Comprehensive analysis of prognostic value and immune infiltration of Regulator of Chromosome Condensation 2 in lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) incidence and mortality take the leading place of most malignancies. Previous studies have revealed the regulator of chromosome condensation 1 (RCC1) family members played an essential role during tumorigenesis. However, its biological functions in LUAD still need further investigation. Methods: Several databases were applied to explore potential effects of RCC1 family members on LUAD, such as Oncomine, GEPIA, and cBioPortal. Real-time PCR and immunohistochemistry were used to verify the expression of RCC2 in stage I LUAD. H1975 and A549 were selected to explore the biological function of RCC2 in cellular malignant phenotype. Results: The expressions of RCC1 and RCC2 showed marked differences in malignant tissue compared to lung tissue. The higher the expression levels of RCC1 or RCC2 in LUAD patients, the shorter their overall survival (OS). In normal lung tissues, RCC1 expression was highly enriched in alveolar cells and endothelial cells. Compare with RCC1, RCC2 expression in normal lung tissue was significantly enriched in macrophages, B cells and granulocytes. Additionally, RCC2 expression level was correlated with multiple immune cell infiltration in LUAD. Moreover, the mutation or different sCNA status of RCC2 exerted influence on multiple immune cell infiltration distribution. We found that the upregulation of RCC1 and RCC2 were obviously related to TP53 mutation. GSEA analysis revealed that RCC2 was involved in the process of DNA replication, nucleotide excision repair and cell cycle, which might affect tumor progression through P53 signaling pathway. We further elucidated that downregulation of RCC2 could dramatically repress the migration and invasion of LUAD cells. Conclusions: The study demonstrated that RCC1 and RCC2 expression were markedly increased in early-stage of LUAD. Patients with high expression of RCC1 or RCC2 had a worse prognosis. Based on our analysis, RCC1 and RCC2 might exert influence on LUAD process through DNA replication, nucleotide excision repair and cell cycle, as well as cells migration and invasion. Different from RCC1, RCC2 also involved in immune infiltration. These analyses provided a novel insight into the identification of diagnostic biomarker.

Identification of Small Airway Epithelium-Related Hub Genes in Chronic Obstructive Pulmonary Disease.

Background: Pulmonary small airway epithelia are the primary site of cellular and histological alterations in chronic obstructive pulmonary disease (COPD), while the potential therapeutic hub genes of pulmonary epithelia are rarely identified to elucidate profound alterations in the progression of the disease. Methods: Microarray dataset of GSE11906 containing small airway epithelia from 34 healthy non-smokers and 33 COPD patients was applied to screen differentially expressed genes (DEGs). Weighted gene correlation network analysis (WGCNA) was further used to identify the hub genes related to clinical features. Moreover, single-cell RNA sequencing data from GSE173896 and GSE167295 dataset were applied to explore the expression and distribution of the hub genes. The expression levels of hub genes in epithelial cells stimulated by cigarette smoke extract (CSE) were detected by RT-qPCR. Results: Ninety-eight DEGs correlated with clinical features of COPD were identified via limma and WGCNA. Eight hub genes (including AKR1C3, ALDH3A1, AKR1C1, CYP1A1, GPX2, CBR3, AKR1B1 and GSR) that might exert an antioxidant role in COPD process were identified. Single-cell transcriptomic analysis indicated that the expressions of AKRAC3, ALDH3A1, GPX2, CBR3 and AKR1B1 were significantly increased in the COPD group when compared with the normal group. Moreover, we found that the expression of ALDH3A1 was the most abundantly expressed in ciliated cells. RT-qPCR results indicated that the majority of candidate novel genes were significantly elevated when the epithelial cells were exposed to CSE. Conclusion: Through integrating limma, WGCNA, and protein-protein interaction (PPI) analysis, a total of eight candidate hub genes of pulmonary airway epithelia were identified in COPD. Moreover, single-cell transcriptomic analysis indicated that ALDH3A1 was enriched in ciliated cells, which may provide a new insight into the pathogenesis and treatment of COPD.

Integrated profiling of endoplasmic reticulum stress-related DERL3 in the prognostic and immune features of lung adenocarcinoma.

