COX6C expression driven by copy amplification of 8q22.2 regulates cell proliferation via mediation of mitosis by ROS-AMPK signaling in lung adenocarcinoma.

Copy number variations (CNVs) play a vital role in regulating genes expression and tumorigenesis. We explored the copy number alterations in early-stage lung adenocarcinoma using high-throughput sequencing and nucleic acid flight mass spectrometry technology, and found that 8q22.1-22.2 is frequently amplified in lung adenocarcinoma tissues. COX6C localizes on the region and its expression is notably enhanced that driven by amplification in lung adenocarcinoma. Knockdown of COX6C significantly inhibits the cell proliferation, and induces S-G2/M cell cycle arrest, mitosis deficiency and apoptosis. Moreover, COX6C depletion causes a deficiency in mitochondrial fusion, and impairment of oxidative phosphorylation. Mechanistically, COX6C-induced mitochondrial deficiency stimulates ROS accumulation and activates AMPK pathway, then leading to abnormality in spindle formation and chromosome segregation, activating spindle assemble checkpoint, causing mitotic arrest, and ultimately inducing cell apoptosis. Collectively, we suggested that copy amplification-mediated COX6C upregulation might serves as a prospective biomarker for prognosis and targeting therapy in patients with lung adenocarcinoma.

ARAP1 negatively regulates stress fibers formation and metastasis in lung adenocarcinoma via controlling Rho signaling.

Small GTPases regulate multiple important cellular behaviors and their activities are strictly controlled by a mass of regulators. The dysfunction or abnormal expression of small GTPases or their regulators was frequently observed in various cancers. Here, we analyzed the expression and prognostic correlation of several GTPases and related regulators based on the TCGA database and found that Ankyrin Repeat and PH Domain 1 (ARAP1), a GTPase activating protein (GAP), is reduced in lung adenocarcinoma tissues compared to normal tissues and displays a positive correlation with overall survival (OS) and progression-free survival (PFS) of patients with lung adenocarcinoma. qPCR and western blot verified that ARAP1 is frequently downregulated in lung adenocarcinoma tumor tissues and cancer cells, and its downregulation might be mediated by epigenetic modification. Moreover, metastatic assays showed that overexpression of ARAP1 significantly inhibits metastasis of lung adenocarcinoma in vitro and in vivo. We further demonstrated that Rho signaling inhibition, mediated by RhoGAP activity of ARAP1, majorly contributes to suppressing migration and invasion of lung adenocarcinoma cancer cells via inhibiting stress fibers formation. In summary, this study indicates that ARAP1 may serve as a potential prognostic predictor and a metastatic suppressor in lung adenocarcinoma via its RhoGAP activity.

LncRNA UCA1 promoted cisplatin resistance in lung adenocarcinoma with HO1 targets NRF2/HO1 pathway.

PURPOSE: Our previous experiments have demonstrated that lncRNA UCA1 (UCA1) promoted cisplatin resistance in lung adenocarcinoma (LUAD). This study aimed to explore the potential downstream target genes regulated by UCA1 and how this downstream gene promotes cisplatin resistance in LUAD. METHODS: Here, we measured the expression level of Heme oxygenase1 (HO1) in LUAD cell lines by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based on UCA1 overexpression cell lines and UCA1 knockdown cell lines. HO1 was knocked down in the UCA1 overexpression cell line, and HO1 was overexpressed in the UCA1 knockdown cell line, and the half maximal inhibitory concentration (IC50) trends were observed by adding cisplatin containing a certain concentration gradient. Cell functional assays were performed to observe the changes in the biological behavior of HO1 after overexpression and knockdown, and the tumorigenic assay in nude mice was performed to verify the effect of UCA1 in regulating the growth and cisplatin resistance of HO1 on LUAD cells in vivo. RESULTS: The results showed that HO1 and UCA1 expression were both upregulated in LUAD tissues and LUAD cisplatin-resistant cell lines, and there was a significant positive correlation between the expression of HO1 and UCA1. In vitro experiments showed that HO1 overexpression could reverse the reduced sensitivity to cisplatin caused by UCA1 knockdown in A549/DDP cells, and HO1 knockdown could reduce cisplatin resistance in A549 UCA1 overexpressing cells. Tumorigenic assays in nude mice further confirmed the role of HO1 in the regulation of UCA1 by activating the NRF2/HO1 pathway against LUAD cisplatin resistance. CONCLUSION: Our findings suggested that UCA1 regulates HO1 targets the UCA1/NRF2-HO1 pathway to exert cisplatin resistance in LUAD.

Establishing a metastasis-related diagnosis and prognosis model for lung adenocarcinoma through CRISPR library and TCGA database.

