Shotgun metagenomics has become a pivotal technology in microbiome research, enabling in-depth analysis of microbial communities at both the high-resolution taxonomic and functional levels. This approach provides valuable insights of microbial diversity, interactions, and their roles in health and disease. However, the complexity of data processing and the need for reproducibility pose significant challenges to researchers. To address these challenges, we developed EasyMetagenome, a user-friendly pipeline that supports multiple analysis methods, including quality control and host removal, read-based, assembly-based, and binning, along with advanced genome analysis. The pipeline also features customizable settings, comprehensive data visualizations, and detailed parameter explanations, ensuring its adaptability across a wide range of data scenarios. Looking forward, we aim to refine the pipeline by addressing host contamination issues, optimizing workflows for third-generation sequencing data, and integrating emerging technologies like deep learning and network analysis, to further enhance microbiome insights and data accuracy. EasyMetageonome is freely available at https://github.com/YongxinLiu/EasyMetagenome.

Metastasis is a principal factor in the poor prognosis of colorectal cancer. Recent studies have found microbial metabolites regulate colorectal cancer metastasis. By analyzing metabolomics data, we identified an essential fecal metabolite citraconate that potentially promotes colorectal cancer metastasis. Next, we tried to reveal its effect on colorectal cancer and the underlying mechanism. Firstly, the response of colorectal cancer cells (HCT116 and MC38 cells) to citraconate was assessed by Cell Counting Kit-8 assay, clonogenic assay, transwell migration and invasion assay. Moreover, we utilized an intra-splenic injection model to evaluate the effect of citraconate on colorectal cancer liver metastasis in vivo. Then molecular approaches were employed, including RNA sequencing, mass spectrometry-based metabolomics, western blot, quantitative real-time PCR, cell ferrous iron colorimetric assay and intracellular malondialdehyde measurement. In vitro, citraconate promotes the growth of colorectal cancer cells. In vivo, citraconate aggravated liver metastasis of colorectal cancer. Mechanistically, downstream genes of NRF2, NQO1, GCLC, and GCLM high expression induced by citraconate resulted in resistance to ferroptosis of colorectal cancer cells. In summary, citraconate promotes the malignant progression of colorectal cancer through NRF2-mediated ferroptosis resistance in colorectal cancer cells. Furthermore, our study indicates that fecal metabolite may be crucial in colorectal cancer development.

Patients carrying mutations in polymerase epsilon/polymerase delta have shown positive responses to immune checkpoint inhibitors. Yet, prospective trials exploring the efficacy in those with polymerase epsilon/polymerase delta mutations are still lacking. A phase II clinical trial was initiated to evaluate the efficacy of toripalimab, a humanized IgG4K monoclonal antibody to human PD-1, in patients with advanced solid tumors with unselected polymerase epsilon/polymerase delta mutations but without microsatellite instability-high. A total of 15 patients were enrolled, 14 of whom were assessed for treatment efficacy. There was a 21.4% overall response rate, with a disease control rate of 57.1%. The median overall survival and median progression-free survival were 17.9 (95% CI 13.5-not reach) months and 2.5 (95% CI 1.4-not reach) months, respectively. For patients with exonuclease domain mutations, the objective response rate was 66.7% (2/3), with a disease control rate of 66.7% (2/3). For those with non-exonuclease domain mutations, the rates were 9.1% (1/11) and 54.5% (6/11), respectively. Notably, patients with PBRM1 gene mutations exhibited a high response rate to toripalimab at 75.0% (3/4). This study showed that neither the exonuclease domain mutations nor non-exonuclease domain mutations could fully predict the efficacy of immunotherapy, urging the need for more investigations to clarify potential immune sensitization differences within polymerase epsilon/polymerase delta mutation variants.

