Expressions of miR-203a and its target genes in hepatocellular carcinoma tissues and their clinical significance / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨肝细胞癌（HCC）患者血清及癌组织中miR-203a和其靶基因的表达及其与患者临床病理特征和预后的关系。方法：利用生物信息学方法从TargetScan、miRDB和PicTar网站预测HCC组织中miR-203a的靶基因，通过双荧光素酶报告基因实验进行验证。选取2018年1月至2019年6月在常州市金坛区第二人民医院手术切除的96例HCC患者的癌和癌旁组织标本、血清和临床资料，以及90例健康体检者的血清作为对照。qPCR法检测血清miR-203a水平，以及HCC组织和癌旁组织中miR-203a及其靶基因表达，比较分析不同临床病理特征HCC患者miR-203a及其靶基因表达。随访3年，采用Kaplan-Meier法进行生存（OS）分析。结果：从数据库筛选出HCC中miR-203a相关的靶基因共10个，包括APC、CDK6、GATA6、HOXD3、IGF1R、IGFBP5、KCNE2、PAQR3、PRMT5和SOSC3。HCC组织中miR-203a和APC、PAQR3 mRNA表达水平均显著低于癌旁组织（均P<0.01），CDK6、GATA6、HOXD3、IGF1R、IGFBP5、KCNE2、PRMT5和SOSC3 mRNA表达水平均显著高于癌旁组织（均P<0.01）；血清miR-203a、HCC组织miR-203a及其靶基因表达均与患者肿瘤临床分期、分化程度、肝功能分级、OS率有关（均P<0.01）。结论：HCC组织中miR-203a呈低表达，miR-203a及靶基因表达均与患者肿瘤临床分期、分化程度、肝功能及远期OS率有关。

MiR-203a-5p Inhibits Multiple Myeloma Cell Proliferation and Cell Cycle Progression via Targeting JAG1 / 中国实验血液学杂志

OBJECTIVE@#To investigate the biological function of miR-203a-5p and the underlying mechanism in multiple myeloma (MM).@*METHODS@#Three miRNA expression profiles (GSE16558, GSE24371 and GSE17498) were downloaded from the GEO database. The three miRNA expression profiles contained 131 MM samples and 17 normal plasmacyte samples. The robust rank aggregation (RRA) method was used to identify the differentially expressed miRNAs between MM and normal plasmacytes. In order to carry out cytological experiments, MM cell line with stable over-expression of miR-203a-5p was constructed with lentivirus. Expression levels of miR-203a-5p in MM cells were quantified by qRT-PCR. The effects of miR-203a-5p on MM cells were investigated using assays of cell viability and cell cycle. Cell proliferation was measured using the Cell Counting kit (CCK)8 assay. The percentage of cells in each cell cycle was measured with a FACSCalibur system. Xenograft tumor models were established to evaluate the role of miR-203a-5p in tumorigenesis in vivo . To elucidate the underlying molecular mechanisms of miR-203a-5p in mediating cell proliferation inhibition and cell cycle arrest in MM, we used TargetScan and miRanda to predict the candidate targets of miR-203a-5p. The potential target of miR-203a-5p in MM cells was explored using the luciferase reporter assay, qRT-PCR, and Western blot.@*RESULTS@#An integrated analysis of three MM miRNA expression datasets showed that the levels of miR-203a-5p in MM were notably downregulated compared with those in normal plasmacytes. Accordingly, the relative expression levels of miR-203a-5p were decreased in MM cell lines. In addition, overexpression of miR-203a-5p inhibited the proliferation and cell cycle progression of RPMI8226 and U266 cells. In vivo experiments demonstrated that upregulation of miR-203a-5p expression could significantly inhibit the tumorigenesis of subcutaneous myeloma xenografts in nude mice. Mechanistic investigation led to the identification of Jagged 1 (JAG1) as a novel and direct downstream target of miR-203a-5p. Interestingly, the reintroduction of JAG1 abrogated miR-203a-5p-induced MM cell growth inhibition and cell cycle arrest.@*CONCLUSION@#Our data demonstrate that miR-203a-5p inhibits cell proliferation and cell cycle progression in MM cells by targeting JAG1, supporting the utility of miR-203a-5p as a novel and potential therapeutic agent for miRNA-based MM therapy.

