Preparation and efficacy evaluation of polyclonal antibodies against serum albumin of five species / 中国生物制品学杂志

@#Objective To prepare polyclonal antibodies against the serum albumin of human,cattle,sheep,pig and horse,and evaluate their efficacy in the identification of human serum albumin(HSA). Methods The specific polypeptides of human,cattle,sheep,pig and horse serum albumin were prepared by bioinformatics and polypeptide synthesis method,which were coupled with keyhole limpet hemocyanin(KLH)to prepare the peptide antigen after the purity was identified by high performance liquid chromatography(HPLC). Male New Zealand white rabbits were immunized with polypeptide antigens of five species subcutaneously,with 2 for each kind of antigen. The antiserum was then obtained and purified by Protein A affinity chromatography to prepare the polyclonal antibody. The titers and specificity of the polyclonal antibodies were determined by ELISA and Western blot respectively,and the prepared five species of serum albumin were used to identify HSA products. Results The synthetic peptides of human,cattle,sheep,pig and horse serum albumin had a purity of over 91%,and the corresponding polyclonal antibodies all had the titer of 1∶160 000,which showed specific binding with the corresponding antigens and effectively identified the HSA products. Conclusion The polyclonal antibodies of human cattle,sheep,pig and horse serum albumin prepared in this study have good specificity and the preparation process is simple and rapid,suitable for the mass production,which lays a foundation of the development of HSA rapid identification kit.

Hsa_circ_0013561 promotes progression of nasopharyngeal carcinoma by activating JAK2/STAT3 signaling pathway

Abstract Objective Nasopharyngeal Carcinoma (NPC) is a malignancy of epithelium of epithelium of the nasopharynx, with the highest incidence of otolaryngeal malignancies. A growing number of studies confirm that Circular RNA (circRNA) plays an important role in tumor development, including Hsa_circ_0013561. This study aims to elucidate the process and mechanism of NPC regulation hsa_circ_0013561. Methods In this study, circRNA expression nodes and subcellular localization in NPC tissues were analyzed by fluorescence in situ hybridization. The expression of hsa_circ_0013561 in NPC cells was further clarified by RT-qPCR. At the same time, the lentivirus vector interfered by hsa_circ_0013561 was constructed and transfected. The cell proliferation was detected by CCK-8 method, EdU assay and plate cloning assay. The cell cycle and apoptosis were detected by flow cytometry, and the cell migration ability was detected by wound healing assay and Transwell assay. Western blotting examined the expression of apoptosis, Epithelial-Mesenchymal Transition (EMT)-associated proteins, and Janus Kinase/Signal Transductor and Activator of Transcription (JAK/STAT) signaling pathway-related proteins. Results The results showed that the expression of hsa_circ_0013561 in NPC samples was significantly upregulated and hsa_circ_0013561 localized in the cytoplasm. After down-regulating hsa_circ_0013561 expression, it significantly inhibited the proliferation and metastasis ability of NPC, inhibited EMT progression, and promoted apoptosis. Further studies showed that interference hsa_circ_0013561 significantly inhibited JAK2/STAT3 signaling pathway activation and induced the expression of apoptosis-related proteins. Conclusion In summary, we found that hsa_circ_0013561 is a pro-tumor circRNA in NPC, which can reduce the activation of JAK2/STAT3 pathway by knocking down hsa_circ_0013561, thereby slowing down the malignant progression of NPC. Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence Level 4.

hsa_circ_0140180 affects the migration and invasion of esophageal squamous cell carcinoma cells by regulating miR-1287-5p / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨hsa_circ_0140180在食管鳞状细胞癌（ESCC）细胞中的表达水平及对其细胞恶性生物学行为的影响与分子机制。方法：收集2018年11月至2019年3月间在南充市中心医院胸心外科手术切除的6对ESCC组织和对应癌旁组织并进行全转录组测序，筛选出在ESCC组织中低表达的hsa_circ_0140180；建立过表达hsa_circ_0140180的TE-1和KYSE30细胞，qPCR法检测hsa_circ_0140180在人正常食管上皮细胞、ESCC细胞中的表达，以及过表达hsa_circ_0140180后TE-1和KYSE30细胞中miR-1287-5p的表达；CCK-8法和FCM检测过表达hsa_circ_0140180对TE-1和KYSE30细胞增殖和周期的影响；划痕实验和Transwell实验检测过表达hsa_circ_0140180对TE-1和KYSE30细胞迁移和侵袭能力的影响，双荧光素酶报告实验验证hsa_circ_0140180与miR-1287-5p的靶向关系。WB法检测过表达hsa_circ_0140180对TE-1和KYSE30细胞中EMT相关蛋白的表达及PI3K-Akt通路的磷酸化水平的影响。结果：转录组测序和qPCR结果显示，hsa_circ_0140180在ESCC组织和细胞中呈低表达（P<0.05或P<0.01），并确认其闭合环状分子特征且定位于细胞质。过表达hsa_circ_0140180能明显抑制ESCC细胞的迁移及侵袭能力（P<0.05），但不影响其增殖和周期。双荧光素酶报告基因实验证实hsa_circ_0140180靶向结合miR-1287-5并负调控其表达（P<0.01）。过表达hsa_circ_0140180能显著上调TE-1和KYSE30细胞中E-cadherin的表达（（P<0.05），而显著下调Snail的表达（P<0.05）和PI3K-Akt通路的磷酸化水平（P<0.01或P<0.001）。结论：hsa_circ_0140180在ESCC细胞及组织中呈低表达，其可能通过调控miR-1287-5p表达来降低PI3K-Akt通路的磷酸化水平和抑制EMT进程，从而影响ESCC细胞的迁移及侵袭能力。

