miR-200a-3p在胆囊癌中的表达及其作用与机制

背景与目的 miR-200a-3p参与了多种肿瘤的生物学过程的调控,但在不同肿瘤中起着不同的作用（促癌基因或抑癌基因）。目前,其在胆囊癌中的作用尚不清楚。因此,本研究探讨miR-200a-3p在胆囊癌中的表达及其对胆囊癌细胞生物学功能的影响与机制。方法 采用qRT-PCR法测定32例胆囊癌及癌旁组织、胆囊癌细胞系（GBC-SD、SGC-996、NOZ）及正常人胆囊上皮细胞系HGBEC中miR-200a-3p的表达。采用Lipofectamine? 3000试剂盒将GBC-SD及NOZ细胞系分别转染miR-200a-3p模拟物（miR-200a-3p过表达组）、miR-200a-3p抑制物序列（miR-200a-3p沉默组）及阴性对照序列（阴性对照组）。MTT实验测定细胞增殖能力,Transwell实验测定细胞侵袭能力,MiRBD/Targetscan7.2/starBase2.0/miRtarbase网站预测miR-200a-3p的下游靶基因,并采用荧光素酶实验验证。Western blot检测上述3组细胞中靶基因与上皮间质转化（EMT）相关分子蛋白（E-cadherin、vimentin）的表达。结果 qRT-PCR结果显示,胆囊癌组织中miR-200a-3p表达量低于癌旁组织,所有胆囊癌细胞系中miR-200a-3p表达量低于正常人胆囊上皮系HGBEC（均P<0.05）。GBC-SD及NOZ细胞系转染后,与各自的阴性对照组比较,两种细胞的miR-200a-3p过表达组miR-200a-3p表达量明显升高、增殖与侵袭能力明显减弱,两种细胞的miR-200a-3p沉默组miR-200a-3p表达量明显降低、增殖与侵袭能力明显增强（均P<0.05）。在线网站预测显示,miR-200a-3p和Notch2存在潜在结合位点;荧光素酶实验显示,miR-200a-3p导致Notch2野生型质粒pmirGLO-Notch2-3''UT WT荧光素酶活性明显降低,而miR-200a-3p对Notch2突变型质粒pmirGLO-Notch2-3''UTR MUT的荧光素酶活性没有影响。Western blot结果显示,两种细胞的miR-200a-3p过表达组与各自的阴性对照组比较,E-cadherin蛋白表达量升高、vimentin蛋白与Notch2蛋白表达量降低,而3种蛋白在两种细胞的miR-200a-3p沉默组则呈相反的变化（均P<0.05）。结论 miR-200a-3p在胆囊癌中表达降低,并可能起了抑癌基因的作用,其抑制胆囊癌细胞增殖和侵袭的机制可能与靶向下调Notch2从而抑制EMT过程有关。     

SIX4和miR-103a-3p在胆囊癌组织中的表达及其对胆囊癌细胞增殖和侵袭的影响

目的 探究SIX同源盒蛋白4（SIX4）、微小RNA（miR）-103a-3p在胆囊癌组织中的表达情况,以及二者对胆囊癌细胞增殖、侵袭的影响。方法 胆囊癌组织及癌旁组织取自永州市中心医院2019年10月至2021年9月32例接受手术治疗的胆囊癌患者,并将体外培养的胆囊癌细胞系GBC-SD分为对照组、mimic NC组、miR-103a-3p mimic组、miR-103a-3p mimic+pc DNA3.1组、miR-103a-3p mimic+SIX4组。实时荧光定量PCR（qRT-PCR）检测SIX4 mRNA、miR-103a-3p水平;蛋白印迹法检测SIX4、增殖细胞核相关抗原Ki-67、增殖细胞核抗原（PCNA）以及基质金属蛋白酶（MMP）2、MMP9蛋白水平;CCK-8法检测细胞增殖情况;Transwell检测细胞侵袭情况。双荧光素酶验证SIX4 mRNA 3’UTR与miR-103a-3p的结合作用。结果 与癌旁组织相比,胆囊癌组织中SIX4 mRNA水平升高,miR-103a-3p水平降低（P<0.05）。miR-103a-3p靶向负调控SIX4。与对照组、mimic NC组比较,miR-103a-3p mimic组细胞中SIX4 mRNA和蛋白水平,OD₄₅₀,侵袭数量,Ki-67、PCNA、MMP2、MMP9蛋白水平降低,miR-103a-3p水平升高（P<0.05）;与miR-103a-3p mimic+pc DNA3.1组比较,miR-103a-3p mimic+SIX4组上述各项指标均被逆转。结论 胆囊癌组织中SIX4水平升高、miR-103a-3p水平降低,过表达miR-103a-3p可能通过靶向抑制SIX4表达,从而抑制胆囊癌细胞增殖、侵袭。     