Effect of miR-203/CREB1 Signaling Regulation Mediated by DNA Methylation on the Proliferation and Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of miR-203/CREB1 signaling regulation mediated by DNA methylation on the proliferation, invasion and apoptosis of multiple myeloma (MM) cells.@*METHODS@#The methylation level of miR-203 in the RPMI 8226 cells was detected by bisulfite sequcucing polymerase chain reaction (BSP). The mRNA expression of miR-203 was measured by quantitative real-time polymerase chain reaction. RPMI 8226 cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). The miR-203 mimic in MM cell line RPMI 8226 was transfected to establish overexpressed miR-203 cell. The proliferation, invasion ability and apoptosis of RPMI 8226 cell was detected by CCK-8 assay, Transwell, and flow cytometry, respectively. The targeting relationship between miR-203 and CREB1 was verified by double luciferase report assay. Western blot was used to detect the expression of CREB1 protein.@*RESULTS@#Hypermethylation of miR-203 promoter region and low expression level of miR-203 mRNA were detected in the RPMI 8226 cells, which showed that demethylation could induce the expression of miR-203. The proliferation and invasion ability of RPMI 8226 cells after treated by 5-Aza-CdR were inhibited, and showed statistical significance as compared with blank control group (both P<0.05)，while the apoptosis rate was promoted (P<0.05). The proliferation, invasion ability and apoptosis of overexpressed miR-203 were the same as the demethylation group. Double luciferase report assay confirmed that CREB1 was the direct target of miR-203. The protein level of CREB1 was inhibited by demethylation and showed statistical significance as compared with control group (P<0.05).@*CONCLUSION@#MiR-203 targeting CREB1 mediated by DNA methylation leads to maintain the malignant biological behaviors of MM cells.

linc00941 regulates the proliferation, invasion and glycolysis of esophageal squamous cell carcinoma via miR-203/CCL2 axis / 中国肿瘤生物治疗杂志

@#[摘要] 目的：探究linc00941 作为ceRNA吸附miR-203 上调CC-趋化因子配体2（CC chemokine ligand 2，CCL2）的表达在食 管鳞癌（esophageal squamous cell carcinoma，ESCC）中的作用机制。方法：选取南阳医学高等专科学校第一附属医院58 例ESCC 患者的癌组织和癌旁组织，其中，男性患者33 例，年龄（49.3±18.6）岁，女性患者25 例，年龄（44.6±20.7）岁。qPCR 法检测 linc00941、miR-203、CCL2 在ESCC 组织和4 株人ESCC 细胞系（EC9706、KYSE30、ECA109 和TE1）以及人正常食管上皮细胞株 HET-1A细胞系中的表达。构建linc00941-wt、linc00941-mut、CCL2-wt、CCL2-mut 质粒并分别与miR-203 NC 或miR-203 模拟物 共转染到293T 细胞中。双荧光素酶报告基因实验验证linc00941、miR-203、CCL2 之间的相互作用。CCK-8 和Transwell 实验检 测细胞的增殖与侵袭能力。乳酸含量检测评价细胞的糖酵解能力。流式细胞术检测细胞的凋亡情况。糖酵解抑制剂2-DG以及 linc00941 共同干预ESCC细胞，以进一步观察linc00941 对ESCC细胞的调控作用。结果：在ESCC组织中和细胞系中linc00941、 CCL2 表达均上调，miR-203 表达下调（均P<0.05）。linc00941 与miR-203、miR-203 与CCL2 的相互作用在ECA109 细胞中得到证 实。下调linc00941 能够抑制ECA109 细胞的增殖、侵袭和糖酵解，并诱导细胞凋亡，该作用被miR-203 抑制剂部分逆转（均 P<0.05）。过表达CCL2 可以部分逆转敲减linc00941 对ECA109 细胞增殖、侵袭、糖酵解和凋亡的影响（均P<0.05）。结论： linc00941 能够吸附miR-203 进而上调CCL2 的表达，促进ESCC细胞的增殖、侵袭和糖酵解，诱导细胞凋亡。linc00941 对ESCC细 胞增殖、侵袭和凋亡的影响可能是通过调控糖酵解实现的。

