DANCR Regulates hESC Differentiation Towards Definitive Endoderm / 中山大学学报(医学科学版)

ObjectiveTo explore the function of DANCR during the differentiation of human embryonic stem cells （hESC） toward definitive endoderm （DE）. MethodsThe in vitro DE differentiation system was established and its efficiency was verified. The correlation between the expression level of DANCR and DE differentiation process was detected. Using lentivirus system， we stably knocked down DANCR in hESC. The shDANCR hESC line was applied to DE differentiation， using qPCR and Western blot to detect the expression of DE marker genes SOX17 and FOXA2， and that of primitive streak marker genes Brachyury （T）， EOMES， MIXL1 and GSC. Dual luciferase reporter assay and qPCR were used to confirm the interaction between DANCR and the WNT pathway during DE differentiation. ResultsThe in vitro differentiation system mimicked DE differentiation efficiently. And the expression of DANCR was gradually downregulated during differentiation. DANCR was efficiently knocked down in the shDANCR hESC line （P < 0.001）. Compared with those in the control group， the expression levels of primitive markers Brachyury （T）， EOMES， MIXL1 and GSC， as well as DE markers SOX17 and FOXA2， were significantly decreased in shDANCR groups （P < 0.05）. Furthermore， the transcriptional activity of the WNT pathway in shDANCR groups was lower than that in the control group （P < 0.05）. And RNA levels of downstream genes of the WNT pathway， FZD5， FZD8， SFRP1， FRZB and ANKRD6， were significantly decreased in shDANCR groups （P < 0.05）. However， differences in protein levels of the TGFβ pathway effectors SMAD2/3 and p-SMAD2 were statistically insignificant in shDANCR and control groups （P > 0.05）. Forced activation of β-CATENIN rescued DANCR knock down-induced deficiency in DE differentiation. ConclusionsThe expression of DANCR decreases during DE differentiation. DANCR may promote DE differentiation through modulating the activity of the WNT pathway.

Study on the Role and Mechanism of METTL3 Mediating the Up-regulation of m6A Modified Long Non-coding RNA THAP7-AS1 in Promoting the Occurrence of Lung Cancer / 中国肺癌杂志

BACKGROUND@#Lung cancer is a major threat to human health. The molecular mechanisms related to the occurrence and development of lung cancer are complex and poorly known. Exploring molecular markers related to the development of lung cancer is helpful to improve the effect of early diagnosis and treatment. Long non-coding RNA (lncRNA) THAP7-AS1 is known to be highly expressed in gastric cancer, but has been less studied in other cancers. The aim of the study is to explore the role and mechanism of methyltransferase-like 3 (METTL3) mediated up-regulation of N6-methyladenosine (m6A) modified lncRNA THAP7-AS1 expression in promoting the development of lung cancer.@*METHODS@#Samples of 120 lung cancer and corresponding paracancerous tissues were collected. LncRNA microarrays were used to analyze differentially expressed lncRNAs. THAP7-AS1 levels were detected in lung cancer, adjacent normal tissues and lung cancer cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The diagnostic value of THAP7-AS1 in lung cancer and the relationship between THAP7-AS1 expression and survival rate and clinicopathological parameters were analyzed. Bioinformatics analysis, methylated RNA immunoprecipitation (meRIP), RNA pull-down and RNA-immunoprecipitation (RIP) assay were used to investigate the molecular regulation mechanism of THAP7-AS1. Cell proliferation, migration, invasion and tumorigenesis of SPC-A-1 and NCI-H1299 cells were determined by MTS, colony-formation, scratch, Transwell and xenotransplantation in vivo, respectively. Expression levels of phosphoinositide 3-kinase/protein kenase B (PI3K/AKT) signal pathway related protein were detected by Western blot.@*RESULTS@#Expression levels of THAP7-AS1 were higher in lung cancer tissues and cell lines (P<0.05). THAP7-AS1 has certain diagnostic value in lung cancer [area under the curve (AUC)=0.737], and its expression associated with overall survival rate, tumor size, tumor-node-metastasis (TNM) stage and lymph node metastasis (P<0.05). METTL3-mediated m6A modification enhanced THAP7-AS1 expression. The cell proliferation, migration, invasion and the volume and mass of transplanted tumor were all higher in the THAP7-AS1 group compared with the NC group and sh-NC group of SPC-A-1 and NCI-H1299 cells, while the cell proliferation, migration and invasion were lower in the sh-THAP7-AS1 group (P<0.05). THAP7-AS1 binds specifically to Cullin 4B (CUL4B). The cell proliferation, migration, invasion, and expression levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphoinositide-3 kinase, catalytic subunit delta (PIK3CD), phospho-phosphatidylinositol 3-kinase (p-PI3K), phospho-protein kinase B (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) were higher in the THAP7-AS1 group compared with the Vector group of SPC-A-1 and NCI-H1299 cells (P<0.05).@*CONCLUSIONS@#LncRNA THAP7-AS1 is stably expressed through m6A modification mediated by METTL3, and combines with CUL4B to activate PI3K/AKT signal pathway, which promotes the occurrence and development of lung cancer.

