Effect of Compound Phylanthus urinaria Ⅱ in Regulating Expression of lncRNA CCAT1 in Hepatocellular Carcinoma HepG2 Cells / 中国实验方剂学杂志

Objective:To explore the anti-hepatoma effect of compound <italic>Phylanthus urinaria</italic> Ⅱ （ CPU Ⅱ） by inhibiting the expression of the long non-coding RNA （lncRNA） colon cancer associated transcript-1 （CCAT1） and restoring the expression of microRNA let-7a. Method:Real-time fluorescence quantitative polymerase chain reaction （PCR） was used to detect the expression of lncRNA CCAT1 in normal liver cells （LO2 cells） and hepatocellular carcinoma HepG2 cells， and the differences in expression between these two types of cells were compared. The methylthiazolyl tetrazolium（MTT） assay was used to detect the proliferation of HepG2 cells after treatment with different concentrations of CPU Ⅱ and 5-fluorouracil（5-FU） for 24， 48 and 72 h. Hepatocellular carcinoma HepG2 cells were cultured <italic>in vitro </italic>and set into three gropes： cell control group， CPU Ⅱ low-dose group （0.8 g·L^-1） and high-dose group （1.6 g·L^-1）. Real-time PCR was used to detect the mRNA expression of lncRNA CCAT1， microRNA let-7a and its target genes high mobility group protein A2（HMGA2）， and N-RAS in each grope. Western blot was used to detect the protein expression of HMGA2， and Cyclin D₁ in each grope. Result:As compared with LO2 cells， expression of lncRNA CCAT1 in HepG2 cells was significantly up-regulated （<italic>P</italic><0.05）. Results of MTT assay showed that the 50% inhibiting concentration（IC₅₀）of CPU Ⅱ and 5-FU on hepatocellular carcinoma HepG2 cells was 1.649， 0.044 648 g·L^-1respectively. As compared with the control group， CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L^-1） significantly inhibited the proliferation of HepG2 cells （<italic>P</italic><0.05）， and the effect was most remarkable in CPU Ⅱ high-dose group （<italic>P</italic><0.05）. The results of Real-time PCR showed that as compared with control group， the expression of lncRNA CCAT1 mRNA was significantly inhibited in CPU Ⅱ high-and low-dose groups （<italic>P</italic><0.05）， and the expression of microRNA let-7a mRNA was obviously up-regulated in high-dose group （<italic>P</italic><0.05）， but the expression of HMGA2 mRNA in CPU Ⅱ high-and low-dose groups as well as the expression of N-RAS mRNA in CPU Ⅱ low-dose group were down-regulated （<italic>P</italic><0.05）. Western blot results showed that as compared with the cell control group， the protein expression of HMGA2 and Cyclin D₁ in CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L^-1） was significantly down-regulated （<italic>P</italic><0.05）. Conclusion:CPU Ⅱ can inhibit the expression of lncRNA CCAT1， recover the expression of microRNA let-7a， and suppress the mRNA and protein expression of related downstream target genes in hepatoma cells line HepG2， thereby inhibiting the proliferation of hepatocellular carcinoma cells and exerting anti-hepatocellular carcinoma effect.

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation

PURPOSE: Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. MATERIALS AND METHODS: This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. RESULTS: Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. CONCLUSION: Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders.

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation

PURPOSE: Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. MATERIALS AND METHODS: This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. RESULTS: Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. CONCLUSION: Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders.

Involvement of miR-Let7A in inflammatory response and cell survival/apoptosis regulated by resveratrol in THP-1 macrophage

