MicroRNA-301b-3p accelerates the growth of gastric cancer cells by targeting zinc finger and BTB domain containing 4

MicroRNAs (miRNAs) have been found to be aberrantly expressed and exert essential roles in the tumorigenesis and progression of gastric cancer (GC). miR-301b-3p has been recognized as a cancer-related miRNA in lung cancer, bladder cancer and hepatocellular carcinoma. However, the function of miR-301b-3p in GC progression and its underlying mechanism have not been studied yet. In this study, we found that miR-301b-3p expression was up-regulated in GC tissues compared to adjacent noncancerous tissues. Furthermore, the elevated levels of miR-301b-3p were detected in GC cell lines (SGC-7901, AGS, MKN-45 and MGC-803) as compared with GES-1 cells. Interestingly, GC tissues from patients with tumor size ≥ 5 cm and advanced tumor stages showed obvious higher levels of miR-301b-3p compared to matched controls. Functionally, miR-301b-3p knockdown prominently inhibited cell proliferation, and induced cell cycle arrest at G1 phase and apoptosis in MGC-803 cells. Meanwhile, ectopic expression of miR-301b-3p conversely regulated these biological behaviors of MKN-45 cells. Next, we found that miR-301b-3p knockdown increased, whereas miR-301b-3p overexpression reduced the expression of zinc finger and BTB domain containing 4 (ZBTB4) in GC cells. Accordingly, luciferase reporter assay identified ZBTB4 as a direct target of miR-301b-3p. ZBTB4 overexpression markedly restrained the growth of MGC-803 cells. More importantly, ZBTB4 silencing partially reversed miR-301b-3p knockdown-induced tumor suppressive effects on MGC-803 cells. In conclusion, we firstly revealed that miR-301-3p was highly expressed in GC and contributed to tumor progression via attenuating ZBTB4, which might provide a novel molecular-targeted strategy for GC treatment.     

LncRNA MEG3靶向miR-181a-5p调控宫颈癌细胞放射敏感性

目的 研究LncRNA MEG3对宫颈癌细胞放射敏感性的影响,并探讨其作用机制。方法 运用qRT-PCR法检测放射抗性和放射敏感性宫颈癌细胞中LncRNA MEG3的表达;将过表达对照组(转染pcDNA 3.1)、过表达LncRNA MEG3组(转染pcDNA 3.1-LncRNA MEG3)、抑制miR-NC组(转染anti-miR-NC)、抑制miR-181a-5p组(转染anti-miR-181a-5p)、过表达LncRNA MEG3+过表达miR-NC组(共转染pcDNA 3.1-LncRNA MEG3和anti-miR-NC)、过表达LncRNA MEG3+过表达miR-181a-5p组(共转染pcDNA 3.1-LncRNA MEG3和anti-miR-181a-5p),均用脂质体法转染至SiHa细胞;克隆形成实验检测细胞的存活分数;流式细胞术检测细胞的凋亡率;双荧光素酶报告基因检测实验检测细胞的荧光活性;Western blot检测细胞中PTEN、p-Akt、Akt的蛋白表达。结果 与放射敏感组相比,放射抗性宫颈癌组织中LncRNA MEG3的表达明显降低(P<0.05),其表达量与宫颈癌细胞的放射敏感性呈正相关;过表达LncRNA MEG3、抑制miR-181a-5p均可显著增强宫颈癌细胞SiHa放射敏感性,促进凋亡(P<0.05);野生型LncRNA MEG3细胞的荧光活性受miR-181a-5p的抑制。过表达miR-181a-5p逆转了LncRNA MEG3对宫颈癌细胞放射增敏和促凋亡作用及对PTEN/Akt信号通路的调控。结论 长链非编码RNA LncRNA MEG3可增强宫颈癌细胞放射敏感性,其机制可能与靶向miR-181a-5p调控PTEN/Akt 信号通路有关,可为提高宫颈癌的预后提供新方向。     

Propofol upregulates miR-320a and reduces HMGB1 by downregulating ANRIL to inhibit PTC cell malignant behaviors