Expression and clinical significance of circ_002082 in breast cancer / 中华内分泌外科杂志

Objective:This paper mainly discusses the expression of hsa_circ_002082 in breast cancer and its clinical significance to breast cancer.Methods:The up-regulated hsa_circ_002082 in breast cancer was screened out through the bioinformatics website, and verified by quantitative reverse transcription PCR (qRT-PCR). The differential expression of hsa_circ_002082 in different clinical index groups was analyzed by one-way ANOVA and t-test, and the ROC curve was used to analyze the value of hsa_circ_002082 in diagnosing breast cancer or identifying clinical indexes.Results:Compared with para-cancerous tissue (1.00±0.21), the expression of hsa_circ_002082 in cancer tissue (1.34±0.25) was higher than that in peripheral blood of healthy people (1.00±0.26), and the expression of hsa_circ_002082 in peripheral blood of breast cancer patients (1.39±0.24) increased (all P<0.05). The AUC value of hsa_circ_002082 for breast cancer diagnosis was 0.8520 ( P<0.05). There were differences in the expression of hsa_circ_002082 in tumor grade, estrogen receptor (ER) positive, carcino-embryonic antigen (CEA) positive, Ki-67 proliferation index, tumor metastasis, and TNM grade (all P<0.05). The relative expression level of hsa_circ_002082 was significantly positively correlated with the level of tumor marker CEA ( r=0.368, P=0.009). The AUC values of hsa_circ_002082 for distinguishing tumor grade, ER positive, CEA positive, Ki-67 proliferation index, tumor metastasis, and TNM grade were 0.8744, 0.7005, 0.7890, 0.8075, 0.8317, and 0.8301, respectively (all P<0.05) . Conclusion:hsa_circ_002082 is highly expressed in breast cancer tissue and peripheral blood, and hsa the potential to be a biomarker for the diagnosis of breast cancer.

The aberrant overexpression of hsa_circ_0078607 in colorectal cancer tissues and serum and its clinical significance / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究hsa_circ_0078607在结直肠癌组织和患者血清中的表达水平及其与结直肠癌患者临床病理特征的关系，评价其能否作为结直肠癌潜在的分子诊断标志物及治疗靶标。方法：收集2018年6月至2022年1月于柳州市人民医院胃肠外科接受结直肠癌切除手术患者的58对癌及癌旁组织标本，收集2020年1月至2022年12月于柳州市人民医院初次确诊的结直肠癌患者、结直肠息肉患者及健康人体检血清共152例；从结直肠癌差异表达circRNA谱中挑选特异性高表达的hsa_circ_0078607作为候选标志物，采用qPCR法检测其在结直肠癌细胞、组织、患者血清及结直肠息肉患者血清中的相对表达量，分析其与临床病理特征的关系。采用ROC曲线评估hsa_circ_0078607对结直肠癌及结直肠息肉的诊断价值。通过Circular RNA Interactome数据库预测与hsa_circ_0078607结合的miRNA，并用Cytoscape 3.9.1软件构建circRNA-miRNA-mRNA调控网络，同时通过GO/KEGG富集分析进一步了解其功能。结果：与癌旁组织或健康人血清相比，hsa_circ_0078607在结直肠癌细胞、组织和血清及息肉患者血清中呈高表达（P<0.001），其中有52例（89.7%）患者癌组织中表达上调，6例（10.3%）表达下调。结直肠癌组织中hsa_circ_0078607的相对表达量与肿瘤位置（P=0.029）、分化程度（P=0.046）和远处转移（P=0.043）有关联。ROC结果显示，在结直肠癌组织和血清中其诊断结直肠癌的AUC分别为0.845 7[95%CI（0.772 8，0.918 6），P<0.000 1]和0.868 3[95%CI（0.790 7，0.945 9），P<0.000 1]；在息肉患者血清中，hsa_circ_0078607诊断结直肠息肉的AUC为0.710 1 [95%CI（0.610 0，0.810 1）]。GO/KEGG富集分析结果表明，hsa_circ_0078607下游的miRNA可能参与RNA聚合酶Ⅱ启动子转录调控、蛋白K48-连锁泛素化、Wnt、Hippo及MAPK信号通路调控等多个生物过程。结论：Hsa_circ_0078607在结直肠癌细胞、组织和血清中呈高表达，其在结直肠癌组织中的表达水平与肿瘤位置、分化程度和远处转移有关联，提示其可作为结直肠癌潜在的分子诊断标志物；其还可能介导结直肠癌的发生发展过程，对发现结直肠癌潜在的治疗靶点有重要意义。

