Identification of necroptosis-associated miRNA signature for predicting prognosis and immune landscape in stomach adenocarcinoma.

PURPOSE: This study aims to identify the potential necroptosis related genes (NRGs)-associated miRNAs signature and explore the impact on the prognosis of stomach adenocarcinoma (STAD). METHODS: Employing rigorous methodologies, we utilized univariate Cox, Lasso and multivariate Cox regression analyses to develop a prognostic signature. Kaplan-Meier (K-M) and ROC curves were applied to assess the prognostic value of signature in a training group and an independent test group. Furthermore, we conducted Gene Set Enrichment Analysis (GSEA) for enrichment of tumor-related pathways. The risk score was calculated for each patient based on the expression of miRNAs which were enrolled in the signature. Patients were stratified into high- and low-risk groups. The immune cell infiltration and immunotherapy were compared between the two groups. Finally, the diagnostic potential of the miRNA was explored by RT-qPCR. RESULTS: We constructed a prognostic model based on 6 NRGs-associated miRNAs. K-M plots underscored superior survival outcomes in the low-risk group. GSEA results revealed the enrichment of several tumor-related pathways in the high-risk group. Notably, CD8+ T cells, Tregs and activated memory CD4+ T cells exhibited negative correlations with the risk score. Additionally, a few immune checkpoint genes, such as CTLA4, PD1 and PD-L1, were significantly upregulated in the low-risk group. Furthermore, the serum expression levels of all these 6 miRNAs were significantly elevated in STAD patients. CONCLUSIONS: Our study identified a robust risk score derived from a signature of 6 NRGs-associated miRNAs, demonstrating high efficacy for prognosis of STAD. These results not only contributed to our understanding of STAD pathogenesis, but also held promise for potential clinical applications, particularly in the realm of personalized immunotherapy for STAD patients.

Comprehensive analysis of cuproptosis-related long noncoding RNA for predicting prognostic and diagnostic value and immune landscape in colorectal adenocarcinoma.

BACKGROUND: Cuproptosis, as a copper-induced mitochondrial cell death, has attracted extensive attention recently, especially in cancer. Although some key regulatory genes have been identified in cuproptosis, the related lncRNAs have not been further studied. Exploring the prognostic and diagnostic value of cuproptosis-related lncRNAs (CRLs) in colon adenocarcinoma and providing guidance for individualized immunotherapy for patients are of great significance. RESULTS: A total of 2003 lncRNAs were correlated with cuproptosis genes and considered as CRLs. We screened 33 survival-associated CRLs and established a prognostic signature base on 7 CRLs in the training group. The patients in the low-risk group had better outcomes in both training group (P < 0.001) and test group (P = 0.016). More exciting, our model showed good prognosis prediction in both stage I-II (P = 0.020) and stage III-IV (P = 0.001). The nomogram model could further improve the accuracy of prognosis prediction. Interestingly, glucose-related metabolic pathways, which were closely related to cuproptosis, were enriched in the low-risk group. Meanwhile, the immune infiltration scores were lower in the high-risk group. The high-risk group was more sensitive to OSI.906 and ABT.888, while low-risk group was more sensitive to Sorafenib. Three lncRNAs, FALEC, AC083967.1 and AC010997.4, were highly expressed in serum of COAD patients, and the AUC was 0.772, 0.726 and 0.714, respectively, indicating their valuable diagnostic value. CONCLUSIONS: Our research constructed a prognostic signature based on 7 CRLs and found three promising diagnostic markers for COAD patients. Our results provided a reference to the personalized immunotherapy strategies.

Integrin alpha 6 is upregulated and drives hepatocellular carcinoma progression through integrin α6ß4 complex.

