Retracted: LINC00707 contributes to hepatocellular carcinoma progression via sponging miR-206 to increase CDK14

Recently, increasing numbers of long noncoding RNAs (lncRNAs) have been found to be aberrantly expressed in various cancers. However, the roles of lncRNAs in hepatocellular carcinoma (HCC) progression is largely unknown. In our current study, we identified that long intergenic nonprotein-coding RNA 707 (LINC00707) was remarkably elevated in HCC cells, indicating that LINC00707 was involved in HCC development. Subsequently, LINC00707 was significantly decreased in HepG2 and Huh7 cells. The in vitro functional assays demonstrated that knockdown of LINC00707 significantly reduced HCC cell proliferation, induced cell apoptosis, and blocked the cell cycle progression. In addition, HCC cell migration and invasion was also greatly inhibited by downregulation of LINC00707. Increasing evidence has indicated that lncRNAs can act as molecular sponges of microRNAs. Currently, we observed that microRNA-206 (miR-206) was dramatically inhibited in HCC cells and LINC00707 can modulate HCC development through sponging miR-206. The binding correlation between LINC00707 and miR-206 was confirmed by dual-luciferase reporter assay, RNA pull down and RNA immunoprecipitation assay in our study. Moreover, cyclin-dependent kinase 14 (CDK14) was predicted as a target of miR-206 and we found that miR-206 suppressed CDK14 levels in HCC cells. Finally, in vivo assays were used and it was proved that silence of LINC00707 can restrain HCC development through modulating miR-206 to upregulate CDK14. In conclusion, it was implied that LINC00707 can lead to HCC progression through sponging miR-206 and modulating CDK14.     

Oncogene PLCE1 may be a diagnostic biomarker and prognostic biomarker by influencing cell cycle,proliferation, migration,and invasion ability in hepatocellular carcinoma cell lines

Hepatocellular carcinoma (HCC) is a lethal malignancy worldwide. HCC has traits of late diagnosis and high recurrence. This study explored potential diagnosis and prognosis significance of phospholipase C epsilon 1 (PLCE1) in HCC. The messenger RNA (mRNA) levels and diagnostic value of PLCE1 were determined by real-time polymerase chain reaction and online databases GEPIA, oncomine, and GSE14520 data set. Survival analysis used the Kaplan–Meier Plotter website. Cell cycle, proliferation, migration, and invasion assays were performed with downregulated PLCE1 expression in HCC-M and HepG2 cell lines. PLCE1 was differentially expressed and highly expressed in tumors and had low expression in nontumor tissues (all p < .05). The diagnostic value of PLCE1 was validated with the datasets (all p < .01, all areas under curves > 0.7). PLCE1 mRNA expression was associated with the overall and relapse-free survival (both p < .05). Functional experiments indicated that downregulation of PLCE1 expression led to increased G1 stage in cell cycle and decreased cell proliferation, migration, and invasion compared with a negative control group (all p ≤ .05). The oncogene PLCE1 was differentially expressed in HCC and non-HCC tissues. It is a candidate for diagnosis and serves as prognosis biomarker. PLCE1 influenced survival by affecting the cell cycle, proliferation, migration, and invasion ability.     

Long noncoding RNA HAND2-AS1 inhibits cancer cell proliferation,migration, and invasion in esophagus squamous cell carcinoma by regulating microRNA-21

Long noncoding RNA (lncRNA) HAND2-AS1 is a well-characterized tumor suppressor in several types of malignancies, while its role in esophagus squamous cell carcinoma (ESCC) is unknown. In this study, we found that lncRNA HAND2-AS1 was downregulated, while microRNA-21 ( miRNA-21) was upregulated in tumor tissues than in adjacent healthy tissues of ESCC patients. Expression levels of lncRNA HAND2-AS1 and miRNA-21 were significantly and inversely correlated in tumor tissues but not in healthy tissues. Plasma levels of lncRNA HAND2-AS1 were lower in ESCC patients than in healthy controls, and downregulation of plasma lncRNA HAND2-AS1 distinguished early stage ESCC patients from healthy controls. lncRNA HAND2-AS1 overexpression resulted in downregulation of miRNA-21 in cells of ESCC cell lines and inhibited cell proliferation, migration, and invasion. miRNA-21 overexpression failed to affect lncRNA HAND2-AS1 expression but significantly attenuated the inhibitory effect of lncRNA HAND2-AS1 overexpression on cancer cell proliferation, migration, and invasion. Therefore, lncRNA HAND2-AS1 may inhibit cancer cell proliferation, migration, and invasion in ESCC by regulating miRNA-21.     