Background: DERL3 has been implicated as an essential element in the degradation of misfolded lumenal glycoproteins induced by endoplasmic reticulum (ER) stress. However, the correlation of DERL3 expression with the malignant phenotype of lung adenocarcinoma (LUAD) cells is unclear and remains to be elucidated. Herein, we investigated the interaction between the DERL3 and LUAD pathological process. Methods: The Cancer Genome Atlas (TCGA) database was utilized to determine the genetic alteration of DERL3 in stage I LUAD. Clinical LUAD samples including carcinoma and adjacent tissues were obtained and were further extracted to detect DERL3 mRNA expression via RT-qPCR. Immunohistochemistry was performed to evaluate the protein expression of DERL3 in LUAD tissues. The GEPIA and TIMER website were used to evaluate the correlation between DERL3 and immune cell infiltration. We further used the t-SNE map to visualize the distribution of DERL3 in various clusters at the single-cell level via TISCH database. The potential mechanisms of the biological process mediated by DERL3 in LUAD were conducted via KEGG and GSEA. Results: It was indicated that DERL3 was predominantly elevated in carcinoma compared with adjacent tissues in multiple kinds of tumors from the TCGA database, especially in LUAD. Immunohistochemistry validated that DERL3 was also upregulated in LUAD tissues compared with adjacent tissues from individuals. DERL3 was preliminarily found to be associated with immune infiltration via the TIMER database. Further, the t-SNE map revealed that DERL3 was predominantly enriched in plasma cells of the B cell population. It was demonstrated that DERL3 high-expressed patients presented significantly worse response to chemotherapy and immunotherapy. GSEA and KEGG results indicated that DERL3 was positively correlated with B cell activation and unfolded protein response (UPR). Conclusion: Our findings indicated that DERL3 might play an essential role in the endoplasmic reticulum-associated degradation (ERAD) process in LUAD. Moreover, DERL3 may act as a promising immune biomarker, which could predict the efficacy of immunotherapy in LUAD.

KCNK3 inhibits proliferation and glucose metabolism of lung adenocarcinoma via activation of AMPK-TXNIP pathway.

Non-small cell lung cancer (NSCLC) is a primary histological subtype of lung cancer with increased morbidity and mortality. K+ channels have been revealed to be involved in carcinogenesis in various malignant tumors. However, TWIK-related acid-sensitive potassium channel 1 (TASK-1, also called KCNK3), a genetic member of K2P channels, remains an enigma in lung adenocarcinoma (LUAD). Herein, we investigated the pathological process of KCNK3 in proliferation and glucose metabolism of LUAD. The expressions of KCNK3 in LUAD tissues and corresponding adjacent tissues were identified by RNA sequencing, quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry. Gain and loss-of-function assays were performed to estimate the role of KCNK3 in proliferation and glucose metabolism of LUAD. Additionally, energy metabolites of LUAD cells were identified by targeted metabolomics analysis. The expressions of metabolic molecules and active biomarkers associated with AMPK-TXNIP signaling pathway were detected via western blot and immunofluorescence. KCNK3 was significantly downregulated in LUAD tissues and correlated with patients' poor prognosis. Overexpression of KCNK3 largely regulated the process of oncogenesis and glycometabolism in LUAD in vitro and in vivo. Mechanistic studies found that KCNK3-mediated differential metabolites were mainly enriched in AMPK signaling pathway. Furthermore, rescue experiments demonstrated that KCNK3 suppressed proliferation and glucose metabolism via activation of the AMPK-TXNIP pathway in LUAD cells. In summary, our research highlighted an emerging role of KCNK3 in the proliferative activity and glycometabolism of LUAD, suggesting that KCNK3 may be an optimal predictor for prognosis and a potential therapeutic target of LUAD.

C1QTNF6 regulated by miR-29a-3p promotes proliferation and migration in stage I lung adenocarcinoma.

OBJECTIVE: C1QTNF6 has been implicated as an essential component in multiple cellular and molecular preliminary event, including inflammation, glucose metabolism, endothelial cell modulation and carcinogenesis. However, the biological process and potential mechanism of C1QTNF6 in lung adenocarcinoma (LUAD) are indefinite and remain to be elucidated. Therefore, we investigated the interaction among the traits of C1QTNF6 and LUAD pathologic process. METHODS: RT-qPCR and western blot were conducted to determine the expression levels of C1QTNF6. RNA interference and overexpression of C1QTNF6 were constructed to identify the biological function of C1QTNF6 in cellular proliferative, migratory and invasive potentials in vitro. Dual-luciferase reporter assay was applied to identify the possible interaction between C1QTNF6 and miR-29a-3p. Moreover, RNA sequencing analysis of C1QTNF6 knockdown was performed to identify the potential regulatory pathways. RESULTS: C1QTNF6 was upregulated in stage I LUAD tissues compared with adjacent non-cancerous tissues. Concurrently, C1QTNF6 knockdown could remarkably inhibit cell proliferation, migratory and invasive abilities, while overexpression of C1QTNF6 presented opposite results. Additionally, miR-29a-3p may serve as an upstream regulator of C1QTNF6 and reduce the expression of C1QTNF6. Subsequent experiments showed that miR-29a-3p could decrease the cell mobility and proliferation positive cell rates, as well as reduce the migratory and invasive possibilities in LUAD cells via downregulating C1QTNF6. Moreover, RNA sequencing analysis demonstrated that the cytokine-cytokine receptor interaction pathway may participate in the process of C1QTNF6 regulating tumor progression. CONCLUSION: Our study first demonstrated that downregulation of C1QTNF6 could inhibit tumorigenesis and progression in LUAD cells negatively regulated by miR-29a-3p. These consequences could reinforce our awareness and understanding of the underlying mechanism and provide a promising therapeutic target for LUAD.

An integrated analysis of the competing endogenous RNA network associated of prognosis of stage I lung adenocarcinoma.

