and migration abilities of human hepatocellular carcinoma (HCC) cells and its mechanism of action. Methods HCC samples and corresponding adjacent tissue samples were collected from 12 patients with HCC who underwent surgical treatment in The Sixth Medical Center of PLA General Hospital from February 2018 to June 2019, and quantitative real-time PCR was used to measure the relative expression level of LNC01309. Quantitative real-time PCR was also used to measure the expression level of LNC01309 in human hepatoma cell lines (HepG2, SNU-398, and Hep3B) and the human immortalized normal liver cell line THLE-2. After LNC01309 was overexpressed in HepG2 cells, the cells were divided into plasmid control group (pEXP-control) and overexpression group (pEXP-LNC01309). CCK-8 assay was used to observe the change in cell proliferation, and wound healing assay and Transwell assay were used to observe migration ability. RNA co-immunoprecipitation was used to detect the interaction between LNC01309 with RBM38, with cells divided into IgG group and RBM38 antibody group, and cycloheximide chase assay was used to analyze the effect of LNC01309 on the stability of RBM38 protein. RBM38 was overexpressed in HepG2 cells to conduct the recovery experiment, and CCK-8 assay, wound healing assay, and Transwell assay were used to observe the changes in cell proliferation and migration abilities. The t -test was used for comparison of continuous data between two groups. Results The mean expression level of LNC01309 in HCC tissue was significantly higher than that in adjacent tissue (4.225±2.285 vs 1.541±0.530, t =3.618, P =0.004), and the relative expression level of LNC01309 in hepatoma cells (HepG2, SNU-398, and Hep3B) was also significantly higher than that in normal hepatocytes (THLE-2) ( t =4.231、6.489、14.480, all P < 0.05). Compared with the control group, HepG2 cells with the overexpression of LNC01309 had significant increases in growth rate (OD 450 value at 96 hours: 1.885±0.107 vs 2.527±0.234, t =4.330, P =0.012) and migration ability (11.65%±2.40% vs 35.66%±4.90%, t =9.837, P < 0.001; 100.00%±3.11% vs 161.00%±35.93%, t =4.399, P =0.005); however, the upregulated proliferation and migration abilities of hepatoma cells induced by LNC01309 overexpression were partially inhibited by RBM38 (OD 450 value at 96 hours: 2.500±0.227 vs 1.913±0.282, t =2.812, P =0.048; 168.00%±9.43% vs 117.20%±18.03%, t =6.622, P < 0.001). Compared with the IgG control group, RBM38 antibody significantly enriched the precipitation of LNC01309 ( t =3.846, P =0.031). The results of cycloheximide chase assay showed that the LNC01309 overexpression group had a significant reduction in the stability of RBM38 protein ( t =8.038, P =0.001). Conclusion The newly identified LNC01309 reduces the stability of RBM38 protein through interaction with RBM38 and promotes the proliferation and migration of HCC cells.

Expression and clinical significance of autophagy-related proteins LC3, P62 and Beclin-1 in hypertrophic scar formation / 中国医师杂志

Objective To explore the differences in autophagic expression levels between hypertrophic scar (HS) tissue and normal skin tissue,and further investigate the relationship between hypertrophic scar formation and autophagy protein expression through the rabbit ear hypertrophic scar model.Methods 30 patients with hypertrophic scar were collected.One hypertrophic scar tissue and one normal skin tissue were harvested.The relative expressions of LC3,P62 and Beclin-1 in each tissue specimen were detected by immunohistochemistry and Western blot.Western blot was used to detect the autophagic-associated protein LC3 (MAPLC3),P62 and Beclin-1 in the hypertrophic scar tissue of rabbit ear and the corresponding normal tissue of rabbit ears at 4 weeks,8 weeks,12 weeks,and 24 weeks,and further explore their clinical significance.Results In vivo,the expression of hypertrophic scar tissue protein LC3 and Beclin-1 was significantly stronger than that in normal skin tissue (P ＜ 0.05).The expression of P62 was significantly weaker than that in normal skin tissue (P ＜ 0.05).In animal experiments,during the process of HS formation,the protein expression of LC3 gradually increased,while the protein expression of P62 gradually decreased;the protein expression of Beclin-1 was higher than that of normal rabbit ears tissue,with statistically significant differences (P ＜ 0.05).Conclusions The expression of LC3 and Beclin-1 in human hypertrophic scar tissues is higher than that in normal tissues.While the expression of P62 is lower than that in normal tissues.That is,the expression of autophagy in human hypertrophic scar tissue showed an upward trend in a certain period of time,and was significantly higher than that in normal tissue.

