BMC Molecular and Cell Biology最新文献

Background: For the purpose of skeletal muscle engineering, primary myoblasts (Mb) and adipogenic mesenchymal stem cells (ADSC) can be co-cultured and myogenically differentiated. Electrospun composite nanofiber scaffolds represent suitable matrices for tissue engineering of skeletal muscle, combining both biocompatibility and stability Although growth differentiation factor 11 (GDF11) has been proposed as a rejuvenating circulating factor, restoring skeletal muscle function in aging mice, some studies have also described a harming effect of GDF11. Therefore, the aim of the study was to analyze the effect of GDF11 on co-cultures of Mb and ADSC on poly-ε-caprolactone (PCL)-collagen I-polyethylene oxide (PEO)-nanofibers.

Results: Human Mb were co-cultured with ADSC two-dimensionally (2D) as monolayers or three-dimensionally (3D) on aligned PCL-collagen I-PEO-nanofibers. Differentiation media were either serum-free with or without GDF11, or serum containing as in a conventional differentiation medium. Cell viability was higher after conventional myogenic differentiation compared to serum-free and serum-free + GDF11 differentiation as was creatine kinase activity. Immunofluorescence staining showed myosine heavy chain expression in all groups after 28 days of differentiation without any clear evidence of more or less pronounced expression in either group. Gene expression of myosine heavy chain (MYH2) increased after serum-free + GDF11 stimulation compared to serum-free stimulation alone.

Conclusions: This is the first study analyzing the effect of GDF11 on myogenic differentiation of Mb and ADSC co-cultures under serum-free conditions. The results of this study show that PCL-collagen I-PEO-nanofibers represent a suitable matrix for 3D myogenic differentiation of Mb and ADSC. In this context, GDF11 seems to promote myogenic differentiation of Mb and ADSC co-cultures compared to serum-free differentiation without any evidence of a harming effect.

Background: Cytochrome P450 4F2 (CYP4F2) enzyme is a member of the CYP4 family responsible for the metabolism of fatty acids, therapeutic drugs, and signaling molecules such as arachidonic acid, tocopherols, and vitamin K. Several reports have demonstrated that the missense variant CYP4F2*3 (V433M) causes decreased activity of CYP4F2 and inter-individual variations in warfarin dose in different ethnic groups. However, the molecular pathogenicity mechanism of missense V433M in CYP4F2 at the atomic level has not yet been completely elucidated.

Methods and results: In the current study, we evaluated the effect of the V433M substitution on CYP4F2 using 14 different bioinformatics tools. Further molecular dynamics (MD) simulations were performed to assess the impact of the V433M mutation on the CYP4F2 protein structure, stability, and dynamics. In addition, molecular docking was used to illustrate the effect of V433M on its interaction with vitamin K1. Based on our results, the CYP4F2*3 variant was a damaging amino acid substitution with a destabilizing nature. The simulation results showed that missense V433M affects the dynamics and stability of CYP4F2 by reducing its compactness and stability, which means that it tends to change the overall structural conformation and flexibility of CYP4F2. The docking results showed that the CYP4F2*3 variant decreased the binding affinity between vitamin K1 and CYP4F2, which reduced the activity of CYP4F2*3 compared to native CYP4F2.

Conclusions: This study determined the molecular pathogenicity mechanism of the CYP4F2*3 variant on the human CYP4F2 protein and provided new information for understanding the structure-function relationship of CYP4F2 and other CYP4 enzymes. These findings will aid in the development of effective drugs and treatment options.

Background: Hypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of some commonly used housekeeping genes, such as GAPDH and PGK1, can confound the relative gene expression analysis. Thus, there is ongoing debate as to which housekeeping gene is appropriate for studies investigating hypoxia-induced cell responses. Specifically, there is still contradicting information for which housekeeping genes are stable in hypoxia cultures of mesenchymal stem cells. In this study, candidate housekeeping genes curated from the literature were matched to RNAseq data of normoxic and hypoxic human adipose-derived stem cell cultures to determine if gene expression was modulated by hypoxia or not. Expression levels of selected candidates were used to calculate coefficient of variation. Then, accounting for the mean coefficient of variation, and normalised log twofold change, genes were ranked and shortlisted, before validating with qRT-PCR. Housekeeping gene suitability were then determined using GeNorm, NormFinder, BestKeeper, comparative[Formula: see text], RefFinder, and the Livak method.

