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ObjectiveTo investigate the expression of RNA binding motif single stranded interacting protein 3 （RBMS3） in epithelial ovarian cancer （EOC） tissues and its relationship with the clinicopathological features and prognosis of EOC. MethodsThe study enrolled the paraffin-embedded tissues from 110 EOC cases and 73 benign epithelial ovarian tumor cases pathologically diagnosed in the first affiliated Hospital of Bengbu Medical College from January 2015 to December 2019. By using anti-RBMS3 polyclonal antibody， the immunohistochemical staining was employed to detect RBMS3 expression in the tissues and then its correlation with the clinicopathological parameters and prognosis of EOC was analyzed. ResultsRBMS3 was expressed in both EOC and benign epithelial ovarian tumor tissues. RBMS3 expression in EOC tissues， significantly related with International Federation of Gynecology and Obstetrics （FIGO） stage， histological grade， CEA levels and survival status， was significantly lower than that in benign epithelial ovarian tumor tissues （P<0.05）. Kaplan–Meier survival curve showed that low RBMS3 expression in EOC patients was correlated with decreased progression-free survival （PFS） and overall survival （OS） （P<0.05）. Univariate analysis showed that RBMS3 expression， FIGO stage， residual lesion size， intestinal metastasis and intraperitoneal implantation were associated with OS of EOC patients （P<0.05）； multivariate analysis showed that low RBMS3 expression and intestinal metastasis were independent risk factors for poor prognosis in EOC patients （P<0.05）. ConclusionsRBMS3 is expressed at low levels in EOC tissues， which is closely related to poor prognosis of EOC patients. RBMS3 may function as a tumor suppressor gene in EOC tissues and can be used as an EOC-independent prognostic marker for targeted therapy against EOC.
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Objective To analyze the RNA binding protein of Toxoplasma gondii (TgDDX39) using bioinformatics technology, and to evaluate the immunogenicity of TgDDX39, so as to provide insights into development of toxoplasmosis vaccines. Methods The amino acid sequences of TgDDX39 were retrieved from the ToxoDB database, and the physicochemical properties, transmembrane structure domain, signal peptide sites, post-translational modification sites, coils, secondary and tertiary structures, hydrophobicity, and antigenic epitopes of the TgDDX39 protein were predicted using online bioinformatics tools, incluiding ProtParam, TMHMM 2.0, SignalP 5.0, NetPhos 3.1, COILS, SOPMA, Phyre2, ProtScale, ABCpred, SYFPEITHI and DNA-STAR. Results TgDDX39 protein was predicted to be an unstable hydrophilic protein with the molecular formula of C2173H3458N598O661S18, which contained 434 amino acids and had an estimated molecular weight of 49.1 kDa and a theoretical isoelectric point of 5.55. The protein was predicted to have an extremely low possibility of signal peptides, without transmembrane regions, and contain 27 phosphorylation sites. The β turn and random coils accounted for 39.63% of the secondary structure of the TgDDX39 protein, and a coiled helix tended to produce in one site. In addition, the TgDDX39 protein contained multiple B and T cell antigenic epitopes. Conclusions Bioinformatics analyses predict that TgDDX39 protein has high immunogenicity and contains multiple antigenic epitopes. TgDDX39 protein is a potential candidate antigen for vaccine development.
