Characterization of m6A methylation modifications and tumor microenvironment infiltration in thyroid cancer

Background Thyroid cancer (TC) is the most common endocrine malignancy worldwide, and immunotherapy is a new cancer treatment that stimulates and enhances the natural ability of the immune system to fight cancer cells. The role of RNA N6-methyladenosine (m6A) related genes in these challenges has recently become a research hotspot, but he potential role of m6A modifications in tumor microenvironment (TME) cell infiltration remains unknown. Purpose There is growing evidence that m6A plays a critical role in the regulation of gene expression by participating in important biological processes. A comprehensive analysis of the m6A regulator-mediated infiltration characteristics of the TME will help advance the understanding of immune regulation in thyroid tumors. Methods This study assessed m6A modification modes in 510 thyroid cancer samples from the Cancer Genome Atlas (TCGA) databases according to a comprehensive set of 24 m6A regulators. In this study, we analyzed the biological characteristics and m6A methylation modification patterns. Based on this, we constructed m6A signatures and analyzed m6A modification features in tumor somatic mutations and TCGA molecular subtypes. Results These modification modes were systematically linked to TME cell infiltration signatures. m6A modification patterns were comprehensively assessed and correlated with immune cell infiltration features in the TME. An unsupervised clustering approach was applied and three distinct m6A modification subtypes and three m6A-associated gene subtypes were identified. Additionally, three distinct m6A methylation modification modes were identified in the thyroid cancer samples. The TME profiles of the identified genetic subtypes were strongly congruent with the immuno-heat and immuno-cold phenotypes... (AU)

Characterization of m6A methylation modifications and tumor microenvironment infiltration in thyroid cancer.

BACKGROUND: Thyroid cancer (TC) is the most common endocrine malignancy worldwide, and immunotherapy is a new cancer treatment that stimulates and enhances the natural ability of the immune system to fight cancer cells. The role of RNA N6-methyladenosine (m6A) related genes in these challenges has recently become a research hotspot, but he potential role of m6A modifications in tumor microenvironment (TME) cell infiltration remains unknown. PURPOSE: There is growing evidence that m6A plays a critical role in the regulation of gene expression by participating in important biological processes. A comprehensive analysis of the m6A regulator-mediated infiltration characteristics of the TME will help advance the understanding of immune regulation in thyroid tumors. METHODS: This study assessed m6A modification modes in 510 thyroid cancer samples from the Cancer Genome Atlas (TCGA) databases according to a comprehensive set of 24 m6A regulators. In this study, we analyzed the biological characteristics and m6A methylation modification patterns. Based on this, we constructed m6A signatures and analyzed m6A modification features in tumor somatic mutations and TCGA molecular subtypes. RESULTS: These modification modes were systematically linked to TME cell infiltration signatures. m6A modification patterns were comprehensively assessed and correlated with immune cell infiltration features in the TME. An unsupervised clustering approach was applied and three distinct m6A modification subtypes and three m6A-associated gene subtypes were identified. Additionally, three distinct m6A methylation modification modes were identified in the thyroid cancer samples. The TME profiles of the identified genetic subtypes were strongly congruent with the immuno-heat and immuno-cold phenotypes. CONCLUSIONS: The results revealed that m6A modifications play an integral role in the diversity and complexity of thyroid carcinomas. Evaluating the m6A modification patterns of individual tumors will create more efficient immunotherapeutic strategies. A comprehensive analysis of the role of TME in thyroid cancer provides a research idea for studying the effect of m6A epigenetics on thyroid tumors and their immune microenvironment.

THBS1, a fatty acid-related metabolic gene, can promote the development of laryngeal cancer.

Laryngeal cancer is the second most prevalent head and neck tumor and it is one of the most common malignancies of the upper respiratory tract. Fatty acid metabolism affects cancer cell biology in several ways, and alterations in fatty acid metabolism are characteristic of both tumorigenesis and metastasis. Despite advances in laryngeal cancer diagnosis and treatment over the years, there has been no significant improvement in survival or mortality. Studying the role of fatty acid metabolism-related genes in laryngeal cancer will facilitate our search for valuable biomarkers to guide prognostic management and treatment selection. We constructed a prognostic risk score model for fatty acid metabolism-related genes by downloading and analyzing laryngeal cancers from the TCGA and GEO databases. We predicted survival outcomes of laryngeal cancer patients using a prognostic risk score model of fatty acid metabolism-related genes and analyzed the resistance of laryngeal cancer in different individuals to multiple drugs. In addition, the relationship between the prognostic risk score model and cellular infiltration characteristics of the tumor microenvironment were investigated. Through the prognostic risk scoring model, the genes with risk-prompting effect and related to prognosis were screened out for further research. Through the study of gene expression levels in the TCGA database, we screened out 120 differentially expressed fatty acid metabolism genes. LASSO-Cox and Cox regression analyses identified nine genes associated with prognosis to construct a prognostic risk score model for genes related to fatty acid metabolism. Both TCGA and GEO confirmed that samples in the high-risk score group had a worse prognosis than those in the low-risk score group. We found significant differences between the high-risk and low-risk groups for 22 drugs (P < 0.05). In addition, we found differences in immune cell infiltration between the different risk score groups. Finally, through the risk assessment model, combined with multiple databases, THBS1, a high-risk and prognosis-related gene, was screened. We also found that THBS1 could promote the migration, invasion and proliferation of laryngeal cancer cells by constructing THBS1 knockout cell lines. In our study, we identified key fatty acid-related genes differentially expressed in laryngeal carcinoma that can be used to adequately predict prognosis using a comprehensive bioinformatic experimental approach. It was also found that THBS1, a high-risk and prognosis-related gene, may regulate the occurrence and development of laryngeal cancer through fatty acid metabolism, which has further helped us to explore the role of fatty acid metabolism genes in laryngeal cancer.