Results: Gene expression levels of 78 candidate genes identified in the literature were analysed in the RNAseq dataset generated from hADSC cultured under Nx and Hx conditions. From the dataset, 15 candidates with coefficient of variation ≤ 0.15 were identified, where differential expression analysis results further shortlisted 8 genes with least variation in expression levels. The top 4 housekeeping gene candidates, ALAS1, RRP1, GUSB, and POLR2B, were chosen for qRT-PCR validation. Additionally, 18S, a ribosomal RNA commonly used as housekeeping gene but not detected in the RNAseq method, was added to the list of housekeeping gene candidates to validate. From qRT-PCR results, 18S and RRP1 were determined to be stably expressed in cells cultured under hypoxic conditions.

Conclusions: We have demonstrated that 18S and RRP1 are suitable housekeeping genes for use in hypoxia studies with human adipose-derived stem cell and should be used in combination. Additionally, these data shown that the commonly used GAPDH and PGK1 are not suitable housekeeping genes for investigations into the effect of hypoxia in human adipose-derived stem cell.

Nasopharyngeal carcinoma is a tumor with high malignancy and poor prognosis, which severely affects the health of the patients. LncRNAs and microRNAs are crucial for the occurrence and development of nasopharyngeal carcinoma, which regulate the progression of nasopharyngeal carcinoma through the ceRNA network. SCARB1 plays an essential role in nasopharyngeal carcinoma. However, the mechanism underlying the regulation of SCARB1 in nasopharyngeal carcinoma through non-coding RNAs remains unclear. Our findings indicated that the SCAT8/miR-125b-5p axis promoted the malignant progression of nasopharyngeal carcinoma by driving the expression of SCARB1. Mechanistically, the expression of SCARB1 could be regulated by the lncRNA, SCAT8 and the microRNA, miR-125b-5p. Moreover, as a ceRNA of miR-125b-5p, SCAT8 can not only regulate the expression of SCARB1, but also regulate the malignant progression of nasopharyngeal carcinoma. Notably, our results reveal a novel ceRNA regulatory network in nasopharyngeal carcinoma, which could serve as a potential target for the diagnosis and treatment of nasopharyngeal carcinoma.

Background: Recent studies have reported that the circadian transcription factor aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2) promotes the metastatic progression of lung adenocarcinoma. However, the molecular mechanisms of ARNTL2 in non-small cell lung cancer (NSCLC) cell growth and proliferation remain to be explored.

Methods: The expression of ARNTL2 and acyl-CoA thioesterase 7 (ACOT7) in lung cancer patients was analyzed based on TCGA database. Gain-of-function of ARNTL2 and ACOT7 was conducted by transfecting the cells with plasmids or lentivirus. Knockdown assay was carried out by siRNAs. Western blot and qRT-PCR were performed to check the protein and mRNA expression. Dual luciferase and ChIP-qPCR assay was applied to check the interaction of ARNTL2 on ACOT7's promoter sequence. Triglyceride level, MDA production, the activity of casapase 3 to caspase 7, and lipid ROS were measured by indicated assay kit. Cellular function was detected by CCK8, colony formation and flow cytometry analysis of cell death and cell cycle.

Results: We demonstrated that ARNTL2 upregulation of ACOT7 was critical for NSCLC cell growth and proliferation. Firstly, overexpression of ARNTL2 conferred the poor prognosis of LUAD patients and supported the proliferation of NSCLC cells. Based on molecular experiments, we showed that ARNTL2 potentiated the transcription activity of ACOT7 gene via direct binding to ACOT7's promoter sequence. ACOT7 high expression was correlated with the worse prognosis of LUAD patients. Gain-of-function and loss-of-function experiments revealed that AOCT7 contributed to NSCLC cell growth and proliferation. ACOT7 regulated the apoptosis and ferroptosis of NSCLC cells, while exhibited no effect on cell cycle progression. ACOT7 overexpression also potentiated fatty acid synthesis and suppressed lipid peroxidation. Lastly, we showed that ARNTL2 knockdown and overexpression inhibited and promoted the cellular triglyceride production and subsequent cell proliferation, which could be reversed by ACOT7 overexpression and knockdown.

Conclusion: Our study illustrated the oncogenic function of ARNTL2/ACOT7 axis in the development of NSCLC. Targeting ARNTL2 or ACOT7 might be promising therapeutic strategies for NSCLC patients with highly expressed ARNTL2.