Physical and Functional Interaction between 5-HT₆ Receptor and Nova-1

5-HT₆ receptor (5-HT₆R) is implicated in cognitive dysfunction, mood disorder, psychosis, and eating disorders. However, despite its significant role in regulating the brain functions, regulation of 5-HT₆R at the molecular level is poorly understood. Here, using yeast two-hybrid assay, we found that human 5-HT₆R directly binds to neuro-oncological ventral antigen 1 (Nova-1), a brain-enriched splicing regulator. The interaction between 5-HT₆R and Nova-1 was confirmed using GST pull-down and co-immunoprecipitation assays in cell lines and rat brain. The splicing activity of Nova-1 was decreased upon overexpression of 5-HT₆R, which was examined by detecting the spliced intermediates of gonadotropin-releasing hormone (GnRH), a known pre-mRNA target of Nova-1, using RT-PCR. In addition, overexpression of 5-HT₆R induced the translocation of Nova-1 from the nucleus to cytoplasm, resulting in the reduced splicing activity of Nova-1. In contrast, overexpression of Nova-1 reduced the activity and the total protein levels of 5-HT₆R. Taken together, these results indicate that when the expression levels of 5-HT₆R or Nova-1 protein are not properly regulated, it may also deteriorate the function of the other.

Clinical Relevance for Serum Cold-Inducible RNA-Binding Protein Level in Alopecia Areata

BACKGROUND: Alopecia areata (AA), a chronic, relapsing hair-loss disorder, is considered to be a T-cell-mediated autoimmune disease. Cold-inducible RNA-binding protein (CIRP) belongs to a family of cold-shock proteins that respond to cold stress, and has been identified as a damage-associated molecular pattern (DAMP) molecule that triggers the inflammatory response. Recent studies have shown that high-mobility group box 1, another DAMP molecule, is elevated in serum and scalp tissue of AA patients, suggesting a relationship between DAMP molecules and the pathogenesis of AA. OBJECTIVE: To investigate the clinical significance of serum CIRP levels in AA. METHODS: The serum levels of CIRP were compared between 68 patients with AA and 20 healthy controls. Additionally, the correlation between CIRP level and various clinical parameters was evaluated. RESULTS: The serum CIRP levels were significantly higher in AA patients compared to healthy subjects. Moreover, there was an association between the serum CIRP level and clinical characteristics, such as disease duration and disease activity. However, there was no significant difference in the serum CIRP level among the clinical types of AA (AA multiplex, alopecia totalis, and alopecia universalis). CONCLUSION: These results suggest that CIRP may play a significant role in the pathogenesis of AA and could be a potential biologic marker for monitoring the disease activity of AA.

Characterization of the mRNA untranslated regions [UTR] of the Trypanosoma cruzi LYT1 isoforms derived by alternative trans-splicing / Caracterización de las regiones no traducidas ARN [UTR] de las isoformas de Trypanosoma cruzi LYT1 derivadas de un trans-empalme alternativo / Caracterização das regiões não-traduzidas do RNA [UTR] das isoformas de Trypanosoma cruzi LYT1 derivadas de uma junção trans-alternativa

Abstract In trypanosomatids, gene expression is mainly regulated at posttranscriptional level, through mechanisms based on the interaction between RNA Binding Proteins [RBPs] and motifs present in the untranslated regions [UTRs] of the mRNAs, which altogether form ribonucleoproteic complexes [RNP] that define the fate of the mRNA. The pre-mRNA derived from the LYT1 gene of Trypanosoma cruzi, is processed by alternative trans-splicing, resulting in different mRNAs which code for the isoforms mLYTl and kLYTl, proteins having differential expression, cellular location and function. The aim of this study was to characterize the 5' and 3' UTRs of the LYT1 mRNAs as the initial step towards the objective of identification of the RBPs responsible for their differential expression. The presence of the two types of 5' UTRs were confirmed in two T. cruzi isolates belonging to the DTU I, thus, corroborating the occurrence of alternative trans-splicing also in the LYT1 gene of this T. cruzi DTU. In addition, for the first time, was unscovered the existence of two types of LYT1 mRNAs transcripts, differing in length by 116 nts, that are generated by alternative polyadenylation. Furthermore, an in-silico analysis of the experimentally obtained UTRs, and ten additional LYT1 sequences retrieved from TritrypDB and GenBank databases, together with a thoroughly search of structural motifs, showed a remarkable conservation of relevant structural motifs previously associated with RNA metabolism in the different UTRs; these elements might be involved in the differential stage-specific expression of each LYT1 isoform.

