Anti-inflammatory effect of chlorogenic acid in Klebsiella pneumoniae-induced pneumonia by inactivating the p38MAPK pathway

IntroductionPneumonia is an inflammation-related respiratory infection and chlorogenic acid (CGA) possesses a wide variety of bioactive properties, such as anti-inflammation and anti-bacteria.AimThis study explored the anti-inflammatory mechanism of CGA in Klebsiella pneumoniae (Kp)-induced rats with severe pneumonia.MethodsThe pneumonia rat models were established by infection with Kp and treated with CGA. Survival rates, bacterial load, lung water content, and cell numbers in the bronchoalveolar lavage fluid were recorded, lung pathological changes were scored, and levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assay. RLE6TN cells were infected with Kp and treated with CGA. The expression levels of microRNA (miR)-124–3p, p38, and mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) in lung tissues and RLE6TN cells were quantified by real-time quantitative polymerase chain reaction or Western blotting. The binding of miR-124–3p to p38 was validated by the dual-luciferase and RNA pull-down assays. In vitro, the functional rescue experiments were performed using miR-124–3p inhibitor or p38 agonist.ResultsKp-induced pneumonia rats presented high mortality, increased lung inflammatory infiltration and the release of inflammatory cytokines, and enhanced bacterial load, while CGA treatment improved rat survival rates and the above situations. CGA increased miR-124–3p expression, and miR-124–3p inhibited p38 expression and inactivated the p38MAPK pathway. Inhibition of miR-124–3p or activation of the p38MAPK pathway reversed the alleviative effect of CGA on pneumonia in vitro.ConclusionCGA upregulated miR-124–3p expression and inactivated the p38MAPK pathway to downregulate inflammatory levels, facilitating the recovery of Kp-induced pneumonia rats.     

Ginsenoside Rh2 inhibits thyroid cancer cell migration and proliferation via activation of miR-524-5p

IntroductionThyroid cancer is an important disease that threatens the health of humans. Ginsenoside Rh2 is known as an anticancer molecule; however, its function in thyroid cancer cells has not been reported. In the present study, we identified that Rh2 treatment of the thyroid cancer cell line K1 inhibited cell migration and proliferation.Material and methodsWe determined the Rh2 function in thyroid cancer cell lines. By RT-PCR, expression of miR-524-5p and related genes were determined. The cell phenotype including cell migration and proliferation were detected after serials treatment. The relevant protein level were checked by Western blot.ResultsInterestingly, we observed that miR-524-5p, a type of miRNA, had lower expression in the thyroid cancer cell lines TPC-1, K1, and NPA than in the normal thyroid cell line Nthyri3-1. Additionally, Rh2 treatment induced miR-524-5p expression. Further examination using overexpression of miR-524-5p identified that the miR-524-5p mimic inhibited cell migration and proliferation of the K1 line. Similar to Rh2-treated cells, the miR-524-5p mimic-expressing cells had increased E-cadherin and reduced vimentin levels compared to the control cells. Next, we examined the relationship between Rh2 and miR-524-5p with respect to thyroid cell migration and proliferation. Treatment with Rh2 and miR-524-5p inhibitor suppressed Rh2 action on K1 thyroid cell migration and proliferation, and the rates were similar to those in control cells, suggesting that Rh2 might induce miR-524-5p expression to inhibit thyroid cancer cell migration and proliferation.ConclusionsOur analyses identified Rh2 and miR-524-5p action on thyroid cancer cell migration and proliferation as well as the linkage between Rh2 and miR-524-5p in thyroid cancer cell development.     

