Rhamnetin and Cirsiliol Induce Radiosensitization and Inhibition of Epithelial-Mesenchymal Transition (EMT) by miR-34a-mediated Suppression of Notch-1 Expression in Non-small Cell Lung Cancer Cell Lines

Radioresistance is a major cause of decreasing the efficiency of radiotherapy for non-small cell lung cancer (NSCLC). To understand the radioresistance mechanisms in NSCLC, we focused on the radiation-induced Notch-1 signaling pathway involved in critical cell fate decisions by modulating cell proliferation. In this study, we investigated the use of Notch-1-regulating flavonoid compounds as novel therapeutic drugs to regulate radiosensitivity in NSCLC cells, NCI-H1299 and NCI-H460, with different levels of radioresistance. Rhamnetin and cirsiliol were selected as candidate Notch-1-regulating radiosensitizers based on the results of assay screening for activity and pharmacological properties. Treatment with rhamnetin or cirsiliol reduced the proliferation of NSCLC cells through the suppression of radiation-induced Notch-1 expression. Indeed, rhamnetin and cirsiliol increased the expression of tumor-suppressive microRNA, miR-34a, in a p53-dependent manner, leading to inhibition of Notch-1 expression. Consequently, reduced Notch-1 expression promoted apoptosis through significant down-regulation of the nuclear factor-κB pathway, resulting in a radiosensitizing effect on NSCLC cells. Irradiation-induced epithelial-mesenchymal transition was also notably attenuated in the presence of rhamnetin and cirsiliol. Moreover, an in vivo xenograft mouse model confirmed the radiosensitizing and epithelial-mesenchymal transition inhibition effects of rhamnetin and cirsiliol we observed in vitro. In these mice, tumor volume was significantly reduced by combinational treatment with irradiation and rhamnetin or cirsiliol compared with irradiation alone. Taken together, our findings provided evidence that rhamnetin and cirsiliol can act as promising radiosensitizers that enhance the radiotherapeutic efficacy by inhibiting radiation-induced Notch-1 signaling associated with radioresistance possibly via miR-34a-mediated pathways.     

Upregulated miR-182 increases drug resistance in cisplatin-treated HCC cell by regulating TP53INP1

Chemotherapy plays a crucial role in hepatocellular carcinoma (HCC) treatment especially for patients with advanced HCC. Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of HCC. However, acquisition of cisplatin resistance is common in patients with HCC, and the underlying mechanism of such resistance is not fully understood. In the study, we focused on identifying the role of miRNAs in chemotherapy resistance after cisplatin-based combination chemotherapy. We assayed the expression level of miR-182 after cisplatin-based chemotherapy in patients with advanced HCC, and defined the biological functions by real-time PCR analysis and CCK-8 assay. We found that miR-182 levels were significantly increased in HCC patients treated with cisplatin-based chemotherapy. miR-182 levels were also higher in cisplatin-resistant HepG2 (HepG2-R) cells than in HepG2 cells. Upregulated miR-182 significantly increased the cell viability, whereas miR-182 knockdown reduced the cell viability during cisplatin treatment. miR-182 inhibition also partially overcame cisplatin resistance in HepG2-R cell. Furthermore, we found that upregulated miR-182 inhibited the expression of tumor suppressor gene TP53INP1 (tumor protein 53-induced nuclear protein1) in vitro. In vivo, miR-182 and TP53INP1 expression was negatively correlated. We finally demonstrated that miR-182 increased cisplatin resistance of HCC cell, partly by targeting TP53INP1. These data suggest that miR-182/TP53INP1 signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of HCC.     

Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance

Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the MDR1 gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR.     