Relationship between the single nucleotide polymorphisms in has-mir-125a-5p rs12975333 and expression of has-mir-125a-5p in Han Chinese female breast cancer patients

Objective To investigate the relationship between the single nucleotide polymorphisms ( SNPs) in has-mir-125a-5p rs12975333 and the expression of has-mir-125a-5p and clinicopathological cheracteristics of female breast cancer in Han Chinese women. Methods Genomic DNA was extracted from peripheral blood lymphocytes.     

DCE-MRI减影联合血清miR-133a-3p及CCR3预测TACE治疗肝细胞癌早期效果

目的 观察动态增强MRI(DCE-MRI)减影联合血清微小RNA-133a-3p(miR-133a-3p)及CC趋化因子受体3(CCR3)预测TACE治疗肝细胞癌(HCC)早期效果的效能。方法 对107例HCC行TACE,1个月后根据实体肿瘤疗效评价标准将其分为疗效良好组(部分缓解+完全缓解)和疗效不良组(稳定+进展)。对比2组术前DCE-MRI减影参数动脉期、门静脉期对比噪声比(CNR)及血清miR-133a-3p、CCR3水平;采用受试者工作特征(ROC)曲线分析CNR联合血清miR-133a-3p及CCR3预测TACE治疗早期效果的效能。结果 62例疗效良好,45例疗效不良。TACE前,疗效不良组动脉期CNR高于、门静脉期CNR低于疗效良好组,血清miR-133a-3p水平低于、CCR3水平高于疗效良好组(P均<0.001)。ROC曲线结果显示,动脉期CNR、门静脉期CNR、血清miR-133a-3p和CCR3以及四者联合预测TACE治疗早期效果的曲线下面积(AUC)分别为0.785、0.761、0.692、0.707及0.919,特异度分别为88.7%、74.2%、51....     

MicroRNA-199a-3p在肾癌中的表达及作用

目的 检测microRNA-199a-3p(miR-199a-3p)在肾癌细胞株和组织中的表达情况并探究miR-199a-3p在肾癌细胞中的作用.方法 利用实时定量RT-PCR检测miR-199a-3p在肾癌细胞和组织中的表达水平;利用miR-199a-3p模拟物转染肾癌细胞786-0上调miR-199a-3p后,通过CCK-8、克隆形成、Transwell以及细胞周期检测来探究其在肾癌细胞中的作用.结果 miR-199a-3p在肾癌细胞中明显低表达,在78%(14/18)的肾癌组织中亦明显低表达;上调miR-199a-3p可显著抑制肾癌细胞的增殖、存活和侵袭并能诱导细胞周期G1期阻滞.结论 我们的研究显示在肾癌中miR-199a-3p明显低表达并参与肾癌的发生、发展,这表明miR-199a-3p具有作为肾癌诊断和治疗靶点的潜能.     

右美托咪定对结直肠癌LOVO细胞侵袭和迁移影响的实验研究

目的:探讨右美托咪定对结直肠癌LOVO细胞侵袭和迁移的影响及其发生机制。方法:以0、50、100和200μg/mL右美托咪定处理48 h后,采用细胞计数(CCK-8)法检测细胞活力,Transwell小室检测细胞侵袭和迁移能力,实时荧光定量聚合酶链反应(PCR)法检测细胞中微小RNA(miR)-196a-5p的表达水平。将miR-196a-5p抑制剂转染至LOVO细胞后,观察下调miR-196a-5p表达对右美托咪定作用下的LOVO细胞活力、侵袭和迁移的影响。结果:与0μg/mL组比较,50、100和200μg/mL组右美托咪定可增强LOVO细胞活力、促进细胞侵袭和迁移并上调细胞中miR-196a-5p表达水平(P<0.05),且呈现一定的浓度依赖性。转染miR-196a-5p抑制剂后,右美托咪定对LOVO细胞活力、细胞侵袭和迁移的促进作用明显减弱(P<0.05)。结论:右美托咪定可通过上调miR-196a-5p表达促进结直肠癌LOVO细胞侵袭和迁移。     

