The novel molecular mechanism of pulmonary fibrosis: insight into lipid metabolism from reanalysis of single-cell RNA-seq databases.

Pulmonary fibrosis (PF) is a severe pulmonary disease with limited available therapeutic choices. Recent evidence increasingly points to abnormal lipid metabolism as a critical factor in PF pathogenesis. Our latest research identifies the dysregulation of low-density lipoprotein (LDL) is a new risk factor for PF, contributing to alveolar epithelial and endothelial cell damage, and fibroblast activation. In this study, we first integrative summarize the published literature about lipid metabolite changes found in PF, including phospholipids, glycolipids, steroids, fatty acids, triglycerides, and lipoproteins. We then reanalyze two single-cell RNA-sequencing (scRNA-seq) datasets of PF, and the corresponding lipid metabolomic genes responsible for these lipids' biosynthesis, catabolism, transport, and modification processes are uncovered. Intriguingly, we found that macrophage is the most active cell type in lipid metabolism, with almost all lipid metabolic genes being altered in macrophages of PF. In type 2 alveolar epithelial cells, lipid metabolic differentially expressed genes (DEGs) are primarily associated with the cytidine diphosphate diacylglycerol pathway, cholesterol metabolism, and triglyceride synthesis. Endothelial cells are partly responsible for sphingomyelin, phosphatidylcholine, and phosphatidylethanolamines reprogramming as their metabolic genes are dysregulated in PF. Fibroblasts may contribute to abnormal cholesterol, phosphatidylcholine, and phosphatidylethanolamine metabolism in PF. Therefore, the reprogrammed lipid profiles in PF may be attributed to the aberrant expression of lipid metabolic genes in different cell types. Taken together, these insights underscore the potential of targeting lipid metabolism in developing innovative therapeutic strategies, potentially leading to extended overall survival in individuals affected by PF.

Silencing FHL2 inhibits bleomycin-induced pulmonary fibrosis through the TGF-ß1/Smad signaling pathway.

OBJECTIVE: This study aimed to investigate the inhibiting effects of FHL2 and Arbutin on cell fibrosis and their possible mechanisms. METHODS: The mRNA expression of FHL2 in pulmonary fibrosis tissues was analyzed by bioinformatics. TGF⁃ß1 induced fibrosis of mouse lung fibroblast (Mlg) and mouse primary pulmonary fibroblast (PPF) in rat's lung fibroblasts. FHL2 siRNA was transfected into Mlg and mouse PPF cells to inhibit FHL2. FHL2, α-smooth muscle actin (α-SMA), collagen 1 (Col I), and Fibronectin (Fn) were detected by qRT-PCR. Western blot expression levels of Smad3, p-Smad3, Smad2, and p-Smad2 proteins in cells. High-throughput drug screening for FHL2 inhibitors and the inhibitory effect of Arbutin on pulmonary fibrosis were validated in cellular and animal models of pulmonary fibrosis. RESULTS: The mRNA expression of FHL2 in lung fiber tissue was increased. Meanwhile, the decrease of FHL2 expression significantly inhibited the cellular fibrosis morphological changes of rat's lung fibroblasts (Mlgs) and primary lung fibroblasts (PPFs). The expression levels of α⁃SMA, Col I, and Fn were decreased. High-throughput screening showed that Arbutin targeted FHL2. Arbutin alleviated bleomycin (BLM)-induced pulmonary fibrosis in rats by inhibiting FHL2 and then the TGF-ß1/Smad signaling pathway. CONCLUSION: Inhibition of FHL2 can effectively reduce the fibrosis process induced by TGF⁃ß1 and bleomycin, and then inhibit the fibrosis.

Short- (30-90 days) and mid-term (1-3 years) outcomes and prognostic factors of patients with esophageal cancer undergoing surgical treatments.