Study on the Mechanism of BMSCs in Regulating NF-κB Signal Pathway by Targeting miR-449a to Improve the Inflammatory Response to Peripheral Nerve Injury

Objective:To evaluate the mechanism of Bone Marrow Mesenchymal Stem Cells (BMSCs) in regulating NF-κB signal pathway by targeting miR-449a.Methods:Stem cells were transfected by over-expressing and inhibiting miR-449a to detect the levels and viability of miR-449a in stem cells after transfection. Stem cells and neurons were co-cultured in vitro to evaluate the in vitro mechanism of stem cells over-expressing miR-449a on neurons.Results:After the addition of neurons, the neuronal activity of miR-449a over-expression group increased significantly, the expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, and the inflammatory cytokines (TNF-α and IL-1β) decreased significantly (P<0.05). In vivo experiments in rats also showed that rats were unresponsive, did not chirp or elude after being stimulated. After stem cell therapy, the weight and response of rats gradually returned to normal levels. miR-449a expression significantly increased in the stem cell + miR-449a over-expression group, expression of NF-κB signal pathway proteins (IκBα, p50, and p65) decreased, inflammatory cytokines (TNF-α and IL-1β) significantly decreased, and cell activity significantly increased (P<0.05).Conclusions:BMSCs can modulate NF-κB signaling pathway by targeting miR-449a, so as to reduce the inflammatory response to peripheral nerve injury and repair nerve injury.     

Notch-1 decreased expression contributes to leptin receptor downregulation in nasal epithelium from allergic turbinates

BackgroundAllergic rhinitis is characterized by a remodeling of nasal epithelium. Since the Notch and TGF-β signaling pathways are known to be involved in cell differentiation and remodeling processes and leptin adipokine has already been identified as a marker for homeostasis in human bronchial and nasal epithelial cells of asthmatics, roles played by these pathways have been investigated for chronic allergic rhinitis.MethodsThe leptin/leptin receptor expression has been investigated in a study with 40 biopsies from allergic (AR, n = 18) and non-allergic (C, n = 22) inferior turbinates, using immunohistochemistry, immunofluorescence staining and RT-PCR. In addition, extracts from in vitro samples prepared from primary cells of inferior turbinates as well as in vitro cultured human nasal epithelial RPMI 2650 cells (ATCC-CCL-30) were also tested for leptin expression and activation of the Notch-1 pathway.ResultsWith regards to AR, in vivo expression levels of both leptin and its receptor significantly decreased in comparison to C. Furthermore, leptin receptor mRNA was significantly reduced in AR as compared to C. Immunofluorescence showed an apparent co-expression of leptin receptor with Notch-1, which was not seen with TGF-β. In vitro, in primary turbinate epithelial cells, the expression of leptin receptor and Notch-1 significantly decreased in AR as compared to C. Moreover, in RPMI 2650 cells, leptin receptor expression was shown to be induced by Notch-1 ligand signaling.ConclusionThus, both the leptin and Notch-1 pathways appear to represent markers for epithelial homeostasis in allergic rhinitis.     

Retracted: In vivo and in vitro effects of microRNA-221 on hepatocellular carcinoma development and progression through the JAK–STAT3 signaling pathway by targeting SOCS3

Hepatocellular carcinoma (HCC), as the third leading cancer-caused deaths, prevails with high mortality, and affects more than half a million individuals per year worldwide. A former study revealed that microRNA-221 (miR-221) was involved in cell proliferation of liver cancer and HCC development. The current study aims to evaluate whether miR-221 targeting SOCS3 affects HCC through JAK–STAT3 signaling pathway. A series of miR-221 mimic, miR-221 inhibitor, siRNA against SOCS3, and SOCS3 plasmids were introduced to SMMC7721 cells with the highest miR-221 expression assessed. The expression of JAK–STAT3 signaling pathway–related genes and proteins was determined by Western blot analysis. Cell apoptosis, viability, migration, and invasion were evaluated by means of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, and transwell assays, respectively. HCC xenograft in nude mice was performed to measure HCC tumor growth. miR-221 was found to be highly expressed but SOCS3 was poorly expressed in HCC tissues. miR-221 expression was correlated with lymph node metastasis (LNM) and tumor node metastasis (TNM) of HCC, and SOCS3 expression was correlated with LNM, differentiation and TNM of HCC. SOCS3 is a target gene of miR-221. MiR-221 mimic or si-SOCS3 exposure was found to induce cell viability, migration, and invasion, and reduce apoptosis. MiR-221 inhibitor was observed to have inhibitory effects on HCC cell proliferation, migration, and invasion. Moreover, the expression of JAK–STAT3 signaling pathway was suppressed by miR-221 inhibitor. Downregulated miR-221 expression could promote its target gene SOCS3 to inhibit the proliferation, invasion and migration of HCC cells by repressing JAK–STAT3 signaling pathway.     

