PM2.5-induced ferroptosis by Nrf2/Hmox1 signaling pathway led to inflammation in microglia.

Particulate matter (PM) has been a dominant contributor to air contamination, which will enter the central nervous system (CNS), causing neurotoxicity. However, the biological mechanism is poorly identified. In this study, C57BL/6J mice were applied to evaluate the neurotoxicity of collected fine particulate matter (PM2.5), via oropharyngeal aspiration at two ambient equivalent concentrations. The Y-maze results showed that PM2.5 exposure in mice would lead to the damage in hippocampal-dependent working memory. In addition, cell neuroinflammation, microglial activation were detected in hippocampus of PM2.5-exposure mice. To confirm the underlying mechanism, the microarray assay was conducted to screen the differentially expressed genes (DEGs) in microglia after PM2.5 exposure, and the results indicated the enrichment of DEGs in ferroptosis pathways. Furthermore, Heme oxygenase-1 (Hmox1) was found to be one of the most remarkably upregulated genes after PM2.5 exposure for 24 h. And PM2.5 exposure induced ferroptosis with iron accumulation through heme degradation by Nrf2-mediated Hmox1 upregulation, which could be eliminated by Nrf2-inhibition. Meanwhile, Hmox1 antagonist zinc protoporphyrin IX (ZnPP) could protect BV2 cells from ferroptosis. The results taken together indicated that PM2.5 resulted in the ferroptosis by causing iron overload through Nrf2/Hmox1 signaling pathway, which could account for the inflammation in microglia.

Functional identification of long non-coding RNAs induced by PM2.5 in microglia through microarray analysis.

As a dominating air pollutant, atmospheric fine particulate matter within 2.5 µm in diameter (PM2.5) has attracted increasing attention from the researchers all over the world, which will lead to various adverse effects on the central nervous system (CNS), yet the potential mechanism is unclear. In this study, the microglia (BV2 cell line) were exposed to different concentrations of PM2.5 (5, 10 and 20 µg/cm2) for 24 h. It was found that PM2.5 could result in adverse effects on microglia such as decreased cell viability, structural damage and even cell death. And it was reported that long non-coding RNAs (lncRNAs) could participate in multitudinous neurological diseases. Therefore, the microarray analysis was conducted in order to disclose the underlying neurotoxicity mechanism of PM2.5 by ascertaining the differentially expressed lncRNAs (DElncRNAs). The consequences indicated that the DElncRNAs were enriched in various biological pathways, including ferroptosis, IL-17 signaling pathway and NOD-like receptor signaling pathway. Moreover, the cis- and trans-regulated mRNAs by DElncRNAs as well as the corresponding transcriptional factors (TFs) were observed, such as CEBPA, MYC, MEIS1 and KLF4. In summary, our study supplies some candidate libraries and potential preventive target against PM2.5-induced toxicity through targeting lncRNAs. Furthermore, the post-transcriptional regulation will contribute to the future research on PM2.5-induced neurotoxicity.

Application of Half-Transected and Self-Pulling Esophagojejunostomy in Total Laparoscopic Gastrectomy for Gastric Cancer: A Safe and Feasible Technique.

Objective: This study introduces a technique for esophagojejunostomy with half transected and self-pulling (HTSP) and evaluates the safety, feasibility, and clinical results of this technique in totally laparoscopic total gastrectomy (TLTG). Materials and Methods: From May 2019 to March 2021, 42 patients (HTSP group) who underwent HTSP-TLTG surgery in the Department of Abdominal Tumor Surgery of Jiangxi Cancer Hospital were included in this study. The control group consisted of 50 patients undergoing conventional TLTG surgery (conventional anastomosis group) performed by the same surgical team from March 2018 to March 2020. The clinical data of the two groups were retrospectively analyzed and compared. Results: The mean operation time of the HTSP-TLTG surgery was 166.7 ± 13.1 minutes and the anastomosis time was 20.8 ± 2.0 minutes, which were significantly shorter than those of traditional TLTG (P < 0.05). There were no significant differences between the two groups in blood loss, time to first exhaust, postoperative hospital stay, and incidence of surgery-related complications. Conclusion: HTSP is a safe and feasible way of endoscopic esophagojejunal anastomosis, which requires a relatively low suture technique under endoscopy, and is suitable for promotion.

