Real-time Fluorescent Quantitative Polymerase Chain Research Articles

Overexpression of mitofusion 2 inhibits acute respiratory distress syndrome pulmonary fibrosis and its mechanism

To study the inhibitory effect of overexpression of mitofusion 2 (Mfn2) protein on acute respiratory distress syndrome (ARDS) pulmonary fibrosis and its mechanism. Human embryo lung fibroblasts (HELF) were cultured in vitro, and digested and passaged when the adherent rate of HELF reached 80%, and then the cells in good condition were selected for experiment. The ARDS cell model was reproduced by 5 mg/L of lipopolysaccharide (LPS, LPS group); 75 mol/L adenovirus vector carrying mitofusion 2 (Adv-Mfn2) was transfected into HELF (Adv-Mfn2+LPS group); at the same time, blank control group (complete medium culture) and Adv-vector+LPS group were set as controls. The cell proliferation was observed by sulforhodamine B (SRB) method at 0, 12, 24, 36 and 48 hours. After Hoechst 33342 staining, the morphological changes were observed under confocal microscope. Western blotting was used to detect the protein expressions of Bcl-2 and caspase-3. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expressions of Bcl-2 and caspase-3. After LPS stimulation for 12-48 hours, the cell proliferation rates in the LPS group increased gradually, which were significantly higher than those in the blank control group [12 hours: (10.75±1.51)% vs. (0.73±1.22)%, 24 hours: (20.09±1.71)% vs. (1.15±1.12)%, 36 hours: (20.58±1.55)% vs. (1.20±1.12)%, 48 hours: (21.30±1.51)% vs. (1.23±1.10)%, all P < 0.01]. There was no statistically significant difference in the cell proliferation rate between the LPS group and the Adv-vector+LPS group. After overexpression of Mfn2, the cell proliferation rates at 12, 24, 36, 48 hours in the Adv-Mfn2+LPS group were (8.93±1.14)%, (10.52±1.24)%, (10.72±1.30)%, and (10.91±1.20)%, which were significantly lower than those in the LPS group (all P < 0.05). Confocal microscopy showed that some cells in the blank control group had nuclei of different sizes, and some nuclei fragmented or shrank to form apoptotic bodies. The nuclei of the cells in the LPS and Adv-vector+LPS groups were round or oval in size, and only a few apoptotic cells appeared. When Mfn2 was overexpressed, there were more apoptotic cells in the visual field in the Adv-Mfn2+LPS group than LPS group. Western blotting and RT-qPCR results showed that Bcl-2 expressions increased significantly after LPS stimulation in the LPS group as compared with the blank control group [Bcl-2 protein (Bcl-2/GAPDH): 0.68±0.01 vs. 0.29±0.01, Bcl-2 mRNA (2-ΔΔCT): 2.23±0.34 vs. 1.00±0.00, both P < 0.01], and caspase-3 expressions decreased significantly [caspase-3 protein (caspase-3/GAPDH): 0.37±0.02 vs. 0.66±0.02, caspase-3 mRNA (2-ΔΔCT): 0.31±0.05 vs. 1.00±0.00, both P < 0.01]. Compared with LPS group, the expressions of Bcl-2 after overexpression of Mfn2 in the Adv-Mfn2+LPS group were down-regulated [Bcl-2 protein (Bcl-2/GAPDH): 0.46±0.01 vs. 0.68±0.01, Bcl-2 mRNA (2-ΔΔCT): 1.45±0.14 vs. 2.23±0.34, both P < 0.01], and the expressions of caspase-3 were up-regulated [caspase-3 protein (caspase-3/GAPDH): 0.54±0.02 vs. 0.37±0.02, caspase-3 mRNA (2-ΔΔCT): 0.88±0.10 vs. 0.31±0.05, both P < 0.01]. Mfn2 protein is involved in ARDS pulmonary fibrosis, which may be related to mitochondrial mediated inhibition of cell proliferation.

CRL4B complex promotes the development of pancreatic cancer by inhibiting secreted frizzled related protein 1

Objective: To investigate the role of CUL4B-RING E3 ubiquitin ligase (CRL4B) complex in pancreatic tumorigenesis and the molecular mechanism. Methods: Pancreatic cells were divided into control group (transfected with negative control lentivirus), shCUL4B group (transfected with CUL4B lentivirus), shDDB1 group [transfected with DNA damage binding protein 1 (DDB1) lentivirus], and shCUL4B+ siSFRP1 group (transfected with CUL4B lentivirus and SFRP1-siRNA). RNA-seq was performed in pancreatic cancer cell lines with CUL4B and DDB1 knocked down respectively, to identify the target genes regulated by CRL4B complex. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of target genes. Chromatin immunoprecipitation (ChIP) assay was used to identify the target genes directly regulated by CUL4B and DDB1. Western blot was used to detect the protein expression levels of the epithelial-mesenchymal transition (EMT) markers. The EdU cell proliferation test was used to detect cell proliferation ability. The scratch repair test and Transwell cell invasion test were used to detect cell migration and invasion ability. Finally, the sequencing data of pancreatic cancer-related tumor samples and normal samples in GEO, TCGA and GTEx databases were used to analyze the expression correlations of CUL4B, DDB1 and their downstream target genes. Results: RNA-seq results showed that target genes regulated by CRL4B complex involved in a number of malignant tumor-related signaling pathways. qRT-PCR results verified that the mRNA expression levels of the target genes of CUL4B or DDB1 knockdown groups were higher than those of the control group, and the difference was statistically significant (P<0.05). ChIP-PCR results showed that CRL4B complex directly bound to the promoter regions of the target genes, NME1 and SFRP1, and the enrichment of monoubiquitination of lysine at 119 of histone H2A (H2AK119ub1) in the promoter region of target gene was reduced after CUL4B knockdown. The proliferation rate in PANC-1 cell line of the control group was (32.10±3.58)%, higher than (13.95±1.66)% in the shCUL4B group and (22.38±0.77)% in the shCUL4B+ siSFRP1 group (P<0.05). The proliferation rate in AsPC-1 cell line of the control group was (35.47±7.80)%, higher than (19.60±3.58)% in the shCUL4B group and (30.09±0.81)% in the shCUL4B+ siSFRP1 group (P<0.05). The scratch repair experiment showed that the migration rate of PANC-1 cell line control group was (53.18±3.70)%, higher than that (17.46±2.62)% in the shCUL4B group and (44.99±9.18)% in the shCUL4B + siSFRP1 group (P<0.05). Western blot showed the expression levels of epithelial markers including α-catenin and γ-catenin in the control group were 1.00±0.03 and 1.01±0.11, respectively, lower than 1.44±0.01 and 1.21±0.06 in the shCUL4B group (P<0.05). The expression levels of mesenchymal markers including fibronectin and vimentin in the control group were 1.01±0.14 and 1.02±0.18, respectively, higher than 1.53±0.13 and 1.22±0.07 in the shCUL4B+ siSFRP1 group (P<0.05). The cell metastasis rate of the control group was (100.00±3.96)%, higher than the (35.49±0.34)% in the shCUL4B group and (107.06±2.77)% in the shCUL4B+ siSFRP1 group, the difference was statistically significant (P<0.05). The expressions of CUL4B and DDB1 were significantly upregulated in the pancreatic cancer tissues, and were negatively correlated with the expression of SFRP1 (r=-0.342 and r=-0.264, respectively). Conclusions: CRL4B complex inhibits the transcription of target gene SFRP1 and promotes the development of pancreatic cancer. Moreover, CRL4B complex is upregulated in pancreatic cancer, which provide a potential of therapeutic target for pancreatic cancer.