Background: Endometriosis cause decreases in life quality and pelvic pain in reproductive-age women. Methylation abnormalities played a functional role in the progression of endometriosis, this study aimed to explore the mechanisms mediated by abnormal methylation in the development of EMS.

Materials and methods: Next-generation sequencing dataset and methylation profiling dataset were used to screen out the key gene SFRP2. Western bolt, Real-time PCR, Aza-2?deoxycytidine treatment, luciferase reporter assay, Methylation-specific PCR , Bisulfite sequencing PCR and lentivirus infection were carried out to detect the methylation status and signaling pathway with the primary epithelial cells. Transwell assay and wound scratch assay were implemented to observe the differences of migration ability with the intervening with the expression of SFRP2.

Results: To define the role of the DNA methylation-regulated genes in the pathogenesis of EMS, we performed both DNA methylomic and expression analyses of ectopic endometrium and ectopic endometrium epithelial cells(EEECs) and found that SFRP2 is demethylated/upregulated in ectopic endometrium and EEECs. The expression of lentivirus carrying SFRP2 cDNA up-regulates the activity of Wnt signaling and the protein expression of ?-catenin in EEECs. SFRP2 impact on the invasion and migration of ectopic endometrium by modulating the activities of the Wnt/?-catenin signaling pathway. The invasion and migration ability of EEECs were significantly strengthened after demethylation treatment including 5-Aza and the knockdown of DNMT1.

Conclusion: In summary, the increased SFRP2 expression-induced Wnt/?-catenin signaling due to the demethylation of the SFRP2 promoter plays an important role in the pathogenesis of EMS, suggesting that SFRP2 might be a therapeutic target for EMS treatment.

Background: Human accelerated regions (HARs) are short conserved genomic sequences that have acquired significantly more nucleotide substitutions than expected in the human lineage after divergence from chimpanzees. The fast evolution of HARs may reflect their roles in the origin of human-specific traits. A recent study has reported positively-selected single nucleotide variants (SNVs) within brain-exclusive human accelerated enhancers (BE-HAEs) hs1210 (forebrain), hs563 (hindbrain) and hs304 (midbrain/forebrain). By including data from archaic hominins, these SNVs were shown to be Homo sapiens-specific, residing within transcriptional factors binding sites (TFBSs) for SOX2 (hs1210), RUNX1/3 (hs563), and FOS/JUND (hs304). Although these findings suggest that the predicted modifications in TFBSs may have some role in present-day brain structure, work is required to verify the extent to which these changes translate into functional variation.

Results: To start to fill this gap, we investigate the SOX2 SNV, with both forebrain expression and strong signal of positive selection in humans. We demonstrate that the HMG box of SOX2 binds in vitro with Homo sapiens-specific derived A-allele and ancestral T-allele carrying DNA sites in BE-HAE hs1210. Molecular docking and simulation analysis indicated highly favourable binding of HMG box with derived A-allele containing DNA site when compared to site carrying ancestral T-allele.

Conclusion: These results suggest that adoptive changes in TF affinity within BE-HAE hs1210 and other HAR enhancers in the evolutionary history of Homo sapiens might. have brought about changes in gene expression patterns and have functional consequences on forebrain formation and evolution.

Methods: The present study employ electrophoretic mobility shift assays (EMSA) and molecular docking and molecular dynamics simulations approaches.

Background: Type I Diabetes mellitus (T1D) is characterized by a specific destruction of β-cells by the immune system. During this process pro-inflammatory cytokines are released in the pancreatic islets and contribute for β-cells demise. Cytokine-induced iNOS activation, via NF-κB, is implicated in induction of β-cells death, which includes ER stress activation. Physical exercise has been used as an adjunct for better glycemic control in patients with T1D, since it is able to increase glucose uptake independent of insulin. Recently, it was observed that the release of IL-6 by skeletal muscle, during physical exercise, could prevent β-cells death induced by pro-inflammatory cytokines. However, the molecular mechanisms involved in this beneficial effect on β-cells are not yet completely elucidated. Our aim was to evaluate the effect of IL-6 on β-cells exposed to pro-inflammatory cytokines.