Integrin α6 (ITGA6) forms integrin receptors with either integrin ß1 (ITGB1) or integrin ß4 (ITGB4). How it functions to regulate hepatocellular carcinoma (HCC) progression is not well-elucidated. We found that ITGA6 RNA and protein expression levels are significantly elevated in human HCC tissues in comparison with paired adjacent nontumor tissues by RNA sequencing, RT-qPCR, Western blotting and immunofluorescence staining. Stable knockdown of ITGA6 with different ITGA6 shRNA expression lentivectors significantly inhibited proliferation, migration and anchorage-independent growth of HCC cell lines in vitro, and xenograft tumor growth in vivo. The inhibition of anchorage-dependent and -independent growth of HCC cell lines was also confirmed with anti-ITGA6 antibody. ITGA6 knockdown was shown to induce cell-cycle arrest at G0/G1 phase. Immunoprecipitation assay revealed apparent interaction of ITGA6 with ITGB4, but not ITGB1. Expression studies showed that ITGA6 positively regulates the expression of ITGB4 with no or negative regulation of ITGB1 expression. Finally, while high levels of ITGA6 and ITGB4 together were associated with significantly worse survival of HCC patients in TCGA data set, the association was not significant for high levels of ITGA6 and ITGB1. In conclusion, ITGA6 is upregulated in HCC tumors and has a malignant promoting role in HCC cells through integrin α6ß4 complex. Thus, integrin α6ß4 may be a therapeutic target for treating patients with HCC.

Down-expression of miR-152 lead to impaired anti-tumor effect of NK via upregulation of HLA-G.

It is known that chronic HBV infection (CHB) is the major risk factor for hepatocellular carcinoma (HCC) because CHB could not only cause liver tumorigenesis but also lead to change of local microenviroment and lower immune response to infected and cancerous cells (immune tolerance). Human leucocyte antigen-G (HLA-G) belongs to a non-classic MHC-I family and was considered to be an immune tolerance molecule, which could bind to immunosuppressive receptors of natural killer cell (NK) and T cells and trigger immunosuppressive signaling. Recently, numerous studies highlighted that microRNAs (miRNAs) were significantly differentially expressed in HCC tumorigenesis, and the expression was tissue-specific, indicating that miRNAs may cause great epigenetic changes in HCC tumorigenesis. In this study, we found that the expression of HLA-G was upregulated by hepatitis B virus (HBV) infection and miR-152; a HLA-G-targeting miRNA was downregulated by HBV infection. And high expression of HLA-G further suppressed NK against cancer cells, providing a new concept that miR-152 was involved in HBV-induced hepatocellular carcinoma.

Serum microRNA expression signatures identified from genome-wide microRNA profiling serve as novel noninvasive biomarkers for diagnosis and recurrence of bladder cancer.

Recent advantages of serum microRNAs (miRNAs) open a new realm of possibilities for noninvasive diagnosis and prognosis of bladder cancer (BC). The aim of our study was to identify serum miRNA expression signatures in patients with BC and establish new models for the diagnosis of BC and recurrence prediction. We performed genome-wide serum miRNA analysis by Miseq sequencing followed by evaluations in the training and validation sets with reverse transcription quantitative real-time PCR assays from serum samples of 250 patients with BC and 240 controls. A six-miRNA panel (miR-152, miR-148b-3p, miR-3187-3p, miR-15b-5p, miR-27a-3p and miR-30a-5p) for the diagnosis of BC was finally developed by multivariate logistic regression model with an area under the receiver operating characteristic curve of 0.899. The corresponding sensitivities of this panel for Ta, T1 and T2-T4 were 90.00, 84.85 and 89.36%, significantly higher than those of urine cytology, which were 13.33, 30.30 and 44.68%, respectively (all at p<0.001). In addition, Kaplan-Meier analysis showed that patients with nonmuscle-invasive BC (NMIBC) with high miR-152 level and low miR-3187-3p level had worse recurrence-free survival (p=0.023 and 0.043, respectively). In multivariate Cox regression analysis, miR-152 was independently associated with tumor recurrence of NMIBC (p=0.028). Our results suggested that a serum miRNA signature may have considerable clinical value in diagnosing BC. Furthermore, expression level of serum miR-152 could provide information on the recurrence risk of NMIBC.

Investigation of cell free BIRC5 mRNA as a serum diagnostic and prognostic biomarker for colorectal cancer.