mTOR/miR-145-regulated exosomal GOLM1 promotes hepatocellular carcinoma through augmented GSK-3β/MMPs

Golgi membrane protein 1 (GOLM1/GP73) is a serum marker of hepatocellular carcinoma (HCC). We have previously shown that mTOR promoted tumorigenesis of HCC through stimulating GOLM1 expression. In this study, we demonstrated that the mammalian target of rapamycin (mTOR) was a negative regulator of microRNA-145 (miR-145) expression. miR-145 inhibited GOLM1 expression by targeting a coding sequence of GOLM1 gene. GOLM1 and miR-145 were inversely correlated in human HCC tissues. GOLM1-enriched exosomes activated the glycogen synthase kinase-3β/matrix metalloproteinases (GSK-3β/MMPs) signaling axis of recipient cells and accelerated cell proliferation and migration. In contrast, miR-145 suppressed tumorigenesis and metastasis. We suggest that mTOR/miR-145/GOLM1 signaling pathway should be targeted for HCC treatment.     

MicroRNA-224 upregulation and AKT activation synergistically predict poor prognosis in patients with hepatocellular carcinoma

Background and aimPrevious evidence has shown that microRNA (miR)-224 may function as an onco-miRNA in hepatocellular carcinoma (HCC) cells by activating AKT signaling. However, little is known about the clinical significance of the combined expression of miR-224 and phosphorylated-AKT (pAKT) on human HCC. The aim of this study was to investigate the synergistical influence of miR-224 and pAKT on clinical characteristics and prognosis in patients with HCC.MethodsOne-hundred and thirty HCC patients who had undergone curative liver resection were selected. In situ hybridization and immunohistochemistry were respectively performed to detect the expression of miR-224 and pAKT in the respective tumors.ResultsCompared with the adjacent nonneoplastic liver tissues, the expression levels of miR-224 and pAKT protein in HCC tissues were both significantly increased (both P < 0.001). In addition, the combined upregulation of miR-224 and pAKT protein was significantly associated with serum AFP (P = 0.01), tumor stage (P = 0.002) and tumor grade (P = 0.008). Moreover, HCC patients highly expressing both miR-224 and pAKT protein had worse 5-year disease-free survival and 5-year overall survival (both P < 0.001). Furthermore, the Cox proportional hazards model showed that the combined upregulation of miR-224 and pAKT protein (miR-224-high/pAKT-high) may be independent poor prognostic factors for both 5-year disease-free survival (P = 0.008) and 5-year overall survival (P = 0.01) in HCC.ConclusionThese results indicate for the first time that miR-224 upregulation and AKT activation may synergistically associate with tumor progression of HCC. The combined high expression of miR-224 and pAKT may be a potential indicator for predicting unfavorable prognosis in HCC patients.     

Circ-CSPP1 knockdown suppresses hepatocellular carcinoma progression through miR-493-5p releasing-mediated HMGB1 downregulation