Interaction between BCSG1 and Hsa-circ-0026352 in genetic susceptibility to invasive breast cancer / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨乳腺癌特异基因1（BCSG1）与Hsa-circ-0026352在浸润性乳腺癌（IBC）遗传易感性中的交互作用。方法：选取2019年6月至2022年5月间武汉市中西医结合医院收治的100例IBC患者作为研究对象，采用免疫组化法检测IBC组织及其相应癌旁组织中BCSG1的表达，将研究对象按照IBC组织中BCSG1蛋白表达的高低分为阴性、弱阳性和强阳性组，统计三组患者的临床病理特征及雌激素受体（ER）、孕激素受体（PR）、表皮生长因子受体-2（HER2）、Hsa-circ-0026352的表达情况，采用Logistic回归方程和最大似然法分析BCSG1表达与上述参数的趋势性和交互作用。结果：与癌旁组织比较，IBC组织中BCSG1蛋白呈高表达（P<0.05）；BCSG1蛋白强阳性表达与淋巴结转移、分化程度、临床分期、HER2表达、 Hsa-circ-0026352表达有关联（P<0.05）；BCSG1强阳性表达与IBC存在交互作用（P<0.05）；BCSG1表达与IBC的交互作用在Hsa-circ-0026352阳性表达中最为显著（趋势P<0.001）；BCSG1表达与IBC的交互作用在临床分期Ⅲ期、低分化程度中最为显著（趋势P<0.001）。结论：BCSG1与IBC患病密切相关，且与Hsa-circ-0026352、临床分期、分化程度存在交互作用，可共同增加IBC患病风险性。

Expression of hsa-miR-422a in hypertrophic scars: a bioinformatics analysis / 中华医学美学美容杂志

Objective:To evaluate the expression level of hsa-miR-422a in hypertrophic scars and to identify the target genes of hsa-miR-422a along with their biological functions using bioinformatics approaches.Methods:From June 2020 to December 2020, tissue samples of 3 hypertrophic scar and 3 normal skin were collected from patients (3 males, 3 females, aged 20-42 years) in Department of Plastic and Reconstructive Surgery, Shanghai Ninth People′s Hospital, Shanghai Jiaotong University School of Medicine. Primary fibroblasts were isolated and cultured. Real-time quantitative PCR was performed to quantify the expression of hsa-miR-422a. To construct a ceRNA network, starbase and Target Scandata bases were utilized to predict genes as well as long noncoding RNAs (lncRNAs) that may sponge hsa-miR-422a. GO and KEGG pathway enrichment analyses were conducted on the target genes of hsa-miR-422a; protein-protein interaction (PPI) networks were constructed to identify the hub genes whose functions were predicted by functional enrichment analyses. The expression of hub genes was validated through real-time quantitative PCR in hypertrophic scars.Results:The expression of hsa-miR-422a was significantly lower in the hypertrophic scar tissue samples and fibroblasts compared to that in the normal skin ( P<0.05). 133 target genes as well as 1033 lncRNAs were predicted by starBase and TargetScandata bases and used to construct an hsa-miR-422a-centered ceRNA network. PPI networks of the target genes revealed 10 hub genes, including MAPK1, GRB2, and IGF1R, which were discovered to be related to protein serine/threonine/tyrosine kinase activity, ubiquitin protein ligase binding, fibroblast growth factor receptor signaling pathway, muscle cell proliferation, and many others; besides, they may be involved in FoxO, mTOR, Toll-like receptor, Ras, MAPK, PI3K-Akt signaling pathways and signaling pathways regulating pluripotency of stem cells. Three hub genes (MAPK1, GRB2, and IGF1R) were significantly upregulated in hypertrophic scars ( P<0.05). Conclusions:hsa-miR-422a is significantly downregulated in the hypertrophic scars and may target hub genes such as MAPK1 in ceRNA networks, ultimately modulating hypertrophic scar formation.

Role and mechanism of miR-548-3p/ DAG1 in the occurrence and malignant transformation of laryngeal carcinoma / Papel y mecanismo de miR-548-3p/DAG1 en la aparición y transformación maligna del carcinoma de laringe

Abstract The AMC-HN-8 cell line and the primary human laryngeal epithelial cell lines were utilized in this work to explore the molecular mechanism of miR-548-3p regulating the gene DAG1 to induce the occurrence and malignant transformation of laryngeal carcinoma. Non-coding RNA miR-548- 3p overexpression plasmid, interference plasmid and blank plasmid were constructed, and the plasmids were transfected into AMC-HN-8 cells, respectively. Meanwhile, a non-transfected plasmid group and a human laryngeal epithelial primary cell group were set up. Five groups of cells were named as NC (Normal control), Model, Ov-miR-548-3p, Sh-miR-548-3p and Blank-plasmid group. The luciferase reporter experiment was used to analyze the regulation characteristics of hsa-miR-548-3p on dystrophin-associated glycoprotein 1 (DAG1). Immunofluorescence was used to analyze the relative expression characteristics of the protein DAG1. The cell cloning experiment was used to analyze the proliferation characteristics of AMC-HN-8. The scratch healing test was used to analyze the migration ability of AMC-HN-8. The transwell test was used to analyze the invasion ability of AMC-HN-8. The RT-PCR was used to analyze the expression level of miR-548-3p. Western blot experiments were used to analyze the expression of protein DAG1, laminin α2 (LAMA2) and utrophin (UTRN). The luciferase report experiment and immunofluorescence test found that the expression of DAG1 and miR-548-3p are positively correlated. Cell cloning, scratching and migration experiments identified that the activity of laryngeal cancer cells was positively correlated with the expression of DAG1. The results of Western blot analysis further strengthened the above conclusions. Through carrying out research on the cellular levels, our work has demonstrated that miR-548-3p regulated the content of protein DAG1, and then further induced malignant transformation of laryngeal carcinoma.