Intervention Mechanism of Biejiajian Wan on Primary Liver Cancer by Regulating lncRNA SNHG5/miRNA-26a-5p/GSK-3β Signal Axis / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Biejiajian Wan in the intervention of primary liver cancer based on long non-coding RNA SNHG5 （lncRNA SNHG5）/micro RNA-26a-5p （miRNA-26a-5p）/glycogen synthase kinase-3β （GSK-3β） signal axis. MethodDouble luciferase reporting assay was used to verify the targeted interaction between lncRNA SNHG5 and miRNA-26a-5p， miRNA-26a-5p， and GSK-3β in HepG2 cells. Nude-mouse transplanted tumor model of human HepG2 were established and randomly divided into model group， Biejiajian Wan low-dose group （0.5 g·kg-1）， medium-dose group （1.0 g·kg-1）， and high-dose group （2.0 g·kg-1）， and sorafenib group （100 mg·kg-1）， with 10 mice in each group. The mice were given intragastric administration of normal saline or drug for 28 days， and the tumor volume was measured at different time. Hematoxylin-eosin （HE） staining was used to observe the histological changes of tumors. The nucleic acid levels of lncRNA SNHG5， miRNA-26a-5p， GSK-3β， and β-catenin mPNA in tumor tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of GSK-3β and β-catenin in tumor tissue were detected by western blot. ResultCompared with the SNHG5-WT （wild type） + miRNA NC （negative control） group， the relative luciferase activities of the SNHG5-WT + miRNA-26a-5p mimic group were decreased （P<0.05）. Compared with the GSK-3β-WT + miRNA NC group， the relative luciferase activity of the GSK-3β-WT + miRNA-26a-5p mimic group was decreased （P<0.05）. Compared with the model group， the tumor volume of Biejiajian Wan low-dose， medium-dose， and high-dose groups was significantly decreased （P<0.05， P<0.01）. Compared with the model group， the cells in the tumor tissue of nude mice in each dose group of Biejiajian Wan were sparsely arranged with necrocytosis， which showed concentration-dependent changes. Compared with the model group， the expression levels of lncRNA SNHG5， GSK-3β， and β-catenin were decreased （P<0.05， P<0.01）， while the expression of miRNA-26a-5p was increased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. Compared with the model group， the protein expression levels of GSK-3β and β-catenin were decreased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. ConclusionBiejiajian Wan may affect the necrosis of liver cancer cells through lncRNA SNHG5/miRNA-26a-5p/GSK-3β signal axis and thus play an anti-tumor role. This research will provide more theoretical basis for the clinical application of Biejiajian Wan.

Construction of competitive endogenous RNA network mediated by lung ischemia-reperfusion core genes / 器官移植

Objective To analyze the core genes of lung ischemia-reperfusion injury and construct a competitive endogenous RNA (ceRNA) network. Methods Original data of GSE145989 were downloaded from the Gene Expression Omnibus (GEO) database as the training set, and the GSE172222 and GSE9634 datasets were used as the validation sets, and the differentially-expressed genes (DEG) were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. Protein-protein interaction (PPI) network was constructed, and the core genes were screened, and the diagnostic values of these core genes and the immune infiltration levels of immune cells were evaluated. The ceRNA network was constructed and validated. The targeted drugs based on ceRNA network were assessed. Results A total of 179 DEG were identified, including 61 down-regulated and 118 up-regulated genes. GO analysis showed that DEGs were associated with multiple biological processes, such as cell migration, differentiation and regulation, etc. They were correlated with cell components, such as vesicle membrane, serosa and membrane raft, etc. They were also associated with multiple molecular functions, such as chemokine receptor, G protein-coupled receptor, immune receptor activity and antigen binding, etc. KEGG pathway enrichment analysis revealed that DEG were involved in tumor necrosis factor (TNF), Wnt, interleukin (IL)-17 and nuclear factor (NF)-κB signaling pathways, etc. PPI network suggested that CD8A, IL2RG, STAT1, CD3G and SYK were the core genes of lung ischemia-reperfusion injury. The ceRNA network prompted that miR-146a-3p, miR-28-5p and miR-593-3p were related to the expression level of CD3G. The miR-149-3p, miR-342-5p, miR-873-5p and miR-491-5p were correlated with the expression level of IL-2RG. The miR-194-3p, miR-512-3p, miR-377-3p and miR-590-3p were associated with the expression level of SYK. The miR-590-3p and miR-875-3p were related to the expression level of CD8A. The miR-143-5p, miR-1231, miR-590-3p and miR-875-3p were associated with the expression level of STAT1. There were 13 targeted drugs for CD3G, 4 targeted drugs for IL-2RG, 28 targeted drugs for SYK and 3 targeted drugs for lncRNA MUC2. No targeted drugs were identified for CD8A, STAT1 and other ceRNA network genes. Conclusions CD8A, IL2RG, STAT1, CD3G and SYK are the core genes of lung ischemia-reperfusion injury. The research and analysis of these core genes probably contribute to the diagnosis of lung ischemia-reperfusion injury and providing novel research ideas and therapeutic targets.

Role of inhibiting lncRNA TUG1 to down⁃regulate nucleotide binding oligomerization domain like receptor protein 1 inflammasome in delaying the progression of Alzheimer’s disease / 解剖学报

