Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury.

BACKGROUND: The mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes. METHODS: A 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein-Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms. RESULTS: A total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: The proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

BIN1 in cancer: biomarker and therapeutic target.

BACKGROUND: The bridging integrator 1 (BIN1) protein was originally identified as a pro-apoptotic tumor suppressor that binds to and inhibits oncogenic MYC transcription factors. BIN1 has complex physiological functions participating in endocytosis, membrane cycling, cytoskeletal regulation, DNA repair deficiency, cell-cycle arrest, and apoptosis. The expression of BIN1 is closely related to the development of various diseases such as cancer, Alzheimer's disease, myopathy, heart failure, and inflammation. PURPOSE: Because BIN1 is commonly expressed in terminally differentiated normal tissues and is usually undetectable in refractory or metastatic cancer tissues, this differential expression has led us to focus on human cancers associated with BIN1. In this review, we discuss the potential pathological mechanisms of BIN1 during cancer development and its feasibility as a prognostic marker and therapeutic target for related diseases based on recent findings on its molecular, cellular, and physiological roles. CONCLUSION: BIN1 is a tumor suppressor that regulates cancer development through a series of signals in tumor progression and microenvironment. It also makes BIN1 a feasible early diagnostic or prognostic marker for cancer.

Exploring potential targets of HPV&BC based on network pharmacology and urine proteomics.

BACKGROUND: Bladder cancer (BC) caused by Human papillomavirus (HPV) infection remains a complex public health problem in developing countries. Although the HPV vaccine effectively prevents HPV infection, it does not benefit patients with BC who already have HPV. METHODS: Firstly, the differential genes of HPV-related BC patients were screened by transcriptomics, and then the prognostic and clinical characteristics of the differential genes were analyzed to screen out the valuable protein signatures. Furthermore, the compound components and targets of Astragali Radix (AR) were analyzed by network pharmacology, and the intersection targets of drug components and HPV_BC were screened out for pathway analysis. In addition, the binding ability of the compound to the Astragali-HPV_BC target was verified by molecular docking and virtual simulation. Finally, to identify potential targets in BC patients through urine proteomics and in vitro experiments. RESULTS: Eleven HPV_BC-related protein signatures were screened out, among which high expression of EGFR, CTNNB1, MYC, GSTM1, MMP9, CXCR4, NOTCH1, JUN, CXCL12, and KRT14 had a poor prognosis, while low expression of CASP3 had a poor prognosis. In the analysis of clinical characteristics, it was found that high-risk scores, EGFR, MMP9, CXCR4, JUN, and CXCL12 tended to have higher T stage, pathological stage, and grade. Pharmacological and molecular docking analysis identified a natural component of AR (Quercetin) and it corresponding core targets (EGFR). The OB of the natural component was 46.43, and the DL was 0.28, respectively. In addition, EGFR-Quercetin has high affinity. Urine proteomics and RT-PCR showed that EGFR was expressed explicitly in BC patients. Mechanism analysis revealed that AR component targets might affect HPV_BC patients through Proteoglycans in the cancer pathway. CONCLUSION: AR can target EGFR through its active component (Quercetin), and has a therapeutic effect on HPV_BC patients.

Rethinking neutrophil extracellular traps.

Neutrophils are a major subset of leukocytes in human circulating blood. In some circumstances, neutrophils release neutrophil extracellular traps (NETs). lnitially, NETs were considered to have a strong antibacterial capacity. However, currently, NETs have been shown to have a pivotal impact on various diseases. Different stimulators induce the production of different types of NETs, and their biological functions and modes of clearance do not appear to be the same. In this review, we will discuss several important issues related to NETs in order to better understand the relationship between NETs and diseases, as well as how to utilize the characteristics of NETs for disease treatment.

[Overexpression of angiotensin-converting enzyme 2 inhibits angiotensin II type 1 receptor expression and signal transducer and activator of transcription 3 phosphorylation in cultured smooth muscle cells].