Aldolase A as a prognostic factor and mediator of progression via inducing epithelial–mesenchymal transition in gastric cancer

Glycolysis is regarded as the hallmark of cancer development and progression, which involves a multistep enzymatic reaction. This study aimed to explore the clinicopathological significance and potential role of glycolytic enzyme aldolase A (ALDOA) in the carcinogenesis and progression of gastric cancer (GC). ALDOA was screened from three paired liver metastasis tissues and primary GC tissues and further explored with clinical samples and in vitro studies. The ALDOA protein level significantly correlated with a larger tumor diameter (P = .004), advanced T stage (P < .001), N stage (P < .001) and lymphovascular invasion (P = .001). Moreover, the expression of ALDOA was an independent prognostic factor for the 5‐year overall survival and disease‐free survival of patients with GC in both univariate and multivariate survival analyses (P < .05). Silencing the expression of ALDOA in GC cell lines significantly impaired cell growth, proliferation and invasion ability (P < .05). Knockdown of the expression of ALDOA reversed the epithelial–mesenchymal transition process. Mechanically, ALDOA could affect the hypoxia‐inducible factor (HIF)‐1α activity as demonstrated by the HIF‐1α response element–luciferase activity in GC cells. Collectively, this study revealed that ALDOA was a potential biomarker of GC prognosis and was important in the carcinogenesis and progression of human GC.     

The discovery of a novel eight-mRNA-lncRNA signature predicting survival of hepatocellular carcinoma patients

Increasing evidence indicates that the expressions of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) undergo a frequent and aberrant change in carcinogenesis and cancer development. But some research was carried out on mRNA-lncRNA signatures for prediction of hepatocellular carcinoma (HCC) prognosis. We aimed to establish an mRNA-lncRNA signature to improve the ability to predict HCC patients’ survival. The subjects from the cancer genome atlas (TCGA) data set were randomly divided into two parts: training data set (n = 246) and testing data set (n = 124). Using computational methods, we selected eight gene signatures (five mRNAs and three lncRNAs) to generate the risk score model, which were significantly correlated with overall survival of patients with HCC in both training and testing data set. The signature had the ability to classify the patients in training data set into a high-risk group and low-risk group with significantly different overall survival (hazard ratio = 4.157, 95% confidence interval = 2.648-6.526, P < 0.001). The prognostic value was further validated in testing data set and the entire data set. Further analysis revealed that this signature was independent of tumor stage. In addition, Gene Set Enrichment Analysis suggested that high risk score group was associated with cell proliferation and division related pathways. Finally, we developed a well-performed nomogram integrating the prognostic signature and other clinical information to predict 3- and 5-year overall survival. In conclusion, the prognostic mRNAs and lncRNAs identified in our study indicate their potential role in HCC biogenesis. The risk score model based on the mRNA-lncRNA may be an efficient classification tool to evaluate the prognosis of patients’ with HCC.     

Expression of serum miR-221 in human hepatocellular carcinoma and its prognostic significance

Objective

To investigate whether the serum miR-221 expression correlates with clinicopathologic features and the prognosis of hepatocellular carcinoma (HCC) patients.

Methods

Four miRNAs (miR-221, miR-222, miR-21 and miR-224) related to HCC were selected in the present study. Serum miRNA expression was investigated in 46 HCC patients and 20 healthy normal controls by using real-time PCR technique, and then correlations between miR-221 expression and the clinicopathological features and prognosis of HCC patients were evaluated.

Results

The four miRNAs were found to be differentially overexpressed in HCC serum samples, and high level of miR-221 expression was correlated with tumor size (P < 0.001), cirrhosis (P = 0.003) and tumor stage (P = 0.016). In addition, Kaplan–Meier survival analysis showed that the overall survival rate of the high miR-221 expression group (27.6%) was significantly lower than that of the low miR-221 expression group (62.3%, P < 0.05).

Conclusions

Serum miR-221, upregulated in HCC, can provide predictive significance for prognosis of HCC patients.     

