miR-98 regulates cisplatin-induced A549 cell death by inhibiting TP53 pathway

To explore the possible microRNAs (miRNAs) in the TP53 pathway and their roles in A549 cell death induced by cisplatin, the miRNAs relative to 3′-untranslated region (3′-UTR) of TP53 were predicted by microRNA analysis softwares, which showed that TP53 expression might be targeted by miR-98, miR-453 and miR-485. Then, GFP was used as a reporter gene to reflect whether the 3′-UTR of TP53 was targeted by the predicted miRNAs. After pcDNA-GFP-UTR was constructed, the GFP expression was estimated in A549 cells by the examination of fluorescence microscopy and flow cytometry. The intensity of fluorescence in the miR-98 and miR-453 groups decreased significantly compared with the control group. The percentage of positive GFP cells in miR-98 and miR-453 groups were 30.24% and 32.58%, respectively, much lower than that of NC group (41.86%). The TP53 expression was inhibited after transfection with miR-98/miR-453 by western blot. As the factors in TP53 pathway, Bcl-2 expression was found to be enhanced, and the expression of miR-34a-c was decreased in A549 cells after miR-98/miR-453 treatment. Moreover, the expression of miR-98 and Bcl-2 was decreased, while miR-34a-c and TP53 was increased after A549 treated with cisplatin. Our study demonstrated that miR-98 and miR-453 down-regulated TP53 expression by targeting the 3′-UTR of TP53, and that cisplatin might inhibit A549 cell growth by miR-98 regulating TP53 pathway. Our results indicated that TP53 relevant miRNAs might be the new targets for gene therapy or new drug design.     

mir-17-5p、mir-92a、let-7b表达水平与非小细胞肺癌顺铂耐药的关系

目的探讨mir-17-5p、mir-92a、let-7b表达水平与非小细胞肺癌顺铂耐药关系。 方法以人非小细胞肺癌细胞系A549及其耐药株A549/DDP为研究对象,采用RT-PCR法检测mir-17-5p、mir-92a及let-7b在细胞中的表达水平,采用cck8检测其细胞存活情况,采用细胞克隆平台方法,检测转染前后细胞的增殖情况,采用流式细胞仪检测转染前后细胞的凋亡情况。 结果（1） A549/DDP细胞mir-17-5p的表达水平是A549细胞的2.11±0.25倍（P＜0.05）;A549/DDP细胞mir-92a的表达水平是A549细胞的 7.40 ± 1.05 倍（P＜0.05）;而A549/DDP 细胞let-7b 的表达水平是A549 细胞的（26.54 ± 2.90）%（P＜0.05）;（2）A549 转染mir-17-5pmimic,mir-92a mimic 以及let-7b inhibitor 后对顺铂的敏感性下降（P＜0.05）;A549/ddp 转染mir-17-5p inhibitor, mir-92a inhibitor以及let-7b mimic后对顺铂的敏感性增加（P＜0.05）;（3）A549转染mir-17-5p mimic,mir-92a mimic以及let-7b inhibitor 后,细胞形成克隆集落数量数量多于对照组（P＜0.05）;而A549/ddp 转染mir-17-5p inhibitor,mir-92a inhibitor 以及 let-7b mimic 后,细胞形成克隆集落数量数量少于对照组（P＜0.05）;（4）A549 转染mir-17-5p mimic,mir-92a mimic 以及let-7b inhibitor后,细胞凋亡率明显低于对照组（P＜0.05）;而a549/ddp转染mir-17-5p inhibitor,mir-92a inhibitor以及let-7b mimic后, 细胞凋亡率明显高于对照组（P＜0.05）。结论Mir-17-5p、mir-92a表达水平升高,let-7b表达水平下降,可以促进肺癌细胞增殖, 抑制其凋亡以及使肺癌细胞对顺铂敏感性下降。      

miR-135b reverses chemoresistance of non-small cell lung cancer cells by downregulation of FZD1