circ_0001821 regulates the proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma A431 cells by targeting miR-203 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的： 探讨circ_0001821通过靶向miR-203调控皮肤鳞状细胞癌（cutaneous squamous cell carcinoma，CSCC）A431细胞增殖、凋亡、迁移及侵袭的分子机制。方法：选取2018年2月至2019年8月海南医学院第二附属医院收治的39例CSCC患者的癌及配对癌旁组织，以及人CSCC细胞系A431，用qPCR法检测CSCC癌和癌旁组织中circ_0001821的表达水平。利用脂质体转染技术，分别将si-circ_0001821、si-NC、miR-203 mimic、miR-NC、si-circ_0001821与anti-miR-NC、si-circ_0001821与anti-miR-203转染至A431细胞，用qPCR法检测转染细胞中circ_0001821和miR-203的表达水平，MTT法、流式细胞术和Transwell实验分别检测A431细胞的增殖、凋亡、迁移及侵袭能力，WB法检测细胞中增殖、凋亡、迁移及侵袭相关蛋白的表达水平。通过Circinteractome数据库预测circ_0001821与miR-203存在结合位点，双荧光素酶报告基因实验验证circ_0001821与miR-203的靶向关系。结果：CSCC组织中circ_0001821的表达水平显著高于癌旁组织（P<0.01）。转染si-circ_0001821可显著降低细胞中circ_0001821的表达水平（P<0.01），降低细胞增殖、迁移和侵袭能力（均P<0.01），提高细胞凋亡率（P<0.01）。双荧光素酶报告基因实验证实，circ_0001821可靶向结合miR-203。转染miR-203 mimic可显著降低A431细胞的增殖、迁移和侵袭能力（均P<0.01），提高细胞凋亡率（P<0.01）。共转染si-circ_0001821与anti-miR-203可明显逆转下调circ_0001821表达对A431细胞增殖、迁移、侵袭及凋亡的调控作用。结论：circ_0001821通过靶向miR-203调控CSCC细胞A431的增殖、迁移、侵袭能力及细胞凋亡。

Expression of miR-203a-3p in pancreatic cancer tissues and cells and its effect on proliferation, migration and invasion of BxPC-3 cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨miR-203a-3p对胰腺癌BxPC-3细胞增殖、迁移和侵袭能力的影响。方法：运用癌症基因组图谱（TCGA）数据库筛选胰腺癌组织和癌旁组织中差异表达的miRNA，分析miRNA高表达与低表达时胰腺癌患者的生存率和临床分期；利用TarBase数据库分析miRNA与癌症相关的GO功能与KEGG通路，利用DIANA Tools、miRDB和TargetScan网站预测miR-203a-3p的靶基因。将miR-203a-3p mimic及NC mimic、miR-203a-3p inhibitor及NC inhibitor转染至BxPC-3细胞，用qPCR法检测胰腺癌细胞和胰腺正常上皮细胞HPNE中miR-203a-3p、miR-192-5p和miR-451a表达水平，以CCK-8法、Transwell小室法和克隆形成实验分别检测BxPC-3细胞的增殖、迁移、侵袭和集落形成能力。结果：通过TCGA数据库筛选出18个胰腺癌组织中差异表达的miRNA，其中miR-203a-3p、miR-192-5p、miR-451a具有物种保守性，且其与胰腺癌临床癌症分期、细胞周期和患者生存率相关（均P<0.05）；生物信息学网站预测显示miR-203a-3p的候选靶基因是PPM1A，PPM1A与多基因存在相互作用。miR-203a-3p、miR-192-5p和miR-451a在BxPC-3和Aspc-1细胞中均高表达（均P<0.01）。miR-203a-3p mimic组BxPC-3细胞中miR-203a-3p表达水平以及细胞增殖、迁移和侵袭能力均显著提高（均P<0.01）；miR-203a-3p inhibitor组细胞中miR-203a-3p表达水平以及细胞增殖、迁移和侵袭能力均显著降低（均P<0.01）。结论：miR-203a-3p在胰腺癌组织及细胞中均高表达，其表达与患者生存和临床分期相关，可调控BxPC-3细胞的增殖、迁移和侵袭能力。

