NEAT1_1 confers gefitinib resistance in lung adenocarcinoma through promoting AKR1C1-mediated ferroptosis defence.

Gefitinib is one of the most extensively utilized epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) for treating advanced lung adenocarcinoma (LUAD) patients harboring EGFR mutation. However, the emergence of drug resistance significantly compromised the clinical efficacy of EGFR-TKIs. Gaining further insights into the molecular mechanisms underlying gefitinib resistance holds promise for developing novel strategies to overcome the resistance and improve the prognosis in LUAD patients. Here, we identified that the inhibitory efficacy of gefitinib on EGFR-mutated LUAD cells was partially dependent on the induction of ferroptosis, and ferroptosis protection resulted in gefitinib resistance. Among the ferroptosis suppressors, aldo-keto reductase family 1 member C1 (AKR1C1) exhibited significant upregulation in gefitinib-resistant strains of LUAD cells and predicted poor progression-free survival (PFS) and overall survival (OS) of LUAD patients who received first-generation EGFR-TKI treatment. Knockdown of AKR1C1 partially reversed drug resistance by re-sensitizing the LUAD cells to gefitinib-mediated ferroptosis. The decreased expression of miR-338-3p contributed to the aberrant upregulation of AKR1C1 in gefitinib-resistant LUAD cells. Furthermore, upregulated long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1_1 (NEAT1_1) sponged miR-338-3p to neutralize its suppression on AKR1C1. Dual-luciferase reporter assay and miRNA rescue experiment confirmed the NEAT1_1/miR-338-3p/AKR1C1 axis in EGFR-mutated LUAD cells. Gain- and loss-of-function assays demonstrated that the NEAT1_1/miR-338-3p/AKR1C1 axis promoted gefitinib resistance, proliferation, migration, and invasion in LUAD cells. This study reveals the effects of NEAT1_1/miR-338-3p/AKR1C1 axis-mediated ferroptosis defence in gefitinib resistance in LUAD. Thus, targeting NEAT1_1/miR-338-3p/AKR1C1 axis might be a novel strategy for overcoming gefitinib resistance in LUAD harboring EGFR mutation.

circNOX4 activates an inflammatory fibroblast niche to promote tumor growth and metastasis in NSCLC via FAP/IL-6 axis.

BACKGROUND: Cancer-associated fibroblasts (CAFs) orchestrate a supportive niche that fuels cancer metastatic development in non-small cell lung cancer (NSCLC). Due to the heterogeneity and plasticity of CAFs, manipulating the activated phenotype of fibroblasts is a promising strategy for cancer therapy. However, the underlying mechanisms of fibroblast activation and phenotype switching that drive metastasis remain elusive. METHODS: The clinical implications of fibroblast activation protein (FAP)-positive CAFs (FAP+CAFs) were evaluated based on tumor specimens from NSCLC patients and bioinformatic analysis of online databases. CAF-specific circular RNAs (circRNAs) were screened by circRNA microarrays of primary human CAFs and matched normal fibroblasts (NFs). Survival analyses were performed to assess the prognostic value of circNOX4 in NSCLC clinical samples. The biological effects of circNOX4 were investigated by gain- and loss-of-function experiments in vitro and in vivo. Fluorescence in situ hybridization, luciferase reporter assays, RNA immunoprecipitation, and miRNA rescue experiments were conducted to elucidate the underlying mechanisms of fibroblast activation. Cytokine antibody array, transwell coculture system, and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the downstream effectors that promote cancer metastasis. RESULTS: FAP+CAFs were significantly enriched in metastatic cancer samples, and their higher abundance was correlated with the worse overall survival in NSCLC patients. A novel CAF-specific circRNA, circNOX4 (hsa_circ_0023988), evoked the phenotypic transition from NFs into CAFs and promoted the migration and invasion of NSCLC in vitro and in vivo. Clinically, circNOX4 correlated with the poor prognosis of advanced NSCLC patients. Mechanistically, circNOX4 upregulated FAP by sponging miR-329-5p, which led to fibroblast activation. Furthermore, the circNOX4/miR-329-5p/FAP axis activated an inflammatory fibroblast niche by preferentially inducing interleukin-6 (IL-6) and eventually promoting NSCLC progression. Disruption of the intercellular circNOX4/IL-6 axis significantly suppressed tumor growth and metastatic colonization in vivo. CONCLUSIONS: Our study reveals a role of the circRNA-induced fibroblast niche in tumor metastasis and highlights that targeting the circNOX4/FAP/IL-6 axis is a promising strategy for the intervention of NSCLC metastasis.