BACKGROUND: BIRC5 (Survivin), a key member of inhibitor of apoptosis family, has been shown in colorectal cancer (CRC) tumorigenesis and progress. This study investigated the expression levels of cell free BIRC5 mRNA in serum of CRC and assess its diagnostic and prognostic potential. METHODS: Levels of cell free BIRC5 mRNA were detected by reverse transcription quantitative real-time PCR in serum of 92 CRC patients and 60 healthy volunteers. RESULTS: Cell free BIRC5 mRNA levels were significantly increased in serum of CRC (P < 0.001), and significantly correlated with tumor differentiation (P = 0.035), regional lymph node metastasis (P < 0.001) and TNM stage (P < 0.001). ROC curve demonstrated an optimal cut-off value of 0.128, providing a sensitivity of 84.8% and a specificity of 80.0% for discriminating CRC from controls. The area under the ROC curve for BIRC5 mRNA was significantly larger than that for CEA (0.855 vs. 0.691, P < 0.001). Furthermore, a significantly higher diagnosis capability was showed when combined BIRC5 mRNA and CEA. High serum BIRC5 mRNA expression has a lower OS, compared with low group (36.4% vs. 73.3%, P = 0.022), and was an independent prognostic factor for CRC. CONCLUSION: Serum cell free BIRC5 mRNA is a promising non-invasive biomarker for diagnosis and prognosis of CRC.

Naringenin nanoparticles targeting cyclin B1 suppress the progression of rheumatoid arthritis-associated lung cancer by inhibiting fibroblast-to-myofibroblast transition.

There is growing evidence of an elevated risk of lung cancer in patients with rheumatoid arthritis. The poor prognosis of rheumatoid arthritis-associated lung cancer and the lack of therapeutic options pose an even greater challenge to the clinical management of patients. This study aimed to identify potential molecular targets associated with the progression of rheumatoid arthritis-associated lung cancer and examine the efficacy of naringenin nanoparticles targeting cyclin B1. Mendelian randomizatio analysis revealed that rheumatoid arthritis has a positive correlation with the risk of lung cancer. Cyclin B1 was significantly upregulated in patients with rheumatoid arthritis-associated lung cancer and was significantly overexpressed in synovial tissue fibroblasts. Furthermore, the overexpression of cyclin B1 in rheumatoid arthritis fibroblast-like synoviocytes, which promotes their proliferation and fibroblast-to-myofibroblast transition, can significantly contribute to the growth and infiltration of lung cancer cells. Importantly, our prepared naringenin nanoparticles targeting cyclin B1 effectively attenuated proliferation and fibroblast-to-myofibroblast transition by blocking cells at the G2/M phase. In vivo experiments, naringenin nanoparticles targeting cyclin B1 significantly alleviated the development of collagen-induced arthritis and lung orthotopic tumors. Collectively, our results reveal that naringenin nanoparticles targeting cyclin B1 can suppress the progression of rheumatoid arthritis-associated lung cancer by inhibiting fibroblast-to-myofibroblast transition. These findings provide new insights into the treatment of rheumatoid arthritis-associated lung cancer therapy.

STEAP2 promotes hepatocellular carcinoma progression via increased copper levels and stress-activated MAP kinase activity.

Six Transmembrane Epithelial Antigen of Prostate 2 (STEAP2) belongs to a family of metalloreductases, which indirectly aid in uptake of iron and copper ions. Its role in hepatocellular carcinoma (HCC) remains to be characterized. Here, we report that STEAP2 expression was upregulated in HCC tumors compared with paired adjacent non-tumor tissues by RNA sequencing, RT-qPCR, Western blotting, and immunostaining. Public HCC datasets demonstrated upregulated STEAP2 expression in HCC and positive association with tumor grade. Transient and stable knockdown (KD) of STEAP2 in HCC cell lines abrogated their malignant phenotypes in vitro and in vivo, while STEAP2 overexpression showed opposite effects. STEAP2 KD in HCC cells led to significant alteration of genes associated with extracellular matrix organization, cell adhesion/chemotaxis, negative enrichment of an invasiveness signature gene set, and inhibition of cell migration/invasion. STEAP2 KD reduced intracellular copper levels and activation of stress-activated MAP kinases including p38 and JNK. Treatment with copper rescued the reduced HCC cell migration due to STEAP2 KD and activated p38 and JNK. Furthermore, treatment with p38 or JNK inhibitors significantly inhibited copper-mediated cell migration. Thus, STEAP2 plays a malignant-promoting role in HCC cells by driving migration/invasion via increased copper levels and MAP kinase activities. Our study uncovered a novel molecular mechanism contributing to HCC malignancy and a potential therapeutic target for HCC treatment.