miR-18a-3p在肝癌中的表达及其调控ADCY1表达对肝癌细胞侵袭及增殖的影响

背景与目的 研究表明多种microRNA（miRNA）可能在肝癌的发生发展中发挥重要作用,其作用机制仍值得进一步研究和探讨。因此,本研究从已报道的肝癌差异表达miRNA中进一步筛选关键miRNA,并验证和探讨其作用机制。方法 从已发表的研究中筛选出肝癌组织及肝癌患者血清/血浆中与正常肝组织及正常血清/血浆中共同的差异表达miRNA;用qRT-PCR在正常肝细胞与肝癌细胞中对筛选出的目标miRNA表达情况进行验证;用过表达和抑制的方法观察目标miRNA对肝癌细胞侵袭能力（Transwell实验）与增殖能力（MTT实验）的影响,以及在30例临床标本中检测目标miRNA的表达并通过KM plotter网站分析其对肝癌患者生存的影响;通过miRDB和GEPIA数据库预测和分析目标miRNA的靶基因,并用逆转实验和双荧光素酶报告实验进一步验证。结果 在肝癌组织（vs.正常肝组织）及肝癌患者血清/血浆（vs.正常人血清/血浆）中共同高表达的miRNA有4个（miR-18a-3p、miR-221-3p、miR-222-3p、miR-224-3p）,共同低表达的miRNA有2个（miR-26a-3p、miR-125b-3p）。qRT-PCR实验证实,与正常肝细胞比较,miR-18a在肝癌细胞中高表达,miR-26a在肝癌细胞中低表达（均P<0.05）。过表达/抑制miR-18a-3p表达能促进/降低肝癌细胞的侵袭及生长能力（均P<0.05）,而过表达/抑制miR-26a-3p对肝癌细胞的侵袭及生长能力影响无不法确定。分析结果显示,ADCY1是miR-18a-3p的靶基因,过表达ADCY1能部分逆转miR-18a-3p对肝癌细胞的上述作用,同时,表达上调的miR-18a-3p能通过结合到ADCY1 mRNA 3''UTR抑制ADCY1的表达。结论 miR-18a-3p可能在肝癌的发生发展中起了关键作用,其在肝癌中表达上调,并能通过抑制下游靶基因ADCY1的表达增强进肝癌细胞的侵袭和增殖能力。     

汉族女性乳腺癌患者has-mir-125a-5p基因的rs12975333位点的单核苷酸多态性及其在癌组织的表达研究

目的 探讨汉族女性乳腺癌患者has-mir-125a-5p基因rs12975333位点单核苷酸多态性(SNP)及has-mir-125a-5p基因在癌组织表达水平与乳腺癌发生发展的关系.方法 选取汉族女性乳腺癌患者289例、乳腺纤维腺瘤49例和相匹配的健康汉族妇女外周血338例,分离淋巴细胞,抽提基因组DNA,采用taqman-MGB探针检测338例乳腺肿瘤及338例健康对照组has-mir-125a-5p的rs12975333位点的单核苷酸多态性;采用茎环Real-time RT-PCR检测289例乳腺癌组织及其正常腺体组织中has-mir-125a-5p的表达水平.结果 乳腺癌组has-mir-125a-5p基因rs12975333位点基因型为GG型273例(94.5%)、GT型16例(5.5%);乳腺纤维腺瘤组及对照组has-mir-125a-5p基因m12975333位点基因型均为GG型;乳腺癌组织has-mir-125a-5p的表达水平(0.19 ±0.04)低于腺体组织(0.37±0.05),差异有统计学意义(P=0.04);携带T等位基因乳腺癌组织中has-mir-125a-5p下调水平较GG基因型表达者更为明显(P=0.022).has-mir-125a-5p的表达降低在发病年龄较晚(P=0.036),ERBB2(P=0.007)、ERBB3(P=0.04)受体表达阴性及存在腋窝淋巴结转移(P=0.001)的乳腺癌组织中更为明显.结论 汉族妇女has-mir-125a-5p基因rs12975333位点的G＞T变异可能与乳腺癌遗传易感性有关;has-mir-125a-5p表达与乳腺癌发生发展相关,是一个潜在的乳腺癌分子标志物.