BACKGROUND: The factors influencing the prognosis of patients with esophageal cancer vary among studies and are still poorly known. AIM: To determine the factors associated with survival in patients with esophageal cancer. METHODS: This retrospective study included patients with esophageal cancer admitted between January 2017 and March 2020 at Heping Hospital Affiliated to Changzhi Medical College. All patients were treated according to the available guidelines. Follow-up was censored in October 2020. Univariable and multivariable Cox regression analyses were used to determine the independent risk factors for overall survival (OS). RESULTS: In total, 307 patients were included. Their median age was 64 (range, 44-79) years, 63.5% were male, and the median disease course was 2 (0.1-36) months. The median tumor size was 3 (0-10) cm. Most patients were T3 (29.6%), N0 (70.0%). Most tumors were grade 2 (48.2%), and 87.3% were squamous cell carcinoma. The in-hospital mortality was 16.9%, the 30-day mortality was 19.9%, and the 90-day mortality was 25.4%. The cumulative OS rates at the last follow-up were 82.1% (95%CI: 67.7%-96.5%) for stage 0/I/II and 47.4% (95%CI: 16.5-78.6%) for stage III/IVA (P < 0.001). The multivariable analysis showed that creatinine levels (HR = 1.02, 95%CI: 1.00-1.03, P = 0.050), pTNM III/IVA (HR = 4.19, 95%CI: 2.19-8.01, P < 0.001), adjuvant radiotherapy and/or chemotherapy (HR = 0.23, 95%CI: 0.11-0.49), and the Comprehensive Complication Index (CCI) (HR = 1.02, 95%CI: 1.004-1.03, P = 0.011) were independently associated with OS. CONCLUSION: The survival of patients with esophageal cancer is poor, especially those with pTNM III/IVA. pTNM stage III/IVA, CCI, and adjuvant therapy (radiotherapy and/or chemotherapy) are independently associated with OS.

mmu-microRNA-92a-3p attenuates pulmonary fibrosis by modulating Cpeb4-mediated Smad2/3 signaling pathway.

Pulmonary fibrosis (PF) is a chronic lung disorder, in which the mechanism of mmu-microRNA (miR)-92a-3p is not elucidated clearly. The present work was proposed to disclose mmu-miR-92a-3p-focused mechanism in PF with cytoplasmic polyadenylation element-binding protein 4 (Cpeb4)/Smad2/3 axis. PF was induced in mice by the intratracheal injection of bleomycin (BLM). Then, the BLM-treated mice were injected with mmu-miR-92a-3p- and/or Cpeb4-related adenovirus vectors. mmu-miR-92a-3p, Cpeb4, and Smad2/3 expression in lung tissues were examined. Alveolar cell apoptosis and collagen deposition in lung tissues and inflammatory factors in serum were observed. The interaction between mmu-miR-92a-3p and Cpeb4 was explored. Lowly expressed mmu-miR-92a-3p and highly expressed Cpeb4 and Smad2/3 were manifested in BLM-induced PF mice. BLM-induced PF mice exhibited enhanced inflammation, alveolar cell apoptosis, and collagen deposition, which would be attenuated by upregulating mmu-miR-92a-3p or downregulating Cpeb4. mmu-miR-92a-3p targeted Cpeb4. Upregulating mmu-miR-92a-3p or downregulating Cpeb4 inactivated the Smad2/3 signaling pathway in BLM-induced PF mice. It is elaborated that mmu-miR-92a-3p attenuates the process of PF by modulating Cpeb4-mediated Smad2/3 signaling pathway, renewing the molecular mechanism of PF.

Vascular Adventitial Fibroblasts-Derived FGF10 Promotes Vascular Smooth Muscle Cells Proliferation and Migration in vitro and the Neointima Formation in vivo.

BACKGROUND: Activation of vascular adventitial fibroblasts (VAFs) upon vascular injury contributes greatly to the medial vascular smooth muscle cells (VSMCs) proliferation, migration and the subsequent neointima formation. A number of factors including fibroblast growth factors (FGFs) have been shown to control VSMC growth, proliferation and phenotypic switching, suggesting that they may function as paracrine signals for VAFs to modulate VSMCs functions. However, little is known about the signaling molecule(s) and its mechanism of action. This study is set to identify which and how FGF family members are involved in VAFs mediated vascular remodeling. METHODS: We used qPCR, Western blot and Immunohistochemistry to observe the spatiotemporal expression of FGF10 and FGFR2 in injured vascular tissue. The proliferation and migration assays of VSMCs were performed in a co-culture system. The activation of signaling pathway was detected by Western blot, immunohistochemistry and immunofluorescence. Hematoxylin-eosin and immunofluorescence were used to assess the effects of exogenous FGF10 and siFGF10 on the neointima formation. RESULTS: The expression of FGF10 and FGFR2 were increased from day 3 through day 14 post injury. FGF10 was significantly upregulated in adventitia, and FGFR2 was detected in both media and neointima after injury. In vitro, FGF10 was most prominently expressed in VAFs and FGFR2 was significantly expressed in VSMCs. Both were regulated by PDGF. Co-culture of VAFs and VSMCs in vitro showed that VAF-derived FGF10 promoted the proliferation and migration of VSMCs. PDGF could synergistically enhance the process. VAF-derived FGF10 can significantly activate the FGFR2 in VSMCs and furthermore significantly activate the downstream MAPK/PI3K-AKT signaling pathways. Delivery of exogenous FGF10 potentiated the neointima formation, while siFGF10 attenuated the neointima formation. CONCLUSION: VAFs-derived FGF10 promoted the proliferation and migration of VSMCs and neointima formation, and FGF10-FGFR2 signaling triggered the activation of MAPK/PI3K-AKT pathways in VSMCs and PDGF synergistically amplified FGF10 signaling.