Up-regulation of miR-182 expression in colorectal cancer tissues and its prognostic value.

PURPOSE: Accumulating evidences indicate that dysregulated microRNAs (miRNA) are involved in cancer tumorigenesis and progression. In the present study, we evaluated the expression of miR-182 in colorectal cancer and adjacent noncancerous tissues and explored its associations with clinicopathological characteristics and prognosis. METHODS: Quantitative real-time PCR was used to analyze the expression of miR-182 in 148 pairs of colorectal cancer and adjacent noncancerous tissues. The relationship between miR-182 expression and clinicopathological characteristics in colorectal cancer tissues was estimated using Mann-Whitney U test or Kruskal-Wallis test, as appropriate. We calculated the survival curves and prognostic values of each variable by the Kaplan-Meier method and Cox proportional hazards regression analysis, respectively. RESULTS: The expression of miR-182 was found up-regulated in colorectal cancer tissues compared with adjacent noncancerous tissues (p < 0.001), and its up-regulation was significantly correlated with large tumor size (p = 0.016), positive regional lymph node metastasis (p = 0.008), and advanced tumor-node-metastasis stage (p = 0.020). Furthermore, Kaplan-Meier analysis demonstrated that high miR-182 expression predicted poor survival (p = 0.001), and Cox proportional hazards risk analysis indicated that miR-182 was an independent prognostic factor for colorectal cancer. CONCLUSIONS: MiR-182 was up-regulated in colorectal cancer tissues and correlated with adverse clinical characteristics and poor prognosis, indicating that miR-182 might be involved in colorectal cancer progression and could be used as a potential prognostic biomarker and therapeutic target in the management of colorectal cancer.

Comprehensive analysis of m6A regulators and relationship with tumor microenvironment, immunotherapy strategies in colorectal adenocarcinoma.

BACKGROUND: The N6-methyladenosine (m6A) RNA modification is the most prevalent and abundant type found in eukaryotic cells. It plays a crucial role in the initiation and progression of cancers. In this study, we aimed to comprehensively investigate the landscape of m6A regulators and their association with tumor microenvironment (TME), immunotherapeutic strategies in colon adenocarcinoma (COAD). RESULTS: The differential expression, mutation, CNV frequency and prognostic value of 27 m6A regulators were systematically analyzed in COAD. Patients were classified into two clusters based on m6A regulators through consistent clustering analysis, with cluster A showing significant survival benefits. Most of the m6A regulators were negatively correlated with immune cells, except for WTAP, IGF2BP3, FTO, ALKBH5, which showed a positive correlation. We developed an m6A scoring system to calculate the m6Ascore for each patient. Patients with a high-m6Ascore had a better outcome, with the AUC of 0.775. An independent cohort of 416 COAD patients acquired from GSE38832 database was used to validate the prognosis prediction ability of m6Ascore. Moreover, the m6Ascore was negatively correlated with infiltration of anti-tumor immune cells. Additionally, patients with a high-m6Ascore responded better to anti-PD1 and anti-CTLA4 therapies, and those with MSI-H had a higher m6Ascore. Finally, we investigated the value of m6Ascore in predicting the response of patients to 15 commonly used drugs. CONCLUSIONS: We comprehensively analyzed m6A regulators in COAD, including RNA expression, CNV changes, mutations and their correlation with TME. Our results showed that the m6A scoring system had significant predictive power for the prognosis of COAD patients, potentially leading to new personalized immunotherapy strategies.

Detection of circulating Bmi-1 mRNA in plasma and its potential diagnostic and prognostic value for uterine cervical cancer.