Abstract：

Objective To investigate the relationship between the single nucleotide polymorphisms (SNPs) in has-mir-125a-5p rs12975333 and the expression of has-mir-125a-5p and clinicopathological cheracteristics of female breast cancer in Han Chinese women. Methods Genomic DNA was extracted from peripheral blood lymphocytes. taqman-MGB assay was used to type breast cancer of 338 cases and 338 controls. Expression levels of has-mir-125a-5p in 289 biopsies were examined using stem-loop real-time RTPCR and the clinicopathological cheracteristics of breast cancer were evaluated. Result The gene frequencies (GG,GT,TT) of rsl2975333 in the patients were GG 273 (94. 5% ), GT 16 (5.5%),TT 0(0%), while in breast fibroadenoma and controls there were GG 49 ( 100% ), 338 (100%). The expression level of has-mir-125a-5p in breast cancer(0. 19 ±0. 04) was lower than that in the matched nontumor adjacent tissue specimens (0. 37 ± 0. 05 ) ( P = 0. 04 ) .The expression level of minor T allele of mature miR-125a in breast cancer patients was lower than that in has-mir-125a-5p-GG carying(P =0.022). The expression of has-mir-125a-5p was down-regulated in primary breast cancer, especially in elder patients ( P = 0. 036) and lymph node metastasis groups (P = 0. 001) and with negative ERBB2 (P = 0. 007), ERBB3 (P =0. 04). Conclusions rs12975333 polymorphisms in has-mir-125a-5p gene may work as a risk factor of breast cancer in Han Chinese women. The altered expression of has-mir-125a-5p might play a role in the pathogenesis and progression of breast carcinoma.     

KCNQ1OT1 mediates keratinocyte migration to promote skin wound healing through the miR-200b-3p/SERP1 axis

BackgroundThe basic functions of keratinocyte are crucial steps during skin wound healing. KCNQ1OT1 long noncoding RNA was found to accelerate the migration and proliferation of keratinocyte in psoriasis. Here, we elucidated the action and mechanism of KCNQ1OT1 in skin wound healing.MethodsExpression levels of genes and proteins were evaluated by quantitative real-time PCR (qRT-PCR) and western blotting. Cell migration was assessed by using scratch and transwell assays. The interaction between miR-200b-3p and KCNQ1OT1 or SERP1 (Stress Associated Endoplasmic Reticulum Protein 1) was confirmed by bioinformatics analysis, dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and pull-down assay.ResultsKCNQ1OT1 had increased significantly in wound edge 1 day and 7 day after injury. Functionally, overexpression of KCNQ1OT1 promoted keratinocyte migration. Mechanistically, KCNQ1OT1/miR-200b-3p/SERP1 constituted a competing endogenous RNA (ceRNA) network in keratinocytes. A series of rescue experiments showed that miR-200b-3p up-regulation in keratinocytes attenuated the pro-migration action of KCNQ1OT1 in cells. Moreover, knockdown of miR-200b-3p could promote keratinocyte migration, which was abolished by SERP1 silencing. KCNQ1OT1 competitively sponged for miR-200b-3p to elevate the expression of its target SERP1.ConclusionKCNQ1OT1 could promote keratinocyte migration by miR-200b-3p/SERP1 axis, suggesting that KCNQ1OT1 might play a crucial role in skin wound healing.     