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Objective:To explore the expression of OGDHL and AU element-rich RNA-binding protein 1 (AUF1) in patients with non-small cell lung cancer (NSCLC) and their correlation with the prognosis of patients.Methods:The clinical data of 96 patients with NSCLC who were diagnosed and underwent surgical treatment in Guang'an People's Hospital from February 2018 to February 2019 were retrospectively analyzed. The surgically resected NSCLC tissues and the paracancerous tissues (4 cm from the tumor edge) were taken, and the immunohistochemical staining was used to detect the protein expressions of OGDHL and AUF1 in all specimens. The relationship between OGDHL and AUF1 proteins and clinicopathological characteristics of NSCLC was analyzed. The 3-year overall survival (OS) rate of patients with different expressions of OGDHL and AUF1 proteins were compared by using Kaplan-Meier method. Multivariate Cox proportional hazards model was used to explore the influencing factors for the prognosis of NSCLC patients.Results:The high expression rate of OGDHL protein in the cancer tissues of NSCLC patients was lower than that in the paracancerous tissues [32.3% (31/96) vs. 62.5% (60/96), χ2 = 45.21, P < 0.001], and the high expression rate of AUF1 protein was higher than that in the paracancerous tissues [68.8% (66/96) vs. 34.4% (33/96), χ2 = 42.36, P < 0.001]. The high expression rate of OGDHL protein in patients with TNM stage Ⅰ-Ⅱ was higher than that in patients with stage Ⅲ-Ⅳ [39.1% (25/64) vs. 18.8% (6/32)], and the high expression rate of OGDHL protein in patients with lymph node metastasis was lower than that in patients without lymph node metastasis [10.3% (3/29) vs. 41.8% (28/67)] (both P < 0.05). The high expression rate of AUF1 protein in patients with stageⅠ-Ⅱ was lower than that in patients with stage Ⅲ-Ⅳ [59.4% (38/64) vs. 87.5% (28/32)], and the high expression rate of AUF1 protein in patients with lymph node metastasis was higher than that in patients without lymph node metastasis [86.2% (25/29) vs. 61.2% (41/67)] (both P < 0.05). The 3-year OS rate of all NSCLC patients was 62.50%. The 3-year OS rate of patients with high expression of OGDHL protein after surgery was higher than that of patients with low expression of OGDHL protein (80.66% vs. 53.33%, P < 0.05), and the 3-year OS rate of patients with high expression group of AUF1 protein after surgery was lower than that of patients with low expression of AUF1 protein (50.00% vs. 76.52%, P < 0.05). The difference in 3-year OS rate of patients with different TNM stage and lymph node metastasis was statistically significant (both P < 0.05), and the 3-year OS rate of patients with TNM stage Ⅲ-Ⅳ and lymph node metastasis was lower than that of patients with stage Ⅰ-Ⅱ and no lymph node metastasis (both P < 0.05). Low expression of OGDHL ( HR = 3.78, 95% CI 1.72-8.31) and high expression of AUF1 ( HR = 3.67, 95% CI 1.73-7.80) were both independent risk factors for the prognosis of NSCLC patients (both P < 0.001). Conclusions:The expression of OGDHL protein is decreased and the expression of AUF1 protein is increased in NSCLC tissues. The expression levels of OGDHL and AUF1 proteins are related to the prognosis of NSCLC patients.
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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.
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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.
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Objective:To investigate the expression of double-stranded RNA-binding protein nuclear factor 45 (NF45) in laryngeal squamous cell carcinoma (LSCC), and the effect of NF45 on the radiation sensitivity of LSCC cells and its mechanism.Methods:NF45 expression in LSCC and adjacent tissues was detected by real-time reverse transcription PCR (qRT-PCR) and immunohistochemical staining. The NF45-ShRNA lentivirus was transfected into Hep-2 cells, and cell transfection efficiency was determined by qRT-PCR and Western blot . Hep-2 cells were randomly divided into the control group, 2 Gy group, sh-NC+2 Gy group and sh-NF45+2 Gy group. Lentivirus infection and 2Gy X-ray irradiation treatment were carried out. Cell proliferation activity was assessed by CCK-8 assay. Apoptosis rate was determined by flow cytometry. Hep-2 cells in each group were treated with mCherry-EGFP-LC3B. The levels of autophagy were detected by immunofluorescence staining. The ratio of autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ/LC3-Ⅰ and the expression levels of Beclin-1 and p62 proteins were determined by Western blot.Results:The expression level of NF45 in LSCC tissues was significantly higher than that in adjacent tissues ( P<0.01). The relative expression levels of NF45 mRNA and protein in Hep-2 cells infected with NF45-shRNA were significantly lower than those in the control and sh-NC groups (all P<0.05). Compared with the control group, the cell proliferation activity was decreased, the apoptosis rate was increased, the intracellular autophagy-lysosome were increased, the ratio of LC3-Ⅱ/LC3-Ⅰ was increased, the relative expression levels of Beclin-1 protein were up-regulated, and the relative expression levels of p62 protein were down-regulated in the 2 Gy, sh-NC+2 Gy and sh-NF45+2 Gy groups (all P<0.05). Compared with the 2 Gy group, the cell proliferation activity was decreased, the apoptosis rate was increased, the intracellular autophagy lysosomes were increased, the LC3-Ⅱ/LC3-Ⅰ ratio was increased, the relative expression of Beclin-1 protein was up-regulated, and the relative expression of p62 protein was down-regulated in the sh-NF45+2 Gy group (all P<0.05). Conclusions:The expression of NF45 is up-regulated in LSCC tissues. Targeted down-regulation of NF45 expression can inhibit the proliferation activity of LSCC cells, promote cell apoptosis, and improve the sensitivity of tumor cells to radiation. The mechanism may be related to the regulation of autophagy levels.