Distinct mechanisms for sebaceous gland self-renewal and regeneration provide durability in response to injury.

Sebaceous glands (SGs) release oils that protect our skin, but how these glands respond to injury has not been previously examined. Here, we report that SGs are largely self-renewed by dedicated stem cell pools during homeostasis. Using targeted single-cell RNA sequencing, we uncovered both direct and indirect paths by which resident SG progenitors ordinarily differentiate into sebocytes, including transit through a Krt5+PPARγ+ transitional basal cell state. Upon skin injury, however, SG progenitors depart their niche, reepithelialize the wound, and are replaced by hair-follicle-derived stem cells. Furthermore, following targeted genetic ablation of >99% of SGs from dorsal skin, these glands unexpectedly regenerate within weeks. This regenerative process is mediated by alternative stem cells originating from the hair follicle bulge, is dependent upon FGFR2 signaling, and can be accelerated by inducing hair growth. Altogether, our studies demonstrate that stem cell plasticity promotes SG durability following injury.

Durability of Plant Fiber Composites for Structural Application: A Brief Review.

Environmental sustainability and eco-efficiency stand as imperative benchmarks for the upcoming era of materials. The use of sustainable plant fiber composites (PFCs) in structural components has garnered significant interest within industrial community. The durability of PFCs is an important consideration and needs to be well understood before their widespread application. Moisture/water aging, creep properties, and fatigue properties are the most critical aspects of the durability of PFCs. Currently, proposed approaches, such as fiber surface treatments, can alleviate the impact of water uptake on the mechanical properties of PFCs, but complete elimination seems impossible, thus limiting the application of PFCs in moist environments. Creep in PFCs has not received as much attention as water/moisture aging. Existing research has already found the significant creep deformation of PFCs due to the unique microstructure of plant fibers, and fortunately, strengthening fiber-matrix bonding has been reported to effectively improve creep resistance, although data remain limited. Regarding fatigue research in PFCs, most research focuses on tension-tension fatigue properties, but more attention is required on compression-related fatigue properties. PFCs have demonstrated a high endurance of one million cycles under a tension-tension fatigue load at 40% of their ultimate tensile strength (UTS), regardless of plant fiber type and textile architecture. These findings bolster confidence in the use of PFCs for structural applications, provided special measures are taken to alleviate creep and water absorption. This article outlines the current state of the research on the durability of PFCs in terms of the three critical factors mentioned above, and also discusses the associated improvement methods, with the hope that it can provide readers with a comprehensive overview of PFCs' durability and highlight areas worthy of further research.

Distinct mechanisms for sebaceous gland self-renewal and regeneration provide durability in response to injury.

Sebaceous glands (SGs) release oils that protect our skin, but how these glands respond to injury has not been previously examined. Here, we report that SGs are largely self-renewed by dedicated stem cell pools during homeostasis. Using targeted single cell RNA-sequencing, we uncovered both direct and indirect paths by which these resident SG progenitors ordinarily differentiate into sebocytes, including transit through a PPARγ+Krt5+ transitional cell state. Upon skin injury, however, SG progenitors depart their niche, reepithelialize the wound, and are replaced by hair follicle-derived stem cells. Furthermore, following targeted genetic ablation of >99% of SGs from dorsal skin, these glands unexpectedly regenerate within weeks. This regenerative process is mediated by alternative stem cells originating from the hair follicle bulge, is dependent upon FGFR signaling, and can be accelerated by inducing hair growth. Altogether, our studies demonstrate that stem cell plasticity promotes SG durability following injury.

Single-cell transcriptomics of human-skin-equivalent organoids.

