ABSTRACT
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.
Subject(s)
Animals , Mice , Oligopeptides/metabolism , Neoplastic Stem Cells , Laryngeal Neoplasms , RNA, Messenger/antagonists & inhibitors , Immunohistochemistry , Blotting, Western , DNA Primers , Reverse Transcriptase Polymerase Chain Reaction , Integrin alphaVbeta3/antagonists & inhibitors , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/genetics , Cell Proliferation , Flow Cytometry , Neovascularization, PathologicABSTRACT
@#[Abstract] Objective: To explore the expression and regulation mechanism of Dickkopf-1 (DKK1) in head and neck squamous cell carcinoma (HNSCC) tissues. Methods: Based on the TCGA database, the relationship of DKK1 expression in HNSCC tissues and its methylation site with patients’prognosis was analyzed. GO and KEGG gene enrichment method were used to analyze the signaling pathways of DKK1 enrichment. STRING was used to analyze the interaction between DKK1 protein and other proteins. TargetScan was used to analyze the miRNAs that regulate the expression of DKK1, and the transcription factors of DKK1 were analyzed with the TRRUST website. Results: DKK1 gene was highly expressed in HNSCC tissues (P<0.01), and its expression level was significantly correlated with the HPV status, age, pathological grade, and clinical stage of patients (all P<0.05); the prognosis of HNSCC patients with high DKK1 expression was poorer than those with low DKK1 expression (P<0.01). There were 19 methylation sites in DKK1, 12 of which were significantly different between cancer tissues and normal tissues (P<0.05), and 11 sites were significantly related to the prognosis of HNSCC (P<0.05). In addition, miRNA, circRNA, lincRNA and transcription factors, etc. also participated in the regulation of DKK1. A total of 5 DKK1-related PPI networks that may involve in the occurrence, development, invasion and metastasis of HNSCC were obtained. Conclusion: DKK1 is highly expressed in HNSCC tissues and is a risk factor for poor prognosis of HNSCC patients. DKK1 plays an important role in the pathogenesis of HNSCC and is expected to become a potential target for HNSCC treatment.