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RBM46 is a germ cell-specific RNA-binding protein required for gametogenesis, but the targets and molecular functions of RBM46 remain unknown. Here, we demonstrate that RBM46 binds at specific motifs in the 3'UTRs of mRNAs encoding multiple meiotic cohesin subunits and show that RBM46 is required for normal synaptonemal complex formation during meiosis initiation. Using a recently reported, high-resolution technique known as LACE-seq and working with low-input cells, we profiled the targets of RBM46 at single-nucleotide resolution in leptotene and zygotene stage gametes. We found that RBM46 preferentially binds target mRNAs containing GCCUAU/GUUCGA motifs in their 3'UTRs regions. In Rbm46 knockout mice, the RBM46-target cohesin subunits displayed unaltered mRNA levels but had reduced translation, resulting in the failed assembly of axial elements, synapsis disruption, and meiotic arrest. Our study thus provides mechanistic insights into the molecular functions of RBM46 in gametogenesis and illustrates the power of LACE-seq for investigations of RNA-binding protein functions when working with low-abundance input materials.
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Animals , Mice , 3' Untranslated Regions/genetics , Cell Cycle Proteins/metabolism , Gametogenesis/genetics , Meiosis/genetics , Nuclear Proteins/genetics , RNA-Binding Proteins/geneticsABSTRACT
Objective:To comprehensively analyze the prognostic prediction value of RNA binding protein, transcription factor gene expression and immune infiltration in hepatocellular carcinoma (HCC).Methods:Common gene sets associated with RNA-binding proteins and transcription factors were screened in TCGA ( n=365) , GSE54236 ( n=78) and GSE14520 ( n=221) datasets. Univariate Cox regression was used for primary screening. The survival regression model was constructed by LASSO-Cox. And a complex index [CIRT=(score-min)/max] was calculated. According to the median of CIRT, the HCC patients were divided into CIRT high group ( n=182) and CIRT low group ( n=182). The differences of prognosis, immune infiltration between the two groups were analyzed. Results:Of 37 prognostically relevant RNA binding protein and transcription factor genes were identified. The prognosis prediction model based on seven selected genes was determined by stepwise regression. Patients in the CIRT high group exhibited a lower percentage of macrophages in M1 ( P=0.032), macrophages in M2 ( P=0.009), resting mast cell ( P<0.001), activated NK cells ( P=0.007), and resting memory CD4 + T cells ( P<0.001), while patients in the CIRT low group showed a lower level of resting dendritic cells ( P=0.048), macrophages in M0 ( P<0.001), neutrophils ( P=0.049), follicular helper T cells ( P=0.004) and regulatory T cells ( P=0.001). GSEA analysis has shown that CIRT high groups were highly enriched in cell cycle, DNA repair pathways in TCGA and GSE14520. In the TCGA cohort, the CIRT low group had better overall survival than the CIRT high group. Analysis of 5-year follow-up data in the TCGA cohort showed that CIRT had a good predictive value for long-term survival of patients with liver cancer (area under receiver operating characteristic curve was 0.71). Conclusion:A novel prognostic index and classifier based on RNA-binding protein expression, transcription factors and immune expression profiles were developed and cross-cohort validated. CIRT could be used as an independent predictor.
