Kaempferol suppresses human gastric cancer SNU-216 cell proliferation, promotes cell autophagy, but has no influence on cell apoptosis

Gastric cancer remains a serious threat to human health worldwide. Kaempferol is a plant-derived flavonoid compound with a wide range of pharmacological activities. This study aimed to investigate the effects of kaempferol on gastric cancer SNU-216 cell proliferation, apoptosis, and autophagy, as well as underlying potential mechanisms. Viability, proliferation, and apoptosis of SNU-216 cells after kaempferol treatment were evaluated using cell counting kit-8 assay, 5-btomo-2′-deoxyuridine incorporation assay, and annexin V-FITC/PI staining, respectively. Quantitative reverse transcription PCR was performed to measure the mRNA expressions of cyclin D1 and microRNA-181a (miR-181a) in SNU-216 cells. Cell transfection was used to down-regulate the expression of miR-181a. The protein expression levels of cyclin D1, bcl-2, bax, caspase 3, caspase 9, autophagy-related gene 7, microtubule-associated protein 1 light chain 3-I (LC3-I), LC3-II, Beclin 1, p62, mitogen-activated protein kinase (MAPK), extracellular regulated protein kinases (ERK), and phosphatidylinositol 3 kinase (PI3K) in SNU-216 cells were detected using western blotting. Results showed that kaempferol significantly suppressed SNU-216 cell viability and proliferation but had no influence on cell apoptosis. Further results suggested that kaempferol significantly induced SNU-216 cell autophagy. The expression of miR-181a in SNU-216 cells after kaempferol treatment was enhanced. Kaempferol significantly inactivated MAPK/ERK and PI3K pathways in SNU-216 cells. Suppression of miR-181a significantly reversed the kaempferol-induced MAPK/ERK and PI3K pathways inactivation in SNU-216 cells. This research demonstrated that kaempferol suppressed proliferation and promoted autophagy of human gastric cancer SNU-216 cells by up-regulating miR-181a and inactivating MAPK/ERK and PI3K pathways.

Dynamic expressions and clinical significance of toll-like receptor 4/microRNA-181a in the pathogenesis of hepatic fibrosis in patients with chronic hepatitis B / 中华传染病杂志

Objective To explore the dynamic expressions and clinical significance of toll-like receptor 4 (TLR4)/microRNA (miRNA)-181a in the pathogenesis of hepatic fibrosis (HF) in patients with chronic hepatitis B (CHB) .Methods CHB patients underwent liver biopsy for fibrosis staging HF (S) .Real-time polymerase chain reaction (PCR) was used to detect the expressions of miRNA-181a in both serum and liver tissue and the expression of TLR4 mRNA in liver tissue .Western blot was used to detect the expression of TLR4 protein in liver tissue . The fibrosis-4 (FIB-4 ) index and aspartate aminotransferase-to-platelet ratio index (APRI ) were calculated for noninvasive evaluation of fibrosis staging .One-way ANOVA ,Mann-Whitney U test ,spearman correlation analysis and receiver operating characteristic (ROC) curve were used for statistical analysis .Results Forty CHB patients were includedin this study ,including 7 with S0 ,6 with S1 ,14 with S2 ,7 with S3 and 6 with S4 .Serum levesl of miRNA-181a (2-ΔΔCt ) in paitents with S0-4 were 1 .00 ± 0 .00 ,0 .68 ± 0 .08 ,1 .60 ± 0 .43 ,2 .32 ± 0 .40 , and 1 .81 ± 0 .22 ,respectively ,showing an overall upward trend (F=207 .242 ,P< 0 .01) and a positive correlation with the severity of HF (r= 0 .754 , P< 0 .01) .The expressions of miRNA-181a ,TLR4 mRNA and TLR4 protein in liver tissues showed an overall increasing trend from S 0 to S4 (F=207 .242 , 110 .390 and 57 .030 ,respectively ,all P<0 .01) .The expression of miRNA-181a in liver tissue showed a positive correlation with both the expression of TLR4 protein in liver tissue and the severity of HF (r=0 .673 and 0 .911 ,respectively ,both P< 0 .01) .There was no significant difference of APRI scores between the severe (S3-4) and non-severe (S0-2) HF groups (Z= -1 .401 ,P>0 .05) .The serum level of miRNA-181a was superior to FIB-4 index for evaluation of the severe HF (S3-4) ,with areas under the ROC curve (AUROC ) of 0 .887 and 0 .695 , respectively , and accuracy of 85 .0％ and 60 .0％ , respectively .Conclusions miRNA-181a may be involved in the regulation of TLR4 signaling pathway so that to affect the progression of HF in CHB patients ,which may be a potential new target for the prevention and early treatment of HF and a non-invasive serum marker for evaluation of HF .