Several methods for generating human-skin-equivalent (HSE) organoid cultures are in use to study skin biology; however, few studies thoroughly characterize these systems. To fill this gap, we use single-cell transcriptomics to compare in vitro HSEs, xenograft HSEs, and in vivo epidermis. By combining differential gene expression, pseudotime analyses, and spatial localization, we reconstruct HSE keratinocyte differentiation trajectories that recapitulate known in vivo epidermal differentiation pathways and show that HSEs contain major in vivo cellular states. However, HSEs also develop unique keratinocyte states, an expanded basal stem cell program, and disrupted terminal differentiation. Cell-cell communication modeling shows aberrant epithelial-to-mesenchymal transition (EMT)-associated signaling pathways that alter upon epidermal growth factor (EGF) supplementation. Last, xenograft HSEs at early time points post transplantation significantly rescue many in vitro deficits while undergoing a hypoxic response that drives an alternative differentiation lineage. This study highlights the strengths and limitations of organoid cultures and identifies areas for potential innovation.

Circular RNA hsa_circ_0002938 (circCRIM1) promotes the progression of esophageal squamous cell carcinoma by upregulating transcription factor 12.

Growing evidence has indicated that circular RNAs (circRNAs) play crucial roles in the tumorigenesis and progression of diverse malignancies. However, the majority of circRNAs involved in esophageal squamous cell carcinoma (ESCC) remain undefined and the exact functions and underlying mechanisms of circRNAs in ESCC still need further exploration. In this study, we identified a novel onco-circRNA hsa_circ_0002938, derived from the exons of cysteine-rich transmembrane BMP regulator 1 (CRIM1) pre-mRNA, referred to as circCRIM1. We found that the expression of circCRIM1 was higher in ESCC tissues, compared to para-carcinoma tissues. Increased expression of circCRIM1 was positively correlated with clinical parameters of ESCC patients including tumor-node-metastasis (TNM) stage, tumor invasion range, and lymph node metastasis. Functionally, the results from the experiments in vitro showed that the knockdown of circCRIM1 suppressed proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in ESCC cells. By conducting bioinformatics algorithms analyses and microRNA (miRNA) rescue experiments, we found that circCRIM1 could act as a competing endogenous RNA (ceRNA) to sponge miR-342-3p in ESCC cells, and thereby upregulated the expression of transcription factor 12 (TCF12), a key regulator promoting the EMT process. Taken together, circCRIM1 facilitates the progression of ESCC by sponging miR-342-3p to regulate TCF12 and promote EMT, and the circCRIM1/miR-342-3p/TCF12 axis may be regarded as a potential predictive biomarker and therapeutic target for treating ESCC.

A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes.

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein-protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context.

Fibroblast activation protein in the tumor microenvironment predicts outcomes of PD-1 blockade therapy in advanced non-small cell lung cancer.

PURPOSE: The identification of robust predictive biomarkers of the response to programmed cell death-1 (PD-1) blockade remains a critical concern. Here, we investigated on fibroblast activation protein (FAP) as a microenvironment-derived biomarker of clinical outcomes of PD-1 blockade therapy, and the correlation between FAP expression and T cell infiltration in advanced non-small cell lung cancer (NSCLC). METHODS: A total of 135 patients with advanced NSCLC who received PD-1 blockade therapy were retrospectively analyzed. The potential associations among FAP expression, CD3 + T cell and CD8 + T cell infiltration, and clinical outcomes of immunotherapy were validated by immunohistochemistry, bioinformatic analyses, and statistical measurements. RESULTS: FAP was widely expressed in advanced NSCLC tissues. FAP was correlated with decreased density of CD8 + T cells (Spearman's rho - 0.32, p < 0.001) and immunosuppressive tumor microenvironment (TME) status. No correlations were detected between FAP and PD-L1 expression or with the density of CD3 + T cells. The patients with higher expression of FAP showed worse response rate (16.4% vs. 38.7%, p < 0.001) and worse progression-free survival (HR = 2.56, 95% CI 1.69-3.87, p < 0.001). In addition, FAP contributed to shortened overall survival in subgroups of the patients with squamous cell lung cancer (p = 0.020), PD-1 blockade monotherapy (p = 0.017), and first-line therapy (p = 0.028). CONCLUSION: FAP is a potential predictive biomarker of resistance to PD-1 blockade. Further investigation is warranted to identify a strategy for targeting FAP to alleviate the immunosuppressive TME and broaden the clinical effectiveness of PD-1 blockade therapy.