BackgroundHepatocellular carcinoma (HCC) accounts for over 80% of primary liver cancers and leads to a high death rate. Research on circular RNAs (circRNAs) suggests that circRNAs are promising biomarkers for cancer treatment. This study aimed to explore the function of a novel circRNA (circ-CSPP1) in HCC.MethodsCirc-CSPP1 was obtained from the microarray data downloaded from the Gene Expression Omnibus (GEO) database. The expression of circ-CSPP1, miR-493-5p and high mobility group box 1 (HMGB1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, colony formation ability, migration and invasion were monitored using cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay and transwell assay, respectively. The protein levels of CyclinD1, Vimentin, matrix metallopeptidase 9 (MMP-9) and HMGB1 were detected by western blot. Xenograft models were established to investigate the function of circ-CSPP1 in vivo. The association between miR-493-5p and circ-CSPP1 or HMGB1 was predicted by the online tool starBase and ensured by dual-luciferase reporter assay.ResultsThe expression of circ-CSPP1 and HMGB1 was elevated, while the expression of miR-493-5p was declined in HCC tissues and cells. Circ-CSPP1 knockdown not only depleted HCC cell proliferation, formation, migration and invasion in vitro but also inhibited tumor growth in vivo. MiR-493-5p was a target of circ-CSPP1, and HMGB1 was a target of miR-493-5p. Rescue experiments presented that miR-493-5p deficiency reversed the effects of circ-CSPP1 knockdown, and HMGB1 overexpression reversed the effects of miR-493-5p restoration. Circ-CSPP1 sponged miR-493-5p to regulate HMGB1 expression.ConclusionKnockdown of circ-CSPP1 suppressed HCC development both in vitro and in vivo by upregulation of miR-493-5p and downregulation of HMGB1, hinting that circ-CSPP1 participated in HCC pathogenesis.     

In vitro and in vivo anti-metastatic effect of the alkaliod matrine from Sophora flavecens on hepatocellular carcinoma and its mechanisms

BackgroundsHepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer with high metastasis and recurrence rates. Hypoxia-induced miRNAs and HIF-1α are demonstrated to play essential roles in tumor metastasis. Matrine (C₁₅H₂₄N₂O), an alkaloid extracted from Sophora flavescens Aiton, has been used as adjuvant therapy for liver cancer in China. The anti-metastasis effects of matrine on HCC and the underlying mechanisms remain poorly understood.PurposeWe aimed to investigate the effects of matrine on metastasis of HCC both in vitro and in vivo, and explored whether miR-199a-5p and HIF-1α are involved in the action of matrine.MethodsMTT method, colony formation, wound healing and matrigel transwell assays were performed to evaluate the effects of matrine on cell proliferation, migration and invasion. Nude mice xenograft model and immunohistochemistry (IHC) assay were employed to investigate the anti-metastatic action of matrine in vivo. Quantitative real-time PCR, western blot and dual luciferase reporter assay were conducted to determine the underlying mechanisms of matrine.ResultsMatrine exerted stronger anti-proliferative action on Bel7402 and SMMC-7721 cells under hypoxia than that in normoxia. Both matrine and miR-199a-5p exhibited significant inhibitory effects on migration, invasion and EMT in Bel7402 and SMMC-7721 cells under hypoxia. Further study showed that miR-199a-5p was downregulated in HCC cell lines, and this microRNA was identified to directly target HIF-1α, resulting in decreased HIF-1α expression. Matrine induced miR-199a-5p expression, decreased HIF-1α expression and inhibited metastasis of Bel7402 and SMMC-7721 cells, while miR-199a-5p knockdown reversed the inhibitory effects of matrine on cell migration, invasion, EMT and HIF-1α expression. In vivo, matrine showed significant anti-metastatic activity in the nude mouse xenograft model. H&E and IHC analysis indicated that lung and liver metastasis nodules were reduced, and the protein expression of HIF-1α and Vimentin were significantly decreased by i.p injection of matrine.ConclusionsMatrine exhibits significant anti-metastatic effect on HCC, which is attributed to enhanced miR-199a-5p expression and subsequently impaired HIF-1α signaling and EMT. These findings suggest that miR-199a-5p is a potential therapeutic target of HCC, and matrine may represent a promising anti-metastatic medication for HCC therapy.     