Involvement of the insulin-like growth factor I receptor and its downstream antiapoptotic signaling pathway is revealed by dysregulated microRNAs in bladder carcinoma

ObjectiveUrothelial carcinoma is one of the most common pathological types of bladder cancer. Several studies have shown that dysregulated microRNAs (miRNAs) play an important role in bladder cancer progression. We performed the present miRNA microarray analysis in samples of urothelial carcinoma of the bladder and adjacent normal bladder tissue from Taiwanese patients to investigate dysregulated miRNAs.Materials and methodsTo detect dysregulated miRNAs in urothelial carcinoma of the bladder, samples of tumor and adjacent normal tissues were collected from 10 patients. Tissue samples from three patients were subjected to miRNA microarray analysis, and the remaining tissue samples from the other seven patients were used to validate the results obtained from the microarray data. Potential targets of these dysregulated miRNAs were identified using online databases, including MicroCosm and TargetScan.ResultsA panel of 30 differentially expressed miRNAs with at least fourfold differences in expression compared with normal controls, including 19 upregulated and 11 downregulated miRNAs, was generated. The expression levels of miR-30a-5p, miR-30a-3p, miR-99a, miR-130b, miR-133b, miR-135b, miR-145, miR-195, miR-204, and miR-214 were experimentally verified using real-time RT-PCR analysis. Using an online miRNA target database, we discovered that these dysregulated miRNAs potentially control components of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway.ConclusionOur results indicate that dysregulated miRNAs may be involved in bladder cancer pathogenesis and are potential biomarkers.     

The combination of microRNA-371a-3p and 375-5p can distinguish viable germ cell tumor and teratoma from necrosis in postchemotherapy retroperitoneal lymph node dissection specimens

BackgroundTo identify a combination of microRNAs (miRNA) to differentiate between viable tumor (V) or teratoma (T) and necrosis/fibrosis (N) in pcRPLND specimens of metastatic germ cell tumor (GCT) patients with residual masses ≥1 cm after chemotherapy. Biomarker guided therapy could reduce overtreatment with pcRPLND in patients with only N.MethodsWe selected 48 metastatic GCT patients who had undergone pcRPLND. V, pure T and N was shown in the resected tissue of 16 patients, respectively. Of these areas total RNA was isolated and miRNA expression was analyzed for miR-371a-3p, 375-3p, and 375-5p using qPCR. ROC analysis was performed for each miRNA and for all combinations in order to determine the discriminatory capacity of V and T vs. N.ResultsOn comparing V vs. N miR-371a-3p achieved the highest fold change (FC) of 31.1 (P=0.023) while for T vs. N miR-375-5p performed best (FC 64.2; P<0.001). Likewise, the most accurate AUC for V was 0.75 using miR-371a-3p, for T 0.80 using miR-375-5p. Combining the best performing miRNAs for V and T resulted in an AUC of 0.94 with a sensitivity of 93.75, specificity of 93.75, PPV of 96.8 and NPV of 83.3.ConclusionsBy combining miR-371a-3p and miR-375-5p in pcRPLND tissue samples V and T could be distinguished from necrosis/fibrosis with great accuracy. This combination of miRNAs might serve as new biomarker in the future, in order to spare miRNA-negative patients from pcRPLND. However, further studies analyzing patient’s serum are needed to confirm the clinical impact of these biomarkers.     

The role of microRNA profiling in prognosticating progression in Ta and T1 urinary bladder cancer

ObjectiveTo analyze microRNA profile in Ta and T1 urinary bladder cancers in combination and separately and to relate this to the risk of later developing higher-stage disease.Materials and methodsFormalin-fixed, paraffin-embedded samples of 44 Ta and 42 T1 bladder cancers representing cases with and without stage progression during follow-up were collected and microRNA expression levels were measured by microarray analysis.ResultsIn a comparison between the progressors and controls, in the Ta/T1 group, miR-10a-5p and miR-31-5p were differentially expressed. miR-10a-5p was also correlated to time to progression (P = 0.00012). In the subgroup analysis, 3 microRNAs, miR-10a-5p, miR-31-5p, and miR-130a-3p, were differentially expressed among Ta tumors and had a fold change of more than 1.5 (P<0.038). The comparison concerning microRNA expression between the progressors and controls in category T1 cancers revealed no significant differences.ConclusionsProfiling revealed that certain microRNAs predicted the risk of developing higher-stage disease among patients with Ta cancers. Lower miR-10a-5p expression in Ta progressing tumors indicates that this microRNA could be important for later malignant potential among this group of patients.     