OBJECTIVE: To explore the effects of recombinated lentiviral angiotensin-converting enzyme 2 (ACE2) vector transfer on the expression of angiotensin II type 1 (AT1) receptor in cultured vascular smooth muscle cells (VSMCs). METHODS: VSMCs were divided into 7 groups: (1) CONTROL: serum-free culture medium; (2) Lentiviral-GFP vector group: Lentiviral-GFP vector (MOI = 10); (3) Ang II group (10(-7) mol/L); (4) Ang II (10(-7) mol/L) + Lentiviral-ACE2 (MOI = 10) group; (5) Ang II (10(-7) mol/L) + Irbesartan (10(-7) mol/L) group ; (6) Ang II (10(-7) mol/L) + irbesartan (10(-7) mol/L) + Lentiviral-ACE2 (MOI = 10) group ; (7) Lentiviral-ACE2 (MOI = 10) group. Ninety-six hours later, the proliferation of VSMCs was determined with CCK-8 Kit. AT1 receptor mRNA and protein expressions were detected with quantitative real-time PCR and Western blot, the signaling pathway of signal transducer and activator of transcription 3 (STAT3) was also detected. RESULTS: ACE2 gene transfer significantly inhibited the VSMCs proliferation in the absence or presence of Ang II. AT1 receptor mRNA and protein expressions were also significantly downregulated in the absence or presence of Ang II. Similar to AT1 receptor mRNA and protein expression changes, STAT3 phosphorylation was also significantly inhibited by ACE2 overexpression. CONCLUSION: Our results suggest that overexpression of ACE2 gene could inhibit the VSMCs proliferation by downregulating AT1 receptor expression and STAT3 phosphorylation. ACE2 could also directly inhibit AT1 receptor in cultured VSMCs.

Decreased expression of CLCA2 and the correlating with immune infiltrates in patients with cervical squamous cell carcinoma: A bioinformatics analysis.

OBJECTIVE: Calcium-activated chloride channel 2 (CLCA2) is closely related to the invasion, metastasis, and prognosis of some common malignant tumors. The present study aimed to evaluate the role of CLCA2 in cervical squamous cell carcinoma (CESC) using bioinformatics analysis. MATERIALS AND METHODS: The mRNA sequencing data and the corresponding clinical data were obtained from Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database respectively. Then univariate analysis of variance was used to analyze the differential mRNA expression of CLCA2 between normal, cervical Intraepithelial neoplasia (CIN), and CESC tissues and clinicopathological characteristics. The Gene Expression Profiling Interactive Analysis (GEPIA) was used to assess the association between CLCA2 and Disease-Free Survival (DFS), overall survival (OS). The Gene Set Enrichment Analysis (GSEA) was used to explore the associated signaling pathways. The Tumor Immune Estimation Resource (TIMER) was used to predict the potential biological roles of CLCA2 in tumor-immune of CESC. RESULTS: CLCA2 expression was significantly decreased in CESC tissues compared with normal and CIN tissues (P < 0.05). Meanwhile, obese patients had lower levels of CLCA2 expression than normal-weight CESC patients (P < 0.05). However, there was no significant difference in the expression level of CLCA2 in patients with different T stage, lymph node status, metastasis, and FIGO stage in CC(P > 0.05). The survival analysis indicated that for DFS, CESC with high CLCA2 expression was associated with better prognoses compared with those with low expression levels (P < 0.05). But for the OS, there was no difference. GSEA revealed that 4 pathways exhibited significant differential enrichment in the CLCA2 high-expression phenotype, including the P53 signaling pathway, the ERBB signaling pathway, the NOTCH signaling pathway, and the ubiquitin-mediated proteolysis. The TIMER reveals the expression of CLCA2 showed a significant inverse association with the number of B cells, Macrophage cells, and Dendritic Cell infiltration. CONCLUSION: The present study indicates that CLCA2 expression may be a potential prognostic marker for patients with CESC.

Weight Function Method for Stress Intensity Factors of Semi-Elliptical Surface Cracks on Functionally Graded Plates Subjected to Non-Uniform Stresses.

In this paper, a weight function method based on the first four terms of a Taylor's series expansion is proposed to determine the stress intensity factors of functionally graded plates with semi-elliptical surface cracks. Cracked surfaces that are subjected to constant, linear, parabolic and cubic stress fields are considered. The weight functions for the surface, deepest and general points on the crack faces of long and deep cracked functionally graded plates are derived, which has never been done before in the literature. The accuracy of the method in this study is then validated by comparing the results with those of finite element modeling. The numerical results indicate that the derived weight functions are highly accurate and robust enough to predict the stress intensity factors for cracked functionally graded plates subjected to non-uniform stress distributions. The weight function method is therefore a time-saving technique and suitable for handling non-uniform stress fields.