Objective To investigate the relieving effects of knockdown of long non-coding RNA(lncRNA)taurine up-regulated gene 1 (TUG1) on inhibiting nucleotide binding oligomerization domain like receptor protein 1 (NLRP1) inflammasome and the progression of Alzheimer’ s disease. Methods Wild-type (WT group, 10 mice) or amyloid precursor protein (APP) / presenilin-1 (PS1) transgenic mice (30 mice) with a genetic background of C57 / BL6 aged 9-10 weeks were used in this study. APP / PS1 transgenic mice were randomly divided into model group, model+lncRNA TUG1 short hairpin RNA (shRNA) group and model + shRNA non target (NT) group (n = 10) . Blood samples, cerebral cortex tissues, primary microglial cells and primary astrocytes were collected from mice 12 weeks of age on day 1 (3-month-old) and 32 weeks of age on day 1 (8-month-old), with 5 mice per group at each time point. Real-time PCR analysis was used to detect the expression levels of lncRNA TUG1 and macrophage migration inhibitory factor (MIF) mRNA in cerebral cortex tissues and primary microglial cells, and C1r and C1s mRNA levels in primary astrocytes of 3-month-old and 8-month-old mice in the above 4 groups, respectively. ELISA was used to determine the MIF in plasma samples of the above 4 groups of mice. Primary microglia and astrocytes from the cerebral cortex of 3-month-old and 8-month-old mice were co-cultured. CCK-8 method was used to determine the proliferation ability of the above cells. Western blotting was used to determine the expression levels of MIF, pro interleukin-1β (pro-IL-1β), apoptosis associated speck-like protein containing a caspase recrult domain(ASC), Caspase-1 (p20), Caspase-1 (full), NLRP1 and NLRP3 in cerebral cortex tissues of 3-month-old and 8-month-old mice. Immunofluorescent staining was used to determine amyloid beta(Aβ) in cerebral cortex of 8-month-old mice. Results At the age of 3-month-old and 8-month-old, compared with the WT group, the relative expression level of lncRNA TUG1 and MIF in cerebral cortex tissues and primary microglia of model group mice was significantly up-regulated, with primary microglial cells and astrocytes proliferation ability enhanced (P0. 05) . There was no significant difference between the model group and the model+shRNA NT group mice of all the above factors (P>0. 05) . Conclusion In APP / PS1 transgenic mice, up-regulation of lncRNA TUG1 and MIF are positively associated with the activation of NLRP1 inflammasome in mice cerebral cortex tissues and primary microglia. Knock-down of lncRNA TUG1 can ameliorate the progression of Alzheimer’ s disease.

Effects of Tao Hong Si Wu decoction on IncRNA expression in rats with occlusion of middle cerebral artery / 中国药理学通报

Aim To screen and study the expression of long non-coding RNA (IncRNA) in rats with middle cerebral artery occlusion (MCAO) with MCAO treated with Tao Hong Si Wu decoction (THSWD) and determine the possible molecular mechanism of THSWD in treating MCAO rats. Methods Three cerebral hemisphere tissue were obtained from the control group, MCAO group and MCAO + THSWD group. RNA sequencing technology was used to identify IncRNA gene expression in the three groups. THSWD-regulated IncRNA genes were identified, and then a THSWD-regu-lated IncRNA-mRNA network was constructed. MCODE plug-in units were used to identify the modules of IncRNA-mRNA networks. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) were used to analyze the enriched biological functions and signaling pathways. Cis- and trans-regulatory genes for THSWD-regulated IncRNAs were identified. Reverse transcription real-time quantitative pol-ymerase chain reaction (RT-qPCR) was used to verify IncRNAs. Molecular docking was used to identify IncRNA-mRNA network targets and pathway-associated proteins. Results In MCAO rats, THSWD regulated a total of 302 IncRNAs. Bioinformatics analysis suggested that some core IncRNAs might play an important role in the treatment of MCAO rats with THSWD, and we further found that THSWD might also treat MCAO rats through multiple pathways such as IncRNA-mRNA network and network-enriched complement and coagulation cascades. The results of molecular docking showed that the active compounds gallic acid and a-mygdalin of THSWD had a certain binding ability to protein targets. Conclusions THSWD can protect the brain injury of MCAO rats through IncRNA, which may provide new insights for the treatment of ischemic stroke with THSWD.

Effect of LncRNA p21 regulating Hippo-YAP signaling pathway on formation of abdominal aortic aneurysm in mice and its mechanism / 中国药理学通报

Aim To investigate the effect of long non- coding RNA p21 (LncRNA p21) regulating Hippo- Yes-associated protein (Hippo-YAP) signaling pathway on the formation of abdominal aortic aneurysm (AAA) in mice. Methods C57BL/6 ApoE

Research progress on non-coding small RNA in oral submucosal fibrosis / 口腔疾病防治

@#Oral submucous fibrosis (OSF) is one of the most common precancerous lesions of the oral mucosa, and its pathogenesis has not been fully elucidated. Small noncoding RNAs (SncRNAs), a class of RNA molecules that do not code for proteins, have been widely reported to be involved in the regulation of a variety of human diseases. An increasing number of studies have shown that a variety of SncRNAs play important roles in the pathogenesis of OSF. Current studies have shown that microRNAs (miRNAs) are involved in OSF disease progression by regulating the expression of related transcription factors and genes or epithelial mesenchymal transformation to regulate the activation of fibroblasts (FBs). Long noncoding RNAs (lncRNAs) that transform growth factor-β/suppressor of mothers against decapentaplegic (TGF-β/Smad) signaling pathways or interact with miRNAs are involved in the development of OSF. Circular RNAs (circRNAs) play a role in OSF by interacting with miRNAs. tRNA-derived small RNAs (tsRNAs) are involved in the progression of various fibrotic diseases, but their specific mechanism of action in OSF still needs to be further explored. In the future, it is still necessary to focus on the targets of SncRNAs mediating OSF progression and explore their function and molecular mechanism in OSF to provide new ideas for the diagnosis and treatment of OSF.