Matrix Metalloproteinases (MMP-2) and Tissue Inhibitors of Metalloproteinases (TIMP-2) in Male Inguinal Hernia Patients at Different Ages.

Collagen metabolism, controlled by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), might be related to inguinal hernia formation. It was reported that the incidence of inguinal hernia and the recurrence rate after inguinal hernia repair were higher in the elderly. The objective of the research was to assess the amounts of MMP-2 and TIMP-2 in patients at different ages in order to examine the relationship between age and inguinal hernia occurrence. The research included 40 primary inguinal hernia male patients, and four groups were created: 50-59years old (A group); 60-69years old (B group); 70-79years old (C group); 80-89years old (D group). We got the samples from anterior rectus sheath fascia. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and immunohistochemistry were applied to estimate the levels of MMP-2 and TIMP-2. The MMP-2 amounts in C and D group were statistically higher than control group (P < 0.05), and the TIMP-2 levels in C and D group were statistically lower than control group (P < 0.05). We found a positive correlation between age and expression levels of MMP-2 (r = 0.537, P < 0.001; r = 0.569, P < 0.001) and a negative correlation between age and TIMP-2 in inguinal hernia patients (r = - 0.759, P < 0.001; r = - 0.759, P < 0.001). Increased MMP-2 and reduced TIMP-2 may have some relationships with higher inguinal hernia incidence of the elderly.

Licarin-B Exhibits Activity Against the Toxoplasma gondii RH Strain by Damaging Mitochondria and Activating Autophagy.

Toxoplasma gondii is an obligate intracellular pathogen that infects warm-blooded animals and humans. However, side effects limit toxoplasmosis treatment, and new drugs with high efficiency and low toxicity need to be developed. Natural products found in plants have become a useful source of drugs for toxoplasmosis. In this study, twenty natural compounds were screened for anti-T. gondii activity by Giemsa staining or real-time fluorescence quantitative polymerase chain reaction (qPCR) in vitro. Among these, licarin-B from nutmeg exhibited excellent anti-T. gondii activity, inhibiting T. gondii invasion and proliferation in a dose-dependent manner, with an EC50 of 14.05 ± 3.96 μg/mL. In the in vivo, licarin-B treatment significantly reduced the parasite burden in tissues compared to no treatment, protected the 90% infected mice from to death at 50 mg/kg.bw. Flow cytometry analysis suggested a significant reduction in T. gondii survival after licarin-B treatment. Ultrastructural changes in T. gondii were observed by transmission electron microscopy (TEM), as licarin-B induced mitochondrial swelling and formation of cytoplasmic vacuoles, an autophagosome-like double-membrane structure and extensive clefts around the T. gondii nucleus. Furthermore, MitoTracker Red CMXRos, MDC, and DAPI staining showed that licarin-B promoted mitochondrial damage, autophagosome formation, and nuclear disintegration, which were consistent with the TEM observations. Together, these findings indicate that licarin-B is a promising anti-T. gondii agent that potentially functions by damaging mitochondria and activating autophagy, leading to T. gondii death.

Exploration of the effect of pulmonary fibrosis on erectile function in rats: A study based on bioinformatics and experimental research.

First, the bioinformatics database was used to predict the potential targets and signaling pathways of pulmonary fibrosis (PF) leading to erectile dysfunction (ED), and bleomycin sulfate was used to create a PF rat model. Then, enzyme-linked immunosorbent assay (ELISA), Western blotting, Real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to detect the expression of sex hormones and related proteins and mRNA, and Hematoxylin and eosin (H&E) staining was used to compare the pathological changes of penile tissue. The results showed that, compared with group A, cyclic guanosine phosphate (cGMP) content in group B decreased, protein kinase CGMP-dependent 1(PKG1) and nitric oxide synthase 3 (eNOS) protein and mRNA expression were down-regulated, and phosphodiesterase 5A (PDE5A) protein and mRNA expression was up-regulated (p<.05); the penile tissue of rats in group B had pathological damage. And there was no change in sex hormone-related indicators in the two groups (p>.05). Therefore, PF inhibits erectile function by inhibiting the cGMP-PKG pathway and reducing the expression of eNOS and PKG1 protein and mRNA. And by up-regulating the expression of PDE5A to impair erectile function.