LncRNA NCK1-AS1 promotes non-small cell lung cancer progression via regulating miR-512-5p/p21 axis

ObjectiveThis study aimed at probing into the effect of lncRNA NCK1-AS1 on proliferation, migration and invasion of non-small cell lung cancer (NSCLC) cells and its regulatory function on miR-512-5p/p21 molecular axis.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the expressions of NCK1-AS1 and miR-512-5p in NSCLC tissues and cell lines. The alterations of cell proliferation, migration, invasion and cell cycle were examined by cell counting kit-8 (CCK-8) assay, BrdU experiment, Transwell experiment and flow cytometry, respectively. The dual-luciferase reporter assay and RNA immunoprecipitation experiment were performed to validate the binding relationships between miR-512-5p and NCK1-AS1, and miR-512-5p the 3'UTR of p21 mRNA. Western blot was used to determine the effects of NCK1-AS1 and miR-512-5p on p21 protein expression.ResultsNCK1-AS1 expression was up-regulated in NSCLC tissues and cells, and its high expression was correlated with shorter overall survival time and faster progression of patients. Overexpression of NCK1-AS1 promoted NSCLC cell proliferation, migration and invasion, and accelerated the cell cycle, whereas NCK1-AS1 siRNA inhibited these malignant biological behaviors, and arrested cell cycle. NCK1-AS1 could bind to miR-512-5p, p21 was verified as a target gene of miR-512-5p, and NCK1-AS1 could up-regulate the expression of p21 in NSCLC cells via repressing miR-512-5p expression.ConclusionNCK1-AS1 promotes NSCLC progression by regulating miR-512-5p/p21 molecular axis.     

Long non-coding RNA LINC01296 promotes progression of oral squamous cell carcinoma through activating the MAPK/ERK signaling pathway via the miR-485-5p/PAK4 axis

IntroductionLong intergenic non-protein-coding RNA 1296 (LINC01296), a newly identified lncRNA, can function as an oncogenic driver to promote the development of multiple carcinomas. However, the effect of LINC01296 on oral squamous cell carcinoma (OSCC) is still unclear.Material and methodsWe determined the expression and role of LINC01296 in OSCC tissues and cell lines. The cell viability, migration and invasion were determined by MTT, wound healing assay and transwell assay, respectively. Flow cytometry was used for detecting cell cycle and apoptosis. The interaction and association between LINC01296, microRNA-485-5p (miR-485-5p) and p21 (RAC1) activated kinase 4 (PAK4) were analyzed by RNA immunoprecipitation (RIP) and luciferase reporter assays. The xenograft mouse model was established to detect the effect of LINC01296 on OSCC tumor growth.ResultsOur study showed that LINC01296 was over-expressed in OSCC tissues and cell lines. The level of LINC01296 was positively correlated with the patient’s tumor node metastasis (TNM) stage and nodal invasion. Knockdown of LINC01296 effectively inhibits cell viability, migration and invasion but promotes cell apoptosis in vitro. The in vivo experiment showed that LINC01296 knockdown inhibited OSCC tumor growth. The following analysis indicated that LINC01296 acted as a ceRNA for miR-485-5p, and PAK4 was identified as a direct target of miR-485-5p. Furthermore, we found that the effects of LINC01296 on OSCC progression were through regulating the expression of PAK4/p-MEK/p-ERK via sponging miR-485-5p.ConclusionsLINC01296 promote the cell cycle, proliferation, migration and invasion, and inhibit apoptosis of OSCC cells through activating the MAPK/ERK signaling pathway via sponging miR-485-5p to regulate PAK4 expression. These results suggested that the LINC01296/miR-485-5p/PAK4 axis was closely associated with OSCC progression. Our study provides a new insight into the molecular pathogenesis of OSCC, and may supply novel biomarkers for diagnosis and therapy of OSCC.     

Circ_0000218 plays a carcinogenic role in laryngeal cancer through regulating microRNA-139-3p/Smad3 axis