Prediction of Overall Survival of Patients with Completely Resected Non-Small Cell Lung Cancer: Analyses of Preoperative Spirometry, Preoperative Blood Tests, and Other Clinicopathological Data.

PURPOSE: Risk stratification of patients with non-small cell lung cancer (NSCLC) is crucial to select the appropriate treatments, but available models for patients with complete resection are unsatisfactory. The purpose of this study was to determine a prediction model based on clinical information, routine physical and blood tests, and molecular markers. PATIENTS AND METHODS: This was a retrospective cohort study of patients who underwent surgical resection for lung cancer between 2009 to 2013. Potential prognostic factors were used to build a full prediction model based on a multivariable Cox regression analysis. A nomogram was constructed. The risk stratification cutoffs for clinical use were determined based on the model. RESULTS: A total of 368 NSCLC patients with R0 resection were included. The final multivariable model indicated that low diffusing capacity of the lung for carbon monoxide (HR=1.66, 95% CI: 1.18-2.34), high platelet-to-lymphocyte ratio (HR=1.42, 95% CI: 1.04-1.95), histology type of squamous cell carcinoma and others (squamous cell carcinoma vs adenocarcinoma, HR=1.40, 95% CI: 1.01-1.96; others vs adenocarcinoma, HR=2.36, 95% CI: 1.15-4.84; P trend=0.001), N>0 status (HR=1.96, 95% CI: 1.42-2.70), high serum carcinoembryonic antigen levels (HR=1.61, 95% CI: 1.13-2.27), and postoperative chemotherapy (HR=0.53, 95% CI: 0.33-0.87) were independently associated with poor OS. The patients were classified into four risk groups according to the nomogram, and the OS was different among the four groups (P<0.05). CONCLUSION: A nomogram was successfully constructed based on a multivariable analysis, and the nomogram can discriminate the OS of patients with NSCLC based on risk categories, but external validation is still necessary.

Correlation between RNA-Seq and microarrays results using TCGA data.

RNA sequencing (RNA-Seq) and microarray are two of the most commonly used high-throughput technologies for transcriptome profiling; however, they both have their own inherent strengths and limitations. This research aims to analyze the correlation between microarrays and RNA-Seq detection of transcripts in the same tissue sample to explore the reproducibility between the techniques. Using data of RNA-Seq v2 and three different microarrays provided by The Cancer Genome Atlas, 11,120 genes of 111 lung squamous cell carcinoma samples were simultaneously detected by the four methods. Then we analyzed the Pearson correlation between microarrays and RNA-Seq. Finally, in the six comparison results, 9984 (89.8%) genes, irrespective of which two methods were used, simultaneously showed the existence of correlation, whereas only 83 (0.1%) genes proved to have no significant correlation in either comparison. In addition, the comparisons between 3266 (29.3%) genes showed high correlation (R≥0.8) in all six comparisons, only for 1643 (14.8%) genes correlation were not as high in either comparison. Meanwhile, transcripts with extreme high or low expression levels were more highly discrepant across the methods. In conclusion, we found that, for most transcripts, the results obtained by RNA-Seq and microarrays were highly reproducible.

Bioinformatics analyses of the differences between lung adenocarcinoma and squamous cell carcinoma using The Cancer Genome Atlas expression data.

The present study aimed to explore gene and microRNA (miRNA) expression differences between lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) were identified by analyzing mRNA and miRNA expression data in normal and cancerous lung tissues that were obtained from The Cancer Genome Atlas database. A total of 778 DEGs and 7 DEMs were identified. Altered gene functions and signaling pathways were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, which revealed that DEGs were significantly enriched in extracellular matrix organization, cell differentiation, negative regulation of toll signaling pathway, and several other terms and pathways. Transcription factor (TF)­miRNA­gene networks in LUAD and LUSC were predicted using the TargetScan, Miranda, and TRANSFAC databases, which revealed the regulatory links among the TFs, DEMs, and DEGs. The central TFs, i.e., the TFs in the middle of the TF­miRNA­gene network, of LUAD and LUSC were similar. Although LUAD and LUSC shared similar miRNAs in the predicted networks, miR­29b­3p was demonstrated to be upregulated only in LUAD, whereas miR­1, miR­105­5p, and miR­193b­5p were altered in LUSC. These findings may improve our understanding of the different molecular mechanisms in non­small cell lung cancers and may promote new and accurate strategies for prevention, diagnosis, and treatment.