胃癌组织中微小RNA-375基因甲基化及其表达与临床病理特征的关系研究

目的:研究胃癌组织中微小RNA-375(miRNA-375)基因甲基化及其表达与临床病理特征的关系。方法:选择从2015年9月到2017年6月在我院接受手术治疗的胃癌患者60例纳入研究对象,将术中切除的癌组织标本60例作为观察组;另选同期在我院治疗的浅表性胃炎患者60例,将经内镜采集到的正常胃黏膜标本60例作为对照组。检测并对比两组患者miRNA-375基因甲基化及表达水平,分析胃癌患者的miRNA-375基因表达水平及甲基化与其病理特征的关系,并采用Spearman法分析miRNA-375基因表达水平、基因甲基化与其分化程度的相关性。结果:观察组miRNA-375基因的表达水平为(0.034±0.021),明显低于对照组的(0.187±0.104),差异有统计学意义(P0.05)。miRNA-375基因甲基化的阳性率为61.67%(37/60),明显高于对照组的20.00%(12/60),差异有统计学意义(P0.05)。低分化胃癌患者的miRNA-375基因表达水平明显低于中、高分化者,差异有统计学意义(P0.05)。低分化胃癌患者的miRNA-375甲基化阳性率明显高于中高分化者,差异有统计学意义(P0.05)。根据Spearman相关性分析发现,胃癌患者的miRNA-375基因表达水平与其分化程度呈正相关(P0.05),基因甲基化与其分化程度呈负相关(P0.05)。结论:胃癌组织中的miRNA-375基因的表达水平明显下降,而基因甲基化的比例明显上升,且二者均与肿瘤的分化程度有关,可考虑在临床上将其作为监测靶点,从而更好地评价患者的病情及预后。     

急性心力衰竭患者血清miRNA-21、MYO、CK-MB与心功能和预后的关系

摘要 目的：探讨急性心力衰竭患者血清miRNA-21、肌红蛋白（MYO）、心肌肌酸激酶同工酶（CK-MB）与心功能和预后的关系。方法：选择2018年2月至2019年2月于我院接诊的95例急性心力衰竭患者为研究对象（病例组）,另选择同期在我院进行检查的80例健康人为对照组,分析两组患者血清miRNA-21、MYO、CK-MB、左心室射血分数（LVEF）、左心房内径（LAD）、左心室舒张末期内径（LVEd）水平变化情况及和预后相关性。结果：病例组患者血清miRNA-21、MYO、CK-MB水平显著高于对照组,差异显著（P＜0.05）;病例组患者LAD、LVEd水平显著高于对照组,LVEF水平显著低于对照组,差异显著（P＜0.05）;II级患者血清miRNA-21、MYO、CK-MB水平显著低于III级、IV级患者,III级患者血清miRNA-21、MYO、CK-MB水平显著低于IV级患者,差异显著（P＜0.05）;预后良好组患者血清miRNA-21、MYO、CK-MB水平显著低于预后不良组,差异显著（P＜0.05）;Spearman相关分析显示,血清miRNA-21、MYO及CK-MB和LVEF之间均呈负相关,血清miRNA-21、MYO及CK-MB和LAD、LVEd之间均呈正相关,（P＜0.05）;Logistic 回归分析结果显示,血清miRNA-21、MYO及CK-MB及LVEF、LAD、LVEd为急性心力衰竭患者预后的影响因素（P＜0.05）。结论：急性心力衰竭患者血清miRNA-21、MYO、CK-MB的表达升高,其与心功能和预后密切相关。     

Prognostic significance of combining high mobility group Box-1 and OV-6 expression in hepatocellular carcinoma

The inflammatory environment and existence of cancer stem cells are critical for progression and intrahepatic recurrence of hepatocellular carcinoma (HCC) after curative resections. Here, we investigated the prognostic significance of combining high mobility group box 1 (HMGB1) expression and hepatic progenitor marker OV6 in hepatocellular carcinoma. Expression of HMGB1 and OV6 was evaluated using immunohistochemistry profiling in tissue microarrays containing samples from 208 HCC patients. Invasive clinical or pathological factors were found in patients with high expression of HMGB1 or OV6. Higher HMGB1 was associated with poorer clinical outcomes, and independently related to elevated 5-year recurrence incidence (85.5% vs. 62.4%, P<0.001). We also found that more OV6 positive staining was correlated with poor prognosis of HCC patients (P<0.001). Notably, expression of HMGB1 was positively correlated with OV6 in density (R²=0.032, P<0.001) and reversely related to HCC outcomes. Abnormal expression of HMGB1 in combination with positive staining of OV6 displayed poorer prognostic performance than single biomarker alone (area under curve (AUC) survival=0.696). Therefore, HMGB1 and OV6 positive staining are promising prognostic parameters for HCC, and we propose that HMGB1 and OV6 may cooperate with each other and predict poor prognosis of HCC.     