Piceatannol induced apoptosis through up-regulation of microRNA-181a in melanoma cells

BACKGROUND: Melanoma took top position among the lethal cancers and, despite there have been some great attempts made to increase the natural life of patients with metastatic disease, long-lasting and complete remissions are few. Piceatannol, owns the similar function as resveratrol, has been defined as an anti-cancer agent playing important role in inhibition of proliferation, migration and metastasis in various cancer. Thus, we aim to investigate the anti-cancer effect and mechanisms of piceatannol in melanoma cells. METHODS: Melanoma cell lines WM266-4 and A2058 were treated either with or without piceatannol. Cell viability and cell apoptosis were assessed by using MTT and Annexin V/PI assay, respectively. Cells were transfected with specific miRNA using Lipfectamine 2000. miRNA bingding ability to 3'-UTR region within specific gene was assed by firefly luciferase analysis. Gene and protein expression was eveluated by qRT-PCR and western blot analysis, respectively. RESULTS: Our study showed that piceatannol inhibited WM266-4 and A2058 cells growth and induced apoptosis. Totally, 16 differentially expressed miRNAs were screened out including 8 up-regulated and 8 down-regulated miRNAs. Expression level of miR-181a is significantly higher in piceatannol-treated cells than normal control and is lower in melanoma cancer tissues than its adjacent normal tissues. Bcl-2 is a target gene of miR-181a. Moreover, silencing of miR-181a reverses the decrease of cell viability induced by piceatannol in WM266-4 and A2058 cells. Taken together, present study uncovered the ability of piceatannol to repress melanoma cell growth and clarified the contribution of miR-181a in the anticancer role of piceatannol. CONCLUSION: The present study proposes that piceatannol can be taken into account to be a hopeful anticancer agent for melanoma.

Expression of miRNA-181 a in human lung adenocarcinoma cells and its effect on cell function / 中国病理生理杂志

AIM:To study the expression of microRNA (miRNA)-181a in different human lung adenocarcinoma cell lines, and to investigate the effect of miRNA-181a on cell function and its mechanism in human lung adenocarcinoma drug resistant cell A549/DDP.METHODS:Real-time PCR was used to detect the expression of miRNA-181a in BEAS-2B cells, A549 cells and A549/DDP cells.The A549/DDP cells were transfected with pGenesil-miRNA-181a eukaryotic ex-pression plasmid.At the same time, the untransfection group and negative transfection group were also set up .The expres-sion of miRNA-181a, cell viability, cell growth inhibition and apoptosis rate during cis-diamminedichloroplatinum ( DDP) treatment, cell cycle, cell invasion, the protein expression of miRNA-181a target genes bcl-2 and p53 in the A549/DDP cells were determined by real-time fluorescence quantitative PCR , MTT assay, flow cytometry, Transwell method and West-ern blot, respectivly.RESULTS:The expression of miRNA-181a in A549 cells and A549/DDP cells was significantly lower than that in BEAS-2B cells, and the lowest expression level was observed in A 549/DDP cells (P<0.05).The expression of miRNA-181a in A549/DDP cells was significantly increased after transfection with pGenesil-miRNA-181a (P<0.05).The cell viability, cell cycle and invasion ability of the A549/DDP cells were inhibited after miRNA-181a transfection (P <0.05).The cell growth inhibition rate and apoptotic rate of the A 549/DDP cells were increased (P<0.05).The expression of Bcl-2 was reduced, but the expression of P53 was increased after transfection with miRNA-181a in A549/DDP cells (P<0.05).CONCLUSION: miRNA-181a may be correlated with the development of human lung adenocarcinoma .miRNA-181a can serve as a new target for treatment of lung cancer .

A pilot study on the relationship between miR-181a and RGCs in retinal ischemia-reperfusion injury model / 中华实验眼科杂志

Background Retinal ischemia-reperfusion (RIR) injury is a common pathologic change.Its mechanism has not been identified.Objective This study was to investigate the relationship of microRNA-181a (miR-181a) ,tumor necrosis factor-α (TNF-α) and retinal ganglial cells (RGCs) in RIR injury.Methods RIR models were induced in 68 rats,then the rats were randomly divided into control group and RIR groups,including 0hour group,24-hour group and 72-hour group by random number table.Predicted target gene TNF-α was chosen,according to M iRanda,Targetscan and miRBase databases.Immunofluorescent labeling, Western blot and quantitative real-time PCR were used to identify the expression levels of miR-181a,TNF-α and RGCs.Immunofluorescent labeling of RGCs in retinal flat mounts was analyzed for RGCs counts.Results Compared with the control group, RGCs densitiy was obviously decreased in 24-hour and 72-hour RIR groups (P＜0.001).The expression level of mir-181a significantly decreased with reperfusion time in the RIR groups (P＜0.05).Futhermore, the expression level of miR181a was positively correlated with RGCs numbers (r=0.995 ,P=0.005).TNF-α and miR-181a were mainly located in inner layers of retina.As opposed to the changes in RGCs numbers and miR-181a expression,TNF-α in 24-hour group was obviously higher than that of the 0-hour group, though there was no statistical significance in overall correlation analysis.Conclusions In RIR,miR-181a may be involved in regulating RGCs apoptosis.TNF-α may be a target gene of miR-181 a.Interventions within 24 hours after reperfusion might be critical.Further study of miR181 a may help to explore new molecular targets for neuroprotection treatment.