Supplementary Figure from A Randomized, Open-Label, Multicenter, Phase 3 Study of High-Dose Vitamin C Plus FOLFOX ± Bevacizumab versus FOLFOX ± Bevacizumab in Unresectable Untreated Metastatic Colorectal Cancer (VITALITY Study)

The genetic basis of colorectal cancer (CRC) and its clinical associations remain poorly understood due to limited samples or targeted genes in current studies. Here, we perform ultradeep whole-exome sequencing on 1015 patients with CRC as part of the ChangKang Project. We identify 46 high-confident significantly mutated genes, 8 of which mutate in 14.9% of patients: LYST, DAPK1, CR2, KIF16B, NPIPB15, SYTL2, ZNF91, and KIAA0586. With an unsupervised clustering algorithm, we propose a subtyping strategy that classisfies CRC patients into four genomic subtypes with distinct clinical characteristics, including hypermutated, chromosome instability with high risk, chromosome instability with low risk, and genome stability. Analysis of immunogenicity uncover the association of immunogenicity reduction with genomic subtypes and poor prognosis in CRC. Moreover, we find that mitochondrial DNA copy number is an independent factor for predicting the survival outcome of CRCs. Overall, our results provide CRC-related molecular features for clinical practice and a valuable resource for translational research.

[This retracts the article DOI: 10.1016/j.omtn.2019.06.003.].

目的 探讨微小RNA-28(miR-28)对非小细胞肺癌细胞株细胞A549和H1299的影响及其作用机制.方法 通过转染miR-28 mimics过表达miR-28,转染miR-28 inhibitor抑制miR-28的表达,其中miR-28 NC作为对照组.通过CCK-8实验、克隆形成试验和细胞周期实验分别评估过表达miR-28、抑制miR-28对非小细胞肺癌细胞增殖能力的影响;通过划痕实验、Transwell迁移及Boyden小室侵袭实验分别评估过表达miR-28、抑制miR-28对非小细胞肺癌细胞迁移与侵袭能力的影响;通过生物信息学软件分析发现Ras相关蛋白1b(RAP1B)可能是miR-28的潜在靶基因并利用荧光素酶报告基因实验进行验证;通过RT-qPCR和Western blot方法检测过表达和抑制miR-28表达后A549和H1299细胞RAP1B的表达.结果 与miR-28 NC比较,miR-28 mimics组A549和H1299细胞的增殖速度明显减慢,迁移及侵袭能力明显减弱,差异均有统计学意义(P<0.05);miR-28 inhibitor组A549和H1299细胞的增殖速度增快,迁移及侵袭能力明显增强,差异均有统计学意义(P<0.05);在非小细胞肺癌细胞中,荧光素酶报告基因实验表明miR-28可以靶向RAP1B基因;与miR-28 NC比较,miR-28 mimics组A549和H1299细胞RAP1B mRNA和RAP1B蛋白的表达明显减少,差异均有统计学意义(P<0.05);miR-28 inhibitor组A549和H1299细胞RAP1B mRNA和RAP1B蛋白的表达明显增加,差异均有统计学意义(P<0.05).结论 miR-28可能通过下调靶基因RAP1B从而抑制非小细胞肺癌细胞的增殖、迁移与侵袭.

阐述了DNA聚合酶ε(polymerase epsilon,POLE)和DNA聚合酶delta 1(polymerase delta 1,POLD1)基因编码的亚基参与DNA复制和校对,总结了POLE/POLD1突变对肿瘤突变负荷以及肿瘤内免疫细胞浸润的影响,综述了POLE/POLD1突变在子宫内膜癌、结直肠癌、肺癌中的研究进展,并讨论了POLE/POLD1突变作为潜在的免疫检查点抑制剂疗效预测分子标志物面临的诸多挑战.