Overexpression of osteopontin in hepatocellular carcinoma and its relationships with metastasis,invasion of tumor cells

Osteopontin (OPN) plays an important role in metastasis and relapse of human cancer. However, the whole story of OPN relating to cancer has been far from clear untill now. To investigate the expression of OPN in hepatocellular carcinoma (HCC) and its relationships with recurrence and metastasis of HCC, normal and malignant liver tissues from patients with HCC were analyzed using immunohistochemical staining. OPN expression was inhibited by small interfering RNA (siRNA) in HCC cells lines, and then colony formation and matrigel invasion were examined. The results showed that expression of OPN was associated with metastasis of HCC with a positive rate of OPN in the tissue of HCC (70.00%), which was highly more obvious than those in paracarcinoma tissue and normal liver tissue (P < 0.01). In HCC cell lines, OPN depletion could reduce formed colony and metastasizing numbers in vitro. In conclusion, Expression of OPN in the tissue of HCC is related to metastasis or metastases. Specific siRNA could decrease expressions of OPN at both mRNA and protein levels, and abates the invasiveness of hepatocellular carcinoma cells, suggesting that OPN might be a promising agent for treatment of metastasis and recurrence of HCC.     

Rce1 suppresses invasion and metastasis of hepatocellular carcinoma via epithelial-mesenchymal transition induced by the TGF-β1/H-Ras signaling pathway

Ras converting enzyme 1 (Rce1) plays an important role in invasion and metastasis of malignancy. However, the mechanism has not yet been fully explored in hepatocellular carcinoma (HCC). Primarily, we investigated the expression of Rce1 and H-Ras influence on patient prognosis through the clinical data. Further, we analyzed the regulatory effects of Rce1/H-Ras signal pathway on the epithelial–mesenchymal transition (EMT) in vitro and in vivo. Finally, we screened out the protein which bonds with Rce1 by CO-IP experiment to discuss the mechanism of Rce1 in EMT of HCC. This research revealed a significantly decreased expression of Rce1 in HCC compared with noncancerous tissues (p < .05). In contrast, H-Ras expression was increased in the tumor. The expression of them was a close association with the differentiation and tumor-node-metastasis (TNM) stage of the tumor (p < .001; p = .035, respectively) and Rce1 was an independent prognostic indicator (95%Cl: 0.193–0.821; p = .013). Through targeted regulation of Rce1 by cDNA or small interfering RNA, results show that the lower expression of Rce1 facilitated EMT and promoted the invasion and metastasis of HCC (p < .05). Furthermore, the CO-IP experiment unfolded that Rce1 could bond with farnesyltransferase-β (FNTB) which mediated the expression of H-Ras. Conclusions: Rce1 inhibits EMT via target regulation H-Ras and suppress the early invasion and metastasis of HCC. It may be a potential therapeutic target and prognostic indicator for HCC.     

Clinical significance of blood-based miRNAs as diagnostic and prognostic nucleic acid markers in breast cancer: Comparative to conventional tumor markers

microRNAs (miRNAs) are implicated in carcinogenesis and their expression in biological fluids offer great potential as nucleic acid markers for cancer detection and progression. Authors investigated the expression level of miRNAs (miRNA-21, miRNA-126, and miRNA-155) to evaluate their role as diagnostic and prognostic markers for breast cancer compared with other commonly used protein-based markers (CEA and CA15-3). Serum samples from patients with breast cancer (n = 96), patients with benign breast lesion (n = 47), and healthy individuals (n = 39) were enrolled for detection of miRNA expression levels and protein-based tumor markers using fluorescent real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Correlation among investigated markers with clinicopathological factors and clinical outcomes were determined. Expression of miRNA-21 and miRNA-155 revealed significant increases in patients with breast cancer compared with both benign and control groups, the same result was reported for tumor markers; on the other hand, miRNA-126 was significantly decreased in breast cancer group as compared with the other two groups. miRNA frequencies were significantly related to clinical staging and histological grading as compared with tumor markers. Patients with breast cancer with increased miRNA-21 and miRNA-155 and decreased miRNA-126 expressions had significantly worse disease-free survival, while only miRNA-21 and miRNA-126 showed poor OS (P< 0.005). In conclusion, investigated miRNAs were superior over tumor markers for the early stage of breast cancer especially those with high-risk factor and their assessment in blood facilitates their role as a potential prognostic molecular marker.     