Effects of different implant surface properties on the biological behavior of Schwann cells.

This study investigated the effects of different implant surface properties on the biological behavior of Schwann cells. Schwann cells (SCs) were cultured on three types of implant surfaces including smooth polished (SMO), sand-blasted, large grit, acid-etched (SLA), and chemically-modified SLA (modSLA). At different time points, the morphology and adhesion of SCs on the implant surfaces were observed by scanning electron microscope. Cell proliferation activity was detected by MTT method. The expression levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were detected by enzyme-linked immunosorbent assay. Changes in the mRNA levels of NGF and BDNF were detected by real-time fluorescent quantitative polymerase chain reaction (PCR). SCs adhered, stretched, and proliferated well on the three types of implant surfaces. On the 3rd, 5th, and 7th days, the OD values of the SMO group were higher than those of the SLA group and the modSLA group, and the difference was statistically significant (P<0.05). On the 3rd day, the expression and mRNA levels of NGF and BDNF in the SLA group and the modSLA group were higher than those in the SMO group (P<0.05); in particular, the levels in the modSLA group were higher than those in the SLA group (P<0.05). Different implant surface properties have different effects on the biological behavior of SCs. Proliferation of SCs is significantly promoted by smooth surface, while secretion and gene expression of neurotrophic factors are significantly promoted by modSLA surface at early stage.

Celastrol Inhibits the Proliferation and Decreases Drug Resistance of Cisplatin- Resistant Gastric Cancer SGC7901/DDP Cells.

This study aimed to determine the effect and mechanism of Celastrol inhibiting the proliferation and decreasing the drug resistance of cisplatin-resistant gastric cancer cells. The objective of this study was to explore the effect and mechanism of Celastrol on proliferation and drug resistance of human gastric cancer cisplatin-resistant cells SGC7901/DDP. The thiazole blue (MTT) method was used to detect the sensitivity of human gastric cancer cisplatinresistant cells SGC7901/DPP to cisplatin and Celastrol to determine the Drug Resistance Index (DRI). According to the half Inhibitory Concentration (IC50) value, the action of the concentration of the following experimental drugs was set to reduce the cytotoxicity. Annexin V-FITC/PI double staining method was used to detect the apoptosis of SGC7901/DDP cells induced by Celastrol. Western Blot was used to examine the expression levels of P-glycoprotein (P-gp), Multidrug Resistance Associated Protein 1 (MRP1), Breast Cancer Resistance Associated Protein (Breast Cancer Resistance)-relative protein (BCRP), and mechanistic Target of Rapamycin (mTOR) pathway-related proteins. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of P-gp, MRP1, and BCRP. (1) Compared with the control group (we set the untreated group as the control group), the proliferation of the SGC7901/DPP cells was significantly inhibited after treating with 0.1-6.4μmol/L Celastrol in a time- and concentration-dependent manner (P<0.05). The Drug Resistance Index (DRI) of the SGC7901/DPP cells to DDP was 5.64. (2) Compared with the control group, Celastrol could significantly inhibit the proliferation and induce the apoptosis of the SGC7901/DPP cells (P<0.05). (3) The mRNA and protein expression levels of P-gp, MRP1, and BCRP in the SGC7901/DPP cells were significantly higher than those in the SGC7901 cells. However, after treating with Celastrol, the expression levels of P-gp, MRP1, and BCRP in the SGC7901/DPP cells were significantly reduced (P<0.05). (4) Compared with the control group, the Celastrol treatment also reduced the expression of the mTOR signaling pathway-related proteins, suggesting that the mTOR signaling pathway may be involved in the process of Celastrol inhibiting the proliferation of the SGC7901/DDP cells and reducing their drug resistance. (5) Significantly, the combination of Celastrol and DDP reduced the expression of P-gp, MRP1, and BCRP in the SGC7901/DPP cells. Celastrol can inhibit the proliferation of the SGC7901/DDP cells, induce their apoptosis, and reduce the expression of drug resistance genes, probably by inhibiting the expression of the proteins related to the mTOR signaling pathway.

Benchmark dose analysis for PAHs hydroxyl metabolites in urine based on mitochondrial damage of peripheral blood leucocytes in coke oven workers in China

ObjectivesThe aim was to explore the dose-response relationship between occupational polycyclic aromatic hydrocarbons (PAHs) exposure and mitochondrial damage in coke oven plants workers. Methods544 workers and 238 healthy people were recruited. The ultra-high performance liquid chromatography was used to determine the level of 1-hydroxypyrene, 1-hydroxynaphthalene, 2-hydroxynaphthalene and 3-hydroxyphenanthrene. The real-time fluorescence quantitative polymerase chain reaction was used to determine the mitochondrial DNA copy number (mtDNAcn). The benchmark dose software was used to analyze the benchmark dose. ResultsThe mtDNAcn in the exposure group was lower than that in the control group. The concentrations of 1-hydroxypyrene, 1-hydroxynaphthalene, 2-hydroxynaphthalene and 3-hydroxyphenanthrene in the exposure group were higher than those in the control group. There is a dose-response relationship between 1-hydroxypyrene, 3-hydroxyphenanthrene and mitochondrial DNA damage. The benchmark dose lower confidence limit (BMDL) of 1-hydroxypyrene were 0.045, 0.004, and 0.058 pg/μg creatinine in the total, male, and female population, respectively. The BMDL of 3-hydroxyphenanthrene were 5.142, 6.099, and 2.807 pg/μg creatinine in the total, male, and female population, respectively. ConclusionsThe BMDL of 1-hydroxypyrene and 3-hydroxyphenanthrene initially explored can provide a reference to establish occupational exposure biological limits.