Prognostic value of visceral pleural invasion in non-small cell lung cancer: A propensity score matching study based on the SEER registry.

BACKGROUND AND OBJECTIVES: Visceral pleural invasion (VPI) is considered a poor prognostic factor in non-small cell lung cancer (NSCLC). We aimed to analyze the effect of VPI on cancer-specific survival, using propensity score matching (PSM) based on the Surveillance, Epidemiology, and End Results database. METHODS: We identified 9901 patients with T1-2N0-2M0 who received segmentectomy, lobectomy, or pneumonectomy. Ten covariates were included in PSM. The effect of VPI on survival was assessed, stratified by nodal status and tumor size. RESULTS: One-thousand and eighty-three pairs of patients were matched with standardized differences of covariates <10% after matching. We found that VPI was associated with a significantly worse survival (3-year survival rate: 84.6% vs. 75.9%, P = 0.005), especially in N0 (P < 0.001), but not in N1 or N2. No significant difference was observed between the extent of VPI. Moreover, VPI conferred a significantly worse survival in tumors >1-2 (P = 0.034) and >2-3 cm (P < 0.001), not ≤1, >3-4, or >4-5 cm in N0. CONCLUSIONS: VPI is a poor prognostic factor; but with increasing tumor size and nodal stage, its negative effect disappears. Our findings support current staging system which assigns higher T-stage for early >1-2 and >2-3 cm tumors.

Prognosis of video-assisted thoracoscopic pulmonary metastasectomy in patients with colorectal cancer lung metastases: an analysis of 154 cases.

PURPOSE: Video-assisted thoracoscopic surgery (VATS) is widely used in thoracic surgery and increasingly applied to pulmonary metastasectomy. The purpose of this study was to identify prognostic factors of patients undergoing VATS pulmonary metastasectomy from colorectal cancer (CRC). METHODS: Between January 2005 and June 2015, a total of 154 patients underwent VATS pulmonary metastasectomy from CRC. Patient demographic data and characteristics of the primary tumor and pulmonary metastasis were analyzed to identify factors significantly correlated with prognosis. RESULTS: The median follow-up period after pulmonary resection was 37 months. The cumulative 5-year overall survival rate after VATS pulmonary metastasectomy from CRC was 71.3%. History of metastasis to other sites (p = 0.035), status of mediastinal lymph nodes (p < 0.001), and preoperative carcinoembryonic antigen (CEA) level (p = 0.013) were identified as independent prognostic factors. Subgroup analysis with a combination of these three independent prognostic factors revealed 5-year OS rates of 91.0, 70.0, 30.3, and 0.0% for patients with zero, one, two, and three risk factors, respectively. Other factors, such as sex, disease-free interval, T stage of primary tumor, and status of lymph node near the primary tumor, were not significantly associated with prognosis. CONCLUSION: VATS pulmonary metastasectomy is efficacious for patients with CRC pulmonary metastases. History of metastasis to other sites, status of mediastinal lymph nodes, and preoperative CEA level were identified as independent prognostic factors. The number of risk factors significantly influenced patient survival.

Identification of reference genes and miRNAs for qRT-PCR in human esophageal squamous cell carcinoma.

It is important to select an appropriate reference gene and miRNA when using quantitative real-time polymerase chain reaction (qRT-PCR) to analyze gene and miRNA expression. However, many commonly used reference genes and miRNAs are not stably expressed and therefore not suitable for normalization or quantification of qRT-PCR data. This study aims to identify appropriate reference genes and miRNAs for use in human esophageal squamous carcinoma qRT-PCR analysis. Using data provided by The Cancer Genome Atlas, we identified DDX5, LAPTM4A, P4HB, RHOA, miR-28-5p, miR-34a-5p, and miR-186-5p as candidate reference genes and miRNAs. We used qRT-PCR to verify the expression levels of these candidates and another seven commonly used reference genes and miRNAs. A set of 50 paired human normal esophageal tissues and squamous cell carcinoma samples were used in the analysis. We then used geNorm and NormFinder to analyze the results. DDX5, LAPTM4A, RHOA, ACTB, RNU48, miR-28-5p, miR-34a-5p, and miR-186-5p were stably expressed, indicating they are suitable for used as references in qRT-PCR analysis of esophageal squamous cell carcinoma. However, expression levels of 18s rRNA, GAPDH, P4HB, 5s rRNA, U6, and RNU6B varied greatly between esophageal normal and squamous cell carcinoma samples, indicating that they are not suitable for use as references in the qRT-PCR analysis of esophageal squamous cell carcinoma.