Near-Infrared Carbonized Polymer Dots for NIR-II Bioimaging.

Carbon dots (CDs) or carbonized polymer dots (CPDs) are an emerging class of optical materials that have exceptional applications in optoelectronic devices, catalysis, detection, and bioimaging. Although cell studies of CPDs have produced impressive results, in vivo imaging requires available CPDs to fluoresce in the near-infrared-II (NIR-II) window (1000-1700 nm). Here, a two-step bottom-up strategy is developed to synthesize NIR-CPDs that provide bright emissions in both NIR-I and NIR-II transparent imaging windows. The designed strategy includes a hydrothermal reaction to form a stable carbon core with aldehyde groups, followed by the Knoevenagel reaction to tether the molecular emission centers. This procedure is labor-saving, cost-efficient, and produces a high yield. The NIR-CPDs enable high-performance NIR-II angiography and real-time imaging of the disease degree of colitis noninvasively. This technology may therefore provide a next-generation synthesis strategy for CPDs with rational molecular engineering that can accurately tune the absorption/emission properties of NIR-emissive CPDs.

Long non-coding RNA SNHG10 upregulates BIN1 to suppress the tumorigenesis and epithelial-mesenchymal transition of epithelial ovarian cancer via sponging miR-200a-3p.

Epithelial ovarian cancer (EOC) is one of the most frequent and fatal gynecologic malignant tumors resulting in an unsatisfying prognosis. Long non-coding RNAs (lncRNAs) play pivotal roles in the tumorigenesis and progression of EOC. However, the profile of lncRNAs involved in EOC remains to be expanded to further improve clinical treatment strategy. In present study, we identified a novel tumor-suppressive lncRNA small nucleolar RNA host gene 10 (SNHG10) in EOC. Kaplan-Meier analysis and COX proportional hazard progression model showed that low expression of SNHG10 was correlated with a poor prognosis of EOC patients. Overexpressing SNHG10 suppressed the proliferation, colony formation, migration, and invasion of EOC cells. Furthermore, SNHG10 was predicted to sponge miR-200a-3p in EOC cells according to the LncBase v.2 experimental module. Then, the binding of SNHG10 and miR-200a-3p was confirmed by performing quantitative real-time PCR (qRT-PCR) and luciferase reporter assays. RNA immunoprecipitation (RIP) showed that SNHG10 and miR-200a-3p occupied the same Ago2 protein to form an RNA-induced silencing complex (RISC). By overlapping the results from the bioinformatics algorithms, tumor-suppressor bridging integrator-1 (BIN1) was found to be a main downstream target of the SNHG10/miR-200a-3p axis. Low expression of BIN1 in EOC tissues was detected by using immunohistochemistry (IHC). Besides, BIN1 and SNHG10 expression was positively correlated in EOC tissues. By performing miRNA rescue experiments, a SNHG10/miR-200a-3p/BIN1 axis and its promoting effects on malignant behaviors and epithelial-mesenchymal transition (EMT) process were verified in EOC cells. Moreover, SNHG10 overexpression significantly suppressed the tumorigenesis and EMT of EOC cells in vivo. Altogether, SNHG10 sponges miR-200a-3p to upregulate BIN1 and thereby exerting its tumor-suppressive effects in EOC. Therefore, the SNHG10/miR-200a-3p/BIN1 axis may act as a potential predictive biomarker and therapeutic target for treating EOC.