microRNA-203 Inhibits Proliferation and Migration of Human Corneal Epithelial Cells by Targeting SRC / 中国生物化学与分子生物学报

Corneal epithelial wound healing (CEWH) is vital for maintaining tissue integrity and transparency, but its detailed molecular mechanism is unclear. Previous our study has shown that the expression of miR-203 is dramatically down-regulated during CEWH. Such changes suggest that miR-203 is an important effector of CEWH. In this study, we confirmed down-regulation of miR-203 during CEWH in mice. In vitro experiments, we transfected miR-203 into human corneal epithelial cells (HCECs) to over-express miR-203. The MTS method, EdU detection, flow cytometry, scratch tests and transwell experiments were used to detect changes in cell proliferation, cell cycle, and cell migration after transfection. As a result, it was found that miR-203 can inhibit the proliferation of HCECs (P<0.01). The cell cycle at the G

SOCS3 regulates mucus hypersecretion via JAK/STAT signal pathway / 安徽医科大学学报

Objective To investigate the SOCS3 regulates mucus hypersecretion via JAK/STAT signal pathway in inflammatory cells.Methods Culture 16HBE cells and divide into three groups:control group,inter leukin(IL)-6-exposed group;IL-6-exposed and microRNA-203-transfered group.The protein levels of JAK1/2,SOCS3 and MUC5AC were measured by Western blot.The mRNA expressions of SOCS3,STAT3 and MUC5AC were detected by Real-time PCR.The protein levels of p-JAK1/2,SOCS3 and MUC5AC were analyzed by ELISA.Results Compared with the control group,the mRNA expressions of SOCS3,STAT3,MUC5AC and the protein levels of p-JAK1/2,SOCS3,MUC5AC were both significantly increased in IL-6-exposed group (P ＜ 0.05);in the IL-6-exposed and miR-203-transfered group,the protein levels of p-JAK1/2,MUC5AC and the mRNA expressions of STAT3 were both significantly increased (P ＜ 0.05),but the mRNA expressions of SOCS3 was almost as much as that in IL-6-exposed group,the protein levels of SOCS3 was significantly decreased (P ＜ 0.05).Conclusion SOCS3 over-express when cells are stimulated by IL-6,and negative-feedback regulates MUC5AC secretion via JAK/STAT signal pathway.

Expression of miR-203 in human tongue carcinoma tissues and its in-fluence on viability and invasion ability of Tca8113 cells / 中国病理生理杂志