Resumen En este trabajo se utilizaron la línea celular AMC-HN-8 y la línea celular epitelial laríngea humana primaria, para explorar el mecanismo molecular regulador del miR-548-3p sobre el gen DAG1 para inducir la aparición y la transformación maligna del carcinoma laríngeo. Se construyeron el plásmido de sobreexpresión de miR-548-3p de ARN no codificante, el plásmido de interferencia y el plásmido en blanco, y los plásmidos se transfectaron en células AMCHN-8 respectivamente. Mientras tanto, se establecieron un grupo de plásmidos no transfectados y un grupo de células primarias epiteliales laríngeas humanas. Se nombraron cinco grupos de células como NC (control normal), modelo, OvmiR-548-3p, Sh-miR-548-3p y grupo de plásmido en blanco. El experimento indicador de luciferasa se utilizó para analizar las características de regulación de hsa-miR-548-3p en la glicoproteína 1 asociada a distrofina (DAG1). Se utilizó inmunofluorescencia para analizar las características de expresión relativa de la proteína DAG1. El experimento de clonación celular se utilizó para analizar las características de proliferación de AMC-HN-8. Se utilizó la prueba de cicatrización por rascado para analizar la capacidad de migración de AMC-HN-8. La prueba de transwell se utilizó para analizar la capacidad de invasión de AMCHN-8. Se utilizó RT-PCR para analizar el nivel de expresión de miR-548-3p. Se usó un experimento de transferencia Western (Western blot) para analizar las expresiones de la proteína DAG1, laminina α2 (LAMA2) y utrofina (UTRN). El experimento de reporte de luciferasa y la prueba de inmunofluorescencia encontraron que la expresión de DAG1 y miR-548-3p están positivamente correlacionadas. Los experimentos de clonación celular, rascado y migración, identificaron que la actividad de las células cancerosas de laringe se correlacionó positivamente con la expresión de DAG1. Los resultados del análisis de transferencia Western fortalecieron aún más las conclusiones anteriores. A través de la investigación a nivel celular, nuestro proyecto ha demostrado que miR-548-3p regula el contenido de la proteína DAG1 y luego induce la transformación maligna del carcinoma de laringe.

Effect of Circular RNA hsa_circ_0067582 on the Proliferation and Invasion Ability of Gastric Cancer Cells / 中国医学科学院学报

Objective To investigate the effects on cell proliferation and invasion of the circular RNA hsa_circ_0067582 in gastric cancer(GC). Methods After hsa_circ_0067582 overexpression (Oe-circ_0067582) plasmid was transfected into AGS and SGC-7901 cells,the cell viability,proliferation,invasion ability,and apoptosis were detected by CCK-8,colony formation and EdU assays,Transwell assay,and flow cytometry,respectively.Western blotting was employed to detect the expression levels of proteins related to the cell apoptosis and epithelial-mesenchymal transition(EMT).The effect of Oe-circ_0067582 on the growth of SGC-7901 cells in nude mice was observed.Bioinformatics tools were used to predict the binding target miRNA of hsa_circ_0067582,and the competing endogenous RNA(ceRNA)regulatory network was established.Finally,functional enrichment was performed to analyze the biological functions of the target genes of the predicted miRNA. Results Compared with the pLO-ciR(empty plasmid)group,the Oe-circ_0067582 group in AGS and SGC-7901 cells attenuated the cell viability(t=7.883,P=0.001;t=5.679,P=0.005),proliferation(t=6.709,P=0.003;t=5.857,P=0.003),and invasion ability(t=7.782,P=0.002;t=6.342,P=0.003)and induced cell apoptosis(t=7.225,P=0.002;t=11.509,P=0.001).Western blotting showed that the Oe-circ_0067582 group in AGS and SGC-7901 cells up-regulated the protein levels of cysteinyl aspartate specific proteinase (Caspase) 3(t=6.863,P=0.002;t=7.024,P=0.001),Caspase 7(t=3.295,P=0.04;t=6.008,P=0.004),Caspase 9(t=4.408,P=0.012;t=6.278,P=0.004),and E-cadherin(t=12.453,P=0.002;t=10.867,P=0.001),while down-regulated those of Vimentin(t=7.242,P=0.002;t=5.694,P=0.004)and N-cadherin(t=6.480,P=0.003;t=7.446,P=0.001).Furthermore,Oe-circ_0067582 significantly inhibited the growth of tumor in the SGC-7901 tumor-bearing nude mice(t=3.526,P=0.017).The prediction based on TargetScan and miRnada suggested that hsa_circ_0067582 can competitively bind to hsa-miR-181b-3p,hsa-miR-337-3p,hsa-miR-421,and hsa-miR-548d-3p.The functional enrichment indicated that the target genes of miRNA were involved in multiple cancer-related biological processes including negative regulation of apoptotic process,gene expression,transcriptional misregulation in cancer,transforming growth factor-β,and p53 signaling pathways. Conclusion Oe-circ_0067582 can inhibit the proliferation and attenuate EMT process to reduce the invasion ability of AGS and SGC-7901 cells,which provides a new target for the treatment of GC.

