miR-29c-3p represses the angiogenesis of esophageal squamous cell carcinoma by targeting SERPINH1 to regulate the Wnt signaling pathway.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is characterized by early metastasis and late diagnosis. miR-29c-3p is confirmed to repress angiogenesis in multiple tumor types. Yet, the functions of miR-29c-3p in the mechanism of ESCC angiogenesis, which were not sufficiently explored previously, were exactly what we investigated here at the molecular level. METHODS: The mRNA level of miR-29c-3p and Serpin peptidase inhibitor clade H member 1 (SERPINH1) in ESCC tissues were assessed via bioinformatics analysis. Thereafter, miR-29c-3p and SERPINH1 (HSP47) mRNA level in ESCC cell lines was evaluated via quantitative real-time polymerase chain reaction. The effects of abnormal miR-29c-3p and SERPINH1 expression on ESCC cell viability, proliferation, migration, invasion, and HUVEC angiogenesis were examined via CCK8, colony formation, transwell, and angiogenesis assays, respectively. The protein levels of SERPINH1, vascular endothelial growth factor-A (VEGFA), Wnt-1, ?-catenin, and p-?-catenin were evaluated via Western blot. Expression of VEGFA secreted by ESCC cells was measured via enzyme-linked immunosorbent assay. Treatment with the Wnt activator BML-284 further revealed the way miR-29c-3p mediated the Wnt signaling pathway and its effects on angiogenesis. RESULTS: Herein, we revealed a decrease of miR-29c-3p expression in ESCC tissues and cells, while the overexpressed miR-29c-3p could remarkably suppress ESCC cell progression, as well as HUVEC angiogenesis. Meanwhile, overexpressed miR-29c-3p notably downregulated VEGFA and repressed the Wnt signaling pathway. Treatment with the Wnt activator BML-284 could reverse the inhibition of HUVEC angiogenesis caused by miR-29c-3p. SERPINH1 was a downstream target of miR-29c-3p. SERPINH1 knockdown suppressed the malignant phenotypes of ESCC cells and impeded the Wnt signaling activation, while such suppression was reversed through miR-29c-3p inhibitor. CONCLUSIONS: We confirmed the mechanism that miR-29c-3p targeted SERPINH1, thus regulating angiogenesis in ESCC through the Wnt signaling pathway. It improves the understanding of angiogenesis in ESCC and offers new ideas for the research of ESCC treatment strategies in the future.

miR-29c-3p represses the angiogenesis of esophageal squamous cell carcinoma by targeting SERPINH1 to regulate the Wnt signaling pathway

Purpose: Esophageal squamous cell carcinoma (ESCC) is characterized by early metastasis and late diagnosis. miR-29c-3p is confirmed to repress angiogenesis in multiple tumor types. Yet, the functions of miR-29c-3p in the mechanism of ESCC angiogenesis, which were not sufficiently explored previously, were exactly what we investigated here at the molecular level. Methods: The mRNA level of miR-29c-3p and Serpin peptidase inhibitor clade H member 1 (SERPINH1) in ESCC tissues were assessed via bioinformatics analysis. Thereafter, miR-29c-3p and SERPINH1 (HSP47) mRNA level in ESCC cell lines was evaluated via quantitative real-time polymerase chain reaction. The effects of abnormal miR-29c-3p and SERPINH1 expression on ESCC cell viability, proliferation, migration, invasion, and HUVEC angiogenesis were examined via CCK8, colony formation, transwell, and angiogenesis assays, respectively. The protein levels of SERPINH1, vascular endothelial growth factor-A (VEGFA), Wnt-1, ?-catenin, and p-?-catenin were evaluated via Western blot. Expression of VEGFA secreted by ESCC cells was measured via enzyme-linked immunosorbent assay. Treatment with the Wnt activator BML-284 further revealed the way miR-29c-3p mediated the Wnt signaling pathway and its effects on angiogenesis. Results: Herein, we revealed a decrease of miR-29c-3p expression in ESCC tissues and cells, while the overexpressed miR-29c-3p could remarkably suppress ESCC cell progression, as well as HUVEC angiogenesis. Meanwhile, overexpressed miR-29c-3p notably downregulated VEGFA and repressed the Wnt signaling pathway. Treatment with the Wnt activator BML-284 could reverse the inhibition of HUVEC angiogenesis caused by miR-29c-3p. SERPINH1 was a downstream target of miR-29c-3p. SERPINH1 knockdown suppressed the malignant phenotypes of ESCC cells and impeded the Wnt signaling activation, while such suppression was reversed through miR-29c-3p inhibitor. Conclusions: We confirmed the mechanism that miR-29c-3p targeted SERPINH1, thus regulating angiogenesis in ESCC through the Wnt signaling pathway. It improves the understanding of angiogenesis in ESCC and offers new ideas for the research of ESCC treatment strategies in the future.