[ ABSTRACT] AIM:To study the expression of miR-203 in tongue carcinoma tissues and the effect of miR-203 over-expression on the viability and invasion ability of Tca 8113 cells.METHODS:Twenty-eight pairs of tongue carcinoma tissues and adjacent nontumor tissues were collected , and the clinicopathological characters were analyzed .miR-203 was detected in the tongue tissues of 28 patients with tongue carcinoma by real-time PCR.miR-203 mimics and scramble were transfected into Tca8113 cells by Lipofectamine 2000.The expression of miR-203 was detected in Tca8113, Tca8113-miR-203 mimics and Tca8113-scramble cells by RT-qPCR.The cell viability was measured by CCK-8 assay.The cell invasion ability was determined by Transwell chamber invasion experiment .RESULTS:miR-203 expression was significantly down-regulated in the tongue carcinoma tissues compared with those in the adjacent nontumor tissues .The expression of miR-203 was associated with TNM stage and lymph node metastasis .Up-regulation of miR-203 inhibited the viability and invasion a-bility of Tca8113 cells.CONCLUSION:miR-203 suppresses the growth and invasion of tongue carcinoma cells .miR-203 may be a potential therapeutic target for treating human tongue cancer .

Serum miR-203 expression level detected by fluorescence quantitative PCR in cervical cancer patients and its significance / 国际检验医学杂志

Objective To establish the method of the SYBR Green Ⅰ real‐time fluorescent quantitative PCR for detecting the se‐rum miR‐203 expression level ,and to detect the serum miR‐203 expression levels in the patients with cervical cancer ,cervical benign diseases and healthy controls .Methods The miR‐203 ,U6 stem loop RT primers and the PCR amplification primers were designed for conducting fluorescence quantitative PCR ,with U6 as the internal relative quantification ,the serum miR‐203 levels were com‐pared among different cervical diseases .Results The established method could specifically detect the amplification signal of serum miR‐203 ,the melting curve was single and PCR products were specific .The serum miR‐203 level in the patients with cervical cancer was significantly higher than that in the patients with benign cervical diseases such as hysteromyoma and cervicitis ,the difference was statistically significant (P<0 .05) .Conclusion The SYBR Green Ⅰ real‐time fluorescent quantitative PCR is a quick ,simple detection method with high sensitivity and good specificity ,which may have a better application prospect in cervical cancer auxiliary diagnosis .

Expressions of miR-203 and its downstream target genes p63 and survivin in psoriatic lesions / 中华皮肤科杂志

Objective To measure the expressions of miR-203 and its downstream target genes p63 and survivin in psoriasis vulgaris lesions.Methods Tissue specimens were collected from lesions of 30 patients with psoriasis vulgaris and normal skin of 30 healthy human controls.Real-time PCR was performed to detect miR-203 mRNA expression with U6 as the internal control,as well as p63 and survivin mRNA expressions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control,and Western blot to measure the protein expressions of miR-203 targets p63 and survivin.Statistical analysis was carried out by t test for intergroup comparisons and by Pearson correlation analysis for the analysis of correlation between miR-203,p63 and survivin expressions in psoriasis vulgaris lesions.Results Compared with the normal control skin,psoriasis vulgaris lesions showed significantly decreased mRNA expression level (2-△△C△△Ct) of miR-203 (0.41 ± 0.11,t =3.16,P ＜ 0.05),but increased mRNA expression levels of p63 (4.79 ± 0.63,t =4.72,P＜ 0.05) and survivin (3.43 ± 0.46,t =4.35,P＜ 0.05).The protein expression levels of p63 and survivin in these lesions were (2.40 ± 0.23) times (t =3.87,P ＜ 0.05) and (3.49 ± 0.14) times (t =4.36,P ＜ 0.05) those in the normal control skin respectively.In psoriasis vulgaris lesions,the mRNA expression level of miR-203 showed a significantly negative correlation with that of survivin (r =-0.36,P ＜ 0.05) and p63 (r =-0.43,P ＜ 0.05).Conclusion miR-203 and its downstream target genes p63 and survivin may participate in the occurrence of psoriasis vulgaris.