Enhancer of zeste homolog 2-mediated paired box 8 methylation promotes gastrointestinal stromal tumor progression through Wnt4 downregulation.

Gastrointestinal stromal tumor (GIST) is a refractory malignant tumor without satisfactory therapy. In recent years, aberrant gene methylation has been highlighted as an inducer for tumor progression. In this study, we explored whether enhancer of zeste homolog 2 (EZH2)-mediated paired box 8 (PAX8) methylation affects GIST development through regulation of Wnt4. A total of 50 cases of GIST tissues were collected and the human GIST cell lines were cultured. PAX8 methylation was examined using MS-PCR. Following loss- and gain-function approaches, GIST cell proliferation, migration, invasion, and apoptosis were examined by CCK-8 assay, Transwell assay and flow cytometry. The expression of proliferation related factors and apoptosis related factors was determined. Finally, xenograft tumors in nude mice were observed to examine in vivo tumorigenicity of GIST cells. Downregulated PAX8 and upregulated EZH2 expression was found in GIST tissues. Overexpression of PAX8 or suppression of PAX8 methylation using DNA methyltransferase inhibitor 5-Aza-dC inhibited the proliferation, migration, and invasion of GIST cells while promoting their apoptosis (diminished PCNA, Ki67 and Bcl-2, elevated Bax, and cleaved caspase-3). EZH2 promoted PAX8 methylation to inhibit its expression. Downregulated PAX8 decreased Wnt4 expression to accelerate GIST progression both in vitro and in vivo. Collectively, EZH2 inhibits PAX8 expression by promoting its methylation, which thus downregulates Wnt4 expression, thereby promoting the development of GIST.

Long non­coding RNA SNHG12 regulates cell proliferation, invasion and migration in endometrial cancer by targeting miR­4429.

Long non­coding RNA small nucleolar RNA host gene 12 (SNHG12) has been demonstrated to be oncogenic. The aim of the present study was to examine the effects of SNHG12 on the progression of endometrial cancer (EC). The expression levels of SNHG12 and microRNA (miR)­4429 were assessed in EC cell lines by reverse transcription­quantitative PCR. Plasmids, including SNHG12 short hairpin RNAs (shRNAs), shRNA negative control (NC), SNHG12 overexpression (OV), OV­NC, miR­4429 mimic and mimic­NC, were transfected into RL95­2 cells. Post­transfection, Cell Counting Kit­8, Transwell Matrigel and wound­healing assays were performed to assess cell proliferation, invasion and migration, respectively. Cell cycle phase distribution was assessed by flow cytometry. The protein expression levels of matrix metalloproteinase (MMP)2 and MMP9 were detected by western blotting. miR­4429 target genes were predicted by bioinformatics analysis using target prediction online tools; the findings of this analysis were verified using a dual­luciferase reporter system. Identified as a target of miR­4429, SNHG12 was overexpressed in EC cell lines with decreased expression of miR­4429. Further experiments demonstrated that SNHG12 silencing and overexpression of miR­4429 markedly suppressed proliferation, migration and invasion of RL95­2 cells, arrested cells in the G1 phase, and markedly downregulated the expression of MMP2 and MMP9. The opposite effects were observed in miR­4429 mimic­transfected RL95­2 cells after SNHG12 was overexpressed. The findings of the present study established the role of SNHG12 and miR­4429 in EC. Therefore, targeting the SNHG12/miR­4429 axis could serve as a potential future therapeutic target for treatment of EC.