MiR-486-5p specifically suppresses SAPCD2 expression, which attenuates the aggressive phenotypes of lung adenocarcinoma cells.

BACKGROUND: MiR-486-5p expression is restrained in lung adenocarcinoma (LUAD). However, much less has been understood on its role in LUAD. We aimed to explore the biofunctions of miR-486-5p in LUAD. METHODS: A differential expression analysis based on The Cancer Genome Atlas-LUAD dataset was done to screen the differently expressed miRNAs and mRNAs. MiR-486-5p and SAPCD2 mRNA expression was analyzed by qRT-PCR, and protein level of SAPCD2 was assayed by western blot. Upregulation and downregulation of miR-486-5p or SAPCD2 were achieved by cell transfection. For cell function assays, the proliferation of cancer cells was examined by MTT assay. Cell apoptosis was assessed by flow cytometry and microscopy. Transwell assay was applied to evaluate cell migration and invasion. A dual-luciferase detection was employed to determine the miRNA-mRNA targeting relationship. RESULTS: MiR-486-5p expression was notably reduced in LUAD tissue and cell lines. Upregulating miR-486-5p restrained the anti-apoptotic and proliferative abilities, as well as cell migratory and invasive phenotypes in LUAD cells. SAPCD2 was determined as one target of miR-486-5p. Also, SAPCD2 forced expression was able to attenuate the inhibitory impacts of miR-486-5p on the malignant phenotypes of LUAD cells. CONCLUSION: MiR-486-5p suppressed cell malignant progression in LUAD by targeting SAPCD2, suggesting that the two may be targets for LUAD treatment.

Construction of a prognostic risk assessment model for lung adenocarcinoma based on Integrin ß family-related genes.

BACKGROUND: Integrin ß (ITGB) superfamily plays an essential role in the intercellular connection and signal transmission. It was exhibited that overexpressing of ITGB family members promotes the malignant progression of lung adenocarcinoma (LUAD), but the relationship between ITGB superfamily and the LUAD prognosis remains unclear. METHODS: In this study, the samples were assigned to different subgroups utilizing non-negative matrix factorization clustering according to the expression of ITGB family members in LUAD. Kaplan-Meier (K-M) survival analysis revealed the significant differences in the prognosis between different ITGB subgroups. Subsequently, we screened differentially expressed genes among different subgroups and conducted univariate Cox analysis, random forest feature selection, and multivariate Cox analysis. 9-feature genes (FAM83A, AKAP12, PKP2, CYP17A1, GJB3, TMPRSS11F, KRT81, MARCH4, and STC1) in the ITGB superfamily were selected to establish a prognostic assessment model for LAUD. RESULTS: In accordance with the median risk score, LUAD samples were divided into high- and low-risk groups. The receiver operating characteristic (ROC) curve of LUAD patients' survival was predicted via K-M survival curve and principal component analysis dimensionality reduction. This model was found to have a favorable performance in LUAD prognostic assessment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of differentially expressed genes between groups and Gene Set Enrichment Analysis (GSEA) of intergroup samples confirmed that the high- and low-risk groups had evident differences mainly in the function of extracellular matrix (ECM) interaction. Risk score and univariate and multivariate Cox regression analyses of clinical factors showed that the prognostic model could be applied as an independent prognostic factor for LUAD. Then, we draw the nomogram of 1-, 3-, and 5-year survival of LUAD patients predicted with the risk score and clinical factors. Calibration curve and clinical decision curve proved the favorable predictive ability of nomogram. CONCLUSION: We constructed a LUAD prognostic risk model based on the ITGB superfamily, which can provide guidance for clinicians on their prognostic judgment.