LncRNA-H19 activates CDC42/PAK1 pathway to promote cell proliferation,migration and invasion by targeting miR-15b in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the main causes of cancer-related death. This study aims to explore the role and underlying mechanism of H19 in HCC.MethodsqRT-PCR detected miR-15b-5p and H19 expression, as well as the mRNA level of EMT-associated genes. Western blotting detected protein level of EMT-associated genes. Immunohistochemistry (IHC) examined CDC42 in HCC tissues. Dual luciferase reporter assay verified the regulatory mechanism among H19, miR-15b and CDC42. Colony formation, wound healing assay, transwell, flow cytometry measured proliferation, migration, invasion and apoptosis, respectively.ResultsH19 and CDC42 were up-regulated while miR-15b was down-regulated in HCC cells and tissues. miR-15b interacted with H19 and CDC42 3′-UTR. H19 knockdown inhibited proliferation, migration and invasion, and increased apoptosis, which was rescued by miR-15b inhibitor. H19 knockdown suppressed CDC42/PAK1 pathway and EMT progress.ConclusionH19 knockdown inhibited proliferation, migration and invasion, and promoted apoptosis of HCC cells via targeting miR-15b/CDC42/PAK1 axis.     

β-Elemene inhibits the metastasis of multidrug-resistant gastric cancer cells through miR-1323/Cbl-b/EGFR pathway

Backgroundβ-Elemene is a natural agent extracted from the traditional Chinese herbal medicine Curcuma wenyujin that is a promising novel plant-derived drug with broad-spectrum anticancer activity. Our previous study identified an enhanced capacity for metastasis in multidrug resistant (MDR) gastric cancer and breast cancer cells. However, the anti-metastatic effects of β-Elemene on MDR cancer cells remain unknown.PurposeIn this study, we posit the hypothesis that β-elemene possesses antimetastatic effects on MDR cancer cells.MethodsCell viability assay was used to assess the resistance of SGC7901/ADR cells and the cytotoxic effects of β-Elemene. Wound healing, transwell assay and lung metastatic mice model were used to the anti-metastasis effects of β-Elemene. MicroRNA microarray analysis was used to explore potential regulated miRNAs. Luciferase reporter assay was used to identify the direct target. Human MMP antibody array, western blot, immunoprecipitation, qRT-PCR analyses and immunohistochemistry were conducted to investigate the underlying anti-metastasis mechanism of β-Elemene.ResultsIn this study, we found that β-Elemene significantly inhibited the metastatic capacity of MDR gastric cells in vivo and in vitro. Mechanistically, we found that β-Elemene regulated MMP-2/9 expression and reversed epithelial-mesenchymal transition. Further studies showed that β-Elemene upregulated Cbl-b expression, resulting in inhibition of the EGFR-ERK/AKT pathways, which regulate MMP-2/9. Additionally, we confirmed that β-Elemene upregulated Cbl-b by inhibiting miR-1323 expression. Finally, we found that numbers of metastatic tumor nodules were significantly decreased in the lungs of nude mice after β-Elemene treatment.ConclusionOur results suggested that β-Elemene inhibits the metastasis of MDR gastric cancer cells by modulating the miR-1323/Cbl-b/EGFR signaling axis.     

PAK1 promotes proliferation,migration and invasion of hepatocellular carcinoma by facilitating EMT via directly up-regulating Snail

BackgroundHepatocellular carcinoma (HCC) is a primary cause of cancer mortality. PAK1 plays key roles in many types of cancers. However, the role of PAK1 in HCC is not clear.MethodsqRT-PCR and Western blotting were used to determine expressions of PAK1, Snail and epithelial mesenchymal transition (EMT)-related proteins. Luciferase reporter assay was used to measure the interaction between PAK1 and Snail. Wound healing, transwell, colony formation assays and flow cytometry were used to assess cell migration, invasion, proliferation and apoptosis. Mouse tumor xenograft model was used to determine the effect of PAK1 on tumor growth in vivo.ResultsPAK1 and Snail were up-regulated in HCC cells. PAK1 knockdown suppressed cell proliferation, migration and invasion, and increased apoptosis of HCC cells. PAK1 knockdown also inhibited tumor growth in vivo. Mechanistically, PAK1 promoted EMT by targeting Snail. Knockdown of PAK1 could up-regulate pro-apoptotic proteins but down-regulate proliferation-related proteins via suppressing β-catenin signaling pathway.ConclusionPAK1 promotes EMT process by increasing Snail, and facilitates progression of HCC by activating β-catenin pathway.     