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Objective To investigate the changes in the expression of cold-inducible RNA-binding protein (CIRBP) in a radiation-induced lung injury model. Methods Thirty male C57BL/6 mice were randomly divided by body weight into control group (no intervention) and model group (single chest X-ray irradiation with a dose of 20 Gy to build a radiation-induced lung injury model). The mice were dissected five weeks after irradiation. Hematoxylin-eosin staining and Masson staining were used to observe the pathological changes of the lung tissue and the deposition of collagen fibers. Immunohistochemistry was used to measure the expression of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the lung tissue. qRT-PCR was used to measure the expression of CIRBP mRNA in the lung tissue. The expression of CIRBP protein in the lung tissue was determined by the immunofluorescence assay and Western blot. Results Compared with the control group, the model group showed significant pulmonary vascular congestion, significant inflammatory cell infiltration, significant thickening of some alveolar septa, significantly increased IL-6 expression [(129.41 ± 5.58) vs (187.22 ± 34.77), t = 3.179, P < 0.05], significantly increased TNF-α expression [(137.52 ± 23.53) vs (187.02 ± 19.16), t = 5.069, P < 0.05], significantly increased CIRBP mRNA expression [(1 ± 0.08) vs (1.97 ± 0.39), t = 3.45, P < 0.05], and significantly increased CIRBP protein expression [(9.32 ± 1.26) vs (14.76 ± 1.61), t = 3.751, P < 0.05], by the immunofluorescence assay; [(1.13 ± 0.17) vs (1.49 ± 0.14), t = 2.819, P < 0.05], by Western blot). Conclusion The expression of CIRBP is significantly increased in the radiation-induced lung injury model, which may be an important pro-inflammatory factor in radiation-induced lung injury.
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RNA binding protein (RBP) plays a key role in gene regulation and participate in RNA translation, modification, splicing, transport and other important biological processes. Studies have shown that abnormal expression of RBP is associated with a variety of diseases. The Musashi (Msi) family of mammals is an evolutionarily conserved and powerful RBP, whose members Msi1 and Msi2 play important roles in the regulation of stem cell activity and tumor development. The Msi family members regulate a variety of biological processes by binding and regulating mRNA translation, stability and downstream cell signaling pathways, and among them, Msi2 is closely related to embryonic growth and development, maintenance of tumor stem cells and development of hematological tumors. Accumulating evidence has shown that Msi2 also plays a crucial role in the development of solid tumors, mainly by affecting the proliferation, invasion, metastasis and drug resistance of tumors, involving Wnt/β-catenin, TGF-β/SMAD3, Akt/mTOR, JAK/STAT, Numb and their related signaling pathways (Notch, p53, and Hedgehog pathway). Preclinical studies of Msi2 gene as a therapeutic target for tumor have achieved preliminary results. This review summarizes the molecular structure, physiological function, role of Msi2 in the development and progression of various solid tumors and the signaling pathways involved.
Subject(s)
Animals , Hedgehog Proteins , Mammals/metabolism , Neoplasms/genetics , Neoplastic Stem Cells , RNA-Binding Proteins/metabolism , Signal TransductionABSTRACT
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.
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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.
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Objective To evaluate the effect of mild hypothermia on the renal ischemia-reperfusion injury (IRI), and the expression profile of RNA-binding motif protein 3(RBM3) and its downstream effector molecules during this process. Methods Eighteen healthy SD male rats were randomly divided into the normal control (NC) group, IRI group and mild hypothermia pretreat (MHP) group, with 6 rats in each group. Serum creatinine level was measured to evaluate the renal function. Hematoxylin-eosin (HE) staining was performed to assess the renal tissue injury. Western blot was used to determine the relative expression levels of RBM3, Yes-associated protein 1(YAP1), nuclear factor E2-related factor 2(Nrf2), B cell-lymphoma-2(Bcl-2) and Bcl-2-associated X protein (Bax) in the kidney tissues. Immunohistochemical staining was employed to further detect the expression levels of RBM3 and YAP1 proteins. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was adopted to detect the cell apoptosis of kidney tissues. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were evaluated to determine the oxidative stress level of kidney tissues. Results Compared with the NC group, the serum creatinine level, the pathological injury score of kidney tissues and the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably lower, the apoptosis rate was remarkably elevated, the MDA content was significantly increased and the SOD activity was dramatically reduced in the IRI and MHP groups (all P < 0.05). Compared with the IRI group, the serum creatinine level and the pathological injury score of kidney tissues were significantly decreased, the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably higher, the apoptosis rate was significantly decreased, the MDA content was significantly decreased and the SOD activity was considerably elevated in the MHP group (all P < 0.05). Conclusions Mild hypothermia may exert protective effect upon renal IRI and it could alleviate cell apoptosis and oxidative stress injury induced by IRI, probably by up-regulating the expression level of RBM3 and its downstream effector molecules of YAP1 and Nrf2.