Expression and clinical significance of bridging intergrator 1 in epithelial ovarian cancer tissues and cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨桥接整合因子1（BIN1）在上皮性卵巢癌（EOC）组织中的表达及其临床意义，以及BIN1对EOC细胞A2780增殖、迁移和侵袭的影响。方法：收集2017年7月至2018年1月河北医科大学第四医院手术切除的67例EOC患者的肿瘤组织及同期因其他妇科疾病手术切除的30例非肿瘤患者的卵巢组织（正常对照组）标本。用免疫组织化学染色法检测EOC组织和非肿瘤卵巢组织中BIN1蛋白的表达水平，χ2检验分析BIN1表达与患者临床病理特征之间的关联，Kaplan-Meier法分析BIN1表达与患者的无病生存期（DFS）和总生存期（OS）之间的关系。用qPCR和WB法检测EOC细胞SKOV3、A2780和人卵巢上皮细胞IOSE80中BIN1 mRNA和蛋白的表达水平。利用基因转染技术将BIN1质粒CMV-MCS-GFP-SV40-Neomycin-BIN1和空载体质粒CMV-MCS-GFP-SV40-Neomycin分别转染到A2780细胞以构建过表达BIN1细胞及其对照，用qPCR和WB法分别检测转染细胞中BIN1 mRNA和蛋白的表达水平，CCK-8、划痕愈合和Transwell实验分别检测过表达BIN1对A2780细胞增殖、迁移和侵袭的影响。结果：EOC组织中BIN1阳性表达率显著低于正常卵巢组织（P<0.01）。BIN1表达与EOC患者较晚的术后病理分期、较差的组织学分级、淋巴结转移及腹膜转移存在正向关联（均P<0.05）；BIN1低表达组患者的DFS和OS均短于BIN1高表达组患者（均P<0.05）。SKOV3和A2780细胞中BIN1 mRNA和蛋白的表达水平均显著低于IOSE80细胞（均P<0.01）；过表达BIN1显著抑制A2780细胞的增殖、迁移和侵袭（P<0.05或P<0.01）。结论：BIN1在EOC组织和细胞中呈低表达状态，与患者的不良预后有关；过表达BIN1可降低EOC细胞A2780的增殖、迁移和侵袭能力。

A comparative study of PD-1 antibody plus chemotherapy and antiangiogenic drug plus chemotherapy in the frontline treatment of advanced lung adenocarcinoma / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨PD-1抗体联合化疗对比抗血管生成药物联合化疗在晚期驱动基因阴性肺腺癌一线治疗中的疗效和安全性。方法：收集2018年3月至2021年8月河北医科大学第四医院收治的141例不可手术切除的ⅢB/ⅢC和Ⅳ期驱动基因阴性肺腺癌患者，回顾性分析PD-1抗体联合化疗对比抗血管生成药物联合化疗在一线治疗中的疗效与安全性。主要研究终点为无进展生存期（PFS），次要终点为客观缓解率（ORR）、疾病控制率（DCR）和不良反应。结果：141例患者均纳入生存分析，中位随访时间为13.0个月（95% CI：12.0～14.0）。PD-1抗体联合化疗组（A组）和抗血管生成药物联合化疗组（B组）的ORR分别为33.33%和27.38%，DCR分别为98.25%和89.29%，差异均无统计学意义。A组和B组的中位PFS分别为8.4个月（95% CI: 7.3～9.9）和6.9个月（95% CI: 6.1～7.7），差异无统计学意义。亚组分析结果显示，ⅢB/ⅢC期、肝或脑转移患者中，A组中位PFS较B组均延长（均P<0.01）。A组和B组不良反应发生率分别为26.32%和14.29%，多数为1~2级。结论：PD-1抗体联合化疗对比抗血管生成药物联合化疗一线治疗晚期驱动基因阴性肺腺癌疗效相当，不良反应可耐受，可成为晚期驱动基因阴性肺腺癌标准一线治疗。

Long non-coding RNA NORAD/miR-224-3p/MTDH axis contributes to CDDP resistance of esophageal squamous cell carcinoma by promoting nuclear accumulation of ß-catenin.