BackgroundNon-small cell lung cancer (NSCLC) chemoresistance usually limits the clinical efficacy of chemotherapeutic approaches. However, few reports have revealed the regulation of miR-135b and Frizzled-1 (FZD1) involved in NSCLC chemoresistance.MethodsTo identify the mechanism of miR-135b and FZD1 in NSCLC chemoresistance and to observe their biological functions, we detected the expression levels of miR-135b and FZD1 by conducting quantitative real-time polymerase chain reaction (RT-qPCR) and modified the expressions of miR-135b and FZD1 by transiently transfecting cells with miR-135b mimics or FZD1-siRNA. The 3′-untranslated region (3′-UTR) of FZD1 combined with miR-135b was verified through dual-luciferase reporter assay.ResultsCompared with that in A549 parental cell lines, the miR-135b expression in drug-resistant lung cancer cell lines (A549/DDP) was decreased and their FZD1 expression was increased. The increased miR-135b expression and silenced FZD1 expression enhanced the sensitivity of resistant cells to cisplatin treatment. The high expression of miR-135b in A549/DDP cells remarkably decreased the mRNA levels of FZD1. FZD1 was further identified as the functional downstream target of miR-135b by directly targeting the 3′-UTR of FZD1.ConclusionThe amplification of miR-135b suppressed NSCLC chemoresistance by directly mediating the FZD1 downregulation.     

miR-802对叉头框转录因子M1抑制A549/DDP细胞顺铂耐药性的靶向调控作用

目的探讨miR-802抑制非小细胞肺癌A549/DDP细胞的顺铂耐药性及其对叉头框转录因子M1(forkhead box protein M1, FoxM1)的靶向调控作用。方法 A549、A549/DDP细胞采用反转录PCR法检测miR-802相对表达量。将A549/DDP细胞分为空白转染组和miR-802过表达组(过表达组),分别转染miR-NC和miR-802类似物(miR-802 mimics),转染24、48、72、96 h后,采用反转录PCR法检测2组细胞miR-802相对表达量,并采用CCK-8法检测2组细胞增殖率;转染48 h后,CCK-8法检测转染后A549/DDP细胞对DDP的敏感性,采用流式细胞仪检测2组细胞凋亡率和细胞周期,采用Western blot法检测转染后A549/DDP细胞内FoxM1蛋白相对表达量。结果 A549/DDP细胞miR-802相对表达量(0.21±0.03)低于A549细胞(0.85±0.12)(P<0.05);转染24、48、72、96 h,过表达组细胞miR-802相对表达量依次增高(P<0.05),且均高于空白转染组(P<0.05);转染48、72、96 h,空白转染组细胞增殖率依次增高(P<0.05),且均高于过表达组(P<0.05);转染48 h,过表达组细胞半数抑制浓度[(35.28±2.17)mg/L]和细胞早期凋亡率[(17.2±1.1)%]均高于空白转染组[(14.22±1.28)mg/L、(9.0±0.8)%](P<0.05),S期和G2/M期细胞比率[(21.30±0.20)%、(8.35±0.33)%]及细胞内FoxM1蛋白相对表达量(0.21±0.04)均低于空白转染组[(27.54±0.52)%、(14.67±0.70)%、0.44±0.06](P<0.05)。结论 miR-802可能通过抑制FoxM1表达而降低非小细胞肺癌A549/DDP细胞的顺铂耐药性。     

Long non-coding RNA CASC2 enhanced cisplatin-induced viability inhibition of non-small cell lung cancer cells by regulating the PTEN/PI3K/Akt pathway through down-regulation of miR-18a and miR-21

Long non-coding RNA cancer susceptibility candidate 2 (lncRNA CASC2) is a tumor suppressor and has been proved to contribute to chemotherapy efficacy. However, the effect of CASC2 on cisplatin cytotoxicity in non-small cell lung cancer (NSCLC) is unclear. The present study aimed to investigate the role of CASC2 in regulating cisplatin cytotoxicity in NSCLC cells. Herein, we found that CASC2 was low-expressed, while miR-18a and miR-21 were over-expressed in NSCLC cell lines. CASC2 enhanced the inhibition effect of cisplatin on cell viability. Down-regulation of miR-18a and miR-21 exhibited the similar effect to CASC2 and mimics of miR-18a and miR-21 displayed the opposite effect to CASC2. MiR-18a and miR-21 were two targets of CASC2 in NSCLC. PTEN was found to be a target of miR-18a and miR-21 in NSCLC. CASC2 overexpression increased PTEN expression level and reduced the ratio of p-Akt/Akt. MiR-18a or miR-21 mimics attenuated the effect of CASC2 overexpression on the PTEN expression and ratio of p-Akt/Akt. The results suggested that CASC2 enhanced cisplatin-induced viability inhibition of NSCLC cells via PTEN/PI3K/Akt pathway through suppressing miR-18a and miR-21 expression.