Immune-Related Long Non-coding RNA Signature and Clinical Nomogram to Evaluate Survival of Patients Suffering Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) turns out to be one of the most prevalent cancer types, leading to a relatively high mortality among worldwide sufferers. In this study, gene microarray data of ESCC patients were obtained from the GEO database, with the samples involved divided into a training set and a validation set. Based on the immune-related differential long non-coding RNAs (lncRNAs) we identified, a prognostic eight-lncRNA-based risk signature was constructed following regression analyses. Then, the predictive capacity of the model was evaluated in the training set and validation set using survival curves and receiver operation characteristic curves. In addition, univariate and multivariate regression analyses based on clinical information and the model-based risk score also demonstrated the ability of the risk score in independently determining the prognosis of patients. Besides, based on the CIBERSORT tool, the abundance of immune infiltrates in tumor samples was scored, and a significant difference was presented between the high- and low- risk groups. Correlation analysis with immune checkpoints (PD1, PDL1, and CTLA4) indicated that the eight-lncRNA signature-based risk score was negatively correlated with PD1 expression, suggesting that the eight-lncRNA signature may have an effect in immunotherapy for ESCC. Finally, GO annotation was performed for the differential mRNAs that were co-expressed with the eight lncRNAs, and it was uncovered that they were remarkably enriched in immune-related biological functions. These results suggested that the eight-lncRNA signature-based risk model could be employed as an independent biomarker for ESCC prognosis and might play a part in evaluating the response of ESCC to immunotherapy with immune checkpoint blockade.

CtBP2 interacts with ZBTB18 to promote malignancy of glioblastoma.

OBJECTIVE: To investigate how the interaction of CtBP2 with ZBTB18 affect glioblastoma (GBM). METHODS: Western blotting was performed to detect CtBP2 and ZBTB18 expression in GBM and normal brain tissues (NBT). U-87 MG cells were transfected with ZBTB18 CRISPR activation plasmid, CtBP2 shRNA with/without ZBTB18 shRNA. The biological characteristics were detected by EdU assay, MTT, Wound-healing, Transwell, TUNEL staining, and Flow cytometry. Furthermore, U-87 MG cells transfected with CtBP2 shRNA and/or ZBTB18 shRNA were injected into the flank region of mice and the tumor volume was measured. The mRNA and protein expression was quantified by qRT-PCR or Western blotting. RESULTS: GBM tissues exhibited increased CtBP2 expression and decreased ZBTB18 expression, which demonstrated a negative correlation in GBM tissues and showed the combined effect on prognosis. Based on immunoprecipitation and immunofluorescence, there was an interaction between CtBP2 and ZBTB18 in U-87 MG cells. CtBP2 shRNA counteracted the effect of ZBTB18 shRNA on inhibiting U-87 MG cell apoptosis, as well as promoting cell proliferation and viability with increased EMT, invasion and migration. Meanwhile, CtBP2 shRNA interact with ZBTB18 to block cells at phase G0/G1 and suppress SHH-GLI1 pathway. CtBP2 shRNA decreased tumor volume, increase ZBTB18 expression in tumor tissues, and inhibit SHH-GLI1 pathway in mice, which could be reversed by ZBTB18 shRNA. CONCLUSION: CtBP2 elevation and ZBTB18 down-regulation were found in GBM, both of which were associated with prognosis of GBM patients. CtBP2 interacted with ZBTB18 to affect biological characteristics of GBM cells, and the tumor growth, which may be related to the SHH-GLI1 pathway.

