Objective: To explore the pathogenic mechanism of the miR-340/high mobility group box 1 (HMGB1) axis in the formation of liver fibrosis. Methods: A rat liver fibrosis model was established by injecting CCl(4) intraperitoneally. miRNAs targeting and validating HMGB1 were selected with gene microarrays after screening the differentially expressed miRNAs in rats with normal and hepatic fibrosis. The effect of miRNA expressional changes on HMGB1 levels was detected by qPCR. Dual luciferase gene reporter assays (LUC) was used to verify the targeting relationship between miR-340 and HMGB1. The proliferative activity of the hepatic stellate cell line HSC-T6 was detected by thiazolyl blue tetrazolium bromide (MTT) assay after co-transfection of miRNA mimics and HMGB1 overexpression vector, and the expression of extracellular matrix (ECM) proteins type I collagen and α-smooth muscle actin (SMA) was detected by western blot. Statistical analysis was performed by analysis of variance and the LSD-t test. Results: Hematoxylin-eosin and Masson staining results showed that the rat model of liver fibrosis was successfully established. Gene microarray analysis and bioinformatics prediction had detected eight miRNAs possibly targeting HMGB1, and animal model validation had detected miR-340. qPCR detection results showed that miR-340 had inhibited the expression of HMGB1, and a luciferase complementation assay suggested that miR-340 had targeted HMGB1. Functional experiments results showed that HMGB1 overexpression had enhanced cell proliferation activity and the expression of type I collagen and α-SMA, while miR-340 mimics had not only inhibited cell proliferation activity and the expression of HMGB1, type I collagen, and α-SMA, but also partially reversed the promoting effect of HMGB1 on cell proliferation and ECM synthesis. Conclusion: miR-340 targets HMGB1 to inhibit the proliferation and ECM deposition in hepatic stellate cells and plays a protective role during the process of liver fibrosis.
Animals ; Rats ; Cell Proliferation ; Collagen Type I/metabolism* ; Fibrosis ; Hepatic Stellate Cells ; HMGB1 Protein/genetics* ; Liver Cirrhosis/pathology* ; MicroRNAs/metabolism*

OBJECTIVE: To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs. METHODS: The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-β1 (TGF-β1) or co-treated with TGF-β1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot. RESULTS: The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-β1 and 5 μmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-β1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-β1 single treatment group (P < 0.05). CONCLUSION A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.
Humans ; Transforming Growth Factor beta1/pharmacology* ; Hepatic Stellate Cells/pathology* ; Liver Cirrhosis/genetics* ; Collagen Type I/pharmacology* ; RNA, Messenger/metabolism*

Objective: To investigate the clinicopathological and cytogenetic features of cryptic COL1A1-PDGFB fusion dermatofibrosarcoma protuberans (CC-DFSP). Methods: Three cases of CC-DFSP diagnosed in West China Hospital, Sichuan University, Chengdu, China from January 2021 to September 2021 were studied. Immunohistochemistry for CD34 and other markers, fluorescence in situ hybridization (FISH) for PDGFB, COL1A1-PDGFB and COL1A1, next-generation sequencing (NGS), reverse-transcriptase polymerase chain reaction (RT-PCR) and Sanger sequencing were performed. Results: There were three cases of CC-DFSP, including two females and one male. The patients were 29, 44 and 32 years old, respectively. The sites were abdominal wall, caruncle and scapula. Microscopically, they were poorly circumscribed. The spindle cells of the tumors infiltrated into the whole dermis or subcutaneous tissues, typically arranging in a storiform pattern. Immunohistochemically, the neoplastic cells exhibited diffuse CD34 expression, but were negative for S-100, SMA, and Myogenin. Loss of H3K27me3 was not observed in the tumor cells. The Ki-67 index was 10%-15%. The 3 cases were all negative for PDGFB rearrangement and COL1A1-PDGFB fusion, whereas showing unbalanced rearrangement for COL1A1. Case 1 showed a COL1A1 (exon 31)-PDGFB (exon 2) fusion using NGS, which was further validated through RT-PCR and Sanger sequencing. All patients underwent extended surgical resection. Except for case 3 with recurrence 2 years after surgical resection, the other 2 cases showed no recurrence or metastasis during the follow-up. Conclusions: FISH has shown its validity for detecting PDGFB rearrangement and COL1A1-PDGFB fusion and widely applied in clinical detection. However, for cases with negative routine FISH screening that were highly suspicious for DFSPs, supplementary NGS or at least COL1A1 break-apart FISH screening could be helpful to identify cryptic COL1A1-PDGFB fusions or other variant fusions.
Female ; Humans ; Male ; Collagen Type I, alpha 1 Chain ; Dermatofibrosarcoma/pathology* ; In Situ Hybridization, Fluorescence ; Oncogene Proteins, Fusion/genetics* ; Proto-Oncogene Proteins c-sis/genetics* ; Skin Neoplasms/pathology* ; Adult