Influence of maggot excretions/secretions on the anti-Pseudomonas aeruginosa effect of neutrophils in patients with diabetic foot ulcer

Objective: To investigate the effects of medical maggot excretions/secretions (ES) on neutrophils phagocytosis and bactericidal effect in patients with diabetic foot ulcer (DFU). Methods: The experimental research method was used. Thirty DFU patients (16 males and 14 females, aged (64±7) years)who were admitted to the Diabetes Foot Center, the Department of Endocrinology of Air Force Hospital of Eastern Theater Command from June to December 2020 and met the inclusion criteria were recruited. Discontinuous percoll gradient centrifugation method was used to separate the neutrophils. Cells from each patient were enrolled into normal saline group and maggot ES group (30 wells in each group), respectively; sterile normal saline and ES with a final mass concentration of 357 μg/mL (the same as below) were added, respectively. After 1 and 2 hour(s) of culture, the phagocytosis rate and phagocytic index of cells were observed and counted under Wright's staining. Ten patients were selected, then the cells of each patient were enrolled into Pseudomonas aeruginosa+neutrophils group and Pseudomonas aeruginosa+neutrophils+maggot ES group (10 wells in each group) and were treated corresponding, respectively. Pseudomonas aeruginosa alone group and Pseudomonas aeruginosa+maggot ES group (10 wells in each group) were set up respectively; Pseudomonas aeruginosa+RPMI 1640 culture medium+sterile normal saline and Pseudomonas aeruginosa+RPMI 1640 culture medium+maggot ES were added, respectively. After 2 hours of culture, the number of viable bacteria colony was counted by plate colony number method. Six, six, and three patients were selected respectively, and the cells of each patient were respectively enrolled into maggot ES group and normal saline group (6, 6, and 3 wells in each group, respectively) and treated accordingly. After 6 hours of culture, real-time fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of interleukin 1β (IL-1β), IL-6, and lysozyme in cells, the content of IL-1β and IL-6 in cell culture supernatant were determined by enzyme-linked immunosorbent assay, and the positive cells expressing lysozyme were observed with immunofluorescence method. Data were statistically analyzed with one-way analysis of variance, paired sample t test, least significant difference test, and Wilcoxon rank sum test. Results: After 1 hour of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (53.5% (49.7%, 58.0%) and 3.18 (2.96, 3.32)) were similar to 52.0% (47.5%, 55.2%) and 3.15 (2.96, 3.25) of normal saline group (Z=-1.701, -1.092, P>0.05). After 2 hours of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (70.0% (66.7%, 72.0%) and 4.47 (4.22, 4.96)) were significantly higher than 58.0% (55.0%, 60.0%) and 4.11 (3.52, 4.24) in normal saline group (Z=-4.786, -4.279, P<0.01). After 2 hours of culture, the number of viable bacteria colony in Pseudomonas aeruginosa+neutrophils group was significantly lower than that in Pseudomonas aeruginosa alone group (P<0.01), and the number of viable bacteria colony in Pseudomonas aeruginosa+neutrophils+maggot ES group was significantly lower than that in Pseudomonas aeruginosa+maggot ES group and Pseudomonas aeruginosa+neutrophils group (P<0.01). After 6 hours of culture, the mRNA expressions of IL-1β, IL-6, and lysozyme of cells in maggot ES group were significantly higher those in normal saline group (t=-3.279, -4.273, -4.763, P<0.05 or P<0.01); the concent of IL-1β and IL-6 in cell culture supernatant of maggot ES group were significantly higher than those of normal saline group (t=-9.526, -6.447, P<0.01); there were significantly more positive cells expressing lysozyme in maggot ES group than in normal saline group. Conclusions: Maggot ES can enhance the phagocytosis and bactericidal effect of neutrophils on Pseudomonas aeruginosa by promoting the production of neutrophils immune defense related cytokines and lysozyme in DFU patients.

MiR-142-3p and HMGB1 Are Negatively Regulated in Proliferation, Apoptosis, Migration, and Autophagy of Cartilage Endplate Cells

Cartilage endplate (CEP) degeneration plays a vital role in the pathological process of intervertebral disc degeneration. It has been previously reported that microRNAs may participate in the occurrence and development of intervertebral disc degeneration through regulating its target genes directly. The regulatory roles of miR-142-3p/HMGB1 in some orthopedic diseases have been determined successively, but there was no report about the degeneration of CEP. Therefore, we aimed to determine the regulation of miR-142-3p/HMGB1 or potential molecular mechanisms on proliferation, apoptosis, migration, and autophagy of CEP cells. The target gene of miR-142-3p was determined by double luciferase assay. We selected ATDC5 cell lines. CCK-8 method was used to detect cell proliferation. Real-time fluorescence quantitative polymerase chain reaction was used to determine gene expression levels, and western blot analysis was used to determine protein expression levels. We chose flow cytometry to measure cell apoptosis and cell cycle. The result of luciferase detection showed that the target gene of miR-142-3p in CEP cells was HMGB1. Knockdown of the miR-142-3p inhibited the expression level of HMGB1, the proliferation and migration of CEP cells, but it promoted apoptosis of CEP cells. In addition, the detection results of the proteins related to apoptosis or autophagy showed that knockdown of miR-142-3p promoted apoptosis and autophagy. The negative regulation of miR-142-3p/HMGB1 can affect the proliferation, apoptosis, migration, and autophagy of CEP cells. Our results provide a new idea for the targeted treatment of CEP degeneration by inhibiting the expression of HMGB1.

MiR-4787-5p Regulates Vascular Smooth Muscle Cell Apoptosis by Targeting PKD1 and Inhibiting the PI3K/Akt/FKHR Pathway.