MTBP Promotes the Invasion and Metastasis of Hepatocellular Carcinoma by Enhancing the MDM2-Mediated Degradation of E-Cadherin.

BACKGROUND: Emerging evidence suggests that MTBP plays a role in cancer development and possibly progression, but its influence on hepatocellular carcinoma (HCC) remains unclear. METHODS: We used real-time PCR and Western blotting to investigate MTBP expression in four HCC cell lines, 120 pairs of tumor and corresponding paracarcinomatous tissues from HCC patients. Immunohistochemistry was performed to examine MTBP expression in HCC and corresponding paracarcinomatous tissues from 120 patients. E-cadherin was only examined in HCC tissues of patients mentioned above. Statistical analyses were applied to evaluate the prognostic value and associations of MTBP expression with clinical parameters. Furthermore, the MTBP gene was overexpressed in HepG2 cell and silenced by siRNA in Hu7 cell, and cell migration and invasion were detected in vitro and in vivo. Moreover, the molecular mechanism of E-cadherin regulation by MTBP was explored. RESULTS: In this study, we first showed that MTBP protein expression is positively correlated with distant metastasis and poor prognosis in HCC patients. We also found that MTBP expression was increased in metastatic cell lines when compared with nonmetastatic cell lines. Consistent with these findings, enhanced expression of MTBP promoted HCC cell invasiveness and metastasis both in vitro and in vivo, whereas the knockdown of MTBP with small interfering RNA resulted in reduced HCC migration and invasion. Ectopic expression of MTBP in HCC cells induced epithelial-to-mesenchymal transition, whereas the silencing of MTBP had the opposite effect. Furthermore, our results show that MTBP and E-cadherin protein expression are inversely correlated in primary HCC tissues. Moreover, our findings indicate that MTBP overexpression decreases E-cadherin expression through the modulation of Mdm2 ubiquitination degradation. CONCLUSIONS: Our data show that MTBP aggravates the invasion and metastasis of HCC by promoting the MDM2-mediated degradation of E-cadherin.

[Prenatal genetic diagnosis of oculocutaneous albinism type II through mutation detection combined with SNPs linkage analysis].

OBJECTIVE: To provide prenatal diagnosis for two families affected with oculocutaneous albinism (OCA), in both of which only 1 pathogenic allele has been identified. METHODS: To determine the clinical classification of OCA through DNA sequencing for TYR, P, TYRP1 and SLC45A2 genes in combination with phenotype analysis. Prenatal diagnosis was carried out by direct sequencing and intragenic SNPs family-based linkage analysis. RESULTS: In the first family, only 1 heterozygous mutation c.1255C>T was found in the proband, which was inherited from her mother. Together with its clinical phenotype, the proband was suspected to have OCA2 Screening of amniotic fluid, however, has found no mutation. With family-based linkage analysis, the fetus was deemed to be an OCA2 carrier. In the second family, again only one heterozygous mutation c.1920_1949 del30bp and ins AACA was found in the proband, which was inherited from her father. Together with its clinical phenotype, the proband was suspected to have OCA2. Screening of amniotic fluid has revealed a heterozygous mutation c.1920_1949 del30bp and ins AACA. By family-based linkage analysis, the fetus was deemed to be an OCA2 carrier. Both fetuses had a normal phenotype at birth. CONCLUSION: Prenatal genetic diagnosis has been provided for the first time for two families affected with OCA, in which only 1 pathogenic mutant allele was detected. The combined mutation detection and SNPs linkage analysis has turned out to be successful.

Novel rare alleles of ABCA1 are exclusively associated with extreme high-density lipoprotein-cholesterol levels among the Han Chinese.