BACKGROUND: Cis-diamminedichloro-platinum (CDDP)-based chemotherapy regimens are the most predominant treatment strategies for patients with esophageal squamous cell carcinoma (ESCC). Dysregulated long non-coding RNAs (lncRNAs) contribute to CDDP resistance, which results in treatment failure in ESCC patients. However, the majority of lncRNAs involved in CDDP resistance in ESCC remain to be elucidated. METHODS: The public Gene Expression Omnibus (GEO) dataset GSE45670 was analysed to reveal potential lncRNAs involved in CDDP resistance of ESCC. Candidate upregulated lncRNAs were detected in ESCC specimens by qRT-PCR to identify crucial lncRNAs. Non-coding RNA activated by DNA damage (NORAD) was selected for further study. Kaplan-Meier analysis and a COX proportional regression model were performed to analyse the potential of NORAD for predicting prognosis of ESCC patients. The role of NORAD in CDDP resistance were determined by conducting gain and loss-of-function experiments in vitro. Fluorescence in situ hybridization (FISH) was performed to determine the subcellular location of NORAD in ESCC cells. A public GEO dataset and bioinformatic algorithms were used to predict the microRNAs (miRNAs) that might be latently sponged by NORAD. qRT-PCR was conducted to verify the expression of candidate miRNAs. Luciferase reporter and Argonaute-2 (Ago2)-RNA immunoprecipitation (RIP) assays were conducted to evaluate the interaction between NORAD and candidate miRNAs. A miRNA rescue experiment was performed to authenticate the NORAD regulatory axis and its effects on CDDP resistance in ESCC cells. Western blotting was conducted to confirm the precise downstream signalling pathway of NORAD. A xenograft mouse model was established to reveal the effect of NORAD on CDDP resistance in vivo. RESULTS: The expression of NORAD was higher in CDDP-resistant ESCC tissues and cells than in CDDP-sensitive tissues and cells. NORAD expression was negatively correlated with the postoperative prognosis of ESCC patients who underwent CDDP-based chemotherapy. NORAD knockdown partially arrested CDDP resistance of ESCC cells. FISH showed that NORAD was located in the cytoplasm in ESCC cells. Furthermore, overlapping results from bioinformatic algorithms analyses and qRT-PCR showed that NORAD could sponge miR-224-3p in ESCC cells. Ago2-RIP demonstrated that NORAD and miR-224-3p occupied the same Ago2 to form an RNA-induced silencing complex (RISC) and subsequently regulated the expression of metadherin (MTDH) in ESCC cells. The NORAD/miR-224-3p/MTDH axis promoted CDDP resistance and progression in ESCC cells by promoting nuclear accumulation of ß-catenin in vitro and in vivo. CONCLUSIONS: NORAD upregulates MTDH to promote CDDP resistance and progression in ESCC by sponging miR-224-3p. Our results highlight the potential of NORAD as a therapeutic target in ESCC patients receiving CDDP-based chemotherapy.

Bimolecular Fluorescence Complementation (BiFC) and Multiplexed Imaging of Protein-Protein Interactions in Human Living Cells.

Deciphering protein-protein interactions (PPIs) in vivo is crucial to understand protein function. Bimolecular fluorescence complementation (BiFC) makes applicable the analysis of PPIs in many different native contexts, including human live cells. It relies on the property of monomeric fluorescent proteins to be reconstituted from two separate subfragments upon spatial proximity. Candidate partners fused to such complementary subfragments can form a fluorescent protein complex upon interaction, allowing visualization of weak and transient PPIs. It can also be applied for investigation of distinct PPIs at the same time using a multicolor setup. In this chapter, we provide a detailed protocol for analyzing PPIs by doing BiFC in cultured cells. Proof-of-principle experiments rely on the complementation property between the N-terminal fragment of mVenus (designated VN173) and the C-terminal fragment of mCerulean (designated CC155) and the partnership between HOXA7 and PBX1 proteins. This protocol is compatible with any other fluorescent complementation pair fragments and any type of candidate interacting proteins.

Long noncoding RNA DGCR5 involves in tumorigenesis of esophageal squamous cell carcinoma via SRSF1-mediated alternative splicing of Mcl-1.

Long noncoding RNAs (lncRNAs) emerge as essential roles in the regulation of alternative splicing (AS) in various malignancies. Serine- and arginine-rich splicing factor 1 (SRSF1)-mediated AS events are the most important molecular hallmarks in cancer. Nevertheless, the biological mechanism underlying tumorigenesis of lncRNAs correlated with SRSF1 in esophageal squamous cell carcinoma (ESCC) remains elusive. In this study, we found that lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) was upregulated in ESCC clinical samples, which associated with poor prognosis. Through RNA interference and overexpression approaches, we confirmed that DGCR5 contributed to promote ESCC cell proliferation, migration, and invasion while inhibited apoptosis in vitro. Mechanistically, DGCR5 could directly bind with SRSF1 to increase its stability and thus stimulate alternative splicing events. Furthermore, we clarified that SRSF1 regulated the aberrant splicing of myeloid cell leukemia-1 (Mcl-1) and initiated a significant Mcl-1L (antiapoptotic) isoform switch, which contributed to the expression of the full length of Mcl-1. Moreover, the cell-derived xenograft (CDX) model was validated that DGCR5 could facilitate the tumorigenesis of ESCC in vivo. Collectively, our findings identified that the key biological role of lncRNA DGCR5 in alternative splicing regulation and emphasized DGCR5 as a potential biomarker and therapeutic target for ESCC.