Vascular smooth muscle cell (VSMC) dysfunction is the main cause of aortic dissection (AD). In this study, we focused on the role and mechanism of miR-4787-5p in regulating VSMC apoptosis. Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of miR-4787-5p in aorta tissues of AD (n = 10) and normal aortic tissues of donors (n = 10). Cell apoptosis was tested by TUNEL assay and Annexin V FITC/PI staining flow cytometry. The expression of PC1 and the PI3K/Akt/FKHR signaling pathway associated proteins in VSMCs was measured by Western blot. We found that the miR-4787-5p was highly expressed in aorta tissues of AD compared with 10 healthy volunteers. Meanwhile, PI3K/Akt/FKHR signaling pathway was inactive in the aortic tissue of AD. The overexpression of miR-4787-5p significantly induced VSMC apoptosis, and miR-4787-5p knockdown showed the opposite results. In addition, polycystic kidney disease 1 gene, which encodes polycystin-1 (PC1), was found to be a direct target of miR-4787-5p in the VSMCs and this was validated using a luciferase reporter assay. Overexpression of PC1 by a lentivirus packaging PC1-overexpression plasmid (LV-PC1) plasmids markedly eliminated the promotion of miR-4787-5p overexpression on VSMC apoptosis. Finally, it was found that miR-4787-5p deactivated the PI3K/Akt/FKHR pathway, as demonstrated by the down-regulation of phosphorylated (p-)PI3K, p-Akt, and p-FKHR. In conclusion, these findings confirm an important role for the miR-4787-5p/polycystic kidney disease 1 axis in AD pathobiology.

The expression of myosin-regulated light chain interacting protein (MYLIP) in lung cancer and its inhibitory effects on lung carcinomas.

BackgroundThis study explored the relationship between myosin-regulated light chain interacting protein (MYLIP) and the prognosis of lung cancer and its effects on the proliferation, migration, and invasion of lung cancer cells.MethodsBioinformatics analyses of databases were conducted to explore the relationship between the expression of MYLIP and the prognosis of lung cancer patients. Real-time fluorescent quantitative polymerase chain reaction and Western blot analyses were used to measure the levels of MYLIP expression. Cell counting kit-8 (CCK8) and cell cloning experiments were used to determine the effects of MYLIP on cell proliferation. The scratch test and invasion experiments were conducted to assess the effects of MYLIP on the migration and invasion of lung cancer cells. Tumor formation experiments were performed in nude mice to determine the effects of MYLIP on tumor growth.ResultsThe mRNA and protein expression of MYLIP in cancer tissues from lung cancer patients were significantly lower than that found in normal adjacent tissues (P<0.05). Bioinformatics analysis showed that lung cancer patients with high MYLIP expression had a better prognosis compared to patients with low MYLIP expression. The results of the CCK8 and cell proliferation experiments revealed that the proliferation ability of lung cancer cells overexpressing MYLIP was significantly lower than that of control cells (P<0.05). The scratch experiment and invasion experiments demonstrated that the scratch closure rate and the cell invasion ability of lung cancer cells overexpressing Experiments in nude mice showed that the tumor-forming ability of lung cancer cells with high expression of MYLIP was weaker than that of the control group, and the tumor growth rate and the tumor weight were also lower than that of the control group (P<0.05).ConclusionsLow levels of MYLIP expression were detected in the cancer tissues of lung cancer patients, and its expression levels were positively correlated with the prognosis of lung cancer. Furthermore, MYLIP had a significant inhibitory effect on the proliferation, migration, and invasion of lung cancer cells, suggesting that MYLIP may be a tumor suppressor gene for lung cancer. The results may have significant potential for clinical applications.

NUSAP1 Accelerates Osteosarcoma Cell Proliferation and Cell Cycle Progression via Upregulating CDC20 and Cyclin A2.

PurposeNucleolar and spindle-associated protein 1 (NUSAP1) is a significant mitotic regulator and has been found to be implicated in carcinogenesis of several cancers. The aim of this study was to explore the functional role and underlying mechanisms of NUSAP1 in osteosarcoma.MethodsWestern blot assay and Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) were employed to assess the expressions of NUSAP1, cell division cycle 20 homologue (CDC20) and cyclin A2 (CCNA2) in osteosarcoma cells. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2′-deoxyuridine (EdU) assay, and flow cytometry was applied for exploring cell cycle. In addition, an osteosarcoma tumor-bearing mouse model was established by injection of transfected osteosarcoma cells. Tumor volume and protein expressions of Ki67 and PCNA were examined. Bioinformatics analysis and immunoprecipitation were used to identify the combination of NUSAP1 with CDC20 and CCNA2.ResultsThe mRNA and protein expression of NUSAP1 were extremely upregulated in osteosarcoma cells. Overexpression of NUSAP1 promoted whereas NUSAP1 silencing suppressed cell proliferation and cell cycle progression in transfected osteosarcoma cells. In osteosarcoma mouse model, NUSAP1 expression affected tumor volume and levels of Ki67 and PCNA. Moreover, CDC20 or CCNA2 silencing inhibited NUSAP1-induced cell proliferation and cell cycle in osteosarcoma cells.ConclusionOur data demonstrated that upregulated NUSAP1 may exacerbate the development of osteosarcoma by accelerating the proliferation and cell cycle process of osteosarcoma cells by binding to CDC20 and CCNA2, suggesting NUSAP1 as a possible therapeutic target for treatment of osteosarcoma.