Resumen En los trypanosomátidos, la expresión génica se regula principalmente en el nivel post-transcripcional mediante mecanismos basados en la interacción entre las proteínas de unión del ARN [RBP] y las figuras presentes en las regiones no traducidas [UTR] de las ARN, que en conjunto forman complejos ribonucleoproteicos [RNP] que definen el destino de la ARN. El pre-ARN derivado del gen LYT1 del Trypanosoma cruzi es procesado por trans-empalme alternativo, dando como resultado diferentes ARN que codifican las isoformas mLYTl y kLYTl, proteínas con expresión diferencial, localización celular y función. El objetivo de este estudio fue caracterizar los 5' y 3' UTR de las ARN LYT1 como el paso inicial hacia la identificación de los RPB responsables de la expresión diferencial. Se confirmó la presencia de los dos tipos de 5' UTR en dos aislantes del T. cruzi pertenecientes al DTU I; de esta forma también se comprobó la ocurrencia del trans-empalme alternativo en el gen LYT1 de este T. cruzi DTU. Además, por primera vez, se pudo demostrar la existencia de dos tipos de transcripciones de ARN LYT1, que difieren en longitud por 116 nts, y son generadas por poliadenilación alternativa. Adicionalmente, se realizó un análisis in-silico de la UTR obtenida experimentalmente, y otras diez secuencias LYT1 recuperadas de las bases de datos TritrypDB y GenBank, junto con una búsqueda exhaustiva de figuras estructuradas, mostrando una notable conservación de los figuras estructurales asociadas con el metabolismo del ARN en los diferentes UTR; estos elementos podrían estar implicados en la expresión diferenciada de la etapa específica de cada isoforma LYT1.

Resumo Nos tripanossomatídeos, a expressão génica é regulada principalmente a nível pós-transcricional mediante mecanismos baseados na interação entre as proteínas de união do RNA [RBPs] e as fugiras presentes nas regiões não-traduzidas [UTRs] do RNA. O pré-RNA derivado do gene LYT1 do Trypanosoma cruzí é processado por uma junção trans-alternativa, resultando em diferentes RNA que codificam as isoformas mLYTl e kLYTl, proteínas com expressão, localização celular e função diferenciadas. O objetivo de este estudo foi caracterizar as 5' e 3' UTRs dos RNAs LYT1 como sendo o passo inicial na identificação das RBPs responsáveis pela expressão diferenciada. A presença dos dois tipos de 5' UTRs foi confirmada em dois isolados de T. cruzí pertencentes ao DTU I; corroborando assim com a ocorrência da junção trans-alternativa no gene LYT1 de este T. crují DTU. Adicionalmente, se demonstrou pela primeira vez a existência de dois tipos de transcrições de RNA LYT1, que se diferenciam em comprimento por 116 nts, e são geradas por poliadenização alternativa. Além disso, realizou-se uma análise in-sílico da UTR obtida experimentalmente e outras dez sequencias LYT1 recuperadas das bases de dados TritrypDB e GenBank, junto com uma busca exaustiva de figuras estruturadas, mostrando uma notável conservação das figuras estruturais associadas com o metabolismo do RNA nas diferentes UTRs. Estes elementos poderiam estar envolvidos na expressão estágio-específica diferenciada de cada isoforma LYT1.

High RNA-binding Motif Protein 3 Expression Is Associated with Improved Clinical Outcomes in Invasive Breast Cancer / 한국유방암학회지