Effect of lncRNA HIF1A-AS1 on chemotherapy sensitivity of vincristine-resistant in retinoblastoma cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the effect of long non-coding RNA-HIF1A-AS1(lncRNA HIF1A-AS1)on the chemotherapy sensitivity of vincristine(VCR)-resistant in retinoblastoma(RB)cells by regulating the expression of hypoxia-inducible factor-1α(HIF-1α).METHODS: The human RB VCR-resistant cell line SO-RB50/VCR was established, expression of lncRNA HIF1A-AS1 in SO-RB50 and SO-RB50/VCR cells were detected by reverse transcription-quantitative real-time PCR(RT-qPCR); inhibition of lncRNA HIF1A-AS1 expression or simultaneous overexpression of HIF-1α in SO-RB50/VCR cells, and then median inhibitory concentration(IC50)of VCR and cell proliferation and apoptosis were detected in SO-RB50/VCR cells; the protein expressions of HIF-1α, multidrug resistance associate protein(MRP)and P-glycoprotein(P-gp)were measured by Western blot.RESULTS: Compared with SO-RB50 cells, the expression levels of lncRNA HIF1A-AS1 and HIF-1α protein in SO-RB50/VCR cells were increased(P&#x003C;0.05); after inhibiting the expression of lncRNA HIF1A-AS1 in SO-RB50/VCR cells, the apoptosis rate was significantly increased(P&#x003C;0.05), optical density(OD450), the IC50 value of VCR on cells and the expression levels of HIF-1α, MRP and P-gp proteins were significantly reduced(P&#x003C;0.05); overexpression of HIF-1α attenuates the inhibitory effect of down-regulated lncRNA HIF1A-AS1 expression on drug resistance in SO-RB50/VCR cells.CONCLUSION: The lncRNA HIF1A-AS1 was highly expressed in SO-RB50/VCR cells, and inhibition of lncRNA HIF1A-AS1 expression reduced VCR resistance in SO-RB50/VCR cells by down-regulating HIF-1α expression.

SLC26A4-AS1 Agrava a Hipertrofia Cardíaca Induzida por AngII Aumentando a Expressão de SLC26A4 / SLC26A4-AS1 Aggravates AngII-induced Cardiac Hypertrophy by Enhancing SLC26A4 Expression

Resumo Fundamento Foi relatado que o RNA 1 antisenso 1 (SLC26A4-AS1) do membro 4 da família de transportadores de soluto 26 está altamente relacionado à hipertrofia cardíaca. Objetivo Esta pesquisa visa investigar o papel e o mecanismo específicos de SLC26A4-AS1 na hipertrofia cardíaca, fornecendo um novo marcador para o tratamento da hipertrofia cardíaca. Métodos Angiotensina II (AngII) foi infundida em cardiomiócitos ventriculares (NMVCs) de camundongos neonatos para induzir hipertrofia cardíaca. A expressão gênica foi detectada por PCR quantitativo em tempo real (RT-qPCR). Os níveis de proteína foram avaliados por western blot. Ensaios funcionais analisaram o papel de SLC26A4-AS1. O mecanismo de SLC26A4-AS1 foi avaliado por imunoprecipitação de proteína de ligação a RNA (RIP), pull-down de RNA e ensaios de luciferase repórter. O valor de p < 0,05 foi identificado como significância estatística. O teste t de Student avaliou a comparação dos dois grupos. A diferença entre os diferentes grupos foi analisada por análise de variância (ANOVA) de uma via. Resultados SLC26A4-AS1 é regulado para cima em NMVCs tratados com AngII e promove hipertrofia cardíaca induzida por AngII. SLC26A4-AS1 regula o membro 4 da família de transportadores de soluto 26 (SLC26A4) por meio do funcionamento como um RNA endógeno competitivo (ceRNA) para modular o microRNA (miR)-301a-3p e o miR-301b-3p em NMVCs. SLC26A4-AS1 promove hipertrofia cardíaca induzida por AngII via regulação para cima de SLC26A4 ou absorção de miR-301a-3p/miR-301b-3p. Conclusão SLC26A4-AS1 agrava a hipertrofia cardíaca induzida por AngII via absorção de miR-301a-3p ou miR-301b-3p para aumentar a expressão de SLC26A4.

Abstract Background It has been reported that solute carrier family 26 members 4 antisense RNA 1 (SLC26A4-AS1) is highly related to cardiac hypertrophy. Objective This research aims to investigate the role and specific mechanism of SLC26A4-AS1 in cardiac hypertrophy, providing a novel marker for cardiac hypertrophy treatment. Methods Angiotensin II (AngII) was infused into neonatal mouse ventricular cardiomyocytes (NMVCs) to induce cardiac hypertrophy. Gene expression was detected by quantitative real-time PCR (RT-qPCR). Protein levels were evaluated via western blot. Functional assays analyzed the role of SLC26A4-AS1. The mechanism of SLC26A4-AS1 was assessed by RNA-binding protein immunoprecipitation (RIP), RNA pull-down, and luciferase reporter assays. The P value <0.05 was identified as statistical significance. Student's t-test evaluated the two-group comparison. The difference between different groups was analyzed by one-way analysis of variance (ANOVA). Results SLC26A4-AS1 is upregulated in AngII-treated NMVCs and promotes AngII-induced cardiac hypertrophy. SLC26A4-AS1 regulates its nearby gene solute carrier family 26 members 4 (SLC26A4) via functioning as a competing endogenous RNA (ceRNA) to modulate the microRNA (miR)-301a-3p and miR-301b-3p in NMVCs. SLC26A4-AS1 promotes AngII-induced cardiac hypertrophy via upregulating SLC26A4 or sponging miR-301a-3p/miR-301b-3p. Conclusion SLC26A4-AS1 aggravates AngII-induced cardiac hypertrophy via sponging miR-301a-3p or miR-301b-3p to enhance SLC26A4 expression.