The value of CEP55 gene as a diagnostic biomarker and independent prognostic factor in LUAD and LUSC.

OBJECTIVE: To investigate the value of CEP55 as a diagnostic marker and independent prognostic factor in lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), and to analyze its co-expression genes and related signaling pathways. METHODS: TCGA database and GEO database were used to analyze the expression of CEP55 in LUAD and LUSC compared with normal tissues. The co-expression genes of CEP55 in LUAD and LUSC were excavated by cBioPortal and enriched by KEGG and GO. Establishing Receiver operating characteristic (ROC) curve to evaluate the value of CEP55 as a diagnostic and prognostic factor. The association between CEP55 expression and the clinicopathological features was evaluated using χ2 tests. ROC curves for diagnosis and prognosis detection were constructed. Prognostic values were analyzed by univariate and multivariate Cox regression models. RESULTS: Compared with normal lung tissues, CEP55 expression was significantly upregulated in both LUAD and LUSC. ROC curve analysis showed that CEP55 could be used as an effective diagnostic target for LUAD (AUC = 0.969) and LUSC (AUC = 0.994). When CEP55 gene was selected as an independent prognostic factor, high expression of CEP55 was more disadvantageous to OS and RFS of LUAD patients (P<0.05), but no significant difference was found in LUSC patients (P>0.05). The number of co-expression genes of CEP55 in LUAD is more than that in LUSC, and is related to cell cycle, DNA replication and P53 signaling pathway. CONCLUSION: CEP55 can be used as a diagnostic marker for LUAD and LUSC, but only as an independent prognostic factor for LUAD rather than LUSC.

MiR-29c-3p Suppresses the Migration, Invasion and Cell Cycle in Esophageal Carcinoma via CCNA2/p53 Axis.

OBJECTIVE: In the present study, we tried to describe the role of miR-29c-3p in esophageal carcinoma (EC) and the relationship of miR-29c-3p with CCNA2 as well as cell cycle, accordingly revealing the potential molecular mechanism across cell proliferation, migration and invasion. METHODS: Expression profiles of EC miRNAs and matched clinical data were accessed from TCGA database for differential and survival analyses. Bioinformatics databases were employed to predict the downstream targets of the potential miRNA, and enrichment analysis was performed on the miRNA and corresponding target gene using GSEA software. qRT-PCR was conducted to detect the expression levels of miR-29c-3p and CCNA2 mRNA in EC tissues and cells, and Western blot was performed for the examination of CCNA2, CDK1 and p53 protein levels. Subsequently, cells were harvested for MTT, Transwell as well as flow cytometry assays to examine cell viability, migration, invasion and cell cycle. Dual-luciferase reporter gene assay and RIP were carried out to further investigate and verify the targeted relationship between miR-29c-3p and CCNA2. RESULTS: MiR-29c-3p was shown to be significantly down-regulated in EC tissues and able to predict poor prognosis. CCNA2 was found to be a downstream target of miR-29c-3p and mainly enriched in cell cycle and p53 signaling pathway, whereas miR-29c-3p was remarkably activated in cell cycle. MiR-29c-3p overexpression inhibited cell proliferation, migration and invasion, as well as arrested cells in G0/G1 phase. As suggested by dual-luciferase reporter gene assay and RIP, CCNA2 was under the regulation of miR-29c-3p, and the negative correlation between the two genes was verified. Silencing CCNA2 could suppress cell proliferation, migration and invasion, as well as activate p53 pathway, even was seen to reverse the inhibitory effect of PFTß on p53. Besides, in the presence of low miR-29c-3p, CCNA2 was up-regulated while p53 was simultaneously inhibited, resulting in the promotion of cell migration, invasion and cell cycle arrest. CONCLUSION: MiR-29c-3p plays a regulatory role in EC tumorigenesis and development. MiR-29c-3p can target CCNA2 to mediate p53 signaling pathway, finally attributing to the inhibition of cell proliferation, migration and invasion, and making cells arrest in G0/G1 phase.