Expression of lncRNA TOB1-AS1 in epithelial ovarian cancer tissues and its clinical significance / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨ERBB2.1转导蛋白反义RNA1（transducer of ERBB2.1 antisense RNA 1，TOB1-AS1）在上皮性卵巢癌（epithelial ovarian cancer，EOC）组织中的表达情况及其临床意义，初步探讨TOB1-AS1对EOC细胞体外增殖、迁移和侵袭的影响。方法：使用TCGA数据库对EOC组织中TOB1-AS1表达情况进行分析；收集2017年7月至2018年1月在河北医科大学第四医院妇科行肿瘤切除并经病理检查证实为EOC的67例患者的肿瘤组织，收集同期因其他妇科疾病接受手术的30例患者的非肿瘤卵巢组织作为对照。采用qPCR法检测EOC组织和非肿瘤卵巢组织中TOB1-AS1的表达水平，χ2检验分析TOB1-AS1的表达与不同临床病理特征之间的相关性，Kaplan-Meier和Cox比例风险回归模型分析患者生存及预后的潜在影响因素。CCK-8实验、划痕实验和Transwell实验分别检测敲低TOB1-AS1表达对EOC细胞SKOV3和A2780增殖、迁移和侵袭的影响。结果：TCGA数据库中资料和qPCR检测结果均显示，在EOC组织中TOB1-AS1的表达水平显著高于非肿瘤卵巢组织（均P<0. 01）。TOB1-AS1的高表达与EOC患者较晚的FIGO分期、较差的组织分级、淋巴结转移及腹膜转移有关（均P<0.05）。Kaplan-Meier生存分析结果显示，TOB1-AS1高表达组患者术后DFS和OS均短于TOB1-AS1低表达组（均P<0.05）。Cox比例风险回归模型分析结果显示，FIGO分期、淋巴结转移、腹膜转移及TOB1-AS1表达是EOC患者预后的独立影响因素（均P<0.05）。TOB1-AS1在EOC细胞系SKOV3、A2780中的表达水平也显著高于正常卵巢上皮细胞系IOSE80（均P<0.01）。细胞功能实验结果显示，敲低TOB1-AS1可抑制SKOV3和A2780细胞的增殖、迁移和侵袭（均P<0.05）。结论：TOB1-AS1在EOC组织中高表达，与患者的不良预后显著相关。TOB1-AS1可能通过促进EOC细胞SKOV3、A2780的增殖、迁移和侵袭来影响EOC的恶性进展。

Future of immune checkpoint inhibitors: focus on tumor immune microenvironment.

Immunotherapy has become a powerful clinical strategy in cancer treatment. Immune checkpoint inhibitors (ICIs) have opened a new era for cancer immunotherapy. Nowadays, the number of immunotherapy drug approvals has increased, with numerous treatment options in clinical and preclinical development. However, there remain some obstacles to improve the efficacy of ICIs further. The tumor immune microenvironment (TIME) consists of cancer cell, immune cells and cytokines, et cetera. The dynamics of TIME determine the efficacies of ICIs. Although the ICIs showed manageable toxicity, immune-related adverse effects (irAEs) are still unignorable for clinicians. Since some primary resistance mechanisms exist in TIME, ICIs can only show effects in individual cancer patients. Even for the patients who responded, acquired resistance will occur to neutralize the effect of ICIs. Understanding how to increase the response rates and overcome the resistance to various classes of ICIs is the key to improving clinical efficacy. Besides the novel ICIs in development, there are some approaches to establish combination therapies are underway to improve further the efficacies of ICIs in treating cancer patients. Here, we describe the complicated TIME and state quo of ICIs to prospect the future of ICIs in cancer treatment.