Expression of hsa_circ_0087378 in esophageal squamous cell carcinoma tissues and its effect of prognosis of patients / 中国肿瘤生物治疗杂志

@# 朱永刚a, b，王明博b，孟令娇a，单保恩a（河北医科大学第四医院 a.科研中心；b.胸外科，河北 石家庄 050011） [摘 要] 目的：探讨 hsa_circ_0087378在食管鳞状细胞癌（ESCC）组织中的表达及其对ESCC患者临床病理指标及预后的影响。方法：通过GEO数据库筛选ESCC组织及相应癌旁组织中差异表达的circRNA，采用qPCR法在10例ESCC组织及相应癌旁组织中进行验证。选取河北医科大学第四医院2015年10月至2016年4月收治的90例ESCC患者癌组织及配对癌旁组织标本，通过FISH法检测hsa_circ_0087378的表达，分析其表达与ESCC患者临床病理指标及预后的关系。结果：GEO数据库及qPCR分析结果显示，hsa_circ_0087378在ESCC组织中表达最明显。FISH结果显示，90例癌旁组织中21例hsa_circ_0087378呈高表达，高表达率为23.3%；90例ESCC组织中有65例hsa_circ_0087378呈高表达，高表达率为72.2%，癌组织中表达明显高于癌旁组织（P<0.05）。hsa_circ_0087378在ESCC组织中的高表达与患者的临床分期和淋巴结转移有关联（P<0.05），hsa_circ_0087378高表达组ESCC患者的5年总生存率明显低于低表达组患者（P<0.05）。结论： hsa_circ_0087378在ESCC组织中呈高表达，且与患者的临床分期与淋巴结转移和预后不良有关，其可能是ESCC的治疗靶点。

Effects of Circular RNA hsa_circ_0001922 on Proliferation, Migration and Invasion of Prostate Cancer Cells and Its Potential Molecular Mechanism / 肿瘤防治研究

Objective To investigate the effect of circular RNA hsa_circ_0001922 on the proliferation, migration and invasion of prostate cancer cell PC-3 and its underlying molecular mechanism. Methods qRT-PCR and RNA FISH were used to detect the expression level and localization of hsa_circ_0001922 in PC-3 cells respectively. After hsa_circ_0001922 was targeted inhibited, clone formation, Transwell assay and scratch assay were used to detect the proliferation, migration and invasion abilities of PC-3 cells. qRT-PCR and Western blot were used to detect the expression levels of EMT pathway-related molecules after inhibiting hsa_circ_0001922. Results The expression of circular RNA hsa_circ_0001922 was increased in PC-3 cells (P < 0.01) and it existed in the cytoplasm and nucleus. The invasion, migration and invasion abilities were significantly weakened (P < 0.05) after inhibiting hsa_circ_0001922; the expression of E-cadherin mRNA increased (P < 0.05) while the mRNA level of Vimentin decreased (P < 0.05). The results of Western blot were consistent with the above (both P < 0.05). Conclusion The circular RNA hsa_circ_0001922 may promote the proliferation, migration and invasion of PC-3 cells through the EMT pathway.

Differential expression of cyclic RNA in peripheral blood mononuclear cells of patients with rheumatoid arthritis / 中华风湿病学杂志

Objective:To investigate the differences in the expression profiles of cyclic RNA (circRNA) in peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) and its clinical significance.Methods:Venous blood were collected from 4 patients with RA (group T) and 4 healthy subjects (group C). The expression profiles of circRNA in PBMCs of the two groups were detected by Arraystar circRNA microarray, and the differentially expressed circRNA was analyzed by clustering analysis. The binding sites for interaction between differentially expressed circRNA and miRNA were predicted, and functional analysis such as geneontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed. quantitative real-time polymerase chain reaction (RT-qPCR) was used to verify the expression of partially differentially expressed circRNA in the two groups of PBMCs, and a circRNA-miRNA-mRNA regulatory network (ceRNA network) was constructed for the target circRNA with significantly differential expression. A receiver operating characteristic curve [receiver operating characteristic curve (ROC)] was established to analyze the potential diagnostic value of target circRNA. SPSS Statistics 23.0 and Graphpad Prism 8.0 were used to analyze the data, and the independent t test was used to analyze the difference between the two groups. Results:① Microarray results showed that, compared with group C, a total of 399 [fold of difference (FC)>1.5, and P<0.05] circRNA were abnormally expressed in PBMCs of group T; including 149 up-regulated and 250 down-regulated. ② Bioinformatics analysis: The prediction of the binding site of circRNA and miRNA suggested that the differentially expressed circRNA in RA might affect the inflammatory response by targeting miR-140-5p, miR-338-5p, and miR-9-5p. GO analysis showed that the differentially expressed circRNA was mainly involved in the intimal-binding organelles, protein metabolism and binding, etc. KEGG pathway analysis showed that most of the involved pathways were related to infection and human immune dysregulation. ③ The results of multi-sample RT-qPCR validation showed that the expression level of hsa_circRNA_009012 in group T was significantly higher than that in group C ( t=-4.417, P<0.01), the expression level of hsa_circRNA_101328 was significantly lower than that in group C ( t=-1.042, P<0.01), and the expression of hsa_circRNA_058230 had no significant change ( t=4.691, P>0.05). ④ ROC curve analysis indicated that hsa_circRNA_009012 had potential value in the diagnosis of RA [area under curve=0.96]. Conclusion:The expression of circRNA in PBMCs of patients with RA is imbalanced, and it may participate in the regulation of the development of RA. Among them, hsa_circRNA_009012 is expected to become a new biological marker for the diagnosis and treatment of RA.