[Efficacy comparison of Sweet versus Ivor-Lewis esophagectomy in the treatment of middle-lower esophageal squamous cell carcinoma].

OBJECTIVE: To compare the short-term efficacy and long-term survival between Sweet and Ivor-Lewis esophagectomy for patients with middle-lower esophageal squamous cell carcinoma. METHODS: Clinicopathologic data of 1 308 patients with middle-lower esophageal squamous cell carcinoma undergoing Sweet or Ivor-Lewis procedures in our department from January 2007 to December 2014 were retrospectively analyzed, including 1 021 patients of Sweet operation (Sweet group) and 287 patients of Ivor-Lewis operation(Ivor-lewis group). Lymph node clearance, lymphatic metastasis, postoperative complication morbidity and long-term survival were compared between the two groups. RESULTS: There were no significant differences in baseline data between the two groups(all P>0.05). There were more lymph nodes resected during the Ivor-Lewis procedure compared with the Sweet procedure (20.8 vs.19.3, P=0.030). Compared with Ivor-Lewis group, the incidence of wound infection in Sweet group was significantly lower[(3.2%(33/1 021) vs. 8.0%(23/287), P=0.000]. Sweet group had a significantly lower rate of delayed gastric emptying[1.9%(19/1 021) vs. 5.2%(15/287), P=0.002] and significantly shorter hospital stay (14.7 days vs. 17.2 days, P=0.029). With respect to other postoperative complications, such as pulmonary complications, cardiac events, anastomotic leakage, vocal cord palsy, chylothorax and pyothorax, the differences between the two groups were not statistically significant. The 5-year survival rate was not significantly different between the two group (54.0% vs. 56.9%, P=0.873). Stratified analysis based on TNM staging showed that no significant difference of 5-year survival rate was found between the two groups in stageI( and stageIII( patients (P>0.05), while the 5-year survival rate of stageII( patients in Sweet group was significantly lower than that in Ivor-Lewis group (56.4%% vs. 70.4%, P=0.039). CONCLUSIONS: For patients with middle-lower esophageal squamous cell carcinoma, Sweet procedure has certain superiority regarding the incidence of wound infection and delayed gastric emptying compared with the Ivor-Lewis procedure. Ivor-Lewis esophagectomy can harvest more lymph nodes. The 5-year survival rate of these two procedures is similar. Sweet procedure is still valuable in clinical practice, especially for stageI( and stageIII( patients, while it requires thorough considerations for stageII( patients.

Is routine dissection of the station 9 lymph nodes really necessary for primary lung cancer?

OBJECTIVES: Mediastinal lymph node dissection is an essential component of lung cancer surgery; however, choosing mediastinal lymph nodes stations to be dissected is subjective. We carried out this research to investigate the need for dissection of station 9 lymph nodes during lung cancer surgery. METHODS: Patients with primary lung cancer who underwent radical surgery between 2010 and 2014 were retrospectively reviewed. Clinical, pathologic, and prognosis data were obtained and analyzed. RESULTS: A total number of 1397 patients were included in this research. The metastasis rate of station 9 was 3.45%, which was significantly lower than other mediastinal stations. This metastasis rate was significantly correlated with pT stage, the lobe where the tumor was located, metastasis status of intrapulmonary lymph nodes, pTNM stage, and most of the other mediastinal lymph node stations. In males or ground glass opacity (GGO) patients, the metastasis of station 9 nodes was more unlikely to occur, even though there was no statistical significance. The staging results of most patients (99.63%) would not be impaired, even if station 9 nodes were not dissected, and the prognostic analysis showed that the metastasis status of station 9 had no significant influence on survival. CONCLUSION: The metastasis rate of station 9 lymph nodes was significantly lower than other mediastinal stations in lung cancer patients. The metastasis status of station 9 had no significant influence on tumor staging or prognosis. Routine dissection of station 9 lymph nodes may not be necessary, especially in patients with a low T stage, upper or middle lobe tumors, or without intrapulmonary lymph node metastasis.