Long non-coding RNA cancer susceptibility candidate 2 (lncRNA CASC2) is a tumor suppressor and has been proved to contribute to chemotherapy efficacy.     

NLK,a novel target of miR-199a-3p,functions as a tumor suppressor in colorectal cancer

We previously reported that miR-199a-3p is a newly biomarker for diagnosis and novel prognostic indicator in colorectal cancer. However, the miR-199a-3p regulatory mechanism and its target genes are still unclear. In our present study, we demonstrated miR-199a-3p could directly target 3′-UTR of NLK gene by luciferase reporter assay and western blot analysis. We detected NLK expressions in 92 colorectal cancer cases to evaluate its clinicopathologic characteristics in colorectal cancer. Our results showed that NLK expression was significantly downregulated in cancer tissues than NATs, and NLK low-expression was associated with lymph node metastasis, venous invasion, liver metastasis and the TNM stage (P < 0.05). Moreover, Kaplan–Meier analysis showed that low expression of NLK correlated with a shorter overall survival rates of patients with CRC (P < 0.05). In vitro, we also found that NLK suppressed the biological behaviors of colorectal cancer cells, including the abilities of cell proliferation, clone formation, wound healing, migration and invasion (P < 0.05), while overexpression of NLK increased the apoptotic rate of colorectal cancer cells. All these results suggested that NLK was an identified miR-199a-3p target gene and functioned as a tumor suppressor gene in colorectal cancer. NLK could be a novel direction for developing diagnostic and therapeutic approaches in colorectal cancer.     

Caprin-1 is a novel microRNA-223 target for regulating the proliferation and invasion of human breast cancer cells

MicroRNAs (miRNAs) are 21–22 nucleotides regulatory small non-coding RNAs that inhibit gene expression by binding to complementary sequences especially the 3’ untranslated region (3’UTR) of mRNA. One miRNA can target many messenger RNAs, leading to a complex metabolic network. Previous studies have shown that miRNA-223 regulates migration and invasion of tumor cells and targets cytoplasmic activation/proliferation-associated protein-1 (Caprin-1). In the present study, we detected the expression of miRNA-223 and Caprin-1 in MCF-7, T-47D and MDA-MB-231 cancer cell lines, and MCF-10A normal breast cell line, and analyzed the role of miRNA-223 in Caprin-1-induced proliferation and invasion of human breast cancer cells. We found that miRNA-223 expression levels are significantly lower in MCF-7, T-47D and MDA-MB-231 cancer cells than in MCF-10A normal breast cells, while Caprin-1 expression is higher in cancer cells than in normal breast cells. The most malignant cancer cell line MDA-MB-231 has the lowest expression of miR-223, but the highest expression of Caprin-1. Further, we found that miR-223 targets the 3’UTR of Caprin-1 miRNA and down-regulates the expression of Caprin-1. We also found that over-expression of Caprin-1 can promote the proliferation and the invasion of breast cancer cells, but miRNA-223 can inhibit the proliferation and the invasion. miRNA-223-induced inhibition can be reversed by ectopic over-expression of Caprin-1. These findings suggest that miR-223 may suppress the proliferation and invasion of cancer cells by directly targeting Caprin-1. Our study also indicates that expression levels of miR-223 and Caprin-1 can be used to predict the state of cancer in breast cancer patient.     

miR-892a regulated PPP2R2A expression and promoted cell proliferation of human colorectal cancer cells

Colorectal cancer (CRC) is one of the most common malignancy cancers in the world. Aberrant microRNA expression is involved in human diseases including cancer. In the present study, we investigated the miR-892a's role in CRC cell proliferation. We found that miR-892a was frequently upregulated in human colorectal cancer tissues and cell lines compared with the matched tumor adjacent tissues and normal colonic cell line FHC. Overexpression of miR-892a promoted cell proliferation and colony formation of CRC. Bioinformatics analysis further revealed PPP2R2A was identified as a potential miR-892a. Overexpression of miR-892a-in SW480 cells reduced PPP2R2A protein expression. Subsequently, data from luciferase reporter assays showed that PPP2R2A 3′-untranslated region (3′-UTR) carried the directly binding site of miR-892a. Furthermore, siRNA-mediated silencing of PPP2R2A blocked the inhibitory effect of miR-892a-in on CRC cell growth. In sum, our data provided compelling evidence that overexpression of miR-892a may provide a selective growth promotion for CRC cells by direct suppression of PPP2R2A expression.     