MicroRNA-30a-3p inhibits the progression of lung cancer via the PI3K/AKT by targeting DNA methyltransferase 3a.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs, involved in pathological and physiological processes via regulating target genes expression. Abnormally expressed miR-30a-3p has been verified in several tumors, such as liver cancer, esophageal cancer and lung cancer. It was reported that DNA methylation plays a critical role in the tumorigenesis of lung cancer through regulated tumor suppressor genes silencing. Nevertheless, the potential mechanism of miR-30a-3p in restoring abnormal DNA methylation patterns is still unclear in lung cancer. Therefore, because the miR-30a-3p is complementary to the 3'-untranslated regions (3'-UTR) of DNA methyltransferase 3A (DNMT3A), we investigated whether miRNA-30a-3p could target DNMT3a to regulate the progression of lung cancer cell. METHODS: qRT-PCR was used to evaluate miR-30a-3p and DNMT3a mRNA expression levels in A549 lung cancer cells and normal cell line BEAS-2B. MiR-30a-3p expression plasmid was transferred into A549 cells. The target of miR-30a-3p was detected by luciferase reporter assay. Western blot was used to measure related protein expression levels. MTT assay was used to measure the proliferation of cells in each group. The cycle and apoptosis of cells were detected by flow cytometry. RESULTS: We found down-regulation of miR-30a-3p mRNA expression and up-regulation of DNMT3a mRNA expression in A549 cells. Overexpression of miR-30a-3p downregulates DNMT3a or blocked DNMT3a by interference vector, significantly inhibited the proliferation and G1/S transition in A549 cells via regulating p38 MAPK pathway, and induced the apoptosis in A549 cells via regulating Bcl-2/Bax protein levels. Furthermore, we observed the opposite phenomenon in A549 cells transfected with both miR-30a-3p and DNMT3a vector. CONCLUSION: Our data show that miR-30a-3p suppressed the progression of lung cancer via regulating p38 MAPK pathway by targeting DNMT3A in A549 cells, indicating that miR-30a-3p might be a novel potential therapeutic strategy in the treatment of lung cancer.

Expression of miR-590 in lung cancer and its correlation with prognosis.

The study aim was to evaluate the association of the expression of serum microribonucleic acid-590 (miR-590) with the risk of lung squamous cell carcinoma (LUSC), clinicopathological staging and prognosis. A total of 237 patients with LUSC and 100 healthy volunteers (control group) were included in the study. Total RNA was extracted from the peripheral blood serum of the subjects, and the expression level of miR-590 was detected by reverse transcription real-time quantitative polymerase chain reaction. The baseline clinicopathological information of LUSC patients was evaluated, and the patients were followed up with the median follow-up of 47 months. Compared with that in the control group, the expression level of serum miR-590 in LUSC patients was significantly decreased [0.532 (0.367- 0.821) vs. 1.63 (0.893-1.347), P<0.001]. The receiver operating characteristic (ROC) curve showed that the value of predicting LUSC risk using miR-590 was high, the area under curve (AUC) was 0.883, and 95% confidence interval (CI) was 0.829-0.934. In addition, the expression level of serum miR-590 was correlated with pathological staging (P=0.022), lymph node metastasis (P=0.012), distant metastasis (P<0.001) and tumor, node and metastasis (TNM) staging (P=0.044). The overall survival (OS) of patients in the serum miR-590 low expression group was significantly lower than that of the serum miR-590 high expression group (P=0.012), and the low expression of miR-590 was an independent risk factor for the prognosis of patients [hazard ratio (HR)=2.152, 95% CI=1.285-3.233, P=0.004]. The results suggested that the expression level of miR-590 can be used as a biomarker for the risk of disease, disease staging and prognosis of LUSC patients.