BACKGROUND/OBJECTIVES: Resveratrol, a natural polyphenol, has multiple functions in cellular responses including apoptosis, survival, and differentiation. It also participates in the regulation of inflammatory response and oxidative stress. MicroRNA-Let-7A (miR-Let7A), known as a tumor suppressor miRNA, was recently reported to play a crucial role in both inflammation and apoptosis. Therefore, we examined involvement of miR-Let7A in the modulation of inflammation and cell survival/apoptosis regulated by resveratrol. MATERIALS/METHODS: mRNA expression of pro-/anti-inflammatory cytokines and sirtuin 1 (SIRT1), and protein expression of apoptosis signal-regulating kinase 1 (ASK1), p-ASK1, and caspase-3 and cleaved caspase-3 were measured, and cell viability and Hoechst/PI staining for apoptosis were observed in Lipopolysaccharide (LPS)-stimulated human THP-1 macrophages with the treatment of resveratrol and/or miR-Let7A overexpression. RESULTS: Pre-treatment with resveratrol (25-200 µM) resulted in significant recovery of the reduced cell viabilities under LPS-induced inflammatory condition and in markedly increased expression of miR-Let7A in non-stimulated or LPS-stimulated cells. Increased mRNA levels of tumor necrosis factor-α and interleukin (IL)-6 induced by LPS were significantly attenuated, and decreased levels of IL-10 and brain-derived neurotrophic factor were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. Decreased expression of IL-4 mRNA by LPS stimulation was also significantly increased by miR-Let7A overexpression co-treated with resveratrol. In addition, decreased SIRT1 mRNA levels, and increased p-ASK1 levels and PI-positive cells by LPS stimulation were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. CONCLUSIONS: miR-Let7A may be involved in the inflammatory response and cell survival/apoptosis modulated by resveratrol in human THP-1 macrophages.

Expression of microRNA-Let-7a in patients of Non-small cell lung cancer and anti-tumor mechanism / 中国免疫学杂志

Objective:To investigate the expression of microRNA-Let-7a in the serum and tumor tissue of non-small cell lung cancer( NSCLC) patients and the effects of cancer cell migration and proliferation.Methods: 50 cases of NSCLC patients in our hospital as the study group,50 healthy volunteers were used as control group,we used Real-time RCR to detect the expression of microRNA-Let-7a in the serum and tumor tissue of NSCLC patients.Using microRNA let-7a mimics transfected into A549,the level of cancer cell migration was observed by transwell,the level of cell proliferation was observed by CCK-8,the level of k-Ras was observed by Real-time RCR and Western blot.Results: The expression of microRNA-Let-7a in the serum and tumor tissue of NSCLC patients was significantly higher than the control group,the difference was statistically significant (P<0.05).After microRNA let-7a transfected into A549,the levels of cancer cell migration and proliferation were significantly decreased,the difference was statistically significant (P<0.05),the mRNA and protein levels of k-Ras were reduced inA549 cells,the difference was statistically significant (P<0.05). Conclusion:The expression of microRNA let-7a is low in the serum and tumor tissue of NSCLC patients,and may weaken the levels of cancer cell migration and proliferation through the Ras signaling pathway.

miR-Let7A Modulates Autophagy Induction in LPS-Activated Microglia

Microglia regulate the secretion of various immunomediators in central nervous system diseases. Microglial autophagy is the crucial process for cell's survival and cytokine productions. Recent studies have reported that several microRNAs are involved in the autophagy system. miR-Let7A is such a microRNA that plays a role in various inflammation responses, and is magnified as a key modulator particularly in the autophagy system. In present study, we investigated whether miR-Let7A is involved in autophagy in activating microglia. Overexpression of miR-Let7A in LPS-stimulated BV2 microglial cells promoted the induction of the autophagy related factors such as LC3II, Beclin1, and ATG3. Our results suggest a potential role of miR-Let7A in the autophagy process of microglia during CNS inflammation.

Effect of microRNA-let-7a on apoptosis in melanoma cell line A375 / 中华皮肤科杂志

Objective To investigate the effect of microRNA-let-7a on apoptosis in melanoma cell line A375 and its mechanism. Methods The expression of microRNA-let-7a was detected in A375 cells and melanocytes by real-time PCR. Then, microRNA-let-7a mimics was transfected into A375 cells followed by the measurement of apoptosis and caspase-3 protein expression by flow cytometry and Western blotting, respectively. Results The expression level of microRNA-let-7a was reduced by 0.462 folds in A375 cells compared with melanocytes. The apoptosis rate was 47.4% in A375 cells transfected with microRNA-let-7a mimics, significantly higher than that in untransfected A375 cells (16.9%). A significant decline was observed in the expression of caspase-3 protein in A375 cells after transfection. Conclusion microRNA-let-7a can promote the apoptosis and downregulate caspase-3 protein expression, in A375 human malignant melanoma cells.