MiR-1 targets PIK3CA and inhibits tumorigenic properties of A549 cells

MicroRNAs are small endogenous RNAs that play important roles in the pathogenesis of human diseases, including malignancy. MicroRNA-1 (miR-1) is downregulated in non-small cell lung cancer (NSCLC); however, the underlying mechanisms by which it suppresses tumorigenesis in NSCLC are largely unknown. We investigated whether phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) was a novel target of miR-1 in the NSCLC cell line A549, and the mechanism of miR-1 inhibition of the tumorigenic properties of A549 cells is discussed. The influence of miR-1 on A549 cells was studied by transfection with miR-1 mimics or inhibitor. MiR-1 overexpression led to downregulation of PIK3CA protein, but not mRNA by western blot and quantitative real-time PCR, respectively. The dual-luciferase reporter assay confirmed that miR-1 targeted PIK3CA directly. PIK3CA downregulation by miR-1 mimics led to a significant reduction of phosphorylated Akt and survivin protein, the downstream targets of the PI3 K/Akt pathway. Cell proliferation was studied using a cell counting kit. Migration and invasion were evaluated by Transwell and Matrigel assays, respectively. Cell cycle and apoptosis were detected by flow cytometry. The results were that miR-1 upregulation inhibited A549 cell proliferation, migration, and invasion. These findings indicate that miR-1 may play an important role in the pathogenesis of NSCLC by regulating PIK3CA through the PI3 K/Akt pathway. Increasing miR-1 expression may provide a novel approach for NSCLC treatment.     

对肿瘤内miR-21表达进行契伦科夫光学成像的新方法:基于HSV1-tk报告基因

目的构建一种基于I型单纯疱疹病毒胸苷激酶（HSV1-tk）的新型报告基因体系,用于对肿瘤内微小核糖核酸（miR-21）的表达进行契伦科夫光学成像。方法将细胞巨病毒（CMV）启动子基因、HSV1-tk基因以及可被miR-21互补结合的三联miR-21靶基因序列,串联构建成报告基因（CMV-HSV1-tk-3×miR-21t）,即将CMV-HSV1-tk-3×miR-21t序列连接到pcDNA3.1质粒载体中并转染A549细胞。向稳定表达上述报告基因的A549细胞（A549T细胞）加入9-[4-[18F]氟-3(羟甲基)丁基]鸟嘌呤（[18F] FHBG）孵育;或采用可互补结合miR-21的梯度剂量反义寡聚miR-21（Anti-miR-21）处理A549T细胞后,加入[18F]FHBG孵育。分别对上述细胞进行契伦科夫光学成像和放射性γ计数。另构建A549T裸鼠皮下移植瘤模型,分为2组分别瘤内注射Anti-miR- 21与对照混合RNA,然后分别经尾静脉注射[18F]FHBG后进行活体契伦科夫光学成像。结果A549T细胞摄取[18F]FHBG后,其光学信号强度、γ计数分别与细胞数量之间呈线性正相关（R2=0.9962、0.9807）;加入Anti-miR-21的剂量与光学信号强度、γ计数之间分别呈剂量依赖性正相关（P < 0.05）;A549T细胞皮下瘤模型成像结果显示,瘤内注射Anti-miR-21与对照RNA的移植瘤对比,肿瘤组织信号更高且视觉对比显著。结论基于microRNA调控的示踪剂摄取相关报告基因体系,本研究成功构建了一种用于对肿瘤内miR-21表达进行契伦科夫光学成像的新方法。      

MicroRNA-29a upregulates MMP2 in oral squamous cell carcinoma to promote cancer invasion and anti-apoptosis