There is a lack of high-quality, large-scale, real-world evidence from patients with metastatic colorectal cancer (mCRC), especially in China. It remains unclear whether efforts to improve the quality of care for mCRC would improve patient survival outcomes in real-world practice. On the basis of an intelligent bigdata platform, we established a large-scale retrospective cohort of mCRC patients. We investigated the temporal changes in the systemic and local treatment (resection, ablation, or radiation to liver, lung, or extrahepatic and/or extrapulmonary metastases) patterns of mCRC, and whether these changes were associated with improved overall survival (OS) over time. Between July 2012 and December 2018, 3403 eligible patients were included in this research. The median OS was 42.8 months (95% confidence interval (CI), 40.7– 46.6) for the entire cohort, 25.6 months (95% CI, 24.7–26.9) for those treated with systemic therapy only, and not reached (95% CI, 78.6 months–not reached) for those receiving local therapy. The utility rate of local therapy increased continuously from 37.9% in 2012–2014 to 46.9% in 2017–2018. A dramatic increase in the utility rate of either cetuximab or bevacizumab was observed since 2017 (39.9%, 43.2%, and 60.3% in 2012–2014, 2015–2016, and 2017–2018, respectively). Compared with 2012–2014, the OS of the entire population significantly improved in 2015–2016 (hazard ratio (HR) = 0.87 (95% CI, 0.78–0.99); P = 0.034), but not for patients receiving systemic therapy only (HR = 0.99 (95% CI, 0.86–1.14); P = 0.889), whereas an improved OS was found in 2015–2018 for both the entire population (HR = 0.75 (95% CI, 0.70–0.81); P < 0.001) and for patients receiving systemic therapy only (HR = 0.83 (95% CI, 0.77–0.91); P < 0.001). In summary, the quality of care for mCRC, as indicated by the utility rate of targeted and local therapies, has been continuously improving over time in this study cohort, which is associated with continuously improving survival outcomes for these patients.

In the REGONIVO study, regorafenib combined with nivolumab was effective in the treatment of microsatellite stable (MSS) metastatic colorectal cancer (mCRC), which indicated anti-angiogenic drugs may enhance the efficacy of immune checkpoint inhibitors. Therefore, we designed a single-arm, single-center, open-label, phase II trial to determine the toxicity and efficacy of SHR-1210 (an anti-PD-1 antibody) plus apatinib in MSS mCRC. The sample size was estimated using a Simon Optimum two stage design. 10 patients were included at the first stage and if one effective patient observed, an additional 19 patients would be added. Patients with MSS mCRC who refractory to second-line treatment or intolerant to standard treatment were given SHR-1210 200 mg every 2 weeks and apatinib 250-375 mg once daily until unacceptable toxicity or disease progression occurred. In our study, the objective response rate was 0% and the disease control rate was 22.2%. The median progression-free survival was 1.83 months (95% confidence interval (CI) 1.80-1.86 months), and the median overall survival was 7.80 months (95% CI 0-17.07). Treatment-related adverse events (AEs) occurred in all patients (100%). The most common treatment-related AEs were hypertension and proteinuria (70% each). Grade 3 AEs were observed in nine patients (9/10, 90%), and the commonest was hypertension (30%). In conclusion, SHR-1210 combined with apatinib has failed to improve the efficacy of treatment of MSS mCRC, and the intolerable toxicity may be the leading cause.

Long non-coding RNAs and microRNAs (miRNAs) have been reported to participate in the progression of non-small-cell lung cancer (NSCLC). Long intergenic non-protein-coding RNA 472 (LINC00472), miR-149-3p, and miR-4270 were found to be involved in tumor activities, suggesting potential roles in NSCLC. Thus, this study aimed to examine the ability of LINC00472 to influence the progression of NSCLC with the involvement of miR-149-3p and miR-4270. Initially, differentially expressed long non-coding RNAs (lncRNAs), downstream regulatory miRNAs, and genes related to NSCLC were identified. Next, the interaction among LINC00472, miR-149-3p and miR-4270, and KLLN and the p53-signaling pathway was determined. The effect of LINC00472 on the expression of E-cadherin, N-cadherin, and Vimentin was examined through gain-of-function and loss-of-function experiments. Lastly, the effects of LINC00472 on NSCLC tumor growth were assessed in vivo. LINC00472 and KLLN were found to exhibit low levels, while miR-149-3p and miR-4270 were highly expressed in NSCLC. In addition, the overexpression of LINC00472 was observed to upregulate KLLN and activate the p53-signaling pathway, which ultimately inhibited the invasion, migration, and EMT of NSCLC cells via miR-149-3p and miR-4270, corresponding to decreased N-cadherin and Vimentin and increased E-cadherin. The overexpression of LINC00472 exerted an inhibitory effect on tumor growth in vivo. Taken together, the key evidence suggests that the overexpression of LINC00472 can downregulate miR-149-3p and miR-4270 to upregulate KLLN and activate the p53-signaling pathway, thus inhibiting the development of NSCLC. This study highlights the potential of LINC00472 as a promising therapeutic target for NSCLC treatment.