OBJECTIVE: To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. METHODS: hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. RESULTS: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. CONCLUSION Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
Pregnancy ; Female ; Humans ; Rabbits ; Animals ; Vascular Endothelial Growth Factor A/metabolism* ; Fibronectins/metabolism* ; Collagen Type I/genetics* ; Tenascin/metabolism* ; Collagen/metabolism* ; Anterior Cruciate Ligament/surgery* ; Mesenchymal Stem Cells ; Tendons/metabolism* ; Fibroblasts/metabolism*

OBJECTIVE: To analyze the clinical phenotype and genetic characteristics of a patient with Alport syndrome. METHODS: A patient with Alport syndrome who had visited the First Affiliated Hospital of Zhengzhou University in November 2020 was selected as the study subject. Clinical data of the patient were collected. High-throughput sequencing was carried out to detect potential variant of the COL4A3, COL4A4 and COL4A5 genes, and Sanger sequencing was carried out for verification of candidate variants in the family. RESULTS: The main clinical manifestations of the patient included hematuria, proteinuria, and impaired hearing. Audiometric testing suggested symmetrical cochlear sensory neural hearing loss on both sides. Renal biopsy revealed mild mesangial proliferative glomerulonephritis. Genetic testing revealed that the patient has harbored compound heterozygous variants of the COL4A4 gene, namely c.940G>A (p.Gly314Ser) and c.3773G>A (p.Gly1258Asp), which were respectively inherited from her father and mother. Neither variant has been reported before, and were predicted to be pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION The c.940G>A (p.Gly314Ser) and c.3773G>A (p.Gly1258Asp) compound heterozygous variants of the COL4A4 gene probably underlay the Alport syndrome in this patient. Above finding has enriched the mutational spectrum of the COL4A4 gene.
Female ; Humans ; Nephritis, Hereditary/genetics* ; Hematuria ; Genetic Testing ; Genomics ; Hearing ; Collagen Type IV/genetics*

OBJECTIVE: To explore the genetic basis for a patient with Alport syndrome (AS) and confirm the existence of a splicing variant. METHODS: An AS patient diagnosed at the Affiliated Hospital of Inner Mongolia Medical University on January 8, 2021 for significant proteinuria and occult hematuria was selected as the study subject. Clinical data was collected. Peripheral blood samples were collected for the extraction of genomic DNA. Whole exome sequencing and Sanger sequencing were carried out to identify potential genetic variants. An in vitro experiment was also conducted to verify the abnormal mRNA splicing. Bioinformatic software was used to analyze the conservation of amino acids of the variant sites and simulate the 3D structure of the variant collagen IV protein. Immunofluorescence and immunohistochemistry were carried out on renal tissues from the patient to confirm the presence of AS kidney injury. RESULTS: The patient, a 21-year-old male, had a 24-hour urine protein of 3.53 g/24 h, which fulfilled the diagnostic criteria for proteinuria. His blood uric acid has also increased to 491 μmol/L. DNA sequencing revealed that he has harbored a c.835-9T>A splice variant of the COL4A5 gene, which was not found in either of his parents. In vitro experiment confirmed that the variant has removed 57 bp from the exon 15 of the mRNA of the COL4A5 gene. The deletion may cause loss of amino acid residues from positions 279 to 297, which in turn may affect the stability of the secondary structure of the α5 chain encoded by the COL4A5 gene. The amino acids are conserved across various species. The result of homology modeling indicated that the trimerization of Col-IV with the mutated α5 chain could be achieved, however, the 3D structure was severely distorted. The AS kidney damage was confirmed through immunofluorescence assays. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.835-9T>A variant was classified as likely pathogenic (PVS1_Moderate+PS3_Moderate+PM2_Supporting+PS2+PP3+PP4). CONCLUSION The c.835-9T>A variant of the COL4A5 gene probably underlay the AS in this patient. In vitro experiment has confirmed the abnormal splicing caused by the variant. Histopathological examination of the kidney tissue has provided in vivo evidence for its pathogenicity. Above finding has expanded the mutational spectrum of the COL4A5 gene.
Humans ; Male ; Young Adult ; Amino Acids ; China ; Collagen Type IV/genetics* ; Exons ; Nephritis, Hereditary/genetics* ; RNA Splicing