FTO Prevents Thyroid Cancer Progression by SLC7A11 m6A Methylation in a Ferroptosis-Dependent Manner.

N6 methyladenosine (m6A) modification serves as a novel epigenetic regulatory mechanism that is heavily implicated in the heredity of tumors. Meanwhile, fat mass and obesity-associated protein (FTO) has the potential to affect the regulation of m6A modification in the mRNA of key oncogenes as well as tumor suppressor genes that facilitate tumor progression. In our study, FTO was downregulated in papillary thyroid carcinoma (PTC) tissues. The role of FTO in PTC was assessed by Cell Counting Kit-8 analysis, cell scratch, migration, invasion experiment, flow cytometry apoptosis analysis, and nude mouse experiment. In addition to RNA-Seq and meRIP-Seq, luciferase reporting and mutation analysis have also identified SLC7A11 as the potential FTO regulatory gene. Moreover, X-ray electron microscopy, glutathione (GSH)/oxidized GSH, GPX, malondialdehyde determination, and western blot helped confirmed that FTO inhibited the development of PTC by downregulating the expression of SLC7A11 through ferroptosis. Finally, a rescue experiment was employed to clarify the relationship between FTO and its specific target gene SLC7A11. FTO is able to inhibit the occurrence of PTC by downregulating SLC7A11 in m6A independently, and it functions as a tumor suppressor gene in PTC. These findings could contribute to our understanding of the tumor malignancy regulated by m6A and might lead to new insights for potential biomarkers and therapeutic targets for the treatment of thyroid papillary carcinoma.

Overexpression of DUOX2 mediates doxorubicin resistance and predicts prognosis of pancreatic cancer.

BACKGROUND: Dysregulation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family is frequently observed in cancers and associated with their development and progression. However, the expression, role, and clinical significance of the NOX family members in pancreatic cancer remain unexplored. METHODS: The expression levels of the 7 NOX family genes were analyzed in Gene Expression Omnibus (GEO) datasets. The messenger RNA (mRNA) expression and gene alterations were explored using The Cancer Genome Atlas (TCGA) data portal. Clinical significance analyses of the NOX family genes were conducted among pancreatic cancer patients. The expression and prognostic value of dual oxidase 2 (DUOX2) were then validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) in an independent validation cohort. The function of DUOX2 was analyzed by gene set enrichment analysis (GSEA) and its effect on the chemosensitivity of pancreatic cancer cells was detected by Cell Counting Kit-8 (CCK-8) assay. RESULTS: Results showed that NOX1, NOX2 (CYBB), NOX4, DUOX1, and DUOX2 were upregulated, while NOX3 and NOX5 were downregulated in pancreatic cancer tissues compared with nontumor tissues. Genomic alteration analysis demonstrated that deregulation of NOX family genes was partially caused by genomic alterations. Survival analyses showed that only DUOX2 was associated with overall survival (OS) and relapse-free survival (RFS) of pancreatic cancer patients. The DUOX2 gene was observed to be markedly overexpressed in pancreatic cancer. In the GSEA results for pancreatic cancer patients, DUOX2 was significantly associated with oxidoreductase activity acting on nicotinamide adenine dinucleotide hydrogen (NADH) or NADPH and uridine 5'-diphospho-glucuronosyltansferase (UDP) glycosyltransferase activity. Knockdown of DUOX2 in pancreatic cancer cells increased their sensitivity to doxorubicin. CONCLUSIONS: Overexpression of DUOX2 is correlated with prognosis and recurrence in pancreatic cancer patients and acts as a good marker for pancreatic cancer course prediction; furthermore, DUOX2 might be a therapeutic target for pancreatic cancer patients.

Identification of prognostic biomarkers related to the tumor microenvironment in thyroid carcinoma.