Although airway fibrosis and epithelial-mesenchymal transition (EMT) contribute to airway remodeling in chronic obstructive pulmonary disease (COPD), the mechanisms underlying their development have not been fully elucidated. In the present study, we aimed to assess heparin-binding epidermal growth factor (HB-EGF) expression in the airways of patients with COPD and to elucidate the possible role of HB-EGF in the pathology of COPD. Sputum and lung tissue HB-EGF expression was evaluated in control subjects and patients with COPD. The relationships between HB-EGF expression, disease severity, collagen deposition (fibrosis), and EMT were investigated. In vitro, human bronchial epithelial (HBE) cells and lung fibroblast cells exposed to the recombinant HB-EGF, collagen deposition and EMT were assessed. We found that sputum HB-EGF expression was significantly increased in patients with COPD compared with non-smokers and smokers without COPD. There was a significant positive correlation between sputum HB-EGF and COPD assessment test (CAT) score. HB-EGF expression was significantly increased in the lung tissue samples of patients with COPD and associated with collagen deposition and N- and E-cadherin, and vimentin expression. In vitro, HB-EGF promoted collagen production in lung fibroblasts. Moreover, HB-EGF induced the EMT process through induction of N-and E-cadherin, and vimentin expression in HBE cells. Collectively, HB-EGF induces airway remodeling by modulating airway fibrosis and pulmonary EMT, and contributes to the COPD severity. The current data may provide insight into the underlying pathogenesis of COPD, in which HB-EGF has an important pathogenic role.

沉默信息调节因子2 (Sirt2) 是1986年Ivy等人在酵母菌中发现的和长寿相关的基因[1],后来发现在哺乳动物中也存在和 Sirt2同源蛋白,命名为SIRT1-SIRT7,其中SIRT1和酵母的 Sirt2同源性最高.SIRT1作为细胞内重要的调节因子,广泛分布于人类多种细胞的胞浆和胞核,参与调节炎症、细胞周期、衰老和凋亡、应激等重要生理过程.与多种肺部疾病的发生发展密切相关,例如慢性阻塞性肺疾病、支气管哮喘(简称哮喘)、肺纤维化、肺癌等.然而,目前SIRT1在哮喘中发病机制的研究报道较少.本文主要从SIRT1与哮喘关系进行综述.

Purpose Fibrosis in peripheral airways is responsible for airflow limitation in chronic obstructive pulmonary disease (COPD). Annexin A1 modulates several key biological events during inflammation. However, little is known about its role in airway fibrosis in COPD. We investigated whether levels of Annexin A1 were upregulated in patients with COPD, and whether it promoted airway fibrosis. Methods We quantified serum Annexin A1 levels in never-smokers (n=12), smokers without COPD (n=11), and smokers with COPD (n=22). Correlations between Annexin A1 expression and clinical indicators (eg, lung function) were assessed. In vitro, human bronchial epithelial (HBE) cells were exposed to cigarette smoke extract (CSE) and Annexin A1 expression was assessed. Primary human lung fibroblasts were isolated from patients with COPD and effects of Annexin A1 on fibrotic deposition of lung fibroblasts were evaluated. Results Serum Annexin A1 was significantly higher in patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines stage III or IV than in those with GOLD stages I or II (12.8±0.8 ng/mL versus 9.8±0.7 ng/mL; p=0.016). Annexin A1 expression was negatively associated with airflow obstruction (forced expiratory volume in one second % predicted; r=−0.72, p<0.001). In vitro, Annexin A1 was significantly increased in CSE-exposed HBE cells in a time- and concentration-dependent manner. Annexin A1 promoted lung fibroblasts proliferation, migration, differentiation, and collagen deposition via the ERK1/2 and p38 mitogen-activated protein kinase pathways. Conclusion Annexin A1 expression is upregulated in patients with COPD and affects lung fibroblast function. However, more studies are needed to clarify the role of Annexin A1 in airway fibrosis of COPD.