BackgroundLaryngeal squamous cell carcinoma (LSCC) accounts for about 85%–90% of all cases of laryngeal cancer. So far, the role and molecular mechanism of circular RNA 0,000,218 (circ_0000218)/microRNA (miR)-139−3p in laryngeal cancer are not clear. The present study aimed to investigate the role and regulatory mechanism of circ_0000218/miR-139−3p in laryngeal cancerin vitro and in vivo.Methodsquantitative real time polymerase chain reaction (qRT-PCR) was used to detect the expression of circ_0000218/miR-139−3p in LSCC cells. Dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to confirm binding sites between miR-139−3p and smad family member 3 (Smad3), and circ_0000218 and miR-139−3p. Cell Counting Kit-8 (CCK-8) and cell apoptosis analysis were used to detect cell viability and apoptosis. Xenograft experiment was performed to show in vivo effect of circ_0000218/miR-139−3p on the growth of LSCC.ResultsCirc_0000218 was highly expressed in LSCC cells. miR-139−3p, lower expressed in LSCC cells, was negatively regulated by circ_0000218 in LSCC cells. Besides, the findings suggested that circ_0000218 silencing inhibited the LSCC cell viability and promoted apoptosis by negatively regulating miR-139−3p expression. Furthermore, the data indicated that miR-139−3p inhibited the viability of LSCC cells and promoted apoptosis, and these effects were reversed by Smad3 over-expression. In addition, the in vivo effects of circ_0000218/miR-139−3p on LSCC were consistent with the in vitro study.Conclusionscirc_0000218 inhibition inhibited the growth of LSCC by targeting miR-139−3p/Smad3 axis. Our present study provided a new target for laryngeal cancer treatment.     

miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.     

LncRNA OGFRP1 promotes angiogenesis and epithelial-mesenchymal transition in colorectal cancer cells through miR-423-5p/CTCF axis

ObjectiveTo investigate the mechanism of lncRNA OGFRP1 affecting angiogenesis and epithelial-mesenchymal transition (EMT) in colorectal cancer (CRC) and provide a new target for the treatment of CRC.MethodsThe expressions of OGFRP1, miR-423-5p, and CTCF were measured in CRC cell lines (HT29, LoVo, HCT116, SW620, and SW480) and normal colonic epithelial cells NCM460. Gain and loss of function experiments were performed on HCT116 and SW620 cells, after which the proliferation, apoptosis, EMT, invasion, and migration of the cells were measured using CCK-8 and colony formation assays, flow cytometry, Western blotting, Transwell, and scratch assay. The transfected cells were incubated with human umbilical vein endothelial cells (HUVECs) to assess angiogenesis using tube formation assay. ELISA was performed to detect VEGF in the conditioned medium of HCT116 and SW620 cells. The interactions among OGFRP1, CTCF and miR-423-5p were validated by dual-luciferase reporter assay.ResultsCRC cell lines had increased expression levels of OGFRP1 and CTCF and a suppressed expression level of miR-423-5p when compared with NCM460 cells. Suppression on OGFRP1 or CTCF and overexpression of miR-423-5p led to inhibited proliferation, EMT, invasion and migration and increased apoptosis of HCT116 and SW620 cells. HUVECs incubated with cells transfected with si-OGFRP1, si-CTCF or miR-423-5p mimic had suppressed angiogenesis ability. The effect of OGFRP1 suppression in CRC cells could be counteracted by miR-423-5p inhibition. Both CTCF and OGFRP1 could bind to miR-423-5p.ConclusionOGFRP1 promotes proliferation, EMT, and angiogenesis in CRC through miR-423-5p/CTCF axis.     

MiR-124-3p inhibits the migration and invasion of Gastric cancer by targeting ITGB3

BACKGROUNDGastric cancer is one of the major malignant tumors in the world. Integrins expressed in cancer cells can promote tumor progression and migration. MiRNAs can inhibit the expression of target genes by directly binding to their mRNAs and can affect various important biological processes. The aim of this study was to investigate the role of miR-124- 3p and ITGB3 in gastric cancer.METHODSRT-PCR and western blot are used to detect the expression of miR-124-3p, ITGB3 and integrin β3 in gastric cancer tissues and cells. The wound healing, CCK-8 assay, transwell migration and invasion assay were performed to determine the cell proliferation, migration and invasion. What’s more, bioinformatics prediction and luciferase assay was conducted to demonstrated the binding efficiency between miR-124-3p and ITGB3.RESULTSWe verified that ITGB3 and miR-124-3p changes the migration and invasion of gastric cancer cells in vitro. The overexpression or silencing of miR-124-3p inhibited or promoted the proliferation, migration and invasion of both selected gastric cancer cells, and ITGB3 is just the reverse. Meanwhile, we validated that ITGB3 is the target of miR-124-3p by bioinformatics prediction and luciferase assay. Lastly, the expression of ITGB3 in 40 pairs of gastric cancer tissues were significantly higher than that in the adjacent normal tissues, while the expression level of miR-124-3p was significantly decreased in cancer tissues.CONCLUSIONSmiR-124-3p inhibits the migration and invasion of Gastric cancer by targeting ITGB3 in gastric cancer cells. Our results suggested that miR-124-3p and ITGB3 may reasonably serve as a promising therapeutic target.     