PURPOSE: Expression of RNA-binding motif protein 3 (RBM3) is induced by hypoxia and hypothermia. Recently, high expression of RBM3 was reported to be associated with a good prognosis in colon cancer, prostate cancer, ovarian cancer, and malignant melanoma. Studies on RBM3 in invasive breast carcinoma (IBC), however, are limited. METHODS: RBM3 expression was examined using a tissue microarray from 361 patients with IBC. Immunohistochemistry was performed for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 to compare the expression of these markers. For scoring of RBM3 expression, NF (nuclear staining fraction)×NI (nuclear staining intensity) was used. The RBM3 expression score was considered indicative of either low (≤4) or high (>4) expression. Western blot analysis was performed on breast cancer cell lines to evaluate RBM3 expression. RESULTS: Of the total 361 samples, 240 (66.5%) exhibited high RBM3 expression. High RBM3 expression was significantly associated with positivity for ER (p < 0.001), PR (p < 0.001), T stage (p < 0.001), histologic grade (p < 0.001), and % Ki-67 staining (p=0.004). Multivariate analysis revealed that high RBM3 expression was closely associated with prolonged disease-free survival (DFS) (p < 0.001) and overall survival (OS) (p < 0.001). Western blot analysis revealed reduced RBM3 expression in HCC1954 (HER2-enriched) and BT-20 (basal-like) cells with an aggressive phenotype. CONCLUSION: High nuclear RBM3 expression is strongly associated with a prolonged DFS and OS. Furthermore, RBM3 expression is closely associated with good prognostic markers such as ER and PR in IBC. High nuclear RBM3 expression is, therefore, a critical biomarker of favorable clinical outcomes in IBC.

RNA-binding proteins and their role in the regulation of gene expression in Trypanosoma cruzi and Saccharomyces cerevisiae

Abstract RNA-binding proteins (RBPs) have important functions in the regulation of gene expression. RBPs play key roles in post-transcriptional processes in all eukaryotes, such as splicing regulation, mRNA transport and modulation of mRNA translation and decay. RBPs assemble into different mRNA-protein complexes, which form messenger ribonucleoprotein complexes (mRNPs). Gene expression regulation in trypanosomatids occurs mainly at the post-transcriptional level and RBPs play a key role in all processes. However, the functional characterization of RBPs in Trypanosoma cruzi has been impaired due to the lack of reliable reverse genetic manipulation tools. The comparison of RBPs from Saccharomyces cerevisiae and T. cruzi might allow inferring on the function of these proteins based on the information available for the orthologous RNA-binding proteins from the S. cerevisiae model organism. In this review, we discuss the role of some RBPs from T. cruzi and their homologues in regulating gene expression in yeast.

RNA Binding Protein as an Emerging Therapeutic Target for Cancer Prevention and Treatment

After transcription, RNAs are always associated with RNA binding proteins (RBPs) to perform biological activities. RBPs can interact with target RNAs in sequence- and structure-dependent manner through their unique RNA binding domains. In development and progression of carcinogenesis, RBPs are aberrantly dysregulated in many human cancers with various mechanisms, such as genetic alteration, epigenetic change, noncoding RNA-mediated regulation, and post-translational modifications. Upon deregulation in cancers, RBPs influence every step in the development and progression of cancer, including sustained cell proliferation, evasion of apoptosis, avoiding immune surveillance, inducing angiogenesis, and activating metastasis. To develop therapeutic strategies targeting RBPs, RNA interference-based oligonucleotides or small molecule inhibitors have been screened based on reduced RBP-RNA interaction and changed level of target RNAs. Identification of binding RNAs with high-throughput techniques and integral analysis of multiple datasets will help us develop new therapeutic drugs or prognostic biomarkers for human cancers.

Expression and clinical significance of RNA-binding protein Musashi1 in colorectal cancer / 国际肿瘤学杂志

Musashi1 (Msi1) is an evolutionary conservative RNA-binding protein (RBP),and it is a stem marker in a variety of organizations,including intestinal,neural system.Msi1 maintains the balance between self-renewal and differentiation.Recently,many researchers report that Msi1 is overexpressed in many types of tumors,especially in colorectal neoplasms,participating in the regulation of cell cycle,proliferation,apoptosis and so on.Msi1 becomes a key regulator of many cancers,which is expected to turn into a new target for cancer therapy.