Hsa_circ_0087354 Regulates Proliferation and Redox State via hsa-miR-199-3p / SLC7A11 in MG-63 Cells / 中国生物化学与分子生物学报

Circular RNAs (circRNAs),a kind of novel non-coding RNAs, have been shown to play animportant role in cellular redox reactions. In the previous study, we found that hsa_circ_0087354 wasclosely related to the cellular redox state by real-time PCR. After overexpression of hsa_circ_0087354, the relative expression of ROS1 were decreased significantly (P < 0. 01), while the levels of SOD1 wereincreased significantly (P < 0. 05) . The activities of SOD and Gpx as well as GSH concentration weresignificantly increased (P < 0. 01), and cell proliferation was promoted in cells (P < 0. 05) . Bioinformatics analysis predicted that there were binding sites between hsa-miR-199-3p and hsa_circ_0087354 as well as solute carrier family 7 member 11 (SLC7A11), which might have a targetedregulatory relationship. Dual luciferase reporter assay confirmed the targeted regulatory relationshipbetween hsa-miR-199-3p with hsa_circ_0087354, and SLC7A11. Overexpressed hsa_circ_0087354 plasmid and ctrl plasmid were constructed, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-NC mimicswere synthesized. Real-time PCR analysis showed that the relative expression of hsa-miR-199-3p was observably decreased (P < 0.01), while the relative expression of SLC7A11 in cells was dramaticallyincreased after transfection of has_circ_0087354 plasmid (P < 0.05) . After transfection with hsa-miR-199-3p, the relative expressions of SLC7A11 were markedly decreased (P < 0.001) . The activities ofSOD and Gpx, GSH concentration (P<0.01), and cell proliferation rate (P < 0.05) were significantlyreduced. In conclusion, hsa_circ_0087354 could enhance the expression of SLC7A11, promote theproliferation of cells and reduce the oxidative stress by adsorption of hsa-miR-199-3p.

Effects of hsa_circ_0045943 targeting miR-106a on the biological characteristics of gastric cancer cells / 西安交通大学学报(医学版)

【Objective】 To investigate the effects of hsa_circ_0045943 targeting miR-106a on the biological characteristics of gastric cancer cells and its mechanism. 【Methods】 Human gastric cancer cells MKN-45， AGS and gastric mucosal epithelial cells GES-1 were cultured； circ_0045943 was detected by real-time polymerase chain reaction. The overexpression and silencing of circ_0045943 adenovirus vectors OE-circRNA and sh-circRNA together with their negative controls OE-NC and sh-NC were constructed and transfected； CCK-8 method was used to detect the proliferation activity of AGS cells after overexpression and silencing of circ_0045943； TUNEL method was used to detect the cell apoptosis； transwell assay was used to detect the cell migration and invasion； and would healing assay was used to detect the cell migration. Starbase database screened the binding site of miR-106a and circ_0045943. Real-time PCR was used to detect the expression of miR-106a， and the expression of circ_0045943 and the changes of miR-106a after the treatment of OE-circRNA and sh-circRNA. 【Results】 Real-time PCR showed that the expression of circ_0045943 decreased in gastric cancer cells MKN-45 and AGS compared to GES-1 （Pboth<0.001）. CCK-8 showed that the absorbance value of AGS cells in OE-circRNA group was lower than that in sh-circRNA group （P24 h<0.01， P48 h<0.001， and P72 h<0.001）. TUNEL showed the number of apoptotic AGS cells increased after overexpression of circ_0045943， but decreased after silencing of circ_0045943. Transwell assay showed that the migration and invasion of AGS cells were lower in OE-circRNA group than in sh-circRNA group （Pboth<0.001）. The wound healing assay showed that the migration rate of AGS cells in OE-circRNA group was the lowest， but was high in sh-circRNA group （P<0.001）. Starbase retrieved that circ_0045943 and miR-106a had complementary binding sequences. Real-time PCR showed that miR-106a was highly expressed in gastric cancer cells （P<0.001）， and the expressions of circ_0045943 and miR-106a significantly differed after treatment with OE-circRNA and sh-circRNA （Pboth<0.001）. With the increase or decrease of circ_0045943， the expression of miR-106a changed in the opposite direction. 【Conclusion】 Circ_0045943 has low expression in gastric cancer， and promoting or inhibiting circ_0045943 expression may regulate the proliferation， apoptosis， migration and invasion of gastric cancer cells by targeting miR-106a.