Bmi-1 is overexpressed in uterine cervical cancer (UCC) and is found to be associated with adverse clinical characteristics and poor prognosis. However, little information is available on the status of circulating Bmi-1 mRNA in UCC. Because circulating cell-free nucleic acids have emerged as a novel class of markers for cancer detection, our research aims to address this question by detecting the circulating Bmi-1 mRNA and to assess its diagnostic and prognostic potential in UCC. Reverse transcription quantitative real-time PCR method was established to detect the circulating Bmi-1 mRNA in plasma of 109 patients with UCC, 138 patients with cervical intraepithelial neoplasia (CIN) and 80 healthy volunteers, and found that it was significantly increased in UCC compared with CINs and healthy controls (all at p < 0.001). Moreover, its high level was significantly correlated with advanced clinical stage (p < 0.001) and positive lymph nodes metastasis (p = 0.002). The area under the receiver operating characteristic curve (AUC) was 0.881, and the optimal cut-off value was 0.057, providing a sensitivity of 69.7% and a specificity of 95.9%. The AUC for circulating Bmi-1 mRNA showed higher diagnosis capability than that for SCC-Ag (p = 0.035) or CA125 (p < 0.001) currently utilized. Kaplan-Meier analysis demonstrated a correlation between increased circulating Bmi-1 mRNA level and reduced disease-free survival (DFS) (p = 0.001) and overall survival (OS) (p = 0.015). And, Cox analysis indicated that it was an independent prognostic factor for DFS and OS. We conclude that circulating Bmi-1 mRNA may be a potential noninvasive molecular marker for diagnosis and prognosis of UCC.

Effects of subcutaneous low molecular weight heparin and intravenous unfractionated heparin on serum S100 concentrations in patients with cerebrovascular diseases.

BACKGROUND: S100 plays an important role in diagnosis and prognosis of acute ischemic stroke. The objective was to investigate the effects of clinical heparins administrations on serum S100 concentrations in patients with cerebrovascular diseases. METHODS: Forty cases were enrolled in this study, including 10 patients with percutaneous carotid intervention receiving intravenous unfractionated heparin (UFH) at the beginning of stenting (group A1), 10 patients with carotid angiography receiving UFH-free therapy (group A2), 10 patients with cerebral infarction receiving subcutaneous low molecular weight heparin (LMWH) anticoagulation therapy (group B1) and 10 patients with cerebral infarction receiving LMWH-free therapy (group B2). S100 concentrations were analyzed before and after heparin administrations. RESULTS: A rapid increase of serum S100 (median, 1.74-fold, p<0.05) was observed in group A1 within 15 min of UFH administration. In group B1, S100 significantly increased (median, 1.44-fold, p<0.05) at 3 h after LMWH injection. CONCLUSIONS: Both intravenous UFH and subcutaneous LMWH administration induced increases in serum S100 concentrations. It should be taken into account when using S100 as a biomarker of cerebrovascular diseases.

Comprehensive characterization of 11 prognostic alternative splicing events in ovarian cancer interacted with the immune microenvironment.

Alternative splicing (AS) events play a crucial role in the tumorigenesis and progression of cancer. Transcriptome data and Percent Spliced In (PSI) values of ovarian cancer patients were downloaded from TCGA database and TCGA SpliceSeq. Totally we identified 1472 AS events that were associated with survival of ovarian serous cystadenocarcinoma (OC) and exon skipping (ES) was the most important type. Univariate and multivariate Cox regression analysis were performed to identify survival-associated AS events and developed the prognostic model based on 11-AS events. The immune cells and different response to cytotoxic T lymphocyte associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) blockers in low-risk and high-risk group of OC patients were analyzed. Ten kinds of immune cells were found up-regulated in low-risk group. Activated B cell, natural killer T cell, natural killer cell and regulatory T cell were associated with survival of OC. The patients in low-risk group had good response to CTLA-4 and PD-1 blockers treatment. Moreover, a regulatory network was established according to the correlation between AS events and splicing factors (SFs). The present study provided valuable insights into the underlying mechanisms of OC. AS events that were correlated with the immune system might be potential therapeutic targets.