Progress of the mechanism of energy metabolism reprogramming regulated by long non-coding RNA in cancer / 肿瘤研究与临床

Although it is widely believed that abnormal energy metabolism exists in cancer cells and affects the biological behavior of cancers, the exact mechanism of energy metabolic reprogramming and specific mechanism of its effect on proliferation, invasion and metastasis of cancer cells have not been clarified. In recent years, studies have shown that long non-coding RNA (lncRNA) can affect energy metabolism, development and progression of cancer cells through binding to specific nucleic acids and proteins at the transcriptional and post-transcriptional stages, and specifically through transcriptional interference, epigenetic regulation of genes, changes in protein activity, competitive binding to microRNA (miRNA) and other related mechanisms. The further study on the mechanism of lncRNA regulating energy metabolism reprogramming of cancer cells is expected to find new markers and targets for diagnosis and treatment of cancer. This paper reviews the current research progress of the mechanism of lncRNA regulating metabolic reprogramming of glucose, fatty acid, protein and nucleotide in cancer, and provides a new idea of lncRNA's regulation of energy metabolism pathways for targeted anticancer therapy.

Clinical value of a cuproptosis-related differential long non-coding RNA scoring formula in clear cell renal cell carcinoma / 肿瘤研究与临床

Objective:To investigate the value of a cuproptosis-related differential long non-coding RNA (lncRNA) scoring formula related to the prognosis of clear cell renal cell carcinoma (ccRCC) patients in the clinical diagnosis, prognosis prediction and treatment options based on bioinformatics.Methods:Gene matrix and clinical data of ccRCC patients were obtained from the Cancer Genome Atlas (TCGA) database (update to 29 March, 2022). The expression data of 539 ccRCC tissues and 72 paracancerous normal tissues were collected from gene matrix; the data of 530 ccRCC were collected from clinical data. Pearson correlation analysis, Wilcoxon signed rank test and univariate Cox proportional risk model were used to analyze the screened cuproptosis-related differential lncRNA related to the prognosis. R software was used to randomly divide 530 ccRCC patients with survival data into training set (266 cases) and validation set (264 cases) according to approximate 1∶1 ratio. LASSO regression analysis was used to construct a cuproptosis-related differential lncRNA scoring formula and cross-validation was performed. Receiver operating characteristic (ROC) curve analysis was used to evaluate the specificity and sensitivity of cuproptosis-related differential lncRNA scoring formula, and the area of the curve (AUC) was calculated. According to the median risk value, all patients were divided into the low-risk group and high-risk group; Kaplan-Meier method was used to analyze the difference in the overall survival (OS) of patients in the low-risk group and high-risk group. T test was used to detect the differences in the risk value of patients with different clinicopathological characteristics. R package rms was used to construct the nomogram for predicting 1-year, 3-year, 5-year OS rates of ccRCC patients, R package pRRophetic was used to predict the half-inhibitory concentration ( IC50) of common targeted drugs such as sorafenib and sunitinib in clinical treatment of ccRCC patients, and IC50 value of patients in low-risk group and high-risk group was compared by using Wilcoxon signed rank test. Tissue samples of 20 ccRCC patients who underwent radical nephrectomy and were diagnosed with pathology and the matched paracancerous normal tissues were collected from the First Hospital of Shanxi Medical University between June 2021 and December 2021. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of key lncRNA in ccRCC tissues. Results:Based on the expression matrix of 10 cuproptosis genes (FDX1, LIAS, LIPT1, DLD, DLAT, PDHA1, PDHB, MTF1, GLS, CDKN2A) of ccRCC patients in TCGA database, 153 cuproptosis-related differential lncRNA related to the prognosis were identified. According to LASSO regression analysis, a scoring formula of 4 cuproptosis-related differential lncRNA related to the prognosis was obtained, risk value was calculated as 0.020×AC015912.3+0.011×AC026401.3+0.063×AC103706.1+(-0.076)×EPB41L4A-DT. All patients were divided into high-risk group (≥0.76) and low-risk group (<0.76) based on the median value (0.76). ROC curve analysis showed that the scoring formula had good prediction accuracy in 1-year, 3-year, 5-year OS rates. In training set, validation set, the total cohort, the OS of patients in the high-risk group was worse than that in the low-risk group (all P < 0.001). The age, pathological degree, tumor staging, risk value calculated by cuproptosis-related differential lncRNA were independent influencing factors of OS (all P < 0.001). There were statistically significant differences in the risk value calculated by cuproptosis-related differential lncRNA scoring formula among patients with different pathological degree, tumor staging, T staging, N staging, M staging (all P < 0.01), while there were no statistically significant differences among patients with different gender and age (all P > 0.05). The established nomogram had good prediction accuracy in the 1-year, 3-year, 5-year OS rates. Sunitinib and sirolimus showed higher sensitivity in the high-risk group; axitinib, sorafenib and pazopanib showed higher sensitivity in the low-risk group. qRT-PCR results showed that relative expression level of AC015912.3 in ccRCC tissues was up-regulated compared with paracancerous tissues (1.00±0.04 vs. 0.68±0.24, t = 6.37, P < 0.01); the relative expression level of AC026401.3 in ccRCC tissues was up-regulated compared with paracancerous tissues (1.00±0.05 vs. 0.64±0.22, t = 7.29, P < 0.01); the relative expression level of AC103706.1 in ccRCC tissues was up-regulated compared with paracancerous tissues (1.00±0.04 vs. 0.64±0.21, t = 7.49, P < 0.01); the relative expression level of EPB41L4A-DT in ccRCC tissues was up-regulated compared with paracancerous tissues (1.00±0.06 vs. 0.73±0.10, t = 10.68, P < 0.01). Conclusions:Cuproptosis-related differential lncRNA scoring formula based on TCGA database can be used as a new marker for clinical diagnosis and prognosis prediction of ccRCC patients, which can help guide the clinical drug treatment of patients and facilitate accurate diagnosis and treatment.