PURPOSE: Existing biomarkers for diagnosing and predicting metastasis of lung adenocarcinoma (LUAD) may not meet the demands of clinical practice. Risk prediction models with multiple markers may provide better prognostic factors for accurate diagnosis and prediction of metastatic LUAD. METHODS: An animal model of LUAD metastasis was constructed using CRISPR technology, and genes related to LUAD metastasis were screened by mRNA sequencing of normal and metastatic tissues. The immune characteristics of different subtypes were analyzed, and differentially expressed genes were subjected to survival and Cox regression analyses to identify the specific genes involved in metastasis for constructing a prediction model. The biological function of RFLNA was verified by analyzing CCK-8, migration, invasion, and apoptosis in LUAD cell lines. RESULTS: We identified 108 differentially expressed genes related to metastasis and classified LUAD samples into two subtypes according to gene expression. Subsequently, a prediction model composed of eight metastasis-related genes (RHOBTB2, KIAA1524, CENPW, DEPDC1, RFLNA, COL7A1, MMP12, and HOXB9) was constructed. The areas under the curves of the logistic regression and neural network were 0.946 and 0.856, respectively. The model effectively classified patients into low- and high-risk groups. The low-risk group had a better prognosis in both the training and test cohorts, indicating that the prediction model had good diagnostic and predictive power. Upregulation of RFLNA successfully promoted cell proliferation, migration, invasion, and attenuated apoptosis, suggesting that RFLNA plays a role in promoting LUAD development and metastasis. CONCLUSION: The model has important diagnostic and prognostic value for metastatic LUAD and may be useful in clinical applications.

Construction and analysis of expression profile of exosomal lncRNAs in pleural effusion in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is a highly malignant tumor with a very low five-year survival rate. In this study, we aimed to identify differentially expressed long-chain non-coding RNA (lncRNAs) and mRNAs from benign and malignant pleural effusion exosomes. METHODS: We used gene microassay and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to detect and verify differentially expressed mRNAs and lncRNAs in benign and malignant pleural effusion exosomes. Gene Ontology (GO) functional significance and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway significance enrichment analyses were performed to identify the difference in biological processes and functions between different mRNAs. We selected the lncRNA ZBED5-AS1 with an upregulated differential fold of 3.003 and conducted a preliminary study on its cellular function. RESULTS: Gene microassay results revealed that 177 differentially expressed lncRNAs were upregulated, and 215 were downregulated. The top 10 upregulated were FMN1, AL118505.1, LINC00452, AL109811.2, CATG00000040683.1, AC137932.1, AC008619.1, AL450344.1, AC092718.6, and ZBED5-AS1. The top 10 downregulated were TEX41, G067726, JAZF1-AS1, AC027328.1, AL445645.1, AL022345.4, AC008572.1, AC123777.1, AC093714.1, and PHKG1. For the mRNAs, 79 were upregulated, and 123 were notably downregulated. GO analysis revealed that the upregulated differential mRNAs were mainly involved in "cellular response to acidic pH" (biological processes), "endoplasmic reticulum part" (cellular components), and "at DNA binding, cyclase activity" (molecular functions). KEGG pathways were found to be related to V. cholerae infection, Parkinson's disease, and cell adhesion molecules. RT-qPCR showed that ZBED5-AS1 was highly expressed in LUAD tissues, cells, and benign and malignant pleural fluid exosomes. Overexpression of ZBED5-AS1 could significantly promote the proliferation, migration, invasion, and colony formation of LUAD cells, and knockdown had the opposite consequence. CONCLUSION: The pleural effusion exosomes from patients with LUAD include several improperly expressed genes, and lncRNA-ZBED5-AS1 is a new biomarker that aids in our understanding of the occurrence and progression of LUAD.

Differential expression and analysis of extrachromosomal circular DNAs as serum biomarkers in lung adenocarcinoma.