RNA interference of human papillomavirus type 16 E7 increases HLA class I antigen expression in HaCaT-E7 cells.

BACKGROUND: High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer. The E7 protein of high-risk HPV disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting an ideal target for development of the immunotherapy in HPV-positive cervical cancers. We previously reported that HPV16 E7 could down-regulate cell-surface HLA class I antigen accompanying decreased expression of transporter associated with antigen processing 1 (TAP-1). The purpose of this study was to determine whether knockdown of HPV16 E7 could up-regulate surface HLA class I antigen expression in HPV16 E7 expressing HaCaT cells (HaCaT-E7). METHODS: An E7-specific small interfering RNA (siRNA) was transfected into the HaCaT-E7 cells, and the expression of HPV16 E7 was measured by real-time reverse transcriptase polymerase chain reaction and Western blot. With the use of flow cytometry analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 expression were detected. RESULTS: It was found that transfection of HPV16 E7-siRNA reduced HPV16 E7 expression as measured on messenger RNA and protein levels. The flow cytometry analysis showed that, compared with mock transfection, a statistically significant increase of approximately 75% in surface HLA class I levels was observed in HaCaT-E7 cells at 72 hours after transfection of E7 siRNA. Moreover, he knockdown of E7 in HaCaT-E7 cells could result in an increase of intracellular TAP-1 expression, which is essential for the expression of HLA class I at cell surface. CONCLUSIONS: Our study showed that the knockdown of HPV16 E7 could increase cell surface HLA class I antigen expression in HaCaT-E7 cells. In addition, for HPV-positive human cervical cancer, our observations indicate that the HPV E7 gene is a target of choice.

Prognostic Signatures of Alternative Splicing Events in Esophageal Carcinoma Based on TCGA Splice-Seq Data.

An alternative splicing (AS) event is a highly complex process that plays an essential role in post-transcriptional gene expression. Several studies have suggested that abnormal AS events were the primary element in the pathological process of cancer. However, few works are dedicated to the study of AS events in esophageal carcinoma (EC). In the present study, clinical information and RNA-seq data of EC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The percent spliced in (PSI) values of AS events were acquired from the TCGA Splice-seq. A total of 183 EC patients were enrolled in this study, and 2,212 AS events were found significantly associated with the overall survival of these patients by univariate Cox regression analysis. The prognostic signatures based on AS events were built by multivariate Cox analysis. Receiver operating characteristic (ROC) curves displayed that the area under the curve (AUC) of the following prognostic signatures, including exon skip (ES), alternate terminator (AT), alternate acceptor site (AA), alternate promoter (AP), alternate donor site (AD), retained intron (RI), and total events, was greater than 0.8, suggesting that these seven signatures had valuable prognosis prediction capacity. Finally, the risk score of prognostic signatures was indicated as an independent risk factor of survival. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to explore the function of splicing factors (SFs) that were associated with AS events. Also, the interactive network between AS events and SFs identified several hub genes and AS events which need further study. This was a comprehensive study that explored prognosis-related AS events and established valuable prognosis signatures in EC patients. The network of interactions between AS events and SFs might offer novel insights into the fundamental mechanisms of tumorigenesis and progression of EC.

Down-regulation of HLA class I antigen in human papillomavirus type 16 E7 expressing HaCaT cells: correlate with TAP-1 expression.

OBJECTIVES: High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer, and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein of high-risk HPV types disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting a role for E7 in tumor immune evasion. We previously reported that HPV-16 E7 expression and down-regulation of HLA class I was highly correlated in cervical lesions. This study was aimed to determine whether HPV-16 E7 oncoprotein could down-regulate surface HLA class I antigen in HPV-16 E7-transfected cells, and whether it had correlation with the expression of the transporter associated with antigen processing (TAP). METHODS: The HPV-16 E7 open reading frame was transfected into HaCaT cells. After G418 selection, resistant colonies were individually picked and expanded into clonal cell lines. Using the fluoresence-activated cell sorting analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 and TAP-2 expressions were detected. RESULTS: Compared with the empty vector control, a statistical significant decrease of approximately 50% in cell surface HLA class I expression was observed in HPV-16 E7 expressing HaCaT cells (P < 0.001). Moreover, the expression of HPV-16 E7 in HaCaT cells resulted in decreased expression of TAP-1 that was essential for HLA class I expression at the cell surface, a statistical significant decrease of approximately 40% compared with that with the empty vector control (P < 0.001). CONCLUSIONS: Our finding demonstrates that HPV-16 E7 down-regulates surface HLA class I antigen, which in part correlates with the decrease of TAP-1.