Effects of long non-coding RNA HAGLR on the prognosis of breast cancer and the construction of competitive endogenous RNA interaction network / 肿瘤研究与临床

Objective:To explore the expression of long non-coding RNA (lncRNA) HAGLR in breast cancer and its effect on the prognosis of breast cancer, and to construct a competitive endogenous RNA (ceRNA) network.Methods:The Atlas of Genetics and Cytogenetics in Oncology and Haematology website was used to search for HAGLR chromosome gene mapping and transcript expression. The lnclocater website was used to predict the subcellular localization of HAGLR, and the differential expression of HAGLR in breast cancer tissues and adjacent tissues was analyzed by using lnCAR database. The patients in lnCAR database were divided into HAGLR high expression group and HAGLR low expression according to HAGLR expression. The Kaplan-Meier method was used to analyze the overall survival (OS) and metastasis-free survival, which was verified by using UCSC Xena database. lnCAR database was used to search the co-expressed genes of HAGLR. The top 200 co-expressed genes were submitted to the Metascape website for Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis, and protein interaction network (PPI) was constructed. Starbase, a bioinformatics online analysis website, was used to predict HAGLR targeting mircoRNA (miRNA) and mRNA that directly encoded proteins. ceRNA network of HAGLR was constructed with Cytoscape3.8 software.Results:HAGLR gene was localized in 2q31.1 and mainly distributed in cytoplasm. The expression level of HAGLR in breast cancer tissues was higher than that in adjacent tissues, and the difference was statistically significant ( P < 0.001). lnCAR database and UCSC Xena database analysis showed that OS in HAGLR high expression group was worse than that in HAGLR low expression group (all P < 0.01). lnCAR database, the metastasis-free survival in HAGLR high expression group was worse than that in HAGLR low expression group ( P = 0.030). Among the top 200 HAGLR co-expressed genes, 129 genes were negatively correlated with HAGLR and 71 genes were positively correlated with HAGLR. KEGG pathway analysis showed that HAGLR was related to metabolic pathways, MAPK signaling pathway, JAK-STAT signaling pathway and cancer pathway. GO annotation analysis showed that HAGLR was mainly enriched in cell cycle, centromeric complex assembly, mitotic progression, protein kinase binding, kinase activity regulation, cell response to DNA damage stimulation and other functions. hsa-miR-130b-3p, hsa-miR-1245b-5p, hsa-miR-182b-5p, hsa-miR-512-3p, hsa-miR-302b-3p, hsa-miR-185b-5p, hsa-miR-106b-5p were HAGLR targeting miRNA. Conclusions:HAGLR is highly expressed in breast cancer tissues, and it may be a biomarker for predicting the prognosis of breast cancer.

Effects of long non-coding RNA RP11-1212A22.4 on the cell viability and invasive ability of ovarian cancer cell lines through targeting miRNA-483-5p / 肿瘤研究与临床

Objective:To investigate the effects of long non-coding RNA (lncRNA) RP11-1212A22.4 on the cell viability and invasive ability of esophageal cancer cell lines by targeting miRNA-483-5p (miR-483-5p).Methods:The expression of RP11-1212A22.4 in esophageal cancer tissues was analyzed by using GEPIA online database. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of RP11-1212A22.4 in human esophageal cancer cell lines EC9706, KYSE30, TE-13, Eca109 and normal esophageal epithelial cell line HET-1A. The lowest expression level of EC9706 cell line in RP11-1212A22.4 was divided into RP11-1212A22.4 group (transfected with pcDNA-RP11-1212A22.4 plasmid) and the control group (transfected with pcDNA-NC plasmid). The cell viability of EC9706 cell was analyzed by using methyl thiazolyl tetrazolium (MTT) method, and the invasion ability of EC9706 cell was detected by using Transwell assay. The targeting relationship between RP11-1212A22.4 and miR-483-5p was verified by using StarBase database prediction and dual luciferase reporter assay. The relative expression level of miR-483-5p of EC9706 cell in two groups was detected by using qRT-PCR. Western blot was used to detect the expressions of cyclin-dependent kinase 6 (CDK6), matrix metalloproteinase 2 (MMP-2), cyclin-dependent kinase 4 (CDK4), and matrix metalloproteinase 9 (MMP-9) proteins in two groups.Results:In GEPIA online database, compared with adjacent tissues, the relative expression level of RP11-1212A22.4 in esophageal cancer tissues was decreased, and the difference was statistically significant ( P < 0.001). The relative expression levels of RP11-1212A22.4 in esophageal cancer cell lines EC9706, KYSE30, TE-13, Eca109 and normal esophageal mucosal epithelial cell line HET-1A were 0.11±0.08, 0.32±0.09, 0.72±0.09, 0.59±0.13 and 0.97±0.12, and the difference was statistically significant ( F = 40.42, P < 0.001). The relative expression levels of RP11-1212A22.4 in EC9706 cells of RP11-1212A22.4 group and the control group were 11.9±2.4 and 1.0±0.3, respectively, and the difference was statistically significant ( t = 8.89, P < 0.001). Compared with the control group, the cell viability of EC9706 cell in RP11-1212A22.4 group was decreased (all P < 0.05). The number of invasive cells in RP11-1212A22.4 group was lower than that in the control group (48±12 vs. 106±22, t = 4.63, P < 0.001). StarBase database prediction and dual luciferase reporter assay both showed that RP11-1212A22.4 targeted miR-483-5p. The relative expression level of miR-483-5p in RP11-1212A22.4 group was lower than that in the control group (0.24±0.11 vs. 1.02±0.23, t = 5.98, P = 0.001). Compared with the control group, the expressions of CDK6, MMP-2, CDK4 and MMP-9 proteins in the RP11-1212A22.4 group were decreased. Conclusions:RP11-1212A22.4 is lowly expressed in esophageal cancer tissues and cell lines, and it inhibits the cell viability and invasive ability of esophageal cancer cells by targeting miR-483-5p.