Abnormal microRNA expression is a common and important feature of human malignancies. Matrix metalloproteinase 2 (MMP2), which has been reported in several cancers, plays important roles in cancer progression. However, the microRNA regulatory mechanism on MMP2 expression remains unclear. In this study, we first detected MMP2 and microRNA-29a (miR-29a) expression in oral squamous carcinoma (OSCC) specimens, which showed that MMP2 was higher in OSCC cancer tissues than adjacent tissues but that miR-29a was lower in OSCC cancer tissues than adjacent tissues. Then, we confirmed that miR-29a, which directly targeted 3′-UTR of MMP2 gene, negatively regulated MMP2 expression by miR-29a transfection and luciferase reporter assay. Exogenous overexpression of miR-29a inhibited OSCC cell invasion and anti-apoptosis significantly in vitro. Whereas, knockdown of miR-29a promoted OSCC cell invasion and induced drug-resistance in vitro. This study suggests that miR-29a plays an inhibiting role in the progression of OSCC, which may be a potentially therapeutic approach in the future.     

MiR-551b-3p靶向USP9X对人肺癌A549细胞凋亡和放射敏感性的影响

目的:探讨miR-551b-3p对肺癌A549细胞凋亡和放射敏感性的影响及分子机制。方法:将miRNC,miR-551b-3p,anti-miR-NC,anti-miR-551b-3p,si-NC,si-USP9X分别转染至A549中,记为miR-NC组、miR-551b-3p组、anti-miR-NC组、anti-miR-551b-3p组、si-NC组、si-USP9X组;将miR-551b-3p分别与pc DNA和pc DNA-USP9X共转染至A549中,记为miR-551b-3p+pc DNA组、miR-551b-3p+pc DNA-USP9X组。采用实时荧光定量PCR(RT-q PCR)检测miR-551b-3p和USP9X的表达水平;蛋白质印迹法检测X染色体连锁的泛素特定蛋白水解酶9(ubiquitin-specific peptidase 9 X-linked,USP9X)、 B细胞淋巴瘤/白血病-2 (B-cell lymphoma/leukemia-2, Bcl-2)、 Bcl-2相关X蛋白(Bcl-2 related X protein,Bax)的表达;荧光素酶报告实验检测miR-551b-3p和USP9X的靶向关系;流式细胞术检测细胞凋亡;细胞克隆集落形成实验检测放射敏感性。结果:肺癌组织和肺癌细胞A549中miR-551b-3p表达水平显著降低,USP9X mRNA和蛋白质表达水平显著升高(均P<0.001)。miR-551b-3p靶向调控USP9X,过表达miR-551b-3p和抑制USP9X表达,细胞凋亡率显著升高,Bcl-2表达水平显著降低,Bax表达水平显著升高,细胞的存活曲线明显下移(均P<0.001)。USP9X过表达逆转了miR-551b-3p过表达对肺癌A549细胞凋亡和放射敏感性的作用。结论:过表达miR-551b-3p促进肺癌A549细胞凋亡,增强肺癌细胞放射敏感性,其机制可能与USP9X有关。     

PVT1 induces NSCLC cell migration and invasion by regulating IL-6 via sponging miR-760

Non-small-cell lung carcinoma (NSCLC) accounts for approximately 80% of lung cancers with a high metastatic potential. Elucidating the mechanism of NSCLC metastasis will provide new promising targets for NSCLC therapy and benefit its prognosis. Plasmacytoma variant translocation 1 (PVT1) has been proven to be overexpressed in NSCLC. Although the oncogenic role of PVT1 in NSCLC has been reported, its mechanism remains unclear. Here, we verified that the knockdown of PVT1 inhibited NSCLC cell migration and invasion, and that its inhibitory role on A549 cells and H1299 cells was antagonized by interleukin-6 (IL-6) treatment. The results revealed that PVT1 regulates IL-6 by sponging miR-760 and identified the binding site of miR-760 in the 3′-UTR of IL-6. In conclusion, a new mechanism was revealed, wherein PVT1 regulates NSCLC cell migration and invasion via miR-760/IL-6, suggesting PVT1/miR-760/IL-6 as promising prognostic biomarkers and therapeutic targets for NSCLC metastasis.     