Prognostic value of polarized macrophages in patients with hepatocellular carcinoma after curative resection

As the most predominant tumour‐infiltrating immune cells, tumour‐associated macrophages (TAMs) are significant for fostering tumour growth, progression and metastasis. CD68‐positive TAMs display dissimilarly polarized programmes comprising CD11c‐positive pro‐inflammatory macrophages (M1) and CD206‐positive immunosuppressive macrophages (M2). The aim of this study is to determine the prognostic significance of diametrically polarized TAMs in hepatocellular carcinoma (HCC) and their application to risk stratification of patients according to their specific prognostic values. This study included 80 consecutive patients with HCC, and we evaluated diametrically polarized functional status of macrophages by immunohistochemical staining of CD68, CD11c and CD206. Prognostic values and clinicopathologic features were assessed in these patients. High versus low CD11c‐positive TAM density (P = 0.005) and low versus high CD206‐positive TAM density (P = 0.002) were associated with better overall survival, whereas CD68‐positive TAM density had no prognostic significance (low versus high, P = 0.065). Furthermore, the presence of these positive staining macrophages did not show any prognostic significance for recurrence‐free survival (all P > 0.05). Multivariate Cox regression analysis identified CD11c‐positive and CD206‐positive TAMs as an independent prognostic factor (P < 0.001, P = 0.031, respectively). Intratumoural infiltration of diametrically polarized TAMs, a novel identified independent prognostic factor for survival in patients with HCC, could be combined with the TNM stage and the Barcelona Clinic Liver Cancer stage to improve a risk stratification system.     

A miRNA-HERC4 pathway promotes breast tumorigenesis by inactivating tumor suppressor LATS1

The E3 ligase HERC4 is overexpressed in human breast cancer and its expression levels correlated with the prognosis of breast cancer patients. However, the roles of HERC4 in mammary tumorigenesis remain unclear. Here we demonstrate that the knockdown of HERC4 in human breast cancer cells dramatically suppressed their proliferation, survival, migration, and tumor growth in vivo, while the overexpression of HERC4 promoted their aggressive tumorigenic activities. HERC4 is a new E3 ligase for the tumor suppressor LATS1 and destabilizes LATS1 by promoting the ubiquitination of LATS1. miRNA-136-5p and miRNA-1285-5p, expression of which is decreased in human breast cancers and is inversely correlated with the prognosis of breast cancer patients, are directly involved in suppressing the expression of HERC4. In summary, we discover a miRNA-HERC4-LATS1 pathway that plays important roles in the pathogenesis of breast cancer and represents new therapeutic targets for human breast cancer.     

miRNA-200b improves hepatic fibrosis induced by CCL4 by regulating toll-like receptor 4 in mice

To study the effect of miRNA-200b on hepatic fibrosis induced by CCl₄ in mice. The C59BL/6 mice were randomly divided into three groups (normal control [NC], CCLR model [Model], and CCl ₄ + miRNA-200b [miRNA]). The hepatic fibrosis was induced by CCl ₄ injected subcutaneously twice per week in Model and miRNA groups. After 6 weeks building model, the mice of miRNA group were injected the miRNA-200b from caudal vein twice per week. The mice of Model and miRNA groups were continuously fed for 3 weeks. The IL-1β, IL-6, and TNF-α concentrations of serum were measured by enzyme-linked immunosorbent assay. The hepatic tissues of difference groups were observed by hematoxylin and eosin (H&E) staining, sirius red staining, Masson staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay and measured toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) proteins expressions by western blot assay. The correlation between miRNA-200b and TLR4 were analyzed by dual luciferase target assay. Compared with NC group, the interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) concentrations of Model group were significantly upregulated (P < 0.05, respectively). With miRNA-200b overexpression, the IL-1β, IL-6, and TNF-α concentrations were significantly suppressed (P < 0.05, respectively). The pathologies were improved by H&E staining, sirius red staining, and Masson staining; meanwhile, the hepatic cell apoptosis rate was significantly suppressed (P < 0.05). The TLR4 and NF-κB protein expressions of miRNA group were significantly suppressed compared with the Model group (P < 0.05, respectively). By dual luciferase target assay, the TLR4 was a target gene of miRNA-200b. The miRNA-200b upregulation improved hepatic fibrosis induced by CCl ₄ via regulation of TLR4 in vivo.     