Fetal lung structures and expression of pulmonary surfactant proteins of B and C and their modulating factors in rat with gestational diabetes mellitus / 中华围产医学杂志

Objective To explore the lung developmental disorder of rats with gestational diabetes mellitus (GDM) via investigating the GDM rat fetal lung structures and expression of pulmonary surfactant proteins (SP)-B,SP-C,thyroid transcription factor (TTF)-1 and pleiomorphic adenoma gene like (PLAGL)-2.Methods Sprague-Dawley rats were used to construct the GDM model.Twenty GDM rats were used as GDM group and 20 normal pregnant rats as control group.Cesarean section was performed on day 21 of gestation and random blood sugar was detected,and fetal rats were counted and weighed.Ultrastructure of the fetal lungs was studied by transmission electron microscopy.Sixty fetal rats were selected randomly in each group,and 360 paraffin sections were made from fetal lungs.One hundred discontinuous paraffin sections were picked up in each group to observe morphological and structural changes under optical microscope.The other one hundred discontinuous paraffin sections were picked up in each group to detect the location and expression of SP-B,SP-C,TTF-1 and PLAGL-2 protein by immunohistochemistry.Nine fetal rats were selected randomly to detect the expression level of SP-B,SP-C,TTF-1 and PLAGL-2 proteins in fetal lung tissues by Western blotting.Twenty seven fetal rats were selected randomly to detect the mRNAs level of SP-B,SP-C,TTF-1 and PLAGL-2 by real-time quantitative polymerase chain reaction.Independent sample t-test was used for statistical analysis.Results The average random blood glucose level in GDM group was significantly higher than that in control group [(26.8± 2.8) vs (4.9± 0.5) mmol/L,t=-34.05,P=0.00].The average weight of fetal rats in GDM group was higher than that in control group [(5.6±0.6) vs (5.2±0.5) g,t=-1.97,P=0.03].Alveolar number (10.1 ± 1.6 vs 12.1 ± 1.3) and alveolar area [(986.9 ± 5.5) vs (1 257.3± 5.0) μ m2] in GDM group was less than that in control group (t=9.84 and 27.53,both P ＜ 0.05).Alveolar septum [(11.5±6.2) vs (9.9±4.3) μm] in GDM group was higher than that in control group (t=-2.17,P ＜ 0.05).Microvillus in type] cells were short and the number of lamellar bodies was significantly decreased in GDM group.SP-B,SP-C,TTF-1 and PLAGL-2 proteins were distributed in the cytoplasm in granular form.The average value of absorbance of SP-B,SP-C,TTF-1 and PLAGL-2 proteins in GDM group was 1.15±0.12,1.23±0.06,0.87±0.21 and 1.21 ±0.18 respectively;and that in control group was 1.22±0.05,1.31 ±0.14,1.12±0.09 and 1.33 ±0.07 respectively.The value in GDM group was lower than that in control group (t=2.40,2.35,4.89,and 2.77 respectively,all P ＜ 0.01).The expression level of SP-B,SP-C,TTF-1 and PLAGL-2 proteins in GDM group was 0.57± 0.09,0.45±0.03,1.50±0.04 and 1.11 ±0.04 respectively;and that in control group was 0.81 ±0.03,0.66±0.04,1.69±0.05 and 1.46±0.07 respectively.The value in GDM group was lower than that in control group (t=1 1.77,11.09,8.80 and 13.37,respectively,all P ＜ 0.01).The mRNA level of SP-B,SP-C,TTF-1 and PLAGL-2 in GDM group was 0.60±0.04,0.79±0.04,0.81 ±0.03 and 0.79±0.05 respectively;and that in control group was 1.06±0.19,1.03±0.24,1.03±0.18 and 1.02±0.19 respectively.The value in GDM group was lower than that in control group (t=6.80,2.98,3.54 and 3.54 respectively,all P ＜ 0.01).Conclusions The protein expression level of SP-B and SP-C in fetal lungs of GDM rats decreases obviously,possibly because of the down-regulation of the gene expression of TTF-1 and/or PLAGL-2.The pathological changes in fetal lungs of GDM rats might be associated with the descending level of SP-B and SP-C protein.

A double-negative feedback loop lin28-let-7 and tumor / 国际肿瘤学杂志

The microRNA let-7 family,functioning as a tumor suppressor,has emerged as a regulator of stem cell differentiation and tumor repression.The RNA-binding protein lin28 is a stem cell pluripotency factor,which contributes to the maintenance of stem cell characteristics and promoting cell malignant transformation.lin28 can block the maturity of let-7 by combining with the precursors of let-7 family miRNAs,which is itself able to repress lin28 by binding to the 3'-UTR of lin28 transcripts,thus forming a feedback loop.The double-negative feedback loop comprising lin28 and let-7 is involved in several biological processes such as differentiation of stem cell,tumorigenesis,invasion and metastasis,drug resistance and relapse,which couldbe a potential target for cancer therapy.