Thyroid Carcinoma (THCA) is the most common endocrine tumor that is mainly treated using surgery and radiotherapy. In addition, immunotherapy is a recently developed treatment option that has played an essential role in the management of several types of tumors. However, few reports exist on the use of immunotherapy to treat THCA. The study downloaded the miRNA, mRNA and lncRNA data for THCA patients from the TCGA database ( https://portal.gdc.cancer.gov/ ). Thereafter, the tumor samples were divided into cold and hot tumors, based on the immune score of the tumor microenvironment. Moreover, the differentially expressed lncRNAs and miRNAs were obtained. Finally, the study jointly constructed a ceRNA network through differential analysis of the mRNA data for cold and hot tumors. The study first assessed the level of immune infiltration in the THCA tumor microenvironment then divided the samples into cold and hot tumors, based on the immune score. Additionally, a total of 568 up-regulated and 412 down-regulated DEGs were screened by analyzing the differences between hot and cold tumors. Thereafter, the study examined the differentially expressed genes for lncRNA and miRNA. The results revealed 629 differentially expressed genes related to lncRNA and 114 associated with miRNA. Finally, a ceRNA network of the differentially expressed genes was constructed. The results showed a five-miRNA hubnet, i.e., hsa-mir-204, hsa-mir-128, hsa-mir-214, hsa-mir-150 and hsa-mir-338. The present study identified the immune-related mRNA, lncRNA and miRNA in THCA then constructed a ceRNA network. These results are therefore important as they provide more insights on the immune mechanisms in THCA. The findings also provides additional information for possible THCA immunotherapy.

Analysis of Risk Factors of Level V Lymphatic Metastasis for Papillary Thyroid Carcinoma with pN1b.

OBJECTIVE: To explore the risk factors of level V lymphatic metastasis in papillary thyroid carcinoma (PTC) patients with pN1b. METHODS: Patients were selected if they presented with a suspicious level III or IV lymph node metastasis and underwent surgery by hemi or total thyroidectomy with a lymph node dissection (levels III, IV, VI, and VII). For these patients, if frozen section showed a positive level III or IV node, then levels II and V nodes were resected. Univariate analysis was performed using the chi-square test for some factors, including age, sex, tumor location, multifocal lesions, tumor size, local invasion of primary focus, status of cervical lymphatic metastasis, TNM staging, tumor deposits (independent tumor nodules), and the metastasis to more than 5 central lymph nodes. Then, the factors with statistical significance indicated by the above univariate analysis underwent multivariate analysis. RESULTS: Univariate analysis indicated that the level V lymphatic metastasis was significantly associated with simultaneous metastases to levels II, III, and IV, simultaneous metastases to levels III and IV, and tumor deposits (all p < 0.05), but it was not significantly associated with age, sex, tumor location, multifocal lesions, tumor size, local invasion of primary focus, other cervical lymphatic metastasis, TNM staging, and the metastases to more than 5 central lymph nodes (all p > 0.05). Multivariate analysis suggested that the simultaneous metastases to levels III and IV and tumor deposits were the risk factors of level V lymphatic metastasis. CONCLUSION: The simultaneous metastases to levels III and IV and tumor deposits are independent risk factors of level V lymphatic metastasis. The patients with pN1b PTC who have simultaneous metastases to levels III and IV or/and tumor deposits may have the risk of level V lymph node metastasis.

Evaluation of MEDAG gene expression in papillary thyroid microcarcinoma: associations with histological features, regional lymph node metastasis and prognosis.

Papillary thyroid microcarcinoma accounts for a large proportion of papillary thyroid carcinoma, especially among new cases. Many  PTMC patients have regional lymph node metastasis, with some experiencing recurrence and even death. However, the risk factors and mechanism by which PTMC relates to these factors are unknown. In this study, differentially expressed genes were identified with microarray from The Cancer Genome Atlas, followed by analysis using the Kyoto Encyclopedia of Genes and Genomes. Immunohistochemistry, immunofluorescence, western blot and Oil Red O staining were carried out to evaluate expression levels and functional alterations. Mesenteric Estrogen Dependent Adipogenesis expression was observed in almost all cases of papillary thyroid microcarcinomas, and the location of expression was associated with histological subtype. High expression was correlated with metastasis and poor disease-free survival. Furthermore, the enrichment analysis indicated that Mesenteric Estrogen Dependent Adipogenesis expression may be associated with metabolic reprogramming to influence metastasis and prognosis. These findings contribute to a better understanding of how Mesenteric Estrogen Dependent Adipogenesis affects metastasis and the prognosis of papillary thyroid microcarcinoma patients and suggest that Mesenteric Estrogen Dependent Adipogenesis expression may be a novel prognostic marker in these patients.

The MicroRNA3686 Inhibits the Proliferation of Pancreas Carcinoma Cell Line by Targeting the Polo-Like Kinase 1.