LncRNA DANCR regulates lymphatic metastasis of bladder cancer via the miR-335/VEGF-C axis

BackgroundSubstantial evidence indicate that long non-coding RNA (lncRNA) and microRNA (miRNA) act as key role in bladder cancer. Differentiation antagonistic ncRNA (DANCR) could be used as a biomarker in the occurrence and development of cancer. This study aims to explore the mechanism of DANCR/miR-335/VEGF-C axis affecting lymphatic metastasis of bladder cancer.MethodsqRT-PCR detects the expression of DANCR in bladder cancer cell lines (SW780, 5637, T24, UM-UC-3) and normal bladder cell lines (SV-HUC-1), and selects T24 cell lines for subsequent experiments. The expression levels of DANCR, miR-335 and VEGF were measured by qRT-PCR, and the dual luciferase reporter gene verified the targeted regulation of DANCR on miR-335 and miR-335 on VEGF. CCK-8, Transwell and Wound healing assay detect the proliferation, invasion and migration ability of bladder cancer cells, Endothelial cell adhesion assay and Western blot further prove the lymphatic metastasis of bladder cancer.ResultsIn this study, DANCR was highly expressed in bladder cancer cell lines. Transfection of si-DANCR significantly inhibits the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells. Dual luciferase assay confirmed that DANCR targets miR-335/VEGF-C. Transfection of miR-335 mimic promotes the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells, overexpression of DANCR eliminates the promotion of miR-335 mimic on bladder cancer cells. Further experiments proved that inhibition of miR-335 and overexpression of VEGF-C can reverse the inhibitory effect of silencing DANCR on bladder cancer cells.ConclusionsIn bladder cancer, DARCR plays an important role, which regulates the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells through the miR-335/VEGF-C molecular axis.     

CircSLC8A1 targets miR-181a-5p/HIF1AN pathway to inhibit the growth,migration and extracellular matrix deposition of human keloid fibroblasts

BackgroundCircular RNAs (circRNAs) are identified as important regulators in human diseases, including keloid. The purpose of this study is to reveal the role and molecular mechanism of circSLC8A1 in keloid formation.MethodsExpression of circSLC8A1, microRNA (miR)-181a-5p, and hypoxia inducible factor 1 alpha inhibitor (HIF1AN) were detected by quantitative real-time PCR. Protein expression of extracellular matrix (ECM) deposition markers and HIF1AN was detected by western blot analysis. Furthermore, the interaction between miR-181a-5p and circSLC8A1 or HIF1AN was confirmed by dual-luciferase reporter assay, RIP assay and RNA pull-down assay.ResultsExpression of circSLC8A1 was downregulated in keloid tissues and HKFs. Overexpression of circSLC8A1 suppressed HKFs proliferation, migration, ECM deposition, and promoted apoptosis. MiR-181a-5p is targeted by circSLC8A1, and its mimic reversed the effect of circSLC8A1 on the biological function of HKFs. HIF1AN was a target of miR-181a-5p, and it was positively regulated by circSLC8A1. Knockdown of HIF1AN also reversed the negatively regulation of circSLC8A1 on the biological functions of HKFs.ConclusionOur data showed that circSLC8A1 regulates the miR-181a-5p/HIF1AN axis to restrain HKFs biological functions, confirming that circSLC8A1 might serve as a novel therapeutic target for keloids.     

Benzodiazepines safeguards nerve cells from the toxicity of lidocaine via miR-133a-3p/EGFR pathway

BackgroundLidocaine was an anesthetic commonly used for analgesia, but the neurotoxicity could not be ignored. However, benzodiazepines could alleviate the toxicity when combined with other drugs.PurposeTo explore the molecular mechanism of benzodiazepines in protecting nerve cells after the induction of lidocaine.MethodsPC12 cells were induced by lidocaine (0 mM, 0.1 mM, 0.5 mM and 1 mM) first and then treated by benzodiazepines (0 μM–200 μM). RT-qPCR assays measured RNA expressions of epidermal growth factor receptor (EGFR) and microRNA-133a-3p (miR-133a-3p) in PC12 cell line, respectively. Western blot was for protein detections of EGFR and caspase-3. Flow cytometry assay assessed apoptosis and cellular viability was validated via Cell Counting Kit-8 (CCK-8) test. Bioinformatics analysis predicted the potential link between miR-133a-3p and EGFR and the binding was verified using the Dual luciferase reporter experiment.ResultsBenzodiazepines increased cellular viability of PC12 cells up to 100 μM while suppressed viability between 100 and 200 μM. Benzodiazepines (0 μM, 10 μM, 50 μM and 100 μM) did not regulate PC12 cell viability but promoted the viability of lidocaine-treated PC12 cells. Lidocaine downregulated miR-133a-3p RNA expression but facilitated EGFR mRNA expression, which was reversed after treated by benzodiazepines. MiR-133a-3p targeted and negatively regulated EGFR expressions in mRNA and protein levels. Furthermore, miR-133a-3p inhibitor and overexpressed EGFR transfection both restrained the decreased PC12 cell viability and prompted cell apoptosis caused by benzodiazepines.ConclusionBenzodiazepines restrained lidocaine-induced toxicity in PC12 cells which secured viability and reduced apoptosis via miR-133a-3p/EGFR pathway.     