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ObjectiveTo investigate the effect of AU-rich element RNA-binding factor 1 (AUF1) on glypican 3 (GPC3) in hepatocellular carcinoma (HCC) and its possible mechanism. MethodsTCGA-HCC gene expression data were downloaded from Broad Institute Genome Data Analysis Center, and finally 371 HCC tissue samples with different etiologies and 50 adjacent tissue samples were included; LCI-HCC gene expression data were downloaded from GSE14520, and 214 patients with hepatitis B-associated HCC who had follow-up data were enrolled. A total of 35 primary liver cancer samples and corresponding adjacent tissue samples were collected from HCC patients who underwent radical surgery in Henan Provincial Cancer Hospital from 2009 to 2013. Immunohistochemistry was used to measure the protein expression of GPC3 and AUF1 in HCC tissue; Western Blot and qRT-PCR were used to measure the expression of GPC3 after AUF1 knockdown or overexpression in hepatoma cell lines; RNA-binding protein immunoprecipitation and RNA turnover assay were used to investigate the potential mechanism of AUF1 in regulating the expression of GPC3. The t-test was used for comparison of quantitative data between two groups, and the chi-square test was used for comparison of rates between two groups; the Kaplan-Meier method was used for survival analysis after surgery, and the log-rank test was used for comparison of survival rates. ResultsIn TCGA and LCI databases, the expression of GPC3 in HCC tissue was significantly higher than that in adjacent tissue (P＜0.05), and in TCGA database, the high expression of GPC3 was associated with the poor prognosis of HCC patients (P＜0.05). Immunohistochemistry showed that both GPC3 and AUF1 proteins are highly expressed in HCC tissue, with a positive expression rate of 77.1% (27/35) and 74.3% (26/35), respectively. In vitro experiment showed that AUF1 knockdown significantly reduced the expression of GPC3 in HepG2 and Huh-7 cells (P＜0.05), while AUF1 overexpression significantly increased the expression of GPC3 (P＜0.05). AUF1 protein could bind to GPC3 mRNA, and AUF1 knockdown reduced the stability of GPC3 mRNA. ConclusionAUF1 is an important post-transcriptional regulator of the GPC3 gene, and the abnormal high expression of AUF1 and GPC3 may be involved in the development and progression of HCC.
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Ischemia-reperfusion injury (IRI) is a common pathophysiological phenomenon, secondary to multiple pathological processes, such as organ transplantation, acute kidney injury and myocardial infarction. IRI may significantly aggravate the severity of diseases and increase the fatality of patients. Aseptic inflammation is one of the critical mechanisms of IRI. Damage-associated molecular pattern (DAMP) is a pivotal substance, which mediates aseptic inflammation. After released into extracellular space, it could effectively activate the immune system, and initiate and maintain the inflammatory responses by binding with pattern recognition receptor (PRR). Neutrophil extracellular trap (NET) is a DNA-based network structure released by neutrophils during the process of inflammatory responses, which contains histones and multiple granular proteins. Recent studies have demonstrated that DAMP and NET may aggravate IRI via aseptic inflammation. In this article, relevant studies of DAMP, NET and their relationship in IRI were reviewed, which was of great significance for understanding the pathophysiological mechanism of IRI and studying the corresponding prevention and treatment strategies.