The effect of RNA binding protein Lsm4 on the proliferation and migration of esophageal carcinoma cell line EC109 / 肿瘤

Objective: To investigate the effect of Lsm4 gene interference on the proliferation and migration of esophageal carcinoma EC109 cells, and to explore its possible mechanism. Methods: The expression of Lsm4 mRNA in esophageal cancer tissues and para-carcinoma tissues was detected by real-time fluorescence quantitative-PCR (RFQ-PCR). After transfection with Lsm4-small interference RNA (siRNA) targeting human Lsm4 gene or a negative control (NC) (NC-siRNA), the expression level of Lsm4 mRNA in EC109 cells was detected by RFQ-PCR, and the abilities of cell proliferation and colony formation were determined by MTT and colony formation assay, respectively. The ability of migration of EC109 cells after transfection with Lsm4-siRNA was examined by Transwell assay, and the expression levels of Lsm4, proliferating cell nuclear antigen (PCNA) and vimentin proteins were detected by Western blotting. Results: The expression level of Lsm4 mRNA in esophageal cancer tissues was higher than that in para-carcinoma tissues (P < 0.05). The expression levels of Lsm4 mRNA and protein of EC109 cells in Lsm4-siRNA transfection group were lower than those in NC-siRNA group (P < 0.01). The abilities of proliferation and colony formation of EC109 cells in Lsm4-siRNA transfection group were inhibited (P < 0.05), while the ability of migration was improved (P < 0.01). As compared with NC group, the expression level of PCNA was down-regulated (P < 0.05), and the expression level of vimentin was up-regulated (P < 0.01). Conclusion: The expression of Lsm4 mRNA is higher in esophageal carcinoma tissues. Lsm4 may regulate the growth and migration of EC109 cells. Copyright © 2014 by TUMOR.

Cross-linking and immunoprecipitation assay and its development / 军事医学

It′s reported that RNA-binding proteins ( RBP) play key roles in post-transcriptional regulation of eukaryotic genes.The aberrations of RBP are associated with a large number of human disorders , particularly autoimmune and neuro-logic diseases .The interaction between RNA and proteins has been widely explored since the development of the method known as RNA immunoprecipitation with differential display or microarray analysis (RIP-ChIP) around the year of 2000. Since then, diverse derivatives of the RIP-ChIP, such as ultraviolet crosslinking and immunoprecipitation ( CLIP), high-throughput sequencing of CLIP cDNA library (HITS-CLIP), photoactivatable -ribonucleoside-enhanced crosslinking and immunoprecipitation ( PAR-CLIP) , and individual nucleotide resolution CLIP ( iCLIP ) have been developed .All these methods have some advantages over the original RIP-ChIP and greatly facilitate the study of RBP-RNA interactions .Addi-tionally , aided by the next-generation sequencing , transcriptome-wide identification of RBP target sites has become possible and the RNA-binding site resolution of RBP has also improved to some degree .We introduced the basic principles and processes of the interactions between proteins and RNA , focusing on the advantages , disadvantages and prospect of the present genome-wide version of CLIP .

Role of microRNA-21 in the proliferation and apoptosis of ovarian epithelial carcinoma cells / 中华妇产科杂志