MircoRNA-129-5p suppresses the development of glioma by targeting HOXC10

BackgroundmiR-129-5p has been reported to be abnormally expressed and plays an important role in the progression of various malignancies. However, its role in gliomas and its exact molecular mechanism need further research.Methods and materialsRT-qPCR was performed to evaluate miR-129-5p and HOXC10 mRNA expression levels in tissues and cell lines. Cell proliferation was detected via Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) and clone formation assays. Luciferase assays were used to validate the binding of seeds between miR-129-5p and HOXC10. A tumor xenograft model was developed to study the effect of miR‐129-5p on glioma growth in vivo.ResultsmiR-129-5p was expressed at low levels in glioma tissues and cell lines. miR-129-5p overexpression inhibited glioma proliferation, migration and invasion. miR-129-5p negatively and directly targeted HOXC10. At the same time, HOXC10 was upregulated in glioma cancer, and HOXC10 knockdown inhibited cell proliferation, migration and invasion.ConclusionmiR-129-5p inhibits glioma development by altering HOXC10 expression and may therefore serve as a new diagnostic marker and therapeutic target for glioma in the future.     

Uterine leiomyosarcomas are characterized by high p16, p53 and MIB1 expression in comparison with usual leiomyomas, leiomyoma variants and smooth muscle tumours of uncertain malignant potential

AIMS: It has been suggested that p16 is overexpressed in uterine leiomyosarcomas in comparison with leiomyomas. In this study, p16 immunohistochemical expression was assessed in a variety of uterine smooth muscle tumours, including usual leiomyomas, leiomyoma variants, smooth muscle tumours of uncertain malignant potential (STUMPs) and leiomyosarcomas. The aim was to ascertain whether there are differences in p16 expression between these groups and whether p16 is of potential value in the assessment of problematic uterine smooth muscle neoplasms. p16 expression was also compared with that of p53 and MIB1. METHODS AND RESULTS: Cases of usual leiomyoma (n = 10), leiomyoma variants (n = 27), STUMP (n = 4) and leiomyosarcoma (n = 22) were subject to p16, p53 and MIB1 immunohistochemistry. For p16, cases were evaluated with respect to both staining distribution and intensity. There was a statistically significant difference in p16 distribution (P < 0.001) and intensity (P = 0.001) between leiomyosarcomas and the other groups. There was no difference in p16 expression between usual leiomyomas, leiomyoma variants and STUMPs. There were also statistically significant differences in p53 (P = 0.014) and MIB1 (P < 0.001) immunoreactivity between leiomyosarcomas and the other groups. CONCLUSIONS: p16 is overexpressed in uterine leiomyosarcomas compared with leiomyomas, benign leiomyoma variants and STUMPs, suggesting that p16 may be implicated in the pathogenesis of malignant uterine smooth muscle neoplasms. p16, in combination with p53 and MIB1, may be of value as an adjunct to morphological examination in the assessment of problematic uterine smooth muscle tumours, although further large-scale studies with follow-up are necessary to confirm this.     