Genome-wide DNA methylation analysis by MethylRad and the transcriptome profiles reveal the potential cancer-related lncRNAs in colon cancer.

Colon cancer (CC) is characterized by global aberrant DNA methylation that may affect gene expression and genomic stability. A series of studies have demonstrated that DNA methylation could regulate the expressions of not only protein-coding genes but also ncRNAs. However, the regulatory role of lncRNA genes methylaton in CC remains largely unknown. In the present study, we systemically characterize the profile of DNA methylation, especially the aberrant methylation of lncRNAs genes using MethylRAD technology. A total of 132 999 CCGG/8487 CCWGG sites were identified as differentially methylated sites (DMSs), which were mainly located on the introns and intergenic elements. Moreover, 1,359 CCGG/1,052 CCWGG differentially methylated genes (DMGs) were screened. Our results demonstrated that aberrant methylation of lncRNA genes occurred most frequently, accounting for 37.5% and 44.3% in CCGG and CCWGG DMGs respectively. In addition, 963 lncRNA DMGs were co-analyzed with 1328 differentially expressed lncRNAs which were identified from TCGA database. We found that 15 lncRNAs might be CC-related lncRNAs. ZNF667-AS1 and MAFA-AS1 were down-regulated in CC, which might be silenced by hypermethylation. Besides, 13 lncRNAs were hypomethylated and up-regulated in CC. Moreover, our results validated the expression and methylation level of CC-related lncRNAs by RT-qPCR and pyrosequencing assay. In conclusion, we performed a genome-wide DNA methylation analysis by MethylRAD to acquire both CCGG and CCWGG DMSs and DMGs in CC. The results screened lncRNA DMSs as potential biomarkers and identified 15 lncRNAs as CC-related lncRNAs. This study provided novel therapy targets and valuable insights into molecular mechanism in tumorigenesis and development of CC.

Novel long non-coding RNA LINC02323 promotes epithelial-mesenchymal transition and metastasis via sponging miR-1343-3p in lung adenocarcinoma.

BACKGROUND: We have previously developed a unique metastasis-associated signature consisting of six long non-coding RNAs (lncRNAs), including a novel lncRNA, namely LINC02323. In the present study, we aimed to investigate the underlying roles of LINC02323 in the migration, invasion and TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung adenocarcinoma (LUAD) cells. METHODS: The distribution of LINC02323 was detected by the nuclear-plasma separation experiment. Cell proliferation was assessd by MTT assay, and cell migration and invation were detected by transwell assays. EMT was detected by RT-qPCR and western blotting. Interaction between miRNA and LINC02323 was predicted by starBase v2.0 and confirmed by the double luciferase reporting system. RESULTS: LINC02323 was distributed in the cytoplasm and nucleus. The overexpression or deletion of LINC02323 did not affect the proliferation of LUAD cells, while significantly affected the migration and invasion of LUAD cells. TGF-ß-induced EMT process was significantly affected by both RNA interference (RNAi) and overexpression of LINC02323. The predicted results showed that there were binding sites between LINC02323 and miR-1343-3p. The expression of LINC02323 was found to be negatively correlated with miR-1343-3p in LUAD by analyzing The Cancer Genome Atlas (TCGA) database. The double luciferase reporting system, RT-qPCR and western blotting experiments confirmed that LINC02323 could bind to miR-1343-3p, which bound to TGF-ß receptor 1 (TGFBR1). Inhibition of miR-1343-3p reversed LINC02323 silencing-mediated suppression of migration, invasion and EMT. CONCLUSIONS: LINC02323 acts as a competing endogenous RNA (ceRNA), which sponged miR-1343-3p to upregulate the TGFBR1 expression and promote the EMT and metastasis in LUAD. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: LINC02323 promotes epithelial-mesenchymal transition and metastasis via sponging miR-1343-3p in lung adenocarcinoma. WHAT THIS STUDY ADDS: LINC02323 is a key molecule in the process of invasion and metastasis of LUAD and might be used as a potential target in metastatic cancer.

