RESUMEN
SUMMARY: This study is to investigate the role and mechanism of RGD peptide in laryngeal cancer stem cells (CSCs). Laryngeal cancer CD133+Hep-2 CSCs were sorted by flow cytometry. RGD peptide was co-cultured with sorted laryngeal CSCs. Cell proliferation was detected with CCK-8 assay. The mRNA levels of VEGF/VEGFR2/STAT 3/HIF-1α were detected with RT-PCR. The proteins of VEGF/ VEGFR2/STAT 3/HIF-1α were detected with Western blot. The sorted CSCs were inoculated into nude mice. Tumor volume was measured. Integrin αvβ3 expression in tumor tissues was analyzed with immunohistochemistry. The results showed that the ratio of CD133+ CSCs to the total number of cells was 1.34±0.87 %, while CD133-non-tumor stem cells accounted for 95.0±5.76 %. The sorted cancer stem cells grew well. The RGD peptide significantly inhibited the proliferation of CD133+Hep-2 laryngeal CSCs in a dose-dependent manner. The RGD peptide significantly inhibited the mRNA of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a concentration-dependent manner. Consistently, the RGD peptide significantly inhibited the protein expression of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a dose-dependent manner. At the same time, in vivo tumor experiments showed that the RGD peptide significantly inhibited tumor volume but not the body weight. Furthermore, RGD peptide significantly inhibited the expression of tumor angiogenesis-related protein integrin αvβ3. Our findings demonstrate that RGD peptide inhibits tumor cell proliferation and tumor growth. The underlying mechanism may that RGD inhibits tumor angiogenesis-related signaling pathways, thus affecting the tumor angiogenesis, and decreasing the progression of human laryngeal CSCs.
Este estudio se realizó para investigar el papel y el mecanismo del péptido RGD en las células madre del cáncer de laringe (CSC). Las CSC CD133+Hep-2 de cáncer de laringe se clasificaron mediante citometría de flujo. El péptido RGD se cocultivó con CSC laríngeas clasificadas. La proliferación celular se detectó con el ensayo CCK-8. Los niveles de ARNm de VEGF/VEGFR2/ STAT 3/HIF-1α se detectaron con RT-PCR. Las proteínas de VEGF/ VEGFR2/STAT 3/HIF-1α se detectaron con Western blot. Las CSC clasificadas se inocularon en ratones nudos. Se midió el volumen del tumor. La expresión de integrina αvβ3 en tejidos tumorales se analizó con inmunohistoquímica. Los resultados mostraron que la proporción de CSC CD133+ con respecto al número total de células fue de 1,34 ± 0,87 %, mientras que las células madre no tumorales CD133 representaron el 95,0 ± 5,76 %. Las células madre cancerosas clasificadas crecieron bien. El péptido RGD inhibió significativamente la proliferación de CSC laríngeas CD133+Hep-2 de una manera dependiente de la dosis. El péptido RGD inhibió significativamente el ARNm de VEGFR2, STAT3 y HIF-1α en CSC laríngeas de manera dependiente de la concentración. De manera consistente, el péptido RGD inhibió significativamente la expresión proteica de VEGFR2, STAT3 y HIF-1α en CSC laríngeas, de manera dependiente de la dosis. Al mismo tiempo, los experimentos con tumores in vivo mostraron que el péptido RGD inhibía significativamente el volumen del tumor pero no el peso corporal. Además, el péptido RGD inhibió significativamente la expresión de la proteína integrina αvβ3 relacionada con la angiogénesis tumoral. Nuestros hallazgos demuestran que el péptido RGD inhibe la proliferación de células tumorales y el crecimiento tumoral. El mecanismo subyacente puede ser que RGD inhiba las vías de señalización relacionadas con la angiogénesis tumoral, afectando así la angiogénesis tumoral y disminuyendo la progresión de las CSC laríngeas humanas.
Asunto(s)
Animales , Ratones , Oligopéptidos/metabolismo , Células Madre Neoplásicas , Neoplasias Laríngeas , ARN Mensajero/antagonistas & inhibidores , Inmunohistoquímica , Western Blotting , Cartilla de ADN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Integrina alfaVbeta3/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Proliferación Celular , Citometría de Flujo , Neovascularización PatológicaRESUMEN
INTRODUCTION AND AIM: This study aimed to confirm the association of the transmembrane 6 superfamily member 2 (TM6SF2) E167K variant with non-alcoholic fatty liver disease (NAFLD) and the degree of steatosis, as well as the additive effect of body mass index (BMI) or the patatin-like phospholipase domain-containing protein 3 (PNPLA3) I148M and TM6SF2 E167K variants in NAFLD. MATERIALS AND METHODS: A total of 158 NAFLD patients and 158 matched controls were recruited. Steatosis was classified as mild, moderate and severe by FibroScan. Associations between the TM6SF2 E167K variant and NAFLD as well as clinical parameters were evaluated. RESULTS: Although the frequency of the T allele was low in the Chinese population (MAF=7.4%), there was still a significant association between the E167K variant and NAFLD (odds ratio=3.379, 95% confidence interval: 1.500-7.612, P=0.003). In particular, the TM6SF2 genotype was also associated with the degree of steatosis (P=0.023). The TM6SF2 variant was associated with increased alanine aminotransferase (ALT) but no other clinical parameters, such as aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lipids. Notably, we also found that an additive effect of the TM6SF2 E167K and PNPLA3 I148M variants in NAFLD. Furthermore, we did not identify an association between the TM6SF2 E167K variant and NAFLD in the non-obese population. CONCLUSION: The TM6SF2 E167K variant was associated with NAFLD in northeast China, and there was an interaction between the PNPLA3 I148M and TMS6F2 E167K variants in NAFLD.