Down regulation of miR-203 in radiation-induced thymic lymphoma promoted cells proliferation and inhibited apoptosis / 中华放射医学与防护杂志

Objective To investigate the role of miR-203 in radiation-induced thymic lymphoma (RITL).Methods A 60Co irradiator was used for total-body irradiation.MicroRNAs(miRNAs) level was assayed by qRT-PCR.Cell proliferation was assayed by MTT assay.Cell apoptosis was examined by fluorescence activated cell sorter (FACS).Dual luciferase reporter assay system was used to detect the 3'UTR reporter.Results MiR-203 was down-regulated in RITL tissues.Overexpression of miR-203 strongly inhibited the proliferation of both NIH3T3 cells and EL4 cells and vice versa.MiR-203 inhibited cells proliferation and induced apoptosis via TANK-binding kinase (TBK1),SLUG (SNAI2) and Cyclin D1 (CCND1).Conclusions Radiation down-regulated the level of miR-203 in thymic,which promoted radiation-induced thymic lymphoma by targeting TBK1,SNAI2 and CCND1.

Enhanced sensitivty of leukemic K562 cell to ursolic acid by miR-203 / 中国药理学通报

Aim To study the influences of miR-203 on ursolic acid (UA ) sensitivity in leukemic K562 cell.Methods In the experimental system,eukaryot-ic expression vector of hsa-miR-203 (PmiR-203 )was transfected into K562 cells using LipofectamineTM 2000.K562 cells were incubated with different con-centration of UA alone or in combination with PmiR-203 .The cell proliferation was analyzed by MTT as-say.The cell apoptosis was measured by double stai-ning with Annexin V and Propidium Iodide.The ex-pression of Bcr/abl protein was detected by Western Blot.Results The miR-203 promoted the sensitivity of UA by up to 1 .55-fold and the IC50 was reduced from 44.3μmol · L-1 to 30.7 μmol · L-1 .The a-mount of apoptotic cells was increased in UA combined with PmiR-203 group (P<0.05).PmiR-203 downreg-ulated the expression of Bcr/abl protein in K562 cells. Conclusion Our results support a substantial role of miR-203 that enhances UA sensitivity in K562 cell and the mechanism appears to be related to the dounregula-tion of Bcr/abl by miR-203 .

MiR-203 inhibits proliferation and invasion of esophageal squamous cell carcinoma / 第二军医大学学报

Objective: To investigate the influence of miR-203 on the proliferation and invasion of human esophageal squamous cell carcinoma cell line Eca109. Methods: Double-stranded mimics of miR-203 were designed and transfected into Eca109 cells with Lipofectamine 2000; Eca109 cells transfected with nonsense microRNA mimics were taken as control. The proliferation ability of Eca109 cells was determined by calculating the cell population doubling time and the percentage of apoptotic cells; the invasion ability of Eca109 cells was determined by Transwell assay. Results: In vitro experiment showed that, compared with the control group, Eca109 cells transfected with miR-203 mimics had a significantly longer cell population doubling time ([26.1±0.5] h vs [24.2±0.6] h, P<0.01), a higher cell apoptotic rate ([4.5±0.4]% vs [3.7±0.4]%, P<0.05), and a lower cell invasion rate ([39.2±5.8]% vs [49.5±6.8]%, P<0.05). Conclusion: Our data shows that miR-203 can inhibit the proliferative and invasive abilities of Eca109 cells, suggesting that miR-203 might be a potential gene therapeutic target of human esophageal squamous cell carcinoma.

MiR-203 inhibits proliferation and invasion of esophageal squamous cell carcinoma / 第二军医大学学报

Objective To investigate the influence of miR-203 on the proliferation and invasion of human esophageal squamous cell carcinoma cell line Eca109.Methods Double-stranded mimics of miR-203 were designed and transfected into Eca109 cells with Lipofectamine 2000;Eca109 cells transfected with nonsense microRNA mimics were taken as control.The proliferation ability of Eca109 cells was determined by calculating the cell population doubling time and the percentage of apoptotic cells;the invasion ability of Eca109 cells was determined by Transwell assay.Results In vitro experiment showed that,compared with the control group,Eca109 cells transfected with miR-203 mimics had a significantly longer cell population doubling time([26.1?0.5]h vs [24.2?0.6]h,P