Decitabine,a DNA methyltransferases inhibitor,induces cell cycle arrest at G2/M phase through p53-independent pathway in human cancer cells

Decitabine (5-aza-2′-deoxycytidine), an inhibitor of DNA methyltransferases, has a wide range of anti-metabolic and anti-cancer activities. Decitabine also induces cell cycle arrest at G2/M phase and apoptosis in human cancer cells. However, the cellular and molecular mechanisms of this cell cycle arrest are poorly understood. In the present study, we investigated the roles of the tumor suppressor p53 and the cyclin-dependent kinase (Cdk) inhibitor p21 following decitabine-induced G2/M arrest in human cancer cells. DNA flow cytometric analyses indicated that decitabine induced a G2/M arrest in AGS gastric and A549 lung carcinoma cell lines, which have wild type p53. Western blot analyses using whole cell lysates from AGS cells demonstrated that decitabine treatment did not change the steady-state level of Cdks and Cdk inhibitor p27, but it partially inhibited expression of cyclin A, cyclin B1, and Cdc25C proteins. However, similar results were found using the A549 cell line, where decitabine induced a dramatic up-regulation of both p53 and p21 expression, and the increased levels of p21 were associated with increased binding of p21 with Cdks, cyclin A, and cyclin B1. Knockdown of p53 by small interfering RNA (siRNA) markedly abolished p53 induction by decitabine in AGS cells, yet p53 siRNA had no attenuating effect on p21 induction. In addition, depletion of p21 expression with siRNA, but not p53, significantly attenuated decitabine-induced G2/M arrest. We also observed that decitabine strongly induced G2/M arrest associated with p21 induction in both p53 allele-null (–/–) HCT116 and wild type p53 (+/+) HCT116 cell lines. Therefore, our data indicated that p21 plays a crucial role in decitabine-induced G2/M arrest and operates in a p53-independent manner.     

m6A甲基转移酶METTL3介导miR-127调控非小细胞肺癌细胞系自噬的机制研究

目的　分析N6 甲基腺苷（m6A）甲基转移酶 3（methyltransferase-like 3, METTL3）和miR-127 在非小细胞肺癌（non small cell lung cancer cells, NSCLC）细胞系中的表达及其相关性,并探究METTL3 介导miR-127 调控非小细胞肺癌自噬的作用机制。方法　采用qRT-PCR 法检测正常肺上皮细胞BEAS-2B 与非小细胞肺癌细胞HCC827,A549 和H460 中METTL3 和miR-127 的表达水平;通过Linked Domics 数据库筛选出肺癌中与miR-127 共表达的基因,并分析METTL3 和miR-127 之间的相关性;选择H460 细胞传代培养至对数生长期后,将浓度接近的细胞随机分为三组,分别转染METTL3-siR,NC-siR 及Control,验证转染后H460 细胞中METTL3 和miR-127 表达;通过吖啶橙染色,Lyso-Tracker Red 染色观察METTL3 对细胞自噬的影响;利用Western blot 检测PTEN,AKT,mTOR,ULK1,Beclin-1等自噬相关蛋白的表达。结果　非小细胞肺癌细胞HCC827,A549,H460 中METTL3 相对表达分别为1.35±0.17,1.54±0.11 和1.78±0.21,明显高于正常肺上皮细胞BEAS-2B 中表达水平（0.91±0.11）,差异有统计学意义(F=34.037,P=0.002)。非小细胞肺癌细胞HCC827,A549,H460 中miR-127 相对表达分别为1.56±0.21,1.85±0.19 和2.11±0.25,较正常肺上皮细胞BEAS-2B 中表达（1.02±0.20）亦显著升高,差异有统计学意义（F=28.152,P=0.005）。肺癌中METTL3 与miR-127 共同表达呈正相关性（r=0.452,P ＜ 0.001）。METTL3-siR 组细胞中METTL3 表达水平（0.61±0.15）较Control 组（1.71±0.28） 和NC-siR 组（1.65±0.19） 显著降低, 差异有统计学意义（F=78.357,P ＜ 0.001）。METTL3-siR 组细胞中miR-127 表达水平（0.48±0.15）较Control 组（2.02±0.33）和NC-siR 组（1.97±0.25）亦显著下降,差异有统计学意义（F=105.216,P ＜ 0.001）;吖啶橙和Lyso-Tracker Red 染色分别观察到METTL3-siR 组细胞酸性自噬小泡增多,自噬溶酶体数量也明显增加。与Control 组和NC-siR 组相比,METTL3-siR 组细胞中PTEN,ULK1,Beclin1 蛋白表达水平显著升高,差异均有统计学意义（F=62.420~175.615,均P<0.001）;p-AKT 和p-mTOR 表达水平显著下降,差异均有统计学意义（F=148.781,87.147,均P<0.001）。结论　METTL3 和miR-127 在非小细胞肺癌细胞系中均呈高表达,且它们之间呈正相关性,沉默METTL3 基因可以抑制miR-127 表达,促进非小细胞肺癌H460 细胞发生自噬,其调控机制可能与PTEN/AKT/mTOR 通路有关。     