Objective To investigate the effect of ALDH1A1 silence on the migration and invasion of nasopharyngeal carcinoma cell lines 5-8F and CNE2.Methods The nasopharyngeal carcinoma 5-8F and CNE2 cell lines were divided into 5-8F shRNA-ALDH1A1 group,5-8F control group,CNE2 shRNA-ALDH1A1 group,and CNE2 control group.The lentiviral pMagic 4.1-ALDH1A1 siRNA and pMagic 4.1-shRNA were used to transfect 5-8F and CNE2 cell lines respectively to construct 5-8F,CNE2,5-8F vector and CNE2 vector cell lines stably interfering the expression of ALDH1A1.The protein expression of ALDH1A1,vascular endothelial growth factor (VEGF),matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated with Western blot in four groups.The counts of migration and invasion cells were detected by Transwell chamber and Boyden chamber assay.Results The expression of ALDH1A1 was significantly lower in 5-8F shRNA-ALDH1A1 group (0.34±0.01) than that in 5-8F vector group (0.75±0.03),and in CNE2 shRNA-ALDH1A1 group (0.24±0.03) than that in CNE2 vector group (0.71±0.03) (P＜0.05).The migration and invasion cell counts were significantly lower in 5-8F shRNA-ALDH1A1 group (5.0±2.6,32.7±2.1) than those in 5-8F vector group (117.6±3.2,105.6±4.0),and in CNE2 shRNA-ALDH1A1 group (34.0±3.0,32.3±1.5) than those in CNE2 vector group (121.0±4.0,108.0±1.7) (P＜0.01).The expressions of VEGF,MMP-2 and MMP-9 were significantly lower in 5-8F shRNA-ALDH1A1 group (0.49±0.03,0.36±0.04,0.31±0.04) than those in 5-8F vector group (0.73±0.02,0.50±0.02,0.89±0.01) (P＜0.01),in CNE2 shRNA-ALDH1A1 group (0.49±0.03,0.36±0.04,0.31±0.04) than those in CNE2 vector group (0.78±0.05,0.67±0.05,0.76±0.04) (P＜0.01).Conclusion The down-regulation of ALDH1A1 could inhibit the migration and invasion of nasopharyngeal carcinoma cells.

BACKGROUND:Lung cancer is the leading cancer-related death worldwide. Patients with lung cancer mainly died of tumor metastasis and invasion. Protein kinase CK2 is an ubiquitous serine/threonine protein kinase and is frequently upregulated in various human tumors. This study aims to explore the effect and molecular mechanism of the invasion and migration of lung adenocarcinoma A549 cells after knock-down of CK2α expression.METHODS:The pSilencerTM 4.1-siCK2α-eGFP of lentiviral-mediated shRNA was constructed. The expression of CK2α was knock-downed, and a stable A549 cell line was established. The invasion and migration of A549 cell line was detected through Transwell and Boyden chamber assays. The protein expression of the PI3K/Akt signaling pathway and mesenchymal-to-epithelial transition (EMT) was evaluated using Western blot analysis.RESULTS:The invasion and migration of A549 cells were significantly inhibited after the knockdown of CK2α expression compared with that in the control group. p-PTEN, Akt, p-Akt473, p-Akt308, p-PDK1, p-c-Raf, and p-GSK-3β were significantly downregulated, whereas PTEN was upregulated. Moreover, vimentin, β-catenin, Snail, MMP2, and MMP9 were significantly downregulated after reducing the CK2α expression.CONCLUSIONS:CK2α might regulate the invasion and migration of A549 cells through the PI3K/Akt/GSK-3β/Snail signaling pathway, which controls EMT in lung adenocarcinoma.
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Objective:To investigate the effect of A549 cell line after down-regulation of protein kinase CK2α expression in lung adenocarcinoma.Methods:We constructed pSilencerTM4.1-shCK2α-eGFP of lentiviral-mediated shRNA.Then we knock-downed expression of CK2α gene and established stable A549 cell line by lentivirus-mediated shRNA.Analyzing the proliferation and apoptosis capacities of A549 cell line by MTT assay,colony formation assay and apoptosis assay.Moreover,we examined proteins expression of Caspase-3,Caspase-8,Bcl-xl and Bcl-2 by Western blot.Results:A549 cell line of down-regualted CK2α expression showed lower proliferation and more promotable apoptosis than control groups.Furthermore,compared to control groups,proteins of Caspase-3,Caspase-8 were significantly up-regulated and Bcl-xl,Bcl-2 were significantly down-regulated.Conclusion:Silence of CK2α expression would inhibit proliferation and activate apoptosis in lung adenocarcinoma A549 cell line.