ObjectiveTo investigate the role and mechanism of microRNA-21 (miR-21) in the proliferation and apoptosis of ovarian epithelial carcinoma cells. MethodsA short-hairpin RNA specifically targeting miR-21 plasmid was constructed, and the recombinant was identified by restriction endonuclease analysis and DNA sequencing. Three experimental groups were included, transfection group (transfected with pSIREN-miR-21 ), negative control group ( transfected with pSIREN-miR-21-neg) and blank control group (without transfection plasmid ). The expression of miR-21 was detected by stem-loop real-time reverse transcription (RT)-PCR in OVCAR3 cells ,and western blot was used to detect the expression of programmed cell death 4 ( PDCD4 ) protein. Tethyl thiazolyl tetrazolium (MTT) and flow cytometry method were used respectively. ResultsRecombinant plasmid (pSIREN-miR-21) was constructed successfully and identified by restriction endonuclease analysis and DNA sequencing. The relative expression level of miR-21 in cells transfection, negative control and blank control group was 0.26 ± 0.08, 1.26 ± 0.21and 1.00 respectively. The level of miR-21 in the cells in transfection group was significantly lower than those in the negtive control and blank control group(P ＜0. 01 ). The gray scale of PDCD4. protein was 1443 ±33,858 ± 19 and 846 ± 16 in the transfection group, negative control and blank control group respectively. The value of PDCD4 in transfection group was higher than other control groups, and there were significantly difference among them( P ＜0. 01 ). Moreover, the optical density of the cells in transfection group was 0. 661 ±0. 015,significantly lower than those in two control groups (0. 848 ± 0. 150 for negative control, 0. 935 ± 0. 133 for blank control,P ＜ 0. 01 ). Forty-eight hours after tranfection, the rate of viable apoptotic cell was significantly higher than negative control and blank control group [(25.821 ± 0. 763 )％ vs.(0. 010 ± 0. 003 ) ％ vs. (0. 238 ± 0. 023) ％ ; P ＜ 0. 01];72 hours after tranfection, the rates of viable apoptotic cell and necrotic cell were all higher than the two control groups [the rate of viable apoptotic cell was ( 30. 480 ±0. 821 ) ％, ( 7. 792 ± 0. 312 ) ％ and ( 7. 033 ± 0. 257 ) ％ respectively ( P ＜ 0. 01 ) ; the rate of necrotic cell was (3.558 ±0.211)％, (1.557 ±0.067)％ and (1.049 ±0.028)％, respectively (P＜0. 01)].ConclusionmiR-21 might play an important role in the proliferation and apoptosis of ovarian epithelial carcinoma cells through negatively control the expression of PDCD4.

The expression of intestinal epithelial stem cell markers in mouse radiation enteritis / 中华消化杂志

Objective To investigate the expression of intestinal epithelial stem cell markers Msil and Hesl in mouse acute radiation enteritis (RE) model. Methods Forty female BALB/c mice were undergone one-time whole abdominal irradiation by 300 cGy/min β-ray. Thirty mice were selected before irradiation (Day 0) and every six mice were euthanized at day 3,5,7 and 14 after irradiation. The small intestinal were detached and the expression of Msil and Hesl were detected in middle part of small intestinal by quantitative RT-PCR, Western blots, and immunohistochemistry. Subsequently,the difference of Msil and Hesl expression was compared. Results The mouse acute RE model was successfully established. At the 5th day after radiation, the results of quantitative RT-PCR indicated that the relative ΔCt value of Msil and Hesl expression were 15. 17 ± 0. 47 and 15. 83 ± 0. 24respectively, the results of Western blot showed that the integrated intensity were 0. 443±0. 055 and 0. 301 + 0. 047 for Msil and Hesl. The expressions were significantly higher than other time points at RNA and protein levels (P<0. 05). The results of immunohistochemistry revealed that Msil and Hesl were highly expressed in intestinal crypts at 5th day and 7th day. Conclusions The expression of Msil and Hesl, upregulated before wound repair in RE model, indicates that intestinal epithelial repair is related to intestinal stem cell proliferation.

The roles of Musashil and Notch signaling pathways in neurogenesis after cerebral ischemia / 国际脑血管病杂志

Musashil is an evolutionarily conserved RNA binding protein. It plays an important role in the aspects of maintaining stem cell state, differentiation and tumorigenesis. Notch signal pathway plays an important role in the proliferation of neural stem cells after cerebral ischemia. This article briefly introduces the structure and molecular characteristics of Musashil and Notch, and reviews the interaction in the neurogenesis of Musashil and Notch signaling pathway after cerebral ischemic reperfusion.

Investigation of survival motor neuron gene deletion in Chinese patients with sporadic amyotrophic lateral sclerosis / 中华神经科杂志

Objective To investigate the correlation between survival motor neuron (SMN) gene deletion and Chinese patients with sporadic amyotrophic lateral sclerosis (SALS).Methods A total of 141SALS patients and 134 unrelated controls were recruited from the Chinese population.Polymerase chain reaction (PCR) and restriction fragment length polymorphisro (RFLP) analysis were performed to screen SMN gene deletion.Frequencies of deletion were coropared by Chi-square test.Results Four patients and 3 controls were detected to have horoozygous SMN2 deletion.The frequencies of SMN2 deletion were 2.84%(4/141) and 2.24% (3/134), respectively, which was not significantly different (χ2= 0.0001, P =1.000).No subjects were found to have homozygous SMN1 deletion.Condusion There is no correlation between SMN gene deletion and Chinese patients with SALS.