Unique metastasis-associated lncRNA signature optimizes prediction of tumor relapse in lung adenocarcinoma.

BACKGROUND: Local relapses and metastases are primary causes of death in lung cancer patients. In the present study, we aimed to develop a prognostic signature based on metastasis-associated lncRNAs in patients with lung adenocarcinoma (LUAD). METHODS: Firstly, the potential metastasis-associated lncRNAs were identified by analyzing high-throughput data from The Cancer Genome Atlas (TCGA), and based on which, an lncRNA signature was constructed for prediction of relapse in LUAD patients using Cox proportional hazards regression analysis. Moreover, the prognostic performance of the lncRNA signature was evaluated using Kaplan-Meier survival analysis, time-dependent receiver operating characteristic (ROC) curve and Cox analysis, respectively. In addition, the potential metastasis-associated function of these six lncRNAs was confirmed by lncRNA over-expression or depletion and in vitro transwell assays in LUAD cells. RESULTS: An lncRNA signature consisting of six most important prognostic factors (LINC01819, ZNF649-AS1, HNF4A-AS1, FAM222A-AS1, LINC02323 and LINC00672) was developed. The signature was an independent predictor for patients' relapse-free survival (RFS), which could provide higher tumor relapse prediction capability compared with the TNM staging system at three years and five years, respectively (P = 0.0209 and P = 0.0468). Furthermore, the combination of this lncRNA signature and TNM stage had better prognostic value than TNM stage alone at three and five years, respectively (P = 0.0006 and P = 0.0096). Additionally, all the lncRNAs of the signature had a regulatory role in the LUAD cell mobility. CONCLUSIONS: This novel six-lncRNA signature had considerable prognostic value for prediction of relapse in LUAD patients. KEY POINTS: Significant findings of the study The unique metastasis-associated lncRNA signature was related to tumor metastasis and prognosis in LUAD patients. What this study adds This signature had considerable prognostic value for prediction of relapse in LUAD patients.

Everolimus Inhibits the Progression of Ductal Carcinoma In Situ to Invasive Breast Cancer Via Downregulation of MMP9 Expression.

PURPOSE: We evaluated the role of everolimus in the prevention of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) progression. EXPERIMENTAL DESIGN: The effects of everolimus on breast cancer cell invasion, DCIS formation, and DCIS progression to IDC were investigated in a 3D cell culturing model, intraductal DCIS xenograft model, and spontaneous MMTV-Her2/neu mouse model. The effect of everolimus on matrix metalloproteinase 9 (MMP9) expression was determined with Western blotting and IHC in these models and in patients with DCIS before and after a window trial with rapamycin. Whether MMP9 mediates the inhibition of DCIS progression to IDC by everolimus was investigated with knockdown or overexpression of MMP9 in breast cancer cells. RESULTS: Everolimus significantly inhibited the invasion of human breast cancer cells in vitro. Daily intragastric treatment with everolimus for 7 days significantly reduced the number of invasive lesions from intraductal DCIS foci and inhibited DCIS progression to IDC in the MMTV-Her2/neu mouse mammary tumor model. Mechanistically, everolimus treatment decreased the expression of MMP9 in the in vitro and in vivo models, and in breast tissues from patients with DCIS treated with rapamycin for 1 week. Moreover, overexpression of MMP9 stimulated the invasion, whereas knockdown of MMP9 inhibited the invasion of breast cancer cell-formed spheroids in vitro and DCIS in vivo. Knockdown of MMP9 also nullified the invasion inhibition by everolimus in vitro and in vivo. CONCLUSIONS: Targeting mTORC1 can inhibit DCIS progression to IDC via MMP9 and may be a potential strategy for DCIS or early-stage IDC therapy.