MicroRNA-139-5p inhibits cell proliferation and invasion by targeting insulin-like growth factor 1 receptor in human non-small cell lung cancer

Introduction: Increasing evidence suggested that microRNAs (miRNAs) play a critical role in tumorigenesis. Decreased expression of miRNA-139-5p has been observed in various types of cancers. However, the biological function of miRNA-139-5p in non-small cell lung cancer (NSCLC) is still largely unknown. Methods: Quantitative real-time PCR (qRT-PCR) was used to explore the expression level of miRNA-139-5p in NSCLC tissues and cell lines. Then, we investigated the role of miRNA-139-5p to determine its potential roles on lung cancer cell proliferation, migration and invasion in vitro. A luciferase reporter assay was performed to confirm the target gene of miRNA-139-5p and the results were validated in renal cancer cells. Results: miRNA-139-5p was significantly decreased in NSCLC tissues and cell lines. Over-expression of miRNA-139-5p could inhibit lung cancer cell proliferation, migration, and invasion in vitro. Furthermore, we identified insulin-like growth factor 1 receptor (IGF1R) as a target of miR-139-5p and miR-139-5p function as a tumor suppressor via targeting IGF1R in NSCLC. Conclusions: Our results indicated that miR-139-5p acts as a tumor suppressor in NSCLC partially via down-regulating IGF1R expression.     

LncRNA linc00460 sponges miR-1224-5p to promote esophageal cancer metastatic potential and epithelial-mesenchymal transition

BackgroundIncreasing studies highlight the crucial role of long non-coding RNAs (lncRNAs) in carcinogenesis of various human cancer types, including esophageal cancer (ESCA). Long intergenic non-coding RNA 00460 (Linc00460), a novel oncogenic lncRNA, has been reported to accelerate ESCA cell growth. This study aimed to investigate the role and possible regulatory mechanism of linc00460 in ESCA metastasis.MethodsBioinformatics analysis and quantitative real time polymerase chain reaction (qRT-PCR) were used to detect linc00460 expression in ESCA. Wound healing assay, Transwell assay and Western blot were utilized to examine migration, invasion and epithelial-mesenchymal transition (EMT) of ESCA cells. The direct binding effect between linc00460 and microRNA-1224-5p (miR-1224-5p) was evaluated by the dual luciferase reporter assay.ResultsIn this study, we discovered that lncRNA linc00460 was obviously over-expressed in ESCA, both in tissues and cell lines. Down-regulation of linc00460 significantly suppressed the metastatic potential (including cell migration and invasion) and EMT of ESCA cells. In addition, miR-1224-5p, a potential tumor suppressor, was negatively correlated with linc00460 in ESCA. Linc00460 and miR-1224-5p could bind directly in ESCA cells. Inhibition of miR-1224-5p partially abrogated the effects of linc00460 decrease on metastatic potential and EMT of ESCA cells.ConclusionsTaken together, linc00460 may function as a molecular sponge to adsorb miR-1224-5p, thereby promoting ESCA metastasis and EMT. Our findings suggest that linc00460/miR-1224-5p is a possible clinical target for ESCA.     

miR-199a-3p targets ETNK1 to promote invasion and migration in gastric cancer cells and is associated with poor prognosis

PurposeTo investigate the prognostic significance of miR-199a-3p and its role in invasion and metastasis in gastric cancer.MethodsmiR-199a-3p expression in 436 formalin-fixed and 39 frozen gastric cancer tissues was investigated by in situ hybridization and RT-PCR, respectively. The role of miR-199a-3p in the migration and invasion of gastric cancer cells was determined in overexpression and inhibitor studies using transwell assays and the SGC-7901, BGC-823 and MGC-803 gastric cancer cells lines. The effect of miR-199a-3p expression on ethanolamine kinase 1 (ETNK1) levels was determined by western botting.ResultsmiR-199a-3p was significantly up-regulated in AGS, SGC-7901, BGC-823 and MGC-803 gastric cancer cells, when compared with GES-1 non-malignant gastric epithelial cells. In situ hybridization studies revealed that human non-tumor gastric mucosa samples were negative for miR-199a-3p expression, while 162 of 436 (37.16%) cases of gastric cancer demonstrated positive expression. miR-199a-3p overexpression was associated with tumor size, Lauren classification, depth of invasion, lymph node and distant metastasis, TNM stage and prognosis. In patients with I, II and III stage tumors, high miR-199a-3p expression was associated with a significantly lower 5-year survival rate. miR-199a-3p overexpression was associated with increased cell migration and invasion. ETNK1 expression was inhibited following miR-199a-3p overexpression in BGC-823 and SGC-7901 cells, and elevated following miR-199a-3p suppression in MGC-803 cells.ConclusionmiR-199a-3p is highly expressed in gastric cancer, and correlates with invasion, metastasis and prognosis. miR-199a-3p regulates the invasion and migration of gastric cancer cells by targeting ETNK1. Consequently, miR-199a-3p may serve as a prognostic indicator in gastric cancer.     

