miR-32-5p inhibits the malignant biological behaviors and the growth of nude mice xenograft of pancreatic cancer PANC-1 cells by targeted down-regulation of EZH2 expression / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究miR-32-5p通过靶向zeste基因增强子人类同源物2（EZH2）对胰腺癌PANC-1细胞恶性生物学行为的影响及其分子机制。方法：利用GEPIA数据库分析胰腺癌组织中EZH2的表达水平及其与患者的预后生存期的关系，并分析miR-32-5p表达与患者临床病理因素的关系。qPCR法检测正常胰腺HPDE6-C7细胞和胰腺癌PANC-1、AsPC-1、SW1990细胞中miR-32-5p和EZH2 mRNA的表达，通过LipofectamineTM 2000将miR-NC、miR-32-5p mimic、miR-32-5p inhibitor、pcDNA-NC、pcDNA EZH2质粒分别转染PANC-1细胞，其分为对照组（不转染）、 miR-NC组（转染miR-NC）、 miR-32-5p mimic组（转染miR-32-5p mimic）、Anti-miR-32-5p组（转染miR-32-5p inhibitor）、miR-NC+pcDNA-NC组（转染miR-NC+pcDNA-NC）、miR-NC+pcDNA EZH2组（转染miR-NC+pcDNA EZH2）、miR-32-5p mimic+pcDNA-NC组（转染miR-32-5p mimic+pcDNA-NC）、miR-32-5p mimic+pcDNA EZH2组（转染miR-32-5p mimic+pcDNA EZH2）。双荧光素酶报告基因实验验证miR-32-5p与EZH2的靶向关系；MTT法及克隆形成实验检测各组细胞的增殖能力，划痕愈合实验检测各组细胞的迁移能力；Transwell小室实验检测各组细胞的侵袭能力，WB法检测各组细胞EZH2、E-cadherin、N-cadherin的表达；裸鼠成瘤实验检测转染后各组PANC-1细胞移植瘤的生长情况，免疫组化染色法观察移植瘤组织中Ki67和MMP-2的表达。结果：GEPIA数据库显示胰腺癌组织中EZH2的表达水平高于癌旁组织，患者预后生存期与EZH2的表达水平呈负相关（均P<0.05）；miR-32-5p表达水平与胰腺癌神经浸润、肿瘤分化程度、TNM分期、淋巴结转移有明显的关联（均P<0.05）；与HPDE6-C7细胞相比，PANC-1、AsPC-1、SW1990细胞中miR-32-5p呈高表达、EZH2 mRNA呈低表达、miR-32-5p表达水平与EZH2表达水平呈负相关（均P<0.05）。miR-32-5p靶向EZH2且抑制其表达（均P<0.05）；过表达miR-32-5p能够下调Ki67、MMP-2、N-cadherin表达水平、上调E-cadherin表达水平，抑制PANC-1细胞的增殖、迁移和侵袭能力，抑制移植瘤质量增加（均P<0.05）。结论：miR-32-5p能够靶向调控EZH2从而影响胰腺癌PANC-1细胞的增殖、迁移、侵袭、EMT进程及裸鼠体内移植瘤的生长。

miR-32-5p regulates the biological behaviors of breast cancer MDA-MB-231 cells by targeting the expression of Dickkopf-related protein 3 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：通过生物信息学手段筛选乳腺癌中差异表达的关键miRNA及其靶基因，干预其在乳腺癌细胞中的表达并观察对乳腺癌细胞功能的影响。方法：利用GEO数据库筛选在乳腺癌中差异表达的miRNA，ENCORI数据库验证差异miRNA的表达，以选定最显著的差异表达miRNA为研究对象；利用Starbase、miRDB和miRWalk数据库预测miR-32-5p的靶基因，利用DAVID数据库对靶基因进行GO分析和KEGG分析，利用String数据库联合Cytoscape3.6.2软件进行PPI网络分析及核心基因的筛选，从核心基因中选择相互联系紧密“度值”最显著的Dickkopf相关蛋白3（DDK3）基因进行后续实验。qPCR检测miR-32-5p在人正常乳腺细胞 MCF10A和人乳腺癌细胞MCF7、MDA-MB-231、MDA-MB-453细胞中的表达。向MDA-MB-231细胞中转染miR-32-5p mimic、miR-32-5p inhibitor及各自的对照（NC）序列，分别用CCK-8法、流式细胞术和Transwell实验检测过表达或抑制miR-32-5p对细胞增殖、凋亡和侵袭的影响。结果：从GEO数据库中获取的两个数据集共识别出两个差异miRNA，ENCORI数据库验证差异miRNA的表达发现miR-32-5p的表达水平与GEO数据库的结果一致，故选择其进行研究；预测得到198个miR-32-5p潜在的靶基因并鉴定出10个核心基因（DKK3、WNT2B、SFRP5、SFRP2、SFRP1、LRP6、WNT6、KREMEN1、NEDD4L、TRIP12），其中DKK3的度值最大可能在乳腺癌中较为重要，于是选择miR-32-5p/DKK3轴进行后续研究。miR-32-5p在3种乳腺癌细胞中的表达水平显著高于正常乳腺细胞（均P<0.01），其中以MDA-MB-231细胞中表达最高。双荧光素酶基因报告实验验证了miR-32-5p与DKK3基因的靶向结合及其对后者表达的负向调控。转染miR-32-5p mimic、miR-32-5p inhibitor后成功提高或抑制了MDA-MB-231细胞中miR-32-5p的表达。与对照组相比，过表达miR-32-5p可抑制MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭（P<0.05或P<0.01），敲低miR-32-5p则起相反的作用（均P<0.01）。结论：miR-32-5p/DKK3轴可能是影响乳腺癌发生发展的关键通路，过表达miR-32-5p能够抑制乳腺癌MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭。