Expression of insulin-like growth factor-II,matrix metalloproteinases,and their tissue inhibitors as predictive markers in the peripheral blood of HCC patients

Background/aim: Elevated relative expression of insulin-like growth factor-II (IGF-II) was observed in hepatocellular carcinoma (HCC) liver tissues with a role in neovascularization and associated with poor prognosis. IGF-II is influenced by the proteolytic cleavage of IGF-binding protein 3 and by matrix metalloproteinases (MMP), which are further regulated by their tissue inhibitors tissue inhibitor of metalloprotienase-1 (TIMP-1). Our aim is to study new molecular markers for HCC.

Patients/methods: RNA was extracted from the peripheral blood for evaluating the relative expression of IGF-II, MMP-9, and TIMP-1 in correlation with clinical staging of 39 HCC patients and 15 healthy controls using TaqMan real-time PCR.

Results: The relative expression of IGF-II and MMP-9 mRNA were significantly elevated in HCC patients compared with healthy controls; P-value <0.0001 for both. There was a significant correlation between MMP-9 and different HCC stages. On the other hand, TIMP-1 was significantly down-regulated in HCC patients; P?=?0.0003 with the elevation of the IGF-II/TIMP-1 ratio. Significant correlation between TIMP-1 and HCC Stage III and Stage IV was found; P-value?=?0.0138.

Conclusion: These results highlight the importance of profiling the expression of IGF-II, MMP-9, and TIMP-1 in the peripheral blood as prognostic molecular biomarkers in HCC.     

microRNA-21 表达水平对新疆地区食管癌患者早期诊断及预后评估的临床价值

目的:探讨microRNA-21表达水平对新疆地区食管癌早期诊断及预后评估的临床价值。方法:收集我院收治的100例食管癌患者,其中哈萨克族患者50例,汉族患者50例,采用RT-PCR以及RT-qPCR的方法对患者血清microRNA-21表达水平进行检测并比较。结果:食管癌患者癌组织中microRNA-21表达水平均高于癌旁组织,差异具有统计学意义(P0.05);有淋巴结转移、ⅡB+Ⅲ期、低分化癌组织中的miRNA-21相对表达量高于无淋巴结转移、I+ⅡA期以及高分化癌组织患者,差异具有统计学意义(P0.05);miRNA-21在不同民族、性别、年龄分组中表达无明显差异(P0.05)。结论:microRNA-21表达水平对新疆地区食管癌患者癌组织中表达水平较高,在有淋巴转移、ⅡB+Ⅲ期以及低分化癌组织中表达水平较高,民族、性别以及年龄对microRNA-21水平的影响较小,因此microRNA-21检测对食管癌的早期诊断及预后判断具有指导意义。     

A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs

The RNA interference (RNAi) technique has been widely used in gene function studies. It is typical to screen for effective siRNAs by knocking down targeted genes since a single gene can be suppressed by several siRNAs to varying degrees. The miRNA-based short hairpin RNA (shRNA) is a natural inducer of RNAi and has been used in siRNA expression strategies. We investigated the potential application of multiple putative microRNA-based shRNAs for gene silencing and studied the inhibition efficiency of exogenous GFP and firefly luciferase (luc) by triple human mir155-based shRNA expression vectors. A total of three candidate siRNA sequences targeted against GFP or luc were selected based on an online prediction program. Single and triple miRNA-155-based shRNAs targeted against GFP or luc were transfected into HEK293 cells mediated by the pcDNA₃ vector with an RNA polymerase II-type CMV (cytomegalovirus) promoter. Comparisons with negative control shRNAs revealed that GFP levels were markedly reduced by the triple miRNA-155-based GFP shRNA by fluorescent microscopy. Consistent results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based GFP shRNA significantly suppressed GFP expression (P < 0.01), without significant differences from the most effective single miRNA-155-based GFP shRNA (P > 0.05). Results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based luc shRNA significantly suppressed luc expression as the most effective single miRNA-155-based luc shRNA (P < 0.05). These studies demonstrated the gene silencing efficiency mediated by the triple putative miRNA-155-based shRNAs. This suggested that multiple miRNA-based shRNAs are quick and valuable strategies for gene silencing.