MicroRNA-129-5p suppresses proliferation,migration and invasion of retinoblastoma cells through PI3K/AKT signaling pathway by targeting PAX6

BackgroundRetinoblastoma (RB) is the most common primary intraocular malignancy in children. Accumulating evidences have clarified that microRNAs (miRNAs) modulated signaling molecules by acting as oncogenes or tumor-suppressor genes in RB. Thus, in our study, we aimed to investigate the function of miR-129-5p in RB cells through PI3K/AKT signaling pathway by targeting PAX6. Two RB cell lines, Y79 and WERI-Rb-1, were selected in our study, followed by transfection of miR-129-5p inhibitor or si-PAX6 to explore the regulatory role of miR-129-5p in RB cell proliferation, invasion and migration.Material and methodsDual-luciferase assay was used for the detection of targeting relationship between miR-129-5p and PAX6. Besides, western blot analysis was applied to detect expression of cell cycle-related factors (CDK2 and Cyclin E) and PI3K/AKT signaling pathway-related factors (p-AKT and AKT). Nude mice tumorigenesis experiment was used to evaluate the effect of miR-129a-5p on RB growth in vivo.ResultsmiR-129-5p was down-regulated in RB cell lines. miR-129-5p directly targeted the 3′-untranslated region of PAX6. Artificial down-regulation of miR-129-5p promoted cell proliferation, migration and invasion in RB cell lines Y79 and WERI-Rb-1, and promoted RB growth in vivo via PI3K/AKT signaling pathway, which could be reversed by transfection with silencing PAX6.ConclusionThis study provides evidences that RB progression was suppressed by overexpressed miR-129-5p via direct targeting of PAX6 through PI3K/AKT signaling pathway, which may provide a molecular basis for better treatment for RB.     

miR-194-5p inhibits SLC40A1 expression to induce cisplatin resistance in ovarian cancer

BackgroundmiR-194-5p has been associated with drug resistance in many cancers. However, the role of miR-194-5p in cisplatin resistance in ovarian cancer is still unclear.Materials and methodsTo study the role and mechanism of miR-194-5p in cisplatin resistance, qRT-PCR was performed to determine the expression of miR-194-5p and SLC40A1 in ovarian cancer. Cell Counting Kit-8 (CCK8) assay was used to analyse cell viability after cisplatin treatment. Dual-luciferase reporter gene assay was performed to examine the relationship between miR-194-5p and SLC40A1. The genes downstream of SLC40A1 were investigated through bioinformatics analysis.ResultsCompared to cisplatin-sensitive ovarian cancer cells, higher miR-194-5p expression and lower SLC40A1 expression were found in cisplatin-resistant ovarian cancer cells. Moreover, this study demonstrated that over-expression of miR-194-5p inhibited SLC40A1 expression, and knockdown of miR-194-5p promoted SLC40A1 expression. In addition, dual-luciferase reporter gene assay further confirmed the negative correlation between miR-194-5p and SLC40A1. Furthermore, we found that over-expression of miR-194-5p resulted in cisplatin resistance. When miR-194-5p and SLC40A1 were simultaneously up-regulated, cisplatin sensitivity increased, while down-regulation of miR-194-5p sensitised ovarian cancer cells to cisplatin. However, when miR-194-5p and SLC40A1 were simultaneously down-regulated, cisplatin sensitivity was decreased. These data suggested that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance. In addition, bioinformatics analysis indicated a positive correlation of SLC40A1 with hephaestin (HEPH), and homeostatic iron regulator (HFE). However, we found that HEPH and HFE were associated with cisplatin resistance, suggesting that their role in drug resistance is induced by miR-194-5p/SLC40A1.ConclusionIn conclusion, we found that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance in ovarian cancer. This study suggests that miR-194-5p could be a potential therapeutic target and a prognostic biomarker for ovarian cancer, with important implications for future research.     