Intervention mechanism of moxa-cone moxibustion at Shu- and Mu-acupoints on the expression of RORγt/Foxp3 in asthma mice: an analysis based on PI3K signaling pathway

To explore the effect of moxa-cone moxibustion at "Feishu" (BL13) and "Zhongfu" (LU1) on the contents of related inflammatory factors in serum and bronchoalveolar lavage fluid (BALF), and the expression of phosphatidy-linositol-3-kinase (PI3K), retinoic acid related orphan receptor γt (RORγt) and fork/wing helix transcription factor 3 (Foxp3) in lung tissue in asthma mice. Sixty male Balb/c mice were divided into normal, model, LY294002 (an inhibitor of PI3K, LY), electroacupuncture (EA) and moxa-cone moxibustion (moxibustion) groups (n=12 in each group). The asthma model was established by intraperitoneal injection of ovalbumin sensitization. The mice in the LY group were injected with LY 294002 (7.5 mg/kg) via the tail vein. EA or moxa-cone moxibustion was applied at BL13 and LU1 in the EA or moxibustion group once daily for 2 weeks. The levels of immunoglobulin E (IgE), interleukin-4 (IL-4) and interferon-γ (IFN-γ) in serum and BALF were detected by enzyme-linked immunosorbent assay. The expressions of PI3K, RORγt and Foxp3 in lung tissue were detected by real-time fluorescent quantitative polymerase chain reaction (QRT PCR) and immunohistochemistry. Compared with the normal group, the levels of IgE and IL-4 in serum and BALF, the expressions of PI3K and RORγt mRNA and protein in lung tissue were significantly increased (P<0.01), while the levels of IFN-γ, and the expressions of Foxp3 mRNA and protein were significantly reduced (P<0.01) in the model group. Compared with the model group, the levels of IgE and IL-4 in serum and BALF, the expressions of PI3K and RORγt mRNA and protein in lung tissue were significantly decreased (P<0.01, P<0.05), and the levels of IFN-γ in serum and BALF, the expressions of Foxp3 mRNA and protein in lung tissue were significantly increased (P<0.01) in the LY, EA and moxibustion groups. Moxa cone moxibustion at Shu- and Mu-acupoints of the lung meridian can reduce airway inflammatory reaction and control asthma attacks in asthma mice, which is closely related to its effects in regulating the expressions of RORγt and Foxp3 through PI3K signaling pathway.

Expression and effect of microRNA-627 in human hypertrophic scar

Objective: To investigate the expression and effect of microRNA-627 (miR-627) in human hypertrophic scar. Methods: The experimental research method was used. From October 2019 to January 2020, hypertrophic scar tissue from 6 patients with hypertrophic scar (2 males and 4 females, aged (34±11) years) and the remaining normal skin tissue from 6 trauma patients (3 males and 3 females, aged (35±13) years) after flap transplantation were collected. The above-mentioned 12 patients were admitted to the General Hospital of Northern Theater Command and met the inclusion criteria. The mRNA expression of miR-627 was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The 3rd to 5th passages of fibroblasts (Fbs) were isolated from hypertrophic scar tissue and cultured for subsequent experiments after identification. Fbs from hypertrophic scar were divided into miR-627 negative control group, miR-627 mimic group, and miR-627 inhibitor group. The corresponding sequences were transfected respectively. At 0 (immediately), 12, 24, 36, and 48 h after transfection, the cell viability was detected by thiazolyl blue method; at 24 h after transfection, the apoptosis was detected by flow cytometry; at 24 h after transfection, the protein expression levels of insulin-like growth factor Ⅰ (IGF-Ⅰ), type Ⅰ collagen, and α smooth muscle actin (α-SMA) were detected by Western blotting. Two batches of Fbs from hypertrophic scar were used, one batch was divided into IGF-Ⅰ wild type+miR-627 negative control group and IGF-Ⅰ wild type+miR-627 mimic group, and the other batch was divided into IGF-Ⅰ mutant+miR-627 negative control group and IGF-Ⅰ mutant+miR-627 mimic group. The corresponding sequences were transfected respectively. At 48 h after transfection, the expressions of luciferase and renal luciferase were detected by luciferase reporter gene detection kit, and the ratio of the two was calculated to reflect the activity of IGF-Ⅰ. Fbs from hypertrophic scar were divided into miR-627 negative control group, miR-627 mimic alone group, and miR-627 mimic+IGF-Ⅰ group, and were transfected with the corresponding sequences respectively. At 24 h after transfection, the protein expression levels of IGF-Ⅰ, type Ⅰ collagen, and α-SMA were detected by Western blotting. The number of samples in cell experiment was 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, independent sample t test, and chi-square test. Results: The expression of miR-627 mRNA in hypertrophic scar tissue was 0.47±0.06, which was significantly lower than 1.12±0.23 in normal skin tissue (t=15.090, P<0.01). At 12, 24, 36, and 48 hours after transfection, the cell viability of miR-627 mimic group was significantly lower than that of miR-627 negative control group (t=9.918, 34.370, 13.580, 61.550, P<0.05 or P<0.01); the cell viability of miR-627 inhibitor group was significantly higher than that of miR-627 negative control group (t=4.722, 8.616, 13.330, 14.000, P<0.05 or P<0.01). At 24 h after transfection, compared with the apoptosis rate (8.42±0.47)% in miR-627 negative control group, (10.89±0.35)% in miR-627 mimic group was significantly higher (t=7.301, P<0.01), and (5.00±0.22)% in miR-627 inhibitor group was significantly lower (t=11.510, P<0.01). At 24 h after transfection, compared with the cell protein expressions of IGF-Ⅰ, type Ⅰ collagen, and α-SMA in miR-627 negative control group, those in miR-627 mimic group were significantly lower (t=25.470, 5.282, 7.415, P<0.01), and those in miR-627 inhibitor group were significantly higher (t=15.930, 8.857, 9.763, P<0.01). At 48 h after transfection, the luciferase/renal luciferase ratio of IGF-Ⅰ of cells in IGF-Ⅰ wild type+miR-627 mimic group was 0.463±0.061, which was significantly lower than 0.999±0.011 in IGF-Ⅰ wild type+miR-627 negative control group (t=16.852, P<0.01); the luciferase/renal luciferase ratio of IGF-Ⅰ of cells in IGF-Ⅰ mutant+miR-627 mimic group was 0.934±0.021, which was similar to 0.930±0.023 in IGF-Ⅰ mutant+miR-627 negative control group (t=1.959, P>0.05). At 24 h after transfection, the protein expressions of IGF-Ⅰ, type Ⅰ collagen, and α-SMA of cells in miR-627 mimic alone group were 1.623±0.070, 1.363±0.042, and 1.617±0.025, which were significantly lower than 2.723±0.045, 2.147±0.067, and 2.533±0.055 in miR-627 negative control group (t=22.831, 7.280, 26.220, P<0.01); the protein expressions of IGF-Ⅰ, type Ⅰ collagen, and α-SMA of cells in mimic+IGF-Ⅰ group were 2.477±0.102, 1.760±0.046, and 2.387±0.049, which were significantly higher than those of miR-627 mimic alone group (t=3.830, 8.286, 3.436, P<0.05 or P<0.01). Conclusions: miR-627 expression in human hypertrophic scars is down-regulated; miR-627 can inhibit the proliferation and promote the apoptosis of Fbs in human hypertrophic scar by targeted inhibition of IGF-Ⅰ expression.