OBJECTIVE: To analysis variants of COL4A5 gene in two Chinese pedigrees affected with Alport syndrome (AS) and provide prenatal diagnosis for them. METHODS: Two unrelated ethnic Han Chinese pedigrees who had visited the First Affiliated Hospital of Zhengzhou University respectively in September 2018 and January 2020 were selected as the study subjects. Clinical data were collected, and genomic DNA was extracted from peripheral venous blood and amniotic fluid samples for genetic testing. Following next generation sequencing, candidate variants of the COL4A5 gene were verified by Sanger sequencing and bioinformatic analysis. The gender of the fetuses was determined by the presence of sex-determining region on Y (SRY). RESULTS: Genetic testing revealed that the proband and a fetus from pedigree 1 had both harbored a c.2723G>A (p.Gly908Glu) variant in exon 32 of the COL4A5 gene, whilst the proband and a fetus from pedigree 2 had both harbored a c.3817G>A (p.Gly1273Asp) variant in exon 44 of the COL4A5 gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were classified as likely pathogenic (PP2+PM2_Supporting). Following exclusion of maternal contamination, PCR amplification of the SRY region indicated that both fetuses were males. CONCLUSION The c.2723G>A (p.Gly908Glu) and c.3817G>A (p.Gly1273Asp) variants of the COL4A5 gene probably underlay the AS in the two pedigrees. Detection of the SRY region can reliably identify the fetal sex, which is conducive to the prenatal diagnosis. Above results have also enriched the mutational spectrum of the COL4A5 gene and provided a reference for correlating the genotype and phenotype of the AS.
Female ; Humans ; Male ; Pregnancy ; Collagen Type IV/genetics* ; East Asian People ; Genetic Testing ; Nephritis, Hereditary/genetics* ; Pedigree ; Prenatal Diagnosis

Objective To explore the impact of sinomenine on bleomycin A5-induced pulmonary fibrosis (PF) in rats and the underlying mechanism. Methods MRC-5 cells were cultured and treated with sinomenine to determine its optimal concentration and time through the MTT assay. Subsequently, MRC-5 cells were incubated with 80 μmol/L sinomenine for 48 hours or transfected with miR-21 mimic/a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1) siRNA prior to sinomenine treatment. The expression of miR-21, ADAMTS-1, collagen type 1 (Col1) and collagen type 3 (Col3) was detected by quantitative real-time PCR (qRT-PCR) and/or Western blot analysis. Thirty SD rats were randomly divided into control group, sinomenine group and sinomenine combined with miR-21 agomir group, with 10 animals in each group. Bleomycin A5 were intratracheally administered to establish the PF model. Then, rats in control group, sinomenine group and sinomenine +miR-21 agomir group were treated with 9 g/L sodium chloride solution, sinomenine and sinomenine+miR-21 agomir, respectively. On day 28, all rats were sacrificed. HE and Masson staining was performed in pulmonary tissue. The expression of ADAMTS-1, Col1 and Col3 in pulmonary tissue were detected by qRT-PCR and/or Western blot analysis. ELISA was used to measure serum procollagen type 1 carboxyterminal propeptide (P1CP) and procollagen type 3 aminoterminal propeptide (P3NP) levels. Results Administration of sinomenine decreased miR-21 levels, up-regulated ADAMTS-1 expression, and promoted Col1 and Col3 degradation in MRC-5 cells. Importantly, interfering with the miR-21/ADAMTS-1 signaling pathway partially reversed the promotive effect of sinomenine on Col1 and Col3 degradation. Treatment of SD rats with sinomenine reduced alveolitis and PF scores, decreased serum P1CP and P3NP levels, up-regulated pulmonary ADAMTS-1 expression, and down-regulated Col1 and Col3 expression. However, these effects were reversed by miR-21 agomir. Conclusion Sinomenine promotes Col1 and Col3 degradation and inhibits PF in rats by miR-21/ADAMTS-1 pathway.
Rats ; Animals ; Pulmonary Fibrosis/genetics* ; Procollagen/metabolism* ; Rats, Sprague-Dawley ; Signal Transduction ; Bleomycin/adverse effects* ; Collagen Type III/metabolism* ; MicroRNAs/metabolism*

Humans ; Nephritis, Hereditary/genetics* ; Astigmatism ; Mutation ; Collagen Type IV/genetics*

In this study, we investigated the effects of Panax notoginseng saponins (PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension (PAH). PAH rat model was established, and male Sprague Dawley (SD) rats were randomly divided into control group, monocrotaline (MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure (mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index (RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1, P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI, pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1, and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10, Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3 were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.
Animals ; Male ; Rats ; Caspase 3/metabolism* ; Collagen ; Disease Models, Animal ; Hypertension, Pulmonary/drug therapy* ; Monocrotaline/adverse effects* ; Panax notoginseng/chemistry* ; Proliferating Cell Nuclear Antigen/pharmacology* ; Pulmonary Arterial Hypertension ; Pulmonary Artery/metabolism* ; Rats, Sprague-Dawley ; Receptor, Notch3/genetics* ; RNA, Messenger ; Saline Solution ; Signal Transduction ; Saponins/pharmacology*