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Objective: To investigate the expression and biological functions of cold inducible RNA binding protein (CIRBP) in renal cancer. Methods: Bioinformatics analysis of microarray in Gene Expression Omnibus (GEO) and gene sequencing data in The Cancer Genome Atlas (TCGA) was used to analyze the expression of CIRBP mRNA in renal cancer, further the expression level of CIRBP gene in 20 cases of renal cancer tissues and 3 kinds of renal cancer cell lines was identified by real-time fluorescent quantitative PCR. The renal cancer 786-O and CAKI-1 cells were transfected with the CIRBP overexpression plasmids, then the cell proliferation viability was detected by MTT assay and cell clone formation assay. The migration ability of renal cancer cells was detected by Transwell chamer, and the expressions of cell migration-related protein N-cadherin, E-cadherin and protein kinase B (PKB, also known as AKT) pathway-related proteins were detected by Western blotting. Results: The expression level of CIRBP in renal cancer tissues was significantly lower than that in the adjacent normal tissues. The prognosis of patients with high expression of CIRBP mRNA was significantly better than that of the patients with low expression of CIRBP. The proliferation and clone formation of renal cancer 786-O and CAKI-1 cells transfected with CIRBP overexpresion plasmids were significantly inhibited (all P 0.01). The number of renal cancer 786-O and CAKI-1 cells migrated through the membrane in CIRBP overexpression group was less than that in the control group (all P 0.01). In the 786-O and CAKI-1 cells with CIRBP overexpression, the expression level of migration-related protein N-cadherin was significantly decreased, the expession level of E-cadherin was significantly increased, while the expressions of AKT pathwayrelated phospho-AKT (p-AKT) and phospho-glycogen synthasc kinase 3β (p-GSK3β) proteins were decreased significantly (all P 0.05). Conclusion: CIRBP is down-regulated in renal cancer, and inhibits the proliferation and migration of renal cancer cells. CIRBP can be used as a potential clinical diagnosis target and prognostic marker for renal cancer.
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To investigate the proliferation and apoptosis of lung adenocarcinoma cells line HI299 by the lentiviral vector mediated RNA binding protein quaking-5 ( QKI-5). Methods GV358 ( up-regulation ) and GV248 ( down-regulation) vectors were used to construct the lentiviral QKI-5 up-regulation vector and down-regulation vector, respectively. The vectors were transfected into 293T cells for lentiviral packaging and viral titer were then determined. Gene sequencing was performed to screen the sequence of vectors. Then Real-time PCR was used to evaluate the expression of QKI-5 mRNA and the proliferation of H1299 cells was examined by colony forming assay after transfection. The apoptosis of HI299 cells was determined by the detection of the expression membrane protein V (annexin V ) and propyl iodide ingot (PI) by flow cytometry. Pro-apoptotic protein Caspae-3 and Caspase-8 were evaluated by Western blotting. Results QKI-5 up-regulation and down-regulation lentiviral vectors were constructed successfully. Compared with the controls, the expression of QKI-5 mRNA of HI299 cells was up-regulated, the cell colony formation was decreased, and the early apoptosis of HI299 cells was increased with the over-expression of Caspase-8 after transfected with up-regulated vector, whereas transfecting with QKI-5 down-regulated vector had opposite effect. Conclusion Lenviral vector mediated QKI-5 could inhibit proliferation and promote apoptosis of lung adenocarcinoma cells through Caspase-8.
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Aim To investigate the effect of long non-coding RNA (LncRNA) HIT on the resistance of ima-tinib (IM) in SUP-B15 cell line of acute lymphoblastic leukemia (ALL) and the related mechanisms. Methods SUP-B15 cells were treated with concentration gradient IM and saline as IMR and control groups. The lentivirus transfected LncRNA HIT shRNAl # and 2# vector in IMR group cells to knock down the HIT expression as IMR shHITl # and 2# group cells. CCK-8 assay was used to detect IM half-inhibitory concentration ( ICjo ). Fluorescence quantitative PCR (qPCR) was applied to detect the expression levels of LncRNA HIT, QKI, 0ct4 and Sox2 mRNA. Western blot was employed to detect the expression levels of QKI, 0ct4 and Sox2 protein. Results Compared with those in control cells, there was significantly higher IM ICjq, higher expression of LncRNA HIT, 0ct4 and Sox2, and lower expression of QKI in groups of IMR, IMR shHITl# and 2# cells. Compared with those in IMR cells, there was significantly lower IM IC∗, lower expression of LncRNA HIT, 0ct4 and Sox2, and higher expression of QKI in groups of IMR shHITl# and 2# cells. The difference was statistically significant (P < 0. 05). Conclusions LncRNA HIT can increase the expression of Sox2 and 0ct4 via inhibiting the expression of QKI protein, and mediating the formation of IM resistance in ALL cells.