Hepatitis A virus cell membrane protein receptor 2 promotes endotoxin tolerance in mouse macrophages

To screen out the potential key genes of endotoxin tolerance (ET), and to provide theoretical and experimental evidence for treatment and prognosis of sepsis. (1) Experiment 1 (gene chip and bioinformatics analysis): ET related data set GSE47783 was downloaded from the Gene Expression Omnibus (GEO). The data set was obtained from lipopolysaccharide (LPS) stimulated mouse macrophages to establish sepsis model (LPS group) and ET model (ET group). IDEP 0.92 software was used to screen differential expressed gene (DEG) between the two groups, analyze gene ontology (GO), and locate the main functions and signaling pathways of differential genes. The protein-protein interaction (PPI) network of DEG was constructed by the Search Tool for the Retrieval of Interacting Genes Database (STRING) to screen core genes hepatitis A virus cell membrane protein receptor 2 (HAVCR2) for following up validation study. (2) Experiment 2 (reproduction of mouse macrophage RAW264.7 model): RAW264.7 cells were cultured in vitro, the ET model (ET group, cells were cultured with 10 μg/L LPS for 24 hours and then with 100 μg/L LPS for 4 hours) and sepsis model (LPS group, cells were cultured with 100 μg/L LPS for 4 hours) were reproduced by LPS stimulation. Phosphate buffer saline (PBS) group was given equal volume of solvent PBS for 4 hours. The mRNA and protein expressions of HAVCR2 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. (3) Experiment 3 (RAW264.7 cells transfected with HAVCR2 lentiviral vector): to further clarify whether HAVCR2 was involved in the formation of ET, after knockdown of HAVCR2 in RAW264.7 cells by lentiviral short hairpin RNA (shRNA) technology, the ET model (HAVCR2--ET group) was constructed again, and the control group (ET group) without knockdown of HAVCR2 was set up. RT-qPCR method was used to detect the mRNA expressions of macrophage polarization key proteins [arginase 1 (ARG1), CD206, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide synthase 2 (NOS2)] in cells. (1) Experiment 1: a total of 1 013 DEG were identified, compared with LPS group, 521 genes were up-regulated and 492 genes were down-regulated in ET group. The function of these DEG was to increase biosynthesis and reduce inflammatory reaction. Signal pathways were mainly enriched in Janus kinase/signal transducers and activators of transcription (JAK/STAT), NOD like receptor, Toll-like receptor (TLR), TNF, hypoxia inducible factor-1 (HIF-1). The first up-regulated HAVCR2 in the ET group was selected as the target of the study. (2) Experiment 2: the results of in vitro experiment showed that the mRNA expression of HAVCR2 after high-dose LPS stimulation was down-regulated as compared with PBS group, and the mRNA expression of HAVCR2 in ET group was significantly higher than that in LPS group (2-ΔΔCT: 1.10±0.10 vs. 0.60±0.10, P < 0.05). The results of Western blotting were consistent with RT-qPCR results. (3) Experiment 3: the mRNA expressions of ARG1 and CD206 in HAVCR2--ET group were significantly lower than those in ET group [ARG1 mRNA (2-ΔΔCT): 0.50±0.10 vs. 1.00±0.10, CD206 (2-ΔΔCT): 0.73±0.10 vs. 1.00±0.10], and the mRNA expressions of TNF-α and IL-1β were significantly higher than those in ET group [TNF-α mRNA (2-ΔΔCT): 2.20±0.10 vs. 1.00±0.10, IL-1β mRNA (2-ΔΔCT): 9.00±0.10 vs. 1.00±0.10], with significant differences (all P < 0.05). There was no significant difference in the expression of NOS2 mRNA between the two groups. HAVCR2 is involved in the regulation of inflammatory factors downstream of sepsis and the formation of ET, which is expected to become a new therapeutic target of sepsis.

Effect of DR4 Demethylation to the Proliferation and Apoptosis of Myeloid Leukemia K562 Cells

To investigate the effect of tumor necrosis factor death receptor (DR) 4 demethylation to the proliferation and apoptosis of myeloid leukemia K562 cells. The logarithmic phase of K562 cells were treated by desitabine (DCA) at 0, 0.8, 1.6 and 3.2 μmol/L, and the cells were divided into control group, DCA low dose group, DCA medium dose group and DCA high dose group respectively. The cells in control group were treated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 0.5 μg/ml for 24 h, and the cells were divided into TRAIL group. The cells in DCA high dose group were treated by TRAIL 0.5 μg/ml for 24 h, and were divided into DCA high dose + TRAIL group. Methylation-specific polymerase chain reaction (MS-PCR) was used to measure the methylation status of the DR4 gene promoter in the control group and DCA low, medium and high dose groups. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to determine the relative expression of DR4 mRNA and protein in the control group and DCA low, medium and high dose groups. Dime- thylthiazole (MTT) method was used to determine the inhibition rate of cell proliferation of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group. Flow cytometry was used to determine the apoptotic rate of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group. The cells in the control group were methylation-positive, the brightness of the methylation bands of the cells in the DCA low, medium, and high dose groups was gradually decreased to disappear, and the DCA high dose group showed negative for methylation. The relative expression of DR4 mRNA and protein in the control group, DCA low, medium and high dose groups was increased sequentially (r=0.624, 0.704). The inhibition rate of cell proliferation of the cells in the control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group was increased sequentially (r=0.653, 0.754, 0.709, 0.725) at 24, 48 and 72 h. DCA can reverse the methylation level of DR4 gene promoter in ML K562 cells and up-regulate the expression of DR4, which may enhance the proliferation inhibition and apoptosis promotion effects of TRAIL on K562 cells.