目的 检测肺癌组织中Livin和Fas/FasL的表达水平,研究它们与临床病理特征的关系及其相互关系,为肺癌的早期诊断、治疗及靶向药物研发提供线索.方法 采用免疫组织化学来检测肺癌组织216例、癌旁肺组织90例、肺良性病变组织45例中Livin、Fas和FasL基因的表达情况.结果 Livin在肺癌组织、癌旁肺组织、良性病变组织中阳性表达分别为:63.43％、14.44％、13.33％,差异有统计学意义(P＜O.01);Fas在肺癌组织、癌旁肺组织、良性病变组织中阳性表达分别为:53.24％、83.33％、86.67％,其差异有统计学意义(P＜0.01);Fas表达与病理学分级相关(P＜0.05);FasL在肺癌中表达、淋巴结转移与病理学分级及TNM分期相关(P＜0.05).结论 研究结果提示Livin和Fas/FasL可能作为凋亡相关的基因参与了肺癌的发生发展.

Large tumor suppressor 2 (LATS2) plays significant roles in tumorigenesis and cancer progression. This study was aimed to analyze the correlation between LATS2 expression and clinicopathologic features and its prognostic significance in non-small cell lung cancer (NSCLC). LATS2 expression was examined in 73 NSCLC clinical specimens and 22 normal lung tissues using immunohistochemistry. Low levels of LATS2 protein were inversely associated with the T classification (P=0.001), N classification (P=0.005) and clinical stage (P=0.001) in NSCLC patients. Patients with lower LATS2 expression had a significantly shorter overall survival than patients with high LATS2 expression. Multivariate analysis suggested that low expression of LATS2 was an independent prognostic indicator (P=0.002) for the survival of patients with NSCLC. Furthermore, overexpression of LATS2 resulted in mobility inhibition in NSCLC cell lines A549 and H1299, and reduced protein level of matrix metalloproteinase-2 (MMP-2) and MMP-9. On the contrary, LATS2 siRNA treatment enhanced cell mobility and increased MMP-2 and MMP-9 protein expression level. In conclusion, low expression of LATS2 is a potential unfavorable prognostic factor and promoted cell invasion and migration in NSCLC.

Purpose: Frizzled-1 (FZD1) is closely associated with development of various tumors, but its expression pattern in lung cancer tissues has not been elucidated enough, including the correlation in clinicopathological features and the prognostic significance of FZD1 of non-small cell lung cancer (NSCLC) patients. Methods: We evaluated immunohistochemical staining for FZD1 protein in 152 cases of NSCLC. We correlated the line of differentiation with outcome using Chi-square test and Kaplan-Meier analysis. Results: High-expression of FZD1 was found in 88/152 (57.9%) of NSCLC patients by immunohistochemical analysis. The expression level of FZD1 was strongly significantly associated with TNM stage (P = 0.006) in NSCLC lesions, but not significantly with other clinicopathological features. On Kaplan-Meier survival curves analysis, low FZD1 expression had shorter overall survival rate and disease-free survival than those with high FZD1 (P = 0.001 and P = 0.004, respectively). Conclusion: These results suggest that low expression of FZD1 might indicate poor prognosis in NSCLC patients and our observations demonstrate that expression of FZD1 serve as a biomarker of disease progression.