Effect of hsa_circ_0001613 on Zika virus replication: Preliminary study / 中国输血杂志

【Objective】 To investigate the effect of transfusion-transmitted Zika virus (ZIKV) on the expression of non-coding circular RNA (hsa_circ_0001613) and the role of hsa_circ_0001613 in Zika virus replication. 【Methods】 Human adenocarcinomic alveolar basal epithelial cells (A549) were seeded on a 12-well plate at 1.8×105/ well and infected with ZIKV at 0.05 MOI. The Total RNAs were isolated every day for 5 days after infection, and the relative expression level of hsa_circ_0001613 was detected by qRT-PCR. In addition, 10nM siRNA-hsa_circ_0001613 was transfected into 2×105/ well A549 cells to specifically knock down the expression level of hsa_circ_0001613. 24h later, the cells were infected with ZIKV (MOI=0.05). Total RNAs were isolated at day 1-5 post-infection, proteins were extracted 96h post-infection. ZIKV replication, relative host antiviral gene expression, and interferon stimulated response element (ISRE) activity were tested using qRT-PCR, western blot and dual luciferase assay, respectively. 【Results】 The relative expression of hsa_circ_0001613 decreased significantly after 1-5 days of ZIKV infection. Knockdown of hsa_circ_0001613 inhibited ZIKV replication. Meanwhile, hsa_circ_0001613 knockdown significantly upregulated IFN-α/β and its downstream interferon-stimulated genes (ISGs) expression, also increased ISRE activity. 【Conclusion】 ZIKV infection significantly suppressed hsa_circ_0001613 expression in A4549 cells. Preliminary study indicated that hsa_circ_0001613 knockdown inhibited ZIKV replication possibly through activating type-Ⅰ IFN signaling pathway as showed by increased ISGs expression and ISRE activity.

Screening of differentially expressed miRNAs in A549 cells induced by 17-AAG-M and its effect on radio-sensitivity / 中华放射肿瘤学杂志

Objective:To screen 17-AAG-M-induced differentially expressed miRNAs in human non-small cell lung cancer (NSCLC) A549 cells under X-ray and evaluate its effect on radio-sensitivity.Methods:A549 cells were treated with 17-AAG-M and 4 Gy. Total RNA was extracted for microarray screening. The expression of the miRNAs of interest in the tumor was observed by public database. The target miRNAs were analyzed by using GO and KEGG pathways, and verified by qPCR. The effect of target miRNAs on the survival rate and proliferation of A549 cells under X-ray was evaluated by MTT and clone formation assays. The radio-sensitivity of the target miRNAs was analyzed by the single-hit multi-target model formula.Results:20 differentially expressed miRNAs were screened. The down-regulated hsa-miR-30a-3p showed a close correlation with lung cancer in the database. It was involved with 50 biological processes including cell proliferation and affected the MAPK signaling pathway, cancer-related pathways and cell cycle, etc. Compared with the 17-AAG-M group, the relative expression level of hsa-miR-30a-3p under the action of 17-AAG-M and X-ray was down-regulated from 2.42 to 0.16. hsa-miR-30a-3p inhibited the survival rate of A549 cells (survival rate: 78.52%) and further decreased to 69.00% under X-ray. Up-regulation of hsa-miR-30a-3p expression inhibited the proliferation of tumor cells and increased the radio-sensitivity of A549 cells. The radio-sensitization ratio was 1.18. The above performance became more obvious under the action of 17-AAG-M.Conclusions:In A549 cells, hsa-miR-30a-3p is differentially expressed under the action of 17-AAG-M and X-ray. Moreover, up-regulation of the expression level of hsa-miR-30a-3p in A549 cells can reduce the viability and proliferation of tumor cells, and increase the radio-sensitivity of tumor cells. The inhibition effect of X-ray combined with 17-AAG-M upon tumors can be strengthened.

hsa-miR-4443 inhibits myocardial fibroblast proliferation by targeting THBS1 to regulate TGF-ß1/α-SMA/collagen signaling in atrial fibrillation

Fibrosis caused by the increase in extracellular matrix in cardiac fibroblasts plays an important role in the occurrence and development of atrial fibrillation (AF). The aim of this study was to investigate the role of hsa-miR-4443 in AF, human cardiac fibroblast (HCFB) proliferation, and extracellular matrix remodeling. TaqMan Stem-loop miRNA assay was used to measure hsa-miR-4443 expression in patients with persistent AF (n=123) and healthy controls (n=100). Patients with AF were confirmed to have atrial fibrosis by late gadolinium enhancement. At the cellular level, after hsa-miR-4443 mimic and inhibitor were transfected with HCFBs, proliferation, apoptosis, migration, and invasion were analyzed. Lastly, hsa-miR-4443-targeted gene and transforming growth factor (TGF)-β1/α-SMA/collagen pathway were evaluated by dual-luciferase reporter assay and western blot, respectively. In patients with AF, hsa-miR-4443 decreased significantly and collagen metabolism level increased significantly. Logistic regression analysis showed that low hsa-miR-4443 level was a risk factor of AF (P<0.001). The receiver operating characteristic curve revealed that hsa-miR-4443 was useful for predicting AF (area under the curve: 0.828, sensitivity: 0.71, specificity: 0.78, P<0.001). In HCFBs, hsa-miR-4443 targeted thrombospondin-1 (THBS1) and downregulated TGF-β1/α-SMA/collagen pathway. The inhibition of hsa-miR-4443 expression promoted HCFB proliferation, migration, invasion, myofibroblast differentiation, and collagen production. The significant reduction of hsa-miR-4443 can be used as a biomarker for AF. hsa-miR-4443 protected AF by targeting THBS1 and regulated TGF-β1/α-SMA/collagen pathway to inhibit HCFB proliferation and collagen synthesis.