Effects of HOTAIR on epithelial mesenchymal transition in HaCaT cells / 中华医学美学美容杂志

Objective:To verify that hypoxia induces epithelial mesenchymal transition in HaCaT cells, and to observe the effect of LncRNA HOTAIR on epithelial mesenchymal transition in HaCaT cells.Methods:From Jan. 2018 to Dec. 2018 in Chinese Academy of Medical Science, HaCaT cells were divided into four groups: group A cultured under normoxia, group B cultured under hypoxia, group C transfected with sh-control cultured under hypoxia, and group D transfected with sh-LncRNA HOTAIR cultured under hypoxia. After 36 hours of culturing, the expression of E-cadherin and vimentin were detected using qPCR and Western blot. The migration of HaCaT cells was evaluated by Transwell assay.Results:The relative quantity of E-cadherin mRNA in the four groups were 3.076±0.271, 1.000±0.089, 1.024±0.222, and 2.595±0.085, while vimentin mRNAs were 1.002±0.183, 4.170±0.279, 4.111±0.477, and 2.412±0.134, respectively. In addition, the Transwll invasion assay showed that numbers of cell migration in the four groups were 32.70±3.93, 125.40±6.26, 120.10±6.79, and 58.24±7.06, respectively.Conclusions:The study suggests that hypoxia promotes epithelial mesenchymal transition in keratinocytes. Furthermore, downregulation of HOTAIR is noted to inhibit epithelial mesenchymal transition of keratinocytes under hypoxia condition.

Mechanism and clinical value of long non-coding RNA in the development of atherosclerosis / 中华检验医学杂志

Long non-coding RNAs (lncRNA) are a class of non-coding RNA with a length of more than 200 nucleotide, it is known that lncRNA can regulate gene expression at epigenetic, transcriptional and post transcriptional levels. Recent studies have shown that lncRNA is an important regulator of atherosclerosis (AS) and plays an important role in the pathogenesis of AS. This paper aims to comprehensively explore the mechanism of lncRNA in AS and summarize new biomarkers for AS diagnosis, we focused on the research progress of some key lncRNA in regulating the function of endothelial cells, smooth muscle cells, and monocytes and macrophages through literature review. Furthermore, this review discusses the potential clinical diagnostic value of lncRNA as biomarker in coronary heart disease (CHD), acute myocardial infarction, and heart failure (HF). LncRNA is expected to become a new class of clinically relevant biomarker, which might be useful for the prediction, diagnosis, prognosis and risk stratification of AS and cardiovascular diseases.

Role of NORAD in ketamine-induced neurotoxicity in mouse hippocampal neurons: association with endoplasmic reticulum stress / 中华麻醉学杂志

Objective:To evaluate the role of long non-coding RNA (lncRNA) NORAD in ketamine-induced neurotoxicity in mouse hippocampal neurons and the relationship with endoplasmic reticulum stress.Methods:Primary mouse hippocampal neurons were isolated and cultured and then divided into 5 groups ( n=36 each) using a random number table method: control group (group C), ketamine group (group K), ketamine+ pcDNA3.1-NORAD plasmid group (group K+ NORAD), ketamine+ control plasmid group (group K+ NC), and ketamine+ NORAD+ tunicamycin group (group K+ NORAD+ TM). Group C was cultured with normal medium for 24 h. Group K was cultured with 40 μmol/L ketamine for 24 h. Group K+ NORAD was transfected with pcDNA3.1-NORAD overexpressing plasmid for 48 h, followed by treatment with 40 μmol/L ketamine for 24 h. Group K+ NC was transfected with pcDNA3.1 (+ ) plasmid for 48 h, followed by treatment with 40 μmol/L ketamine for 24 h. Group K+ NORAD+ TM was transfected with pcDNA3.1-NORAD overexpressing plasmid, 24 h later endoplasmic reticulum stress activator tunicamycin 1 μg/ml was added and the neurons were cultured for 24 h, and then ketamine 40 μmol/L was added and the neurons were cultured for another 24 h. Cell viability was detected by CCK-8 assay. The amount of lactate dehydrogenase (LDH) released was analyzed. Cell apoptosis was determined by TUNEL and flow cytometry methods. The NORAD expression was detected by real-time polymerase chain reaction. The expression of endoplasmic reticulum stress-related proteins protein kinase R-like ER kinase (PERK), phosphorylated PERK (p-PERK) and C/EBP homologous protein (CHOP) was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the amount of LDH released, percentage of apoptotic neurons and apoptosis rate were increased, NORAD expression was down-regulated, CHOP expression was up-regulated, and p-PERK/PERK was increased in group K ( P<0.05). Compared with group K, the cell viability was significantly increased, the amount of LDH released, percentage of apoptotic neurons and apoptosis rate were decreased, NORAD expression was up-regulated, CHOP expression was down-regulated, and p-PERK/PERK was decreased in group K+ NORAD ( P<0.05), and no significant change was found in the parameters mentioned above in group K+ NC ( P>0.05). Compared with group K+ NORAD, the cell viability was significantly decreased, the amount of LDH released, percentage of apoptotic neurons and apoptosis rate were increased, CHOP expression was up-regulated, and p-PERK/PERK was increased ( P<0.05), and no significant change was found in the NORAD expression in group K+ NORAD+ TM ( P>0.05). Conclusions:Over-expressed NORAD can alleviate ketamine-induced neurotoxicity in mouse hippocampal neurons via inhibition of the endoplasmic reticulum stress.