LINC00662 Promotes Oral Squamous Cell Carcinoma Cell Growth and Metastasis through miR-144-3p/EZH2 Axis

PurposeLong non-coding RNA (lncRNA) is identified as an important regulator involved in oral squamous cell carcinoma (OSCC) tumorigenesis. This study aimed to investigate the functional role and underlying mechanism of LINC00662 in OSCC.Materials and MethodsThe expression levels of LINC00662, miR-144-3p, and enhancer of zeste homolog 2 (EZH2) mRNA were quantified with quantitative real-time polymerase chain reaction in OSCC tissues and cell lines. Western blot analysis was used to assay the expression levels of E-cadherin, Vimentin, and EZH2. Cell proliferation, migration, and invasion were monitored by cell counting kit-8 and Transwell assays. Dual-luciferase reporter and RNA immunoprecipitation assays were employed to verify the regulatory relationship between LINC00662 and miR-144-3p.ResultsThe expression of LINC00662, positively associated with the increased TNM stage and lymph node metastasis of the patients, was up-regulated in OSCC tissues and cells. The overexpression of LINC00662 facilitated the proliferation, migration, and invasion of OSCC cells. MiR-144-3p could bind to LINC00662, and the promoting effect of LINC00662 overexpression was counteracted by miR-144-3p mimic. Moreover, EZH2 expression was negatively regulated by miR-144-3p and positively regulated by LINC00662. The silencing of EZH2 attenuated the promoting effects of overexpression of LINC00662 on cell proliferation, migration, invasion, and epithelial-mesenchymal transition.ConclusionLINC00662, as an oncogenic lncRNA of OSCC, accelerates OSCC progression by repressing miR-144-3p expression and increasing EZH2 expression.     

miR-141-3p promotes proliferation and metastasis of nasopharyngeal carcinoma by targeting NME1

PurposeThis study aimed to investigate the expression and biological function of miR-141-3p in nasopharyngeal carcinoma (NPC) via targeting neoplasm metastasis 1 (NME1).Materials and methodsThe expression of miR-141-3p and NME1 in 5-8F, C666-1, CNE-1, CNE-2, 6-10B and NP69 nasopharyngeal epithelial cells were detected using real-time Polymerase Chain Reaction (real-time PCR) and western blot, respectively. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8), and the metastasis was detected using Transwell. The binding of miR-141-3p to NME1 was detected by dual luciferase reporter gene detection system. The effects of miR-141-3p on tumor growth were also determined in vivo.ResultsThe results showed that the expression of miR-141-3p significantly increased in various tumor cell lines and the expression of NME1 was higher in NP69 cells and lower in 5-8F cells, which had significant negative correlation. Furthermore, the expression of NME1 was significantly reduced after transfection of miR-141-3p and miR-141-3p promoted cell proliferation and metastasis. The double luciferase reporter gene detection system confirmed that NME1 was the target gene of miR-141-3p. Knockout of NME1 promoted the proliferation and metastasis of NP69 or 6-10B cells and the activation of p-Akt, which were abrogated by miR-141-3p. In vivo, the tumor volumes and weights in the miR-141-3p group significantly increased followed by down-regulation of NME1 and activation of p-Akt.ConclusionsWe confirmed that miR-141-3p promotes the proliferation and metastasis of NPC by targeting NME1.     