DNA Methylation-mediated Repression of miR-941 Enhances Lysine (K)-specific Demethylase 6B Expression in Hepatoma Cells

MicroRNAs (miRNAs) have been shown to play important roles in carcinogenesis. However, their underlying mechanisms of action in hepatocellular carcinoma (HCC) are poorly understood. Recent evidence suggests that epigenetic silencing of miRNAs through tumor suppression by CpG island hypermethylation may be a common hallmark of human tumors. Here, we demonstrated that miR-941 was significantly down-regulated in HCC tissues and cell lines and was generally hypermethylated in HCC. The overexpression of miR-941 suppressed in vitro cell proliferation, migration, and invasion and inhibited the metastasis of HCC cells in vivo. Furthermore, the histone demethylase KDM6B (lysine (K)-specific demethylase 6B) was identified as a direct target of miR-941 and was negatively regulated by miR-941. The ectopic expression of KDM6B abrogated the phenotypic changes induced by miR-941 in HCC cells. We demonstrated that miR-941 and KDM6B regulated the epithelial-mesenchymal transition process and affected cell migratory/invasive properties.     

FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway

The fucosyltransferase (FUT) family is the key enzymes in cell-surface antigen synthesis during various biological processes such as tumor multidrug resistance (MDR). The aim of this work was to analyze the alteration of FUTs involved in MDR in human hepatocellular carcinoma (HCC) cell lines. Using mass spectrometry (MS) analysis, the composition profiling of fucosylated N-glycans differed between drug-resistant BEL7402/5-FU (BEL/FU) cells and the sensitive line BEL7402. Further analysis of the expressional profiles of the FUT family in three pairs of parental and chemoresistant human HCC cell lines showed that FUT4, FUT6 and FUT8 were predominant expressed in MDR cell lines. The altered levels of FUT4, FUT6 and FUT8 were responsible for changed drug-resistant phenotypes of BEL7402 and BEL/FU cells both in vitro and in vivo. In addition, regulating FUT4, FUT6 or FUT8 expression markedly modulated the activity of the phosphoinositide 3 kinase (PI3K)/Akt signaling pathway and MDR-related protein 1 (MRP1) expression. Inhibition of the PI3K/Akt pathway by its specific inhibitor wortmannin, or by Akt small interfering RNA (siRNA), resulted in decreased MDR of BEL/FU cells, partly through the downregulation of MRP1. Taken together, our results suggest that FUT4-, FUT6- or FUT8-mediated MDR in human HCC is associated with the activation of the PI3K/Akt pathway and the expression of MRP1, but not of P-gp, indicating a possible novel mechanism by which the FUT family regulates MDR in human HCC.     

Upregulation of microRNA-372 associates with tumor progression and prognosis in hepatocellular carcinoma

MicroRNA-372 (miR-372) has been demonstrated to play a crucial role in cellular proliferation and apoptosis of cancer cells. However, its effects in hepatocellular carcinoma (HCC) have not been explored. The aim of this study was to investigate the clinical significance of miR-372 in human HCC. Quantitative RT-PCR was performed to detect miR-372 expression in HCC clinical samples and cell lines. Then, Kaplan–Meier and Cox proportional regression analyses were performed to determine the association of miR-372 expression with survival of HCC patients. Moreover, the effects of miR-372 on tumorigenicity of HCC cell lines were evaluated by in vitro assays. miR-372 expression in HCC tissues was significantly higher than in the corresponding normal adjacent liver tissues (P < 0.001). There was a correlation between miR-372 upregulation and advanced TNM stage of HCC patients (P = 0.02). In addition, HCC patients with higher miR-372 expression had significantly poorer recurrence-free survival (P = 0.006) and overall survival (P = 0.001). Multivariate analysis revealed that high miR-372 expression was an independent predictor of poor prognosis (for recurrence-free survival: Hazard Ratio [HR] 6.826, P = 0.01; for overall survival: HR 9.533, P = 0.008). Moreover, in vitro assays demonstrated that the ectopic expression of miR-372 may significantly promote the cellular proliferation, invasion, and migration of HCC cell lines. Our findings showed that miR-372 may serve as a potent prognostic marker for tumor recurrence and survival of HCC patients. Furthermore, miR-372 has been identified as a promoter for tumorigenicity of HCC cells, suggesting that it might be a prospective therapeutic target for HCC.     