Hsa_circ_0002162 has a critical role in malignant progression of tongue squamous cell carcinoma through targeting miR-33a-5p

The aim of this study was to explore the effect of hsa_circ_0002162 on regulating cell proliferation, apoptosis, and invasion, and investigate its potential target microRNA (miRNA) in tongue squamous cell carcinoma (TSCC). Hsa_circ_0002162 expression was detected in human TSCC cell lines and human oral keratinocytes (HOK) cell line. Cell proliferation, apoptosis, invasion, and candidate target miRNA expressions were detected in hsa_circ_0002162 knockdown-treated CAL-27 cells and hsa_circ_0002162 overexpression-treated SCC-9 cells. In the rescue experiment, miR-33a-5p knockdown plasmid was transfected into hsa_circ_0002162 knockdown-treated CAL-27 cells, while miR-33a-5p overexpression plasmid was transfected into hsa_circ_0002162 overexpression-treated SCC-9 cells. Subsequently, cell proliferation, apoptosis, and invasion were detected, and then luciferase reporter assay was performed. Hsa_circ_0002162 expression was increased in human TSCC cell lines SCC-9, CAL-27, HSC-4, and SCC-25 compared with HOK. In CAL-27 cells, hsa_circ_0002162 knockdown inhibited cell proliferation and invasion and promoted apoptosis. In SCC-9 cells, hsa_circ_0002162 overexpression enhanced cell proliferation and invasion and suppressed apoptosis. Furthermore, a negative regulation of hsa_circ_0002162 on miR-33a-5p (but not miR-302b-5p and miR-545-5p) was observed. In the rescue experiment, miR-33a-5p knockdown increased cell proliferation and invasion, and decreased apoptosis in hsa_circ_0002162 knockdown-treated CAL-27 cells, whereas miR-33a-5p overexpression decreased cell proliferation and invasion, but increased apoptosis in hsa_circ_0002162 overexpression-treated SCC-9 cells. The luciferase reporter assay showed the direct binding of hsa_circ_0002162 to miR-33a-5p. In conclusion, hsa_circ_0002162 had an important role in malignant progression of TSCC through targeting miR-33a-5p.

Downregulation of hsa_circ_0000936 sensitizes resistant glioma cells to temozolomide by sponging miR-1294

Glioma is one of the most aggressive forms of brain tumor and is hallmarked by high rate of mortality,metastasis and drug resistance. Herein, we explore the role of circular RNA (circRNA) hsa_circ_0000936 inthe resistance of glioma cells to temozolomide (TMZ). In this study, Relative changes in gene expression levelswere compared using qRT-PCR. The role of hsa_circ_0000936 was characterized by cell count kit -8 assay andflow cytometry. Luciferase reporter assay was carried out for target validation.We found that hsa_circ_0000936was upregulated in glioma tissues as compared to their adjacent normal tissues. Increased expression ofhsa_circ_0000936 was found in the glioma tissues of patients showing resistance to TMZ compared with thatof patients showing sensitivity to TMZ. The upregulation of hsa_circ_0000936 was also confirmed in TMZresistant glioma cells. miR-1294 was downregulated in TMZ-resistant glioma cells and identified as a directtarget of hsa_circ_0000936. Downregulation of hsa_circ_0000936 increased the sensitivity of TMZ-resistantglioma cells towards TMZ. Moreover, restoration of miR-1294 could abrogate the promoting effect ofhsa_circ_0000936 on TMZ resistance in TMZ-resistant glioma cells. In conclusion, downregulation ofhsa_circ_0000936 sensitizes TMZ-resistant glioma cells to TMZ by sponging miR-1294, suggesting thathsa_circ_0000936 may be a potential target for overcoming the resistance of glioma cells to TMZ.

The MicroRNA hsa-let-7g Promotes Proliferation and Inhibits Apoptosis in Lung Cancer by Targeting HOXB1

PURPOSE: The goal of this study was to explore the effects of hsa-let-7g on cell proliferation and apoptosis, and elucidate its role in lung cancer development.MATERIALS AND METHODS: The expression levels of has-let-7g and HOXB1 in tissues and cells were measured by qRT-PCR. An inhibitor of hsa-let-7g or one targeting a control messenger RNA were transfected into A549 and H1944 lung cancer cells, and the effects of hsa-let-7g dysregulation on cell viability and apoptosis were analyzed using CCK-8 and apoptosis detection assays. HOXB1 was confirmed as the target gene of hsa-let-7g, based on luciferase reporter assay results. The relationship between hsa-let-7g and HOXB1 was confirmed by co-transfection of inhibitors of hsa-let-7g and HOXB1 followed by Western blot, CCK-8, and apoptosis detection assays.RESULTS: We observed high expression of hsa-let-7g in lung cancer tissues compared to the corresponding normal tissues, and generally higher expression of hsa-let-7g in patients with advanced tumor classification. The results of CCK-8 and apoptosis detection experiments showed that the inhibition of hsa-let-7g significantly inhibited proliferation of A549 and H1944 cells, but also promoted apoptosis. HOXB1 is a specific target of hsa-let-7g, and downregulation of HOXB1 in lung cancer cells reversed the suppressive effects caused by knocking down hsa-let-7g.CONCLUSION: These data collectively suggest that the expression of hsa-let-7g inhibits lung cancer cells apoptosis and promotes proliferation by down-regulating HOXB1. The results from this study demonstrate the potential of hsa-let-7g/HOXB1 axis as a therapeutic target for the treatment of lung cancer.