The Polo-like kinase 1 (PLK1) is one member of the so-called Polo-like kinase family which plays an important role in tumorigenesis. By analyzing the potential complementary microRNA (miRNA) targeting sequence of PLK1, we identified that miRNA-3686 (hereby and thereafter mir3696) could be the potential regulator for PLK1. Real-time PCR demonstrated that the mir3686 has a relatively higher expression in the immortalized pancreas cell HPDE6C7 than pancreas carcinoma derived cell line PANC1. The upregulation of mir3686 in HPDE6C7 cell corresponded with the low expression of PLK1 as well. Both luciferase based reporter assay and evaluation of endogenous PLK1 expression demonstrated that mir3686 regulated PLK1, which confirms our speculation. Moreover, we found that transfection of mir3686 in PANC1 cell could lead to proliferation inhibition and promote apoptosis. Further analysis demonstrated that mir3686 transfection in PANC1 cell also inhibited cell invasion, and clone formation in cell invasion assay and clonogenic cell survival assay, respectively. In contrast, inhibition of mir3686 expression in HPDE6C7 cell enhanced the capability of proliferation, cell invasion and clone formation. Taken together, our results indicated that mir3686 could target PLK1 to inhibit the cell proliferation in pancreas cancer derived cell line and mir3686 could be a new therapeutic target for pancreas cancer treatment.

Inhibition of acute lung injury by rubriflordilactone in LPS-induced rat model through suppression of inflammatory factor expression.

The present study demonstrates the effect of rubriflordilactone on lipopolysaccharide (LPS)-induced acute kidney injury in rats and MLE-15 cells. LPS administration in rats resulted in formation of edema which was inhibited by pretreatment with rubriflordilactone. The pulmonary tissues of LPS administered rats and MLE-15 cells showed a significant increase in the expression of matrix metalloproteinase-9, interleukin-6 and inducible nitric oxide synthase. However, rubriflordilactone treatment prior to LPS administration caused a significant reduction in the expression of these factors at a concentration of 10 nm/kg. Analysis of the Sirtuin 1 (Sirt1) expression revealed significant (P=0.002) reduction on exposure to LPS in MLE-15 cells. However, rubriflordilactone treatment at 10 nm/ml concentration before LPS exposure caused inhibition of LPS induced reduction in Sirt1 expression. Silencing of Sirt1 by siRNA in MLE-15 cells led to inhibition of increased Sirt1 expression by rubriflordilactone in LPS administered rats. These findings suggest that rubriflordilactone inhibits LPS induced acute lung injury in rats and MLE-15 cells through promotion of Sirt1 expression.

miR-639 promotes the proliferation and invasion of breast cancer cell in vitro.

Breast cancer is characterised by an elevated capacity for tumour invasion and lymph node metastasis, but the cause remains to be determined. Recent studies suggest that microRNAs can regulate the evolution of malignant behaviours by regulating multiple target genes. In this study, we have first confirmed that miR-639 is up-regulated in metastatic breast cancer tissues and cell line with highly invasive capacity. Furthermore, we provided evidence to demonstrate that up-regulation of miR-639 contributes breast cancer invasion and metastasis. These data reveal a key role of miR-639 in breast cancer metastasis and support biological and clinical links between miR-639 and breast cancer.

[Correlation of FHIT expression to cell proliferation and metastasis of laryngeal squamous cell carcinoma].

BACKGROUND & OBJECTIVE: Fragile histidine triad (FHIT) gene, a candidate tumor suppressor gene, located at 3pl4.2, spans the most common fragile site, FRA3B, in human genomes. It is altered in many human cancers, such as head and neck squamous cell carcinoma. This study was to investigate correlation of FHIT expression to cell proliferation and metastasis of laryngeal squamous cell carcinoma (LSCC). METHODS: Expression of FHIT protein, and proliferating cell nuclear antigen (PCNA) was detected by SP immunohistochemistry in 41 specimens of LSCC and their matched adjacent non-cancerous epithelium (NCE). RESULTS: Positive rates of FHIT in NCE, and LSCC were 100.0% (41/41), and 46.3% (19/41) (P< 0.01); in LSCC of stage I-II, and stage III-IV were 69.6% (16/23), and 16.7% (3/18) (P< 0.01); in lymph node metastasis group, and no metastasis group were 20.0% (3/15), and 61.5% (16/26) (P< 0.05). PCNA labeling indexes in NCE, and LSCC were (9.98+/-2.34)%, and (50.71+/-13.64)% (P< 0.01). In LSCC, expression of FHIT negatively correlated with PCNA (P< 0.05). CONCLUSION: Low expression of FHIT might play an important role in cell proliferation and metastasis of LSCC, FHIT might be a useful marker for evaluating biological behaviors of LSCC.