BackgroundOur previous study states that propofol suppresses proliferation and migration of papillary thyroid cancer (PTC) cells by downregulation of lncRNA ANRIL. This study intended to probe the downstream mechanism of ANRIL in PTC with potential microRNAs (miR) and genes.MethodsANRIL expression was detected in normal thyroid epithelial cells (Nthy-ori 3-1) and PTC cells (TPC-1, FTC-133, K1 and BCPAP). ANRIL expression was inhibited in TPC-1 and BCPAP cells to explore the effects of si-ANRIL in PTC malignant behaviors. The gain-and loss-of functions of ANRIL/miR-320a were performed to measure their roles in PTC. Levels of ANRIL, miR-320a, HMGB1, apoptosis- and Wnt/β-catenin and NF-κB pathways-related proteins were measured. Dual-luciferase reporter gene assay and RNA pull-down assay were applied to verify ANRIL/miR-320a/HMGB1 relation. si-ANRIL was transplanted into xenograft tumors in nude mice.ResultsANRIL was upregulated in TPC-1 and BCPAP cells. miR-320a targeted HMGB1, and ANRIL bound to miR-320a. In TPC-1 and BCPAP cells, si-ANRIL prevented PTC cell malignant behaviors, and inactivated the Wnt/β-catenin and NF-κB pathways; while si-ANRIL + miR-320a inhibition showed opposite trends. Overexpressing miR-320a promoted malignant behaviors of TPC-1 cells. In 6 μg/mL propofol-treated TPC-1 cells, miR-320a inhibition weakened propofol’s inhibitory effects on PTC cell growth. After ANRIL inhibition, the volume and weight of xenograft tumors were decreased.ConclusionPropofol upregulated miR-320a and reduced HMGB1 by downregulating ANRIL and inactivating the Wnt/β-catenin and NF-κB pathways, thus preventing PTC cell malignant behaviors. This study may offer new insights in PTC prevention and treatment.     

糖尿病肛瘘lncRNA芯片分析及CNC图搭建＊

目的 研究糖尿病肛瘘创面特异表达LncRNA与mRNA基因功能之间调控网络。方法 用基因芯片技术对糖尿病肛瘘创面和普通肛瘘创面组织中差异性表达的LncRNA及mRNA进行基因表达谱检测，筛选的标准为2倍差异及P < 0.05,再进行差异表达分析，然后对差异表达的mRNAs进行KEGG通路分析及Pathway Map展示，挑选出显著mRNA指标，将这些显著mRNA指标进行q-PCR验证，得到有意义的阳性指标，再将阳性指标与差异LncRNA交集得出LncRNA-mRNA共表达网络，并基于LncRNA-mRNA共表达网络挑选出不同LncRNA进行功能验证。结果 将芯片标准化后分析差异表达的长链非编码RNA和mRNA，发现上调差异有502个，下调差异有1204个；mRNA分析发现上调差异621个，下调差异505个，KEGG通路分析发现上调和下调的通路均有10条；通过分别对上调、下调最明显的通路进行Pathway Map展示，挑选出显著mRNA指标8个：BMP2、IFNB1、IL6、IL18、PIK3CB、SMAD7、SMAD9、β-actin，分别将其进行q-PCR验证，得到有意义的阳性指标个5个：BMP2、IL6、IL18、PIK3CB、SMAD7，将5个阳性指标和差异LncRNA交集得出LncRNA-mRNA共表达网络，并基于LncRNA-mRNA共表达网络挑选出不同LncRNA进行功能验证。结论 挑选出的20个LncRNA（NR_125383、T323486、ENST00000582334、TCONS_00018312、ENST00000418393、TCONS_00017190、TCONS_00019532、ENST00000601559、ENST00000566575、NR_109882、NR_026913、T301537、NR_109774、ENST00000415536、ENST00000610000、ENST00000412485、ENST00000573220、T175957、ENST00000580756、TCONS_00014747）所调控的mRNA居于LncRNA-mRNA共表达网络，其在各个领域当中均有不同程度的报道，为后续的功能和机制研究提供了方向和重点，为加速慢性难愈合和创面愈合的研究提供了新的思路，为开发促愈药物提供基础研究借鉴。     