Results: Pre-treatment with IL-6 sensitized INS-1E cells to cytokine-induced cell death, increasing cytokine-induced iNOS and Caspase-3 expression. Under these conditions, however, there was a decrease in cytokines-induced p-eIF2-α but not p-IRE1expression, proteins related to ER stress. To address if this prevention of adequate UPR response is involved in the increase in β-cells death markers induced by IL-6 pre-treatment, we used a chemical chaperone (TUDCA), which improves ER folding capacity. Use of TUDCA increased cytokines-induced Caspase-3 expression and Bax/Bcl-2 ratio in the presence of IL-6 pre-treatment. However, there is no modulation of p-eIF2-α expression by TUDCA in this condition, with increase of CHOP expression.

Conclusion: Treatment with IL-6 alone is not beneficial for β-cells, leading to increased cell death markers and impaired UPR activation. In addition, TUDCA has not been able to restore ER homeostasis or improve β-cells viability under this condition, suggesting that other mechanisms may be involved.

Background: Hypoxia is considered a critical contributor to renal cell carcinoma progression, including invasion and metastasis. However, the potential mechanisms by which it promotes invasion and metastasis have not yet been clarified. The purpose of this study was to investigate the role and mechanism of hypoxia-induced renal cell carcinoma and provide evidence-based medical proof for improvements to postoperative nursing of renal cell carcinoma patients. A total of 64 patients with renal cell carcinoma were divided into the observation group (nursing based on oxygen administration) and the control group (conventional nursing). Renal function indexes, serum inflammatory factors, and tumor markers were evaluated. The human renal cell carcinoma cell line A498 under hypoxia/normoxia was used as an experimental model in vitro and the biological characteristics and mitochondrial function of the cells were assessed.

Results: Nursing based on oxygen administration decreased the value of renal function indexes, serum inflammatory factors, and tumor markers in renal cell carcinoma patients. Hypoxia was found to induce A498 cell invasion, migration, and the release of inflammatory cytokines, while repressing human solute carrier family 14 member 1 gene expression. Elevated levels of solute carrier family 14 member 1 expression induced mitochondrial reactive oxygen species accumulation, diminished the intracellular adenosine triphosphate level, and destroyed both mitochondrial membrane potential integrity and mitochondrial morphology. Overexpression of the solute carrier family 14 member 1 gene could abolish hypoxia-induced invasion, reduce the migration of A498 cells, inhibit the hypoxia-induced release of inflammatory cytokines, and arrest the cell cycle at the G1/S checkpoint.

Conclusions: These data reveal that nursing based on oxygen administration can improve the clinical efficacy of renal cell carcinoma therapies, being safe and effective. The results elucidate a mechanism wherein the solute carrier family 14 member 1 gene participates in the occurrence and development of hypoxia-induced renal cell carcinoma in a mitochondria-dependent manner.

Background: Mechanical therapies, such as distraction osteogenesis, are widely used in dental clinics. During this process, the mechanisms by which tensile force triggers bone formation remain of interest. Herein, we investigated the influence of cyclic tensile stress on osteoblasts and identified the involvement of ERK1/2 and STAT3.

Materials and methods: Rat clavarial osteoblasts were subjected to tensile loading (10% elongation, 0.5 Hz) for different time periods. RNA and protein levels of osteogenic markers were determined using qPCR and western blot after inhibition of ERK1/2 and STAT3. ALP activity and ARS staining revealed osteoblast mineralization capacity. The interaction between ERK1/2 and STAT3 was investigated by immunofluorescence, western blot, and Co-IP.

Results: The results showed that tensile loading significantly promoted osteogenesis-related genes, proteins and mineralized nodules. In loading-induced osteoblasts, inhibition of ERK1/2 or STAT3 decreased osteogenesis-related biomarkers significantly. Moreover, ERK1/2 inhibition suppressed STAT3 phosphorylation, and STAT3 inhibition disrupted the nuclear translocation of pERK1/2 induced by tensile loading. In the non-loading environment, inhibition of ERK1/2 hindered osteoblast differentiation and mineralization, while STAT3 phosphorylation was elevated after ERK1/2 inhibition. STAT3 inhibition also increased ERK1/2 phosphorylation, but did not significantly affect osteogenesis-related factors.

Conclusion: Taken together, these data suggested that ERK1/2 and STAT3 interacted in osteoblasts. ERK1/2-STAT3 were sequentially activated by tensile force loading, and both affected osteogenesis during the process.