BACKGROUND: High-density lipoprotein (HDL) is a major plasma lipoprotein directly associated with cholesterol metabolism. The ATP binding cassette transporter 1 gene (ABCA1) is one of the major genes modulating plasma levels of HDL-cholesterol (HDL-C). Rare alleles of ABCA1 associated with extreme HDL-C concentrations have not been previously investigated in the Chinese. METHODS: Blood samples were collected from 470 subjects whose HDL-C concentrations were within the top 5% of the distribution, 335 subjects in the lowest 5%, and 220 within the range 5%-95%. First, we sequenced all exons of the ABCA1 gene from 50 subjects from the group with extremely high HDL-C, and 50 from the group with extremely low HDL-C concentrations. Next, in the remaining subjects, we genotyped the non-synonymous variants identified exclusively with either extreme group. RESULTS: Four novel non-synonymous alleles were identified; all were rare. Alleles c.3029C>T (p.Ala1010Val) and c.5399A>G (p.Asn1800Ser) were found exclusively in the low group, c.2031C>A (p.Asp677Glu) and c.2660G>T (p.Cys887Phe) exclusively in the high group. CONCLUSIONS: Our results show that some rare alleles of ABCA1 are associated with marked phenotypes, supporting the "rare-variant common-disease" hypothesis. Certain alleles also provide tools for identifying individuals at high risk of dyslipidaemia, allowing for early therapeutic intervention.

Molecular genetic diagnostics of Prader-Willi Syndrome: a validation of linkage analysis for the Chinese population.

Prader-Willi Syndrome (PWS) is a genetic disorder that is difficult to detect, particularly at an early age. PWS is caused by disruption of normal, epigenetically controlled gene function in the chromosome 15q11-q13 region. Clinical symptoms are difficult to diagnose in infants and only become clearer at later ages as the patients develop hyperphagia and morbid obesity. Molecular genetic tests are able to definitively diagnose PWS and allow early diagnosis of the syndrome. High resolution cytogenetic testing, methylation-specific PCR (MS-PCR), and linkage analysis are routinely used to diagnose PWS. To establish a linkage analysis method for Chinese patients, this study identified a useful set of STR markers in the typical PWS deletion and adjacent area, for linkage analysis in two Chinese families with PWS offspring. Using this method, the authors confirmed that one patient had a paternal deletion in chromosome 15q11-q13 and the other patient had maternal uniparental heterodisomy of chromosome 15. MS-PCR and high resolution chromosome G-banding also confirmed this diagnosis. This linkage analysis method can detect both deletion and uniparental disomy, thus providing valuable information for genetic counseling and the opportunity to analyze the relationship between the genotype and phenotype of PWS.

[Prenatal diagnosis of oculocutaneous albinism type II and discovery of two novel mutations].

OBJECTIVE: To investigate the genotype of oculocutaneous albinism type II (OCA2) and perform prenatal gene diagnosis for OCA2. METHODS: Peripheral blood samples were collected from a 9-year-old girl with OCA and her parents, the mother being pregnant. PCR, automatic sequence analysis and denaturing high performance liquid chromatography (DHPLC) were used to analyze the TYR gene and P gene so as to screen the OCA genes. Amniocentesis was conducted when the mother was 20-week pregnant and the amniotic cells underwent screening of the 2 specific mutations. Peripheral blood samples were collected from 100 healthy persons without phenotype of OCA to undergo genetic analysis. RESULTS: The TYR gene of the proband did not show any mutation, and 2 new mutations were found in her P gene: p. N476D (c. 1426 A>G) and p.Y827H (c.2479T>C). Her father and mother were heterozygote of Y827H and N476D respectively. Based on these findings the amniotic cells underwent sequencing of enlarged fragments of the exons 14 and 24 containing the mutation sites. The result showed that the fetus only got the maternal N476D mutation and didn't get the paternal Y827H mutation. So the fetus was predicted to be a carrier of OCA2 with normal appearance. The baby was born normal later as predicted. None of these 2 mutations was found in the 100 healthy persons. CONCLUSION: This is a success of prenatal gene diagnosis of OCA2. Two novel mutations of the P gene related to OCA have been discovered.