SRSF1-dependent alternative splicing attenuates BIN1 expression in non-small cell lung cancer.

Decreased bridging integrator 1 (BIN1) expression has great significance in promoting the progression of malignant tumors. Reduced messenger RNA expression is partly due to aberrant alternative splicing (AS). However, the AS status of BIN1 and its correlation with BIN1 inactivation in non-small cell lung cancer (NSCLC) remains poorly defined. Here we reported that BIN1 inactivation was not related to DNA methylation in NSCLC. Importantly, BIN1 with exon 12A inclusion (BIN1+12A isoform), the most frequent aberrant splicing variant in tumors was also observed in NSCLC, and might be accounted for BIN1 inactivation. Furthermore, we showed that the aberrant splicing of BIN1 was under the control of serine and arginine-rich factor 1 (SRSF1) in NSCLC. In addition, colony formation assay showed that BIN1+12A isoform could abolish the tumor-inhibiting ability of BIN1 in NSCLC cells. Meanwhile, transwell, wound healing and apoptosis experiments demonstrated that the occurrence of BIN1+12A could abrogate the invasion suppressing activity and proapoptotic property of BIN1 in NSCLC. Significantly, we also found that BIN1+12A isoform neutralized the tumor-suppressing functions of BIN1 via affecting its subcellular localization. Altogether, these data revealed an aberrant splicing phenomenon which abated the expression and tumor-inhibiting activity of BIN1 in NSCLC, and the related mechanisms were associated with SRSF1.

miR-142-5p inhibits the invasion and migration of lung adenocarcinoma H1650 cells by affecting epithelial mesenchymal transformation / 中国肿瘤生物治疗杂志

@# Objective: To study the expression of miR-142-5p in lung adenocarcinoma tissues, and to explore its effect on proliferation, invasion, migration and epithelieal-mesenchymal transition (EMT) of H1650 cells and the potential mechanisms. Methods:Atotal of 107 pairs of lung adenocarcinoma tissues and corresponding para-cancerous tissues from patients, who underwent tumor resection and were pathologically confirmed at the Department of Thoracic Surgery, the Fourth Hospital of Hebei Medical University between Jan. 2014 and Jan. 2015, were collected for this study; in addition, human lung adenocarcinoma cell lines (H1650, HCC827, A549, H1975, PC9) and human bronchial epithelial BEAS-2B cells were also used in this study. qPCR was used to detect the expression of miR-142-5p in lung adenocarcinoma tissues and cell lines. The correlation between expression of miR-142-5p and clinical features was analyzed.After transfection with miR-142-5p mimics or miR-negative control (miR-NC) plasmid, the proliferation, invasion and migration of H1650 cells were detected with CCK-8, Transwell invasion assay and Wound healing assay, respectively. The bioinforamtics tool was used to predict the target genes of miR-142-5p, and Luciferase reporter gene assay was performed to validate the regulation of miR-142-5p on target gene. Western blotting (WB) was used to detect the expressions of cyclin-dependent kinase 5 (CDK5) and EMTrelated protein. Results: Compared to Para-cancerous tissues and BEAS-2B cells, the expression of miR-142-5p was lower in lung adenocarcinoma tissues and cell lines (all P<0.01). Of the 107 cases of lung adenocarcinoma tissues, 61 cases (57.01%) showed decreased miR-142-5 expression, which was correlated with the TNM stage and lymph node metastasis (both P<0.01). Transfection of miR-142-5p mimics significantly up-regulated the expression of miR-142-5p and decreased the proliferation, invasion and migration of H1650 cells (all P<0.05 or P<0.01). Bioinformatics showed that CDK5 was a target gene of miR-142-5p. Luciferase reporter gene assay and WB validated that miR-142-5p could significantly down-regulate CDK5 expression in H1650 cells, up-regulate the expression of E-cadherin and down-regulate the expressions of N-cadherin, Twist and Snail in H1650 cells (all P<0.01). Conclusion: miR-142-5p is low expressed in lung adenocarcinoma tissues and cell lines; it suppresses the EMT process to inhibit, invasion and migration of H1650 cells via down-regulating the expression of CDK5.