BACKGROUND: Extrachromosomal circular DNAs (eccDNAs) increase the number of proto-oncogenes by enhancing oncogene expression to promote tumorigenesis. However, there are limited reports on differential eccDNA expression and analysis in lung cancer, especially in lung adenocarcinoma (LAD). METHODS: Three LAD and three corresponding NT tissues samples were used for eccDNA next-generation sequencing analysis, and an additional 20 were used for quantitative PCR (qPCR) evaluations. We further performed qPCR amplification using serum samples from LAD patients and healthy medical examiners. RESULTS: eccDNAs from LAD samples were mainly 200-1000 bp in length. Gene annotation analysis revealed that most eccDNAs were derived from chromosomes 1 and 2. The top-ten increased and top-ten decreased eccDNAs in LAD tissues were CircD-ARPC1B, CircD-ARPC1A, CircD-FAM49B, CircD-SDK1, CircD-KCNG1, CircD-POLR2F, CircD-SS18L1, CircD-SLC16A3, CircD-CSNK1D, CircD-KCTD1, and CircD-TMIGD2, CircD-PDIA5, CircD-VAV2, CircD-GATAD2A, CircD-CAB39L, CircD-KHDC1, CircD-FOXN3, CircD-SULT2B1, CircD-DPP9, and CircD-CSNK1D. qPCR demonstrated that the expression of CircD-DZRN3 was higher in LAD tissues than in normal lung tissues, whereas CircD-LGR6 and CircD-UMODL1 expression levels were lower in LAD than in normal lung tissues. Furthermore, the serum CircD-PDZRN3 level increased, while CircD-LGR6 decreased in LAD. Receiver operating characteristic (ROC) analysis showed that area under curve (AUC) of serum CircD-PDZRN3 (0.991), CircD-LGR6 (0.916) was higher than that of serum carcinoembryonic antigen (CEA) (0.825), CY211 (cytokeratin 19 fragment) (0.842), SCCA(squamous cell carcinoma antigen) (0.857) for the diagnosis of LAD. CONCLUSIONS: Our study first showed that several eccDNAs were aberrantly expressed in LAD, among which CircD-PDZRN3 and CircD-LGR6 clearly distinguished LAD patients from healthy controls, indicating their potential as biomarkers.

CircRAPGEF5 Promotes the Proliferation and Metastasis of Lung Adenocarcinoma through the miR-1236-3p/ZEB1 Axis and Serves as a Potential Biomarker.

Lung adenocarcinoma (LAD) is a common malignancy; however, its underlying molecular mechanism is unclear. Circular RNAs (circRNAs) serve as significant cancer regulators. The overexpression of circRAPGEF5 in LAD tissues and cells indicated that it may be involved in promoting LAD progression. Analysis of 61 LAD tissues revealed that circRAPGEF5 was related to lymph node metastasis. Functionally, circRAPGEF5 promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition of LAD cells in vitro and promoted LAD cells growth in vivo. Mechanistically, dual-luciferase reporter assays confirmed direct interaction of circRAPGEF5, miR-1236-3p, and ZEB1. miR-1236-3p was upregulated and ZEB1 expression reduced after circRAPGEF5 knockdown, and the proliferation, migration, and invasion of LAD cells was inhibited. circRAPGEF5 was significantly overexpressed in LAD cell exosomes, and co-culture experiments showed that exosomal circRAPGEF5 enhanced the metastatic ability of LAD cells. Further experiments found that serum exosomal circRAPGEF5 was overexpressed in LAD; moreover, the area under the receiver operator characteristic curve of exosomal circRAPGEF5 was superior to that of serum carcinoembryonic antigen (CEA). Jointly detected serum exosomal circRAPGEF5 and serum CEA had better diagnostic performance than when detected individually. Thus, exosomal circRAPGEF5 could promote the proliferation and metastasis of LAD via the miR-1236-3p/ZEB1 axis and serum exosomal circRAPGEF5 may serve as a promising biomarker for LAD.

Mechanistic study of lncRNA UCA1 promoting growth and cisplatin resistance in lung adenocarcinoma.

AIM: This study aimed to explore the mechanism of LncRNA urothelial carcinoma-associated 1 (UCA1) promoting cisplatin resistance in lung adenocarcinoma (LUAD). METHOD: The UCA1 expression level in LUAD cell lines was detected by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). We overexpressed UCA1 in A549 cells and downregulated UCA1 in A549/DDP cells by the lentivirus­mediated technique. Subsequently, in vitro, and in vivo functional experiments were performed to investigate the functional roles of UCA1 in the growth and metastasis of LUAD cell lines. Furthermore, RNA pulldown, mass spectrometry, and RNA immunoprecipitation technique were performed to analyze various downstream target factors regulated by UCA1. RESULTS: The results revealed a higher UCA1 expression level in A549/DDP cells and LUAD tissues than in A549 cells and adjacent cancer tissues. UCA1 expression was significantly associated with distant metastasis, clinical stage, and survival time of patients with LUAD. UCA1 overexpression significantly increased the proliferation, invasion, clone formation, and cisplatin resistance ability and enhanced the expression levels of proliferating cell nuclear antigen and excision repair cross-complementing gene 1 in A549 cells. However, these trends were mostly reversed after the knockdown of UCA1 in A549/DDP cells. Tumorigenic assays in nude mice showed that UCA1 knockdown significantly inhibited tumor growth and reduced cisplatin resistance. Enolase 1 was the RNA-binding protein (RBP) of UCA1. CONCLUSION: Based on the results, we concluded that UCA1 promoted LUAD progression and cisplatin resistance and hence could be a potential diagnostic marker and therapeutic target in patients with LUAD.

Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy.

Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.