BACKGROUND: Accumulating evidence indicates that long non-coding RNAs (lncRNAs) are involving in the tumorigenesis and metastasis of lung cancer. The aim of the study is to systematically characterize the lncRNA-associated competing endogenous RNA (ceRNA) network and identify key lncRNAs in the development of stage I lung adenocarcinoma (LUAD). METHODS: Totally, 1,955 DEmRNAs, 165 DEmiRNAs and 1,107 DElncRNAs were obtained in 10 paired normal and LUAD tissues. And a total of 8,912 paired lncRNA-miRNA-mRNA network was constructed. Using the Cancer Genome Atlas (TCGA) dataset, the module of ME turquoise was revealed to be most relevant to the progression of LUAD though Weighted Gene Co-expression Network Analysis (WGCNA). RESULTS: Of the lncRNAs identified, LINC00639, RP4-676L2.1 and FENDRR were in ceRNA network established by our RNA-sequencing dataset. Using univariate Cox regression analysis, FENDRR was a risk factor of progression free survival (PFS) of stage I LUAD patients (HRs = 1.69, 95%CI 1.07-2.68, P < .050). Subsequently, diffe rential expression of FENDRR in paired normal and LUAD tissues was detected significant by real-time quantitative (qRT-PCR) (P < 0.001). CONCLUSIONS: This study, for the first time, deciphered the regulatory role of FENDRR/miR-6815-5p axis in the progression of early-stage LUAD, which is needed to be established in vitro and in vivo.

Identification of a ZC3H12D-regulated competing endogenous RNA network for prognosis of lung adenocarcinoma at single-cell level.

BACKGROUND: To identify hub genes from the competing endogenous RNA (ceRNA) network of lung adenocarcinoma (LUAD) and to explore their potential functions on prognosis of patients from a single-cell perspective. METHODS: We performed RNA-sequencing of LUAD to construct ceRNA regulatory network, integrating with public databases to identify the vital pathways related to patients' prognosis and to reveal the expression level of hub genes under different conditions, the functional enrichment of co-expressed genes and their potential immune-related mechanisms. RESULTS: ZC3H12D-hsa-miR-4443-ENST00000630242 axis was found to be related with LUAD. Lower ZC3H12D expression was significantly associated with shorter overall survival (OS) of patients (HR = 2.007, P < 0.05), and its expression was higher in early-stage patients, including T1 (P < 0.05) and N0 (P < 0.05). Additionally, ZC3H12D expression was higher in immune cells displayed by single-cell RNA-sequencing data, especially in Treg cells of lung cancer and CD8 T cells, B cells and CD4 T cells of LUAD. The functional enrichment analysis showed that the co-expressed genes mainly played a role in lymphocyte activation and cytokine-cytokine receptor interaction. In addition, ZC3H12D was associated with multiple immune cells and immune molecules, including immune checkpoints CTLA4, CD96 and TIGIT. CONCLUSION: ZC3H12D-hsa-miR-4443-ENST00000630242 ceRNA network was identified in LUAD. ZC3H12D could affect prognosis of patients by regulating mRNA, miRNA, lncRNA, immune cells and immune molecules. Therefore, it may serve as a vital predictive marker and could be regarded as a potential therapeutic target for LUAD in the future.

Comprehensive Analysis of Aquaporin Superfamily in Lung Adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is the most predomintnt lung cancer subtype with increasing morbidity and mortality. Previous studies have shown that aquaporin (AQP) family genes were correlated with tumor progression and metastasis in several kinds of malignancies. However, their biological behaviors and prognostic values in LUAD have not been comprehensively elucidated. Methods: RNA sequencing and real-time reverse transcription PCR (RT-PCR) were used to assess AQP1/3/4/5 gene expressions in LUAD patients using GEPIA and UALCAN databases. And then Kaplan-Meier analysis, cBioPortal, Metascape, GeneMANIA, TISIDB, and TIMER were utilized to determine the prognostic value, mutation frequency, and immune cell infiltration of AQP family members in LUAD. Results: We found that AQP3 expression was significantly elevated and AQP1 expression was markedly reduced in LUAD patients, whereas the expression levels of AQP4 and AQP5 exhibited no significant changes. The Kaplan-Meier survival analysis indicated that the higher expressions of AQP1/4/5 were related to longer overall survival (OS). Of interest, AQP3 was significantly correlated with the clinical tumor stage and lower AQP3 expression showed favorable prognosis in stage I LUAD patients, which indicated that AQP3 may be a potential prognostic biomarker for patients. Through functional enrichment analysis, the functions of these four AQPs genes were mainly involved in the passive transport by aquaporins, water homeostasis, and protein tetramerization. Moreover, AQP1/3/4/5 expression was strongly associated with tumor-infiltrating lymphocytes (TILs) in LUAD. Conclusion: AQP3 can be used as a prognosis and survival biomarker for stage I LUAD. These findings may provide novel insights into developing molecular targeted therapies in LUAD.