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ObjectiveMild hypothermia was an effective way of cerebral resuscitation after cardiac arrest. The expression of cold-induced RNA binding protein (CIRP) was significantly enhanced when the temperature was lowered. This study was to evaluate the effects and the mechanisms of CIRP inhibition on hippocampal neurological and mitochondria function after mild hypothermia in a rat model of cardiac arrest.MethodsFive male Sprague-Dawley rats were injected with AAV9 in the hippocampus, 1 μL on each side, speeding 0.2 μL/min. The expression of GFP was observed by fluorescence microscopy after 2w. Sixty rats were randomly divided into 5 groups (n= 12 for each group): sham operation group, model group, mild hypothermia group, mild hypothermia + CIRP inhibition group and mild hypothermia + normal control group. Injection of AAV9 was performed on mild hypothermia + CIRP inhibition group, same amount of empty vector on mild hypothermia + normal control group, while normal saline on the other groups. Animal models of global cerebral IR were established by transesophageal cardiac pacing inducing cardiac arrest followed by cardiopulmonary resuscitation at 2w after injection. Cooling to 32-34℃ was initiated and the temperature was maintained for 6h on mild hypothermia groups. NDS score, HE staining and pyramidal cell counting on hippocampal CA1 area were performed at 72h after reperfusion. At 24h after reperfusion, mitochondrial structure of pyramidal cells in hippocampal CA1 was observed under electronic microscope and the expressions of CIRP, dynamin-related protein 1 (Drp1) and cytochrome C (Cyt-C) were detected by Western blot.ResultsThe NDS score of model group was decreased, the number of pyramidal cells was reduced, and the mitochondria were severely damaged. The NDS score of mild hypothermia group was increased, and the number of pyramidal cells was increased (all P<0.05), and mitochondrial damage was reduced compared with model group. In mild hypothermia + CIRP inhibition group, the NDS score was no significant difference compared with mild hypothermia + normal control group and model group, and the number of pyramidal cells was lower than that in mild hypothermia + normal control group [(27.2±4.9) vs (50.2±4.4), P<0.05], similar to model group (25.2±3.8), the damage of mitochondria was severe. After 2 weeks of AAV9 injection, GFP was widely expressed in the hippocampus. The expression of CIRP in mild hypothermia + CIRP inhibition group was respectively small compared with sham operation group [(0.14±0.03) vs (0.03±0.01),P<0.05], which was successfully inhibited by injection of AAV9. The expression of CIRP in model group (0.25±0.05) was significantly higher than that in sham operation group. The expression of CIRP in mild hypothermia group (0.37±0.08) and mild hypothermia + normal control group (0.39±0.04) were higher than that in model group (all P<0.05). The trends of Drp1 and Cyt-C expression were the same, in model group was higher than that in sham operation group, in mild hypothermia group was lower than that in model group, in mild hypothermia + CIRP inhibition group was higher than in mild hypothermia + normal control group (all P<0.05); There were no significant differences between model group and mild hypothermia + CIRP inhibition group, and between mild hypothermia group and mild hypothermia + normal control group.ConclusionInhibition of CIRP expression in hippocampus can weaken the protective effects of mild hypothermia on neurons in a rat model of cardiac arrest. The mechanism of those effects might be association with mitochondrial division.
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The outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. Currently, there is no specific viral protein-targeted therapeutics. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein remains unclear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the -sheet core. Complemented by binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.