Retracted Article: MiR-182-5p and miR-96-5p increased hepatocellular carcinoma cell mobility,proliferation and cisplatin resistance partially by targeting RND3

We investigated whether miR-182-5p or miR-96-5p could increase hepatocellular carcinoma (HCC) development by targeting Rho Family GTPase 3 (RND3) gene expression. The expression levels of miR-182-5p, miR-96-5p and mRNA/protein of RND3 in non-HCC liver tissue, HCC tissue and adjacent tissue specimens were evaluated by RT-qPCR and western blot. Patient-derived HCC cell culture was established, and miR-182-5p or miR-96-5p agomir or antagomir treatment was performed to mimic the overexpression or knockdown of the two miRNAs. HCC cell mobility in vitro was monitored by trans-well migration and invasion assay, while HCC cell growth in vitro was evaluated by cell viability, proliferation and apoptosis assay. HCC cell apoptosis was further investigated by caspase-3/-8/-9 activity assay. MiR-182-5p and miR-96-5p were significantly upregulated in HCC tissue specimens compared with non-HCC or adjacent tissue specimens, inversely correlating to RND3 mRNA expression level. Treatment with miR-182-5p or miR-96-5p agomir significantly reduced RND3 mRNA/protein expression level in HCC cells. MiR-182-5p- or miR-96-5p-targeting RND3 mRNA was verified by luciferase reporter assay and AGO2-RNA immunoprecipitation assay. MiR-182-5p or miR-96-5p agomir treatment significantly rescued HCC cell migration and invasion in vitro that were repressed by RND3 overexpression, during which ROCK1 and ROCK2 inhibition were involved. MiR-182-5p or miR-96-5p agomir treatment also increased HCC cell proliferation and cisplatin resistance in vitro, which could be antagonized by RND3 overexpression or ROCK inhibition. Thus, miR-182-5p and miR-96-5p increased HCC cell mobility, proliferation and cisplatin resistance in vitro partially by targeting RND3.

We investigated whether miR-182-5p or miR-96-5p could increase hepatocellular carcinoma (HCC) development by targeting Rho Family GTPase 3 (RND3) gene expression.     

miR-142-3p靶向调控mll-af4融合基因的实验研究

本研究探讨mll-af4融合基因的表观遗传学调控机制,以筛选靶向调控mll-af4融合基因的microRNA。利用Targetscan在线分析软件预测特异性靶向mllaf4基因3′端非翻译区域（3′-untranslated region,3′UTR）的microRNA,采用PCR方法从1例健康供者DNA中扩增mll-af4基因3′UTR序列,插入经EcoRI和PstI双酶切的荧光素酶报告载体pGL3-M,采用脂质体SuperFect包裹荧光素酶重组质粒及microRNA表达质粒转染293T细胞,应用双荧光素酶检测试剂盒测定荧光素酶活性。miR-142-3p mimics以脂质体Hiperfect转染至RS4;11细胞,选用实时定量PCR及Western blot检测mll-af4 mRNA及蛋白的表达。结果表明,成功构建了含有1935bp、2104bp和1371bp的mll-af4基因3′UTR序列的荧光素酶报告重组质粒pGL3-AF4-3′UTR,并通过酶切及基因测序方法鉴定得到证实。荧光素酶报告实验提示,miR-142组荧光素酶活性明显低于对照组。RS4;11细胞中过表达miR-142-3p可以明显下调MLL-AF4融合蛋白及mRNA表达。结论：miR-142-3p通过靶向结合mll-af4基因3′UTR的结合位点特异调控mll-af4基因表达。