MicroRNA-146b-5p suppresses cholangiocarcinoma cells by targeting TRAF6 and modulating p53 translocation

BackgroundIn view of the poor prognosis and high mortality of cholangiocarcinoma, there is a need for new therapeutic strategies. This study aims to reveal the biological function of miR-146b-5p in cholangiocarcinoma cell and its possible mechanism.MethodsThe expression level and prognostic information on miR-146b-5p in cholangiocarcinoma were obtained in TCGA database. The biological function of miR-146b-5p on proliferation and vitality of cholangiocarcinoma cell HUCCT-1 was examined by EdU and MTT assay, and the apoptosis of HUCCT-1 cells transfected with miR-146b-5p mimic, mimic control, inhibitor, inhibitor control was detected by flow cytometry analysis. The western blot was done to evaluate the effect of miR-146b-5p targeting substrate and the expression of p53 in whole-cell protein and mitochondria fractions.ResultsOur finding revealed that miR-146b-5p expression in patients with CHOL was lower than the normal group(p＜0.001). MiR-146b-5p expression was down-regulated in human cholangiocarcinoma HUCCT-1 and RBE cells compared to normal control HIBEC and other cancer cells. The miR-146b-5p mimic could inhibit HUCCT-1 cell proliferation (p＜0.05) and promote HUCCT-1 cell apoptosis significantly (p＜0.05). The results of western blot showed that miR-146b-5p mimic could directly target TRAF6 3′UTR region and up-regulate the expression of p53 in mitochondria and miR-146b-5p inhibitor could down-regulated the level of p53 in mitochondria.ConclusionMiR-146b-5p is a cholangiocarcinoma suppressor by inhibiting cell proliferation and promoting cell apoptosis with targeting TRAF6, possibly via modulating p53 translocation to mitochondria.     

miR-424-5p promotes the proliferation and metastasis of colorectal cancer by directly targeting SCN4B

BackgroundColorectal cancer (CRC) is one of the most common malignancies worldwide usually diagnosed at advanced stages which causes poor prognosis of patients. Therefore, novel diagnostic biomarkers and therapeutic targets are urgently needed.Materials and methodsmiR-424-5p was identified through integrated analysis of three public databases. Loss-of-function experiments in HT29 and SW480 cells and mouse xenograft models were performed to explore the regulatory role of miR-424-5p in CRC. Bioinformatics analysis was used for predicting targets of miR-424-5p and its functional and pathway enrichment analysis.ResultsmiR-424-5p expression was significantly upregulated in CRC tissues and cell lines and associated with prognosis of CRC patients. Experiments in vitro and in vivo showed miR-424-5p promotes CRC cell proliferation and metastasis by directly inhibiting SCN4B. Besides, CRC cells secret miR-424-5p into peripheral blood through exosomes and circulating exosomal miR-424-5p could discriminate CRC patients with early stage from healthy people with AUC value of 0.82.ConclusionsmiR-424-5p serves as an oncogene in CRC and circulating exosomal miR-424-5p is a novel potential diagnostic biomarker of CRC patients.     

miR-15a-5p抑制人肝癌SMMC-7721细胞增殖与迁移

目的:观察高表达的miR-15a-5p对人肝细胞癌SMMC-7721细胞增殖和迁移能力的影响。方法:化学合成加入EcoRⅠ和HindⅢ酶切位点的miR-15a-5p寡聚核苷酸,并进行测序确认;利用pcDNA6.2-GW/EmGFP质粒构建miR-15a-5p真核表达载体,瞬时转染SMMC-7721细胞,实时荧光定量PCR检测miR-15a-5p的表达;CCK-8法和台盼蓝染色活细胞计数检测SMMC-7721细胞的增殖能力,划痕实验检测细胞迁移能力的变化。结果:设计的miR-15a-5p序列与寡核苷酸测序结果匹配达100%;真核表达质粒瞬转后人肝癌SMMC-7721细胞miR-15a-5p的表达量与对照组相比显著增加(P0.05);miR-15a-5p高表达SMMC-7721细胞的增殖能力与对照组相比均显著下降(P0.05);miR-15a-5p高表达组细胞迁移速度低于对照组。结论:高表达的miR-15a-5p可抑制人肝癌SMMC-7721细胞的增殖和迁移能力。