Mechanism of miR-32-5p targeting TOB1 gene in regulating radiosensitization, migration and invasion of colorectal cancer cells / 中华放射肿瘤学杂志

Objection@#To investigate the effect of miR-32-5p on the radiosensitivity, migration and invasion of colorectal cancer cells and the underlying mechanism.@*Methods@#Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured. The colorectal cancer cells were divided into the non-transfected and transfected groups (transfected with anti-miR-NC, anti-miR-32-5p, pcDNA, pcDNA-TOB1, anti-miR-32-5p+ si-NC and anti-miR-32-5p+ si-TOB1, respectively). The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot. The radiosensitivity of the transfected cells was determined by colony formation assay. The migration and invasion ability of the transfected cells were detected by Transwell assay. Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot.@*Results@#Compared with human colonic epithelial cells, the expression of miR-32-5p was significantly up-regulated, whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells (all P<0.05). Compared with the anti-miR-NC, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group. Compared with the pcDNA group, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group. Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein. Compared with the anti-miR-32-5p+ si-NC group, the quantity of cell migration and invasion was significantly increased (both P<0.05) and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+ si-TOB1 group.@*Conclusions@#Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion. The underlying mechanism might be related to the targeted up-regulation of TOB1 expression.

Mechanism of miR-32-5p targeting TOB1 gene in regulating radiosensitization, migration and invasion of colorectal cancer cells / 中华放射肿瘤学杂志

Objection To investigate the effect of miR-32-5p on the radiosensitivity,migration and invasion of colorectal cancer cells and the underlying mechanism.Methods Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured.The colorectal cancer cells were divided into the non-transfected and transfected groups (transfected with anti-miR-NC,anti-miR-32-5p,pcDNA,pcDNA-TOB1,anti-miR-32-5p+si-NC and anti-miR-32-5p+si-TOB1,respectively).The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot.The radiosensitivity of the transfected cells was determined by colony formation assay.The migration and invasion ability of the transfected cells were detected by Transwell assay.Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot.Results Compared with human colonic epithelial cells,the expression of miR-32-5p was significantly up-regulated,whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells (all P＜0.05).Compared with the anti-miR-NC,the quantity of cell migration and invasion was significantly decreased (both P＜0.05) and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group.Compared with the pcDNA group,the quantity of cell migration and invasion was significantly decreased (both P＜0.05) and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group.Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein.Compared with the anti-miR-32-5p+si-NC group,the quantity of cell migration and invasion was significantly increased (both P＜0.05) and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+si-TOB1 group.Conclusions Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion.The underlying mechanism might be related to the targeted up-regulation of TOB1 expression.

lncRNA XIST regulates the malignant biological behaviors of colorectal cancer HCT-8 cells through miR-32-5p/EZH2 molecular axis / 中国肿瘤生物治疗杂志

@#Objective: To explore the mechanism of lncRNA XIST (XIST) regulating the biological behaviors of colorectal cancer HCT-8 cells via miR-32-5p/EZH2 (enhancer of Zeste homolog 2) axis. Methods:Atotal of 28 pairs of cancer tissues and corresponding para-cancerous tissues form colorectal cancer patients with complete clinical data were collected from the Colorectal and Anal Surgery, Xiangya Hospital of Central South University during July 2014 and August 2018. The expression levels of lncRNA XIST and miR-325p in colorectal cancer tissues and cell lines were detected by qPCR. The targeted relationship between lncRNA XIST, miR-32-5p and EZH2 was verified by dual luciferase reporter gene, and the expression level of EZH2 was further detected by WB. The proliferation, migration and apoptosis of HCT-8 cells were detected by CCK-8, Transwell and flow cytometry with Annexin V-FITC/PI staining, respectively. Results: lncRNAXIST was highly expressed in colorectal cancer tissues and cell lines with the highest expression in HCT-8 cells (P<0.05 or P<0.01). Dual luciferase reporter gene assay validated that lncRNA XIST negatively regulated miR-32-5p (P<0.05), and EZH2 was a target gene of miR-32-5p. Knockdown of lncRNAXIST inhibited proliferation and migration and induced apoptosis of HCT-8 cells (P<0.05 or P<0.01). Further experiments demonstrated that knockdown of lncRNA XIST up-regulated the expression of miR-32-5p and further down-regulated the expression level of EZH2, thereby inhibiting the proliferation and migration of HCT-8 cells and inducing apoptosis. Conclusion: lncRNAXIST promotes proliferation, migration and inhibits apoptosis of HCT-8 cells via miR-325p/EZH2 axis.