LncRNA MALAT1 Promotes Lung Cancer Proliferation and Gefitinib Resistance by Acting as a miR-200a Sponge

IntroductionLung cancer is a major public health problem, as the second causes of cancer related death worldwide, with relatively low survival rates, and accessible drug resistance. Long non-coding RNAs (LncRNAs) have been identified as activator in lung cancer with elusive mechanisms. We aimed to detect the regulation of LncRNA MALAT1 in the proliferation and gefitinib resistance in lung cancer cells.MethodsMALAT1 in A549 and HCC 1299 human lung adenocarcinoma cell lines was silenced by shRNA or overexpressed using plasmid, and the cell viability and cell proliferation were evaluated by MTT assay and soft agar colony formation assay. RNA levels were detected by RT-PCR, and the protein expression was measured by western blot. The binding between MALAT1 and miR-200a was validated by luciferase reporter assays using pSi-Chech 2 vectors.ResultsThe cell viability and proliferation of A549 cells transfected with MALAT1 shRNA were significantly lower than the control. The MALAT1 expression in gefitinib resistant A549 cells was upregulated. miR-200a significantly inhibited the fluorescence of pSi-Check 2 vector with MALAT1 gene, suggesting the direct binding between MALAT1 and miR-200a. In addition, LncRNA MALAT1 promotes ZEB1 expression in A549 cells.ConclusionOur study showed that MALAT1 promoted the proliferation and gefitinib resistance of lung cancer cells by sponging miR-200a, which regulates expression of ZEB1 in the A549 cells. This MALAT1/miR-200a axis could serve as new therapeutic target for lung cancer treatment.     

Overexpression of F-box and WD repeat domain containing 7 prevents tumor growth of bladder cancer cells through regulating SREBP1a

BackgroundThis study aimed to explore the effect of F-box and WD repeat domain containing 7 (FBXW7) overexpression on bladder cancer, and to determine the regulatory effect of FBXW7 on sterol regulatory element-binding protein 1a (SREBP1a) in bladder cancer.MethodsThe function of F-box and FBXW7 in tumor growth of bladder cancer cells was investigated using in vivo and in vitro models. We constructed and transfected FBXW7 overexpression vectors into T24 and J82 bladder cancer cells. After transfections, the expression of FBXW7 at messenger RNA (mRNA) and protein levels in human bladder cancer cells was confirmed. To verify the effect of FBXW7 on tumor growth, cell proliferation and migration were detected in the tumor cells after overexpression of FBXW7. Additionally, an in vivo tumor model was produced by inoculating the tumor cells and the effect of FBXW7 was evaluated. Finally, the regulation mechanisms were determined.ResultsOur data showed that overexpression of FBXW7 in J28 and T24 cells significantly inhibited the migration and proliferation of J82 and T24 cells, arrested the cells at G0/G1 phase, and up-regulated phosphorylation of glycogen synthase kinase-3β, while suppressing SREBP1a expression. In vivo data also showed that FBXW7 overexpression triggered apoptosis of tumor cells, prevented the pathological changes of tumor tissues, up-regulated p-GSK3β expression, and suppressed SREBP1a expression. In addition, an interaction between FBXW7 and SREBP1a was confirmed by co-immunoprecipitation.ConclusionsTogether, our data indicate that overexpression of FBXW7 prevents tumor growth of bladder cancer cells, likely through suppressing SREBP1a expression.