肝癌组织LncRNA TINCR表达水平对术后长期生存的影响

目的 探讨肝癌组织中LncRNA TINCR 表达水平对术后长期生存的影响。方法 回顾性分析2013 年4 月～2016 年2 月间在本院接受手术治疗的157 例肝细胞肝癌患者的临床资料。RT-PCR 法检测肝癌标本内LncRNA TINCR 表达水平，采用ROC 曲线和Kaplan-Meier 法分析LncRNA TINCR 表达水平对肝癌术后长期生存的影响。结果 肝癌组织LncRNA TINCR 表达水平对术后长期生存预测的曲线下面积（AUC）为0.812，特异度为73.77%，灵敏度为79.17%，最佳判读值为1.89（P＜0.0001）。根据ROC 曲线分析结果，将肝癌组织LncRNA TINCR 相对表达水平大于1.89 的93 例（59.24%）患者纳入高表达组，而肝癌组织LncRNA TINCR 相对表达水平小于或等于1.89的64 例（40.76%）患者纳入低表达组。Kaplan－Meier 法生存分析发现高表达组术后3 年内有76 例患者死亡，3 年总生存率为18.28%（17/93）；低表达组术后3 年内有20 例患者死亡，3 年总生存率为68.75%（44/64），低表达组3 年总生存率明显优于高表达组（P＜0.0001，两组间死亡风险比为3.7534，95%可信区间为2.5158～5.6000）。结论 肝癌组织LncRNA TINCR 表达水平与肝癌术后长期生存显著相关，LncRNA TINCR 表达水平升高则预示着预后不佳。     

The knockdown of MALAT1 inhibits the proliferation,invasion and migration of hemangioma endothelial cells by regulating MiR-206 / VEGFA axis

AimLncRNA MALAT1 is involved in regulation of angiogenesis, however, its expression and mechanism in infantile hemangioma (IH) are less reported. The study aimed to investigate MALAT1 in IH and to reveal the potential mechanism of MALAT1 acting on IH.MethodsIsolated form IH tissue, human CD31⁺ hemangioma endothelial cells (HemECs) were cultured and sorted by magnetic-activated cell sorting (MACS). Quantitative real-time (qRT)-PCR was performed to detect the expressions of MALAT1, miR-206 and VEGFA. The correlations among MALAT1, miR-206 and VEGFA were confirmed by bioinformatics analysis and dual-luciferase reporter assay. The effects of MALAT1, miR-206 and VEGFA on cell proliferation were detected by cell counting kit-8 (CCK-8) and cell colony formation assay. Flow cytometry, wound scratch, Transwell and Tube formation assay were performed to determine cell apoptosis, migration, invasion and vasoformation, respectively. Apoptosis-related proteins were determined by Western blot.ResultsThe results showed that MALAT1 and VEGFA were high-expressed and miR-206 was low-expressed in IH tissues. SiMALAT1 negatively affected the cell proliferation, migration, invasion and vasoformation of HemECs and promoted apoptosis of HemECs. Moreover, Bcl-2 expression was significantly inhibited and the expressions of Bax and c cleaved-3 were greatly promoted. MALAT1 directly targeted and inhibited the expression of miR-206, and VEGFA was predicted to be the target gene for miR-206. SiMALAT1 suppressed the cell proliferation, migration, invasion and vasoformation of HemECs through modulating miR-206/VEGFA axis.ConclusionKnock-down of MALAT1 inhibits the growth of HemECs through regulating miR-206/VEGFA axis, indicating that MALAT1 is a potential therapeutic mechanism for the treatment of IH.     