MiR-490-5p inhibits the stemness of hepatocellular carcinoma cells by targeting ECT2

To explore the targeting relationship between miR-490-5p and ECT2 in hepatocellular carcinoma (HCC) and the influences of miR-490-5p and ECT2 on the stemness of HCC cells. The expressions of miR-490-5p and ECT2 in HCC tissues and adjacent tissues were identified by quantitative real-time polymerase chain reaction (qRT-PCR). The relationships between the expression levels of miR-490-5p/ ECT2 and the overall/disease-free survival (OS/DFS) of patients with HCC were evaluated using correlative curves. In addition, the targeting relationship between miR-490-5p and ECT2 was predicted by TargetScan and verified by dual-luciferase reporter assay. Plasmid transfection was used for overexpression of ECT2 in HepG2 cells, and transfection efficiency was verified by qRT-PCR. Cell Counting Kit-8 assay and cell sphere-formation assay were conducted to detect the proliferation and sphere-formation ability of HCC cells, respectively. Cell populations with different cell transfections were sorted using flow cytometry. The expression levels of proteins in the stem cell signaling pathway were determined using Western blot analysis. MiR-490-5p was remarkably downregulated, yet ECT2 was upregulated in HCC tissues compared with adjacent tissues. MiR-490-5p expression was positively correlated with OS and DFS of patients with HCC, which were otherwise negatively correlated with ECT2 expression. ECT2 was validated to be the downstream target of miR-490-5p. Overexpression of miR-490-5p restrained the sphere formation ability, stemness, and proliferation of HCC cells. MiR-490-5p repressed the stemness of HCC cells through inhibiting the expression of ECT2. MiR-490-5p may be an underlying therapeutic target in HCC treatment.     

A multiple-targets alkaloid nuciferine overcomes paclitaxel-induced drug resistance in vitro and in vivo

ObjectiveMultidrug resistance (MDR) is the major barrier to the successful treatment of chemotherapy. Compounds from nature products working as MDR sensitizers provided new treatment strategies for chemo-resistant cancers patients.MethodsWe investigated the reversal effects of nuciferine (NF), an alkaloid from Nelumbo nucifera and Nymphaea caerulea, on the paclitaxel (PTX) resistance ABCB1-overexpressing cancer in vitro and in vivo, and explored the underlying mechanism by evaluating drug sensitivity, cell cycle perturbations, intracellular accumulation, function and protein expression of efflux transporters as well as molecular signaling involved in governing transporters expression and development of MDR in cancer.ResultsNF overcomes the resistance of chemotherapeutic agents included PTX, doxorubicin (DOX), docetaxel, and daunorubicin to HCT-8/T and A549/T cancer cells. Notably, NF suppressed the colony formation of MDR cells in vitro and the tumor growth in A549/T xenograft mice in vivo, which demonstrated a very strong synergetic cytotoxic effect between NF and PTX as combination index (CI) (CI<0.1) indicated. Furthermore, NF increased the intracellular accumulation of P-gp substrates included DOX and Rho123 in the MDR cells and inhibited verapamil-stimulated ATPase activity. Mechanistically, inhibition of PI3K/AKT/ERK pathways by NF suppressed the activation of Nrf2 and HIF-1α, and further reduced the expression of P-gp and BCRP, contributing to the sensitizing effects of NF against MDR in cancer.ConclusionThis novel finding provides a promising treatment strategy for overcoming MDR and improving the efficiency of chemotherapy by using a multiple-targets MDR sensitizer NF.