RESUMO
To investigate the effect of piperine on the disorder of glucose metabolism in the cell model with insulin resistance (IR) and explore the molecules mechanism on intervening the upstream target of AMPK signaling pathway. The insulin resistance models in HepG2 cells were established by fat emulsion stimulation. Then glucose consumption in culture supernatant was detected by GOD-POD method. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of leptin(LEP) and adiponectin(APN) in culture supernatant; Real-time quantitative PCR was used to assess the mRNA expression of APN and LEP; and the protein expression levels of LepR, AdipoR1, AdipoR2 and the activation of AMPK signaling pathway were detected by Western blot analysis. The results showed that piperine, rosiglitazone and AMPK agonist AICAR could significantly elevate the glucose consumption in insulin resistance cell models, enhance the level of APN, promote APN mRNA transcripts and increase the protein expression of Adipo receptor. Meanwhile,AMPKα mRNA and Ñ-AMPKα protein expressions were also increased in piperine treated cells, but both LEP mRNA expression and LepR protein expressions were decreased in piperine treated group. The results indicated that piperine could significantly ameliorate the glucose metabolism disorder in insulin resistance cell models through regulating upstream molecules (APN and LEP) of AMPK signaling pathway, and thus activate the AMPK signaling pathway.
Assuntos
Alcaloides/farmacologia , Benzodioxóis/farmacologia , Resistência à Insulina , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais , Quinases Proteína-Quinases Ativadas por AMP , Glucose/metabolismo , Células Hep G2 , HumanosRESUMO
Aberrantly expressed miRNAs widely participate in the signaling cascades of colorectal carcinogenesis. The present study aimed to identify a potential miRNA that serves as effective biomarker for colorectal cancer (CRC). The expression of estrogen receptorß (ERß) was explored using immunohistochemistry. The possible miRNAs targeting ERß were predicted by TargetScan, and their expression patterns were validated using real time PCR. Dual luciferase reporter assays were performed to determine the potential binding of miR-129 in the 3' untranslated region (3'UTR) of ERß. In vitro scratch assays and flow cytometry assays were conducted to determine the role of miR-129 on colon cancer cell migration and apoptosis. Proteins related to cell proliferation were determined using western blots. Compared with adjacent non-cancer tissues, the protein level of ERß was significantly decreased in CRC tissues, and compared with NC the level of miR-129 was significantly increased in blood and tissue samples. Dual luciferase reporter assays demonstrated that ERß was a direct target gene of miR-129. Further study showed that inhibition of miR-129 decreases HCT116 cell migration and enhances cell apoptosis. More importantly, we found that the silencing of ERß significantly decreased the activation of caspase3 but increased the protein expression of PCNA. Interestingly, miR-129 inhibitor-induced protein expression pattern changes could be reversed by the siRNA targeting ERß. The high expression level of circulating miR-129 in the tissue and blood samples of CRC patients contributes to aberrant colon cancer cell proliferation and migration mainly by targeting ERß.
Assuntos
Proliferação de Células/genética , Neoplasias Colorretais/genética , Receptor beta de Estrogênio/genética , MicroRNAs/sangue , Regiões 3' não Traduzidas/genética , Apoptose/genética , Biomarcadores Tumorais/sangue , Movimento Celular/genética , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , RNA Interferente Pequeno/administração & dosagemRESUMO
To investigate the effect of piperine on the disorder of glucose metabolism in the cell model with insulin resistance (IR) and explore the molecules mechanism on intervening the upstream target of AMPK signaling pathway. The insulin resistance models in HepG2 cells were established by fat emulsion stimulation. Then glucose consumption in culture supernatant was detected by GOD-POD method. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of leptin(LEP) and adiponectin(APN) in culture supernatant; Real-time quantitative PCR was used to assess the mRNA expression of APN and LEP; and the protein expression levels of LepR, AdipoR1, AdipoR2 and the activation of AMPK signaling pathway were detected by Western blot analysis. The results showed that piperine, rosiglitazone and AMPK agonist AICAR could significantly elevate the glucose consumption in insulin resistance cell models, enhance the level of APN, promote APN mRNA transcripts and increase the protein expression of Adipo receptor. Meanwhile,AMPKα mRNA and р-AMPKα protein expressions were also increased in piperine treated cells, but both LEP mRNA expression and LepR protein expressions were decreased in piperine treated group. The results indicated that piperine could significantly ameliorate the glucose metabolism disorder in insulin resistance cell models through regulating upstream molecules (APN and LEP) of AMPK signaling pathway, and thus activate the AMPK signaling pathway.