长链非编码RNA HIF1A-AS2在结直肠癌组织中的表达及临床意义

目的检测LncRNA HIF1A-AS2在结直肠癌组织中的表达,并探讨其临床意义。 方法选取2012年12月至2015年12月行结直肠癌患者根治术的114例结直肠癌患者临床病理资料;收集其癌组织及相对应的癌旁组织(距病灶2 cm)和正常组织(距癌灶边缘>5 cm)标本。通过qRT-PCR检测结直肠癌组织、癌旁组织及癌旁正常组织中HIF1A-AS2相对表达水平。取114例入组患者HIF1A-AS2表达水平的平均数,将HIF1A-AS2表达水平大于等于平均数的89例纳入高表达组,其余纳入低表达组;分析HIF1A-AS2表达与临床病理特征之间的关系;Kaplan-Meier生存曲线分析两组患者术后5年生存情况,Log-rank检验比较生存差异;COX比例风险回归模型分析预后不良的独立危险因素;P<0.05为差异有统计学意义。 结果结直肠癌组织中HIF1A-AS2相对表达量高于癌旁组织及正常组织(P<0.001)。HIF1A-AS2表达与临床分期及组织分化程度显著相关(P<0.05)。HIF1A-AS2高表达组患者术后5年总生存率低于HIF1A-AS2低表达组(Log-rank χ²＝5.037, P＝0.025)。COX多因素分析显示淋巴结转移、临床分期Ⅳ期、中-低分化、有远处转移及HIF1A-AS2显著高表达是影响结直肠癌患者预后不良的独立危险因素(P<0.05)。 结论结直肠癌组织中HIF1A-AS2显著高表达,与临床病理及预后密切相关,是影响结直肠癌患者预后不良的危险因素之一。     

LINC01224靶向miR-125b对口腔鳞癌细胞增殖及凋亡的影响

摘要：目的?探讨长链非编码RNA(LncRNA)LINC01224是否通过调控微小RNA-125b(miR-125b)的表达，从而影响口腔鳞癌(OSCC)细胞增殖及凋亡。方法?采用实时荧光定量聚合酶链反应(qRT-PCR)检测OSCC患者癌组织及癌旁组织中LINC01224的表达水平；体外培养OSCC细胞系CAL-27，将si-NC、si-LINC01224、si-LINC01224与anti-miR-NC、si-LINC01224与anti-miR-125b转染至CAL-27细胞；甲基噻唑基四唑(MTT)试验检测细胞增殖能力；流式细胞术检测细胞周期与细胞凋亡率；双荧光素酶报告试验验证LINC01224与miR-125b的靶向结合关系；western blot检测半胱氨酰天冬氨酸特异性蛋白酶3前体蛋白(pro-caspase-3)、增殖标记蛋白细胞增殖核抗原67(Ki67)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(clv-caspase-3)、P21蛋白的表达水平。结果?与癌旁组织相比，OSCC患者癌组织中LINC01224的表达水平(1.00±0.05 vs 2.43±0.17)显著升高(t=94.345，P<0.05)，miR-125b的表达水平(0.98±0.06 vs 0.22±0.02)显著降低(t=14.838，P<0.05)；与si-NC组比较，si-LINC01224组细胞存活率[(100.02±6.73)% vs (47.94±4.69)%]显著降低(t=19.047，P<0.05)，G1期细胞比例[(31.03±3.01)% vs (42.29±4.12)%]显著增加(t=6.615，P<0.05)，S期细胞比例[(34.18±3.38)% vs (23.49±2.57)%]显著减少(t=7.553，P<0.05)，细胞凋亡率[(8.10±0.92)% vs (24.17±1.74)%]显著升高(t=24.494，P<0.05)，Ki67、pro-caspase-3蛋白水平显著降低(P<0.05)，P21、clv-caspase-3蛋白水平显著升高(P<0.05)；双荧光素酶报告试验证实LINC01224与miR-125b靶向结合；与si-LINC01224+anti-miR-NC组比较，si-LINC01224+anti-miR-125b组细胞存活率[(48.03±4.57)% vs (90.01±5.59)%]显著升高(t=17.442，P<0.05)，细胞凋亡率[(24.11±1.58)% vs (12.81±1.12)%]显著降低(t=17.504，P<0.05)，G1期细胞比例[(42.27±4.10)% vs (35.09±3.18)%]显著减少(t=4.151，P<0.05)，S期细胞比例[(23.53±2.54)% vs (30.03±2.96)%]显著增加(t=4.999，P<0.05)，pro-caspase-3、Ki67蛋白水平显著升高(P<0.05)，clv-caspase-3、P21蛋白水平显著降低(P<0.05)。结论?LINC01224能够靶向调控miR-125b的表达，从而促进OSCC细胞增殖及抑制细胞凋亡。