Expression of cyclophilin A in oral squamous cell carcinoma and its effect on cell proliferation and invasion

To investigate the expression of cyclophilin A (CyPA) in oral squamous cell carcinoma (OSCC) and explore the effect of downregulating the expression of CyPA gene on the proliferation and invasion of SCC-25 cells. A total of 77 cases of patients with OSCC were selected. The expression levels of CyPA proteins in OSCC and adjacent normal tissues were evaluated. SCC-25 cells were cultured and divided into the CyPA interference sequence group, negative control group, and blank group. The expression levels of CyPA mRNA and protein in cells were detected by using real-time fluorescent quantitative polymerase chain reaction and Western blot, respectively. Cell proliferation was detected by using methyl thiazolyl tetrazolium and plate colony formation assays. Cell invasion was detected by using Transwell assay. The positive expression rate of CyPA protein in OSCC tissues was 76.62%, which was higher than that in adjacent tissues (P<0.05). The positive expression rate of CyPA protein in TNM stage T3+T4, clinical stage Ⅲ+Ⅳ, moderately or poorly differentiated lymph node metastasis was increased (P<0.05). Compared with the negative control and blank groups, the CyPA interference sequence group had decreased relative expression levels of CyPA mRNA and protein (P<0.05); optical density va-lues of cells at 24, 48, 72, and 96 h (P<0.05); and number of cell colonies and invasive cells (P<0.05). The CyPA protein is highly expressed in OSCC tissues, and the downregulation of CyPA gene expression in SCC-25 cells can reduce cell proliferation and inhibit cell invasion.

Molecular Characterization and Expression Profiling of Chemosensory Proteins in MaleEogystia hippophaecolus(Lepidoptera: Cossidae)

AbstractEogystia hippophaecolus Hua, Chou, Fang et Chen (Lepidoptera: Cossidae) is a notorious carpenterworm pest of sea buckthorn, Hippophae rhamnoides L. (Elaeagnaceae). Chemosensory proteins (CSPs) are thought to be responsible for initial biochemical recognition during olfactory perception by the insect. We examined the structure, function, and expression profiles of these proteins in four structures (e.g., antennae, labipalp, legs, and external genitalia) of male adults. Molecular weight, isoelectric point, hydrophilicity and hydrophobicity of proteins, and signal peptide prediction of 18 E. hippophaecolus CSPs (EhipCSPs) were investigated via software. Expression profiles in the four male structures were analyzed by fluorescence quantitative real-time polymerase chain reaction. Bioinformatics analysis showed that most EhipCSPs are low-molecular-weight proteins with hydrophobic regions and a high proportion of alpha-helices, consistent with the general characteristics of insect CSPs. Eight EhipCSPs (EhipCSP2, EhipCSP5, EhipCSP7, and EhipCSP13–17) were predominantly expressed in the labipalp (P &amp;lt; 0.01), and three (EhipCSP6, EhipCSP8, and EhipCSP9) were predominantly expressed in legs (P &amp;lt; 0.01). We speculate that these proteins may be related to contact sensations, host recognition, and other functions. Two EhipCSPs (EhipCSP4 and EhipCSP11) were highly expressed in the external genitalia (P &amp;lt; 0.01), suggesting that they may be involved in spousal positioning or mating activities. Most EhipCSPs were differentially expressed in the four structures, with wide overall expression, indicating an important role in olfactory recognition in multiple tissues. Our findings establish the foundation for further investigation of EhipCSPs and potential development of nonpesticide control measures.

Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit.

Auxin response factors (ARFs) play important roles in various plant physiological processes; however, knowledge of the exact role of ARFs in plant responses to water deficit is limited. In this study, SlARF4, a member of the ARF family, was functionally characterized under water deficit. Real-time fluorescence quantitative polymerase chain reaction (PCR) and β-glucuronidase (GUS) staining showed that water deficit and abscisic acid (ABA) treatment reduced the expression of SlARF4. SlARF4 was expressed in the vascular bundles and guard cells of tomato stomata. Loss of function of SlARF4 (arf4) by using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 (CRISPR/Cas 9) technology enhanced plant resistance to water stress and rehydration ability. The arf4 mutant plants exhibited curly leaves and a thick stem. Malondialdehyde content was significantly lower in arf4 mutants than in wildtype plants under water stress; furthermore, arf4 mutants showed higher content of antioxidant substances, superoxide dismutase, actual photochemical efficiency of photosystem II (PSII), and catalase activities. Stomatal and vascular bundle morphology was changed in arf4 mutants. We identified 628 differentially expressed genes specifically expressed under water deficit in arf4 mutants; six of these genes, including ABA signaling pathway-related genes, were differentially expressed between the wildtype and arf4 mutants under water deficit and unlimited water supply. Auxin responsive element (AuxRE) elements were found in these genes’ promoters indicating that SlARF4 participates in ABA signaling pathways by regulating the expression of SlABI5/ABF and SCL3, thereby influencing stomatal morphology and vascular bundle development and ultimately improving plant resistance to water deficit.