Research progress on the role of non-coding RNA in chemotherapy resistance of osteosarcoma / 中华骨科杂志

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. With the emergence of chemotherapy resistance in recent years, the survival rate of osteosarcoma patients has reached a bottleneck. Therefore, exploration of its chemoresistance mechanism is one of the popular research directions currently. Non-coding RNA (ncRNA) is a class of RNA without the ability to encode proteins, which is classified into microRNA, long non-coding RNA, and circular RNA based on length and shape. With the development of high-throughput sequencing technology, there is increasing evidence that some non-coding RNAs are abnormally upregulated or downregulated in osteosarcoma cells and affect the response of osteosarcoma to four commonly used chemotherapeutic drugs (methotrexate, doxorubicin, cisplatin and ifosfamide) through mechanisms such as regulation of apoptosis, cell cycle, signaling pathways, intracellular drug concentration, and cellular autophagy. Therefore, thesenon-coding RNAs are expected to be novel targets for osteosarcoma treatment. In this paper, the current studies were searched and reviewed on the above three non-coding RNAs mediating chemoresistance in osteosarcoma, aiming to provide a reference for breaking the bottleneck of survival rate of osteosarcoma patients.

Expression profiling of cell-derived exosomal lncRNAs resistant to concurrent chemoradiotherapy in nasopharyngeal carcinoma / 中华放射肿瘤学杂志

Objective:To screen the key exosomal long non-coding RNAs (lncRNAs) molecules that cause nasopharyngeal carcinoma cells to develop chemoradiotherapy resistance.Methods:In vitro, a model of concurrent chemoradiotherapy for human nasopharyngeal carcinoma cells was constructed, and the continuous shock method of high-dose concurrent chemoradiotherapy was used to induce the establishment of chemoradiotherapy-resistant nasopharyngeal carcinoma cell lines, and its resistance formation was verified. Exosomes produced by chemoradiotherapy-resistant cell lines and respective mother cell lines for nasopharyngeal carcinoma were extracted and identified. Finally, biochip technology was used to detect the differential expression levels of exosomal lncRNAs. Results:After 10 repeated treatments of concurrent chemoradiotherapy, CNE-1 CRR and CNE-2 CRR were successfully obtained. Compared with the mother cell lines, CNE-1 CRR and CNE-2 CRR had a tendency to transform from epithelial to interstitial morphology, and the number of cell clones was higher, and the values of average lethal dose (D 0), quasi-threshould dose (D q), survival fraction after 2 Gy irradiation (SF 2) and cell survival rate were higher. Nasopharyngeal carcinoma cells were detected by PCR chip of exosomal lncRNAs. Compared with their respective mother cell lines, 18 lncRNAs in CNE-1 CRR exosomes were significantly up-regulated and 31 lncRNAs were significantly down-regulated, and 15 lncRNAs were significantly up-regulated and 38 lncRNAs were significantly down-regulated in CNE-2 CRR exosomes. CNE-1 CRR also had similar expression profiles to CNE-2 CRR. Conclusion:There are significantly up-regulated and down-regulated lncRNAs in the exosomes of CNE-1 CRR and CNE-2 CRR.

Clinical efficacy of autologous platelet rich gel in the treatment of type 2 diabetic foot patients and its effect on the expression of MALAT1 in peripheral blood mononuclear cells / 中国医师杂志

Objective:To observe the clinical efficacy of autologous platelet rich gel (APG) in the treatment of type 2 diabetic foot (DF) patients and the effect of APG on the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in peripheral blood mononuclear cells (PBMCs).Methods:A total of 62 patients with DF admitted to the Affiliated Hospital of Kangda College of Nanjing Medical University from February 2021 to May 2022 were randomly divided into a control group (30 cases) and an observation group (32 cases) using a random number table method. The control group received ultrasound debridement and dressing change treatment, while the observation group received ultrasound debridement combined with APG treatment. After 6 weeks of treatment, the effective rate, transcutaneous oxygen partial pressure (TcPO 2), and serum tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), hypoxia inducible factor α (HIF-1 α)and the level of MALAT1 expression in PBMCs of the two groups of patients were observed. The Pearson correlation analysis was used to investigate the relationship between the expression change of MALAT (△ MALAT1) and the total effective rate of treatment. Results:The total effective rate of the observation group was higher than that of the control group [93.75%(30/32) vs 73.33%(22/30), P<0.05]. After treatment, the systolic blood pressure (SBP), diastolic blood pressure (DBP), cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), fasting blood glucose (FPG), glycosylated hemoglobin (HbA 1c), urinary microalbumin/creatinine (UACR), uric acid (UA), white blood cells (WBC), TNF- α and IL-6 of both groups had decreased compared to before; HIF-1 α, VEGF and MALAT1 increased compared to before treatment (all P<0.05); After treatment, there was a statistically significant difference in UA, HIF-1α, VEGF, and MALAT1 between the observation group and the control group (all P<0.05). Pearson correlation analysis showed that Δ MALAT1 in DF patients was negatively correlated with TNF -α ( r=-0.61, P=0.02), IL-6 ( r=-0.52, P=0.04), WBC ( r=-0.53, P=0.03), and positively correlated with VEGF ( r=0.58, P=0.03) and HIF-1α ( r=0.54, P=0.03). The total effective rate of DF treatment was higher in the high change group of△ MALAT [88.37%(38/43) vs 73.68%(14/19), P<0.05]. There was no statistically significant difference in the incidence of adverse reactions between the two groups ( P>0.05). Conclusions:APG can significantly upregulate the expression of MALAT, improve wound tissue blood perfusion, wound angiogenesis, and inflammatory response, promote ulcer healing, and changes in MALAT expression can help determine the prognosis of DF.