- progression in HNSCC. - This study was to investigate its expression pattern, clinicopathological significance and biological roles in HNSCC.. - Methods: HNSCC data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) was used to indicate the detailed expression pattern and outcome association of FOXD1, while Western Blot assay to detect FOXD1 level in a panel of HNSCC cell lines as well as immunocytochemistry to explore FOXD1 protein abundance and sublocation. - Bioinformatic analysis to find out which biological function and cancer-related pathways of FOXD1 associated genes involved in.. - siRNA-mediated FOXD1 knock-down significantly inhibited cell proliferation, migration and invasion and induced apoptosis in HNSCC cells. - Further analysis of GSEA, GO and KEGG showed that FOXD1 expression was significantly associated with oncological function and cancer-related pathways.. - To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - The main inducers of HNSCC include environmental risk, virus infection and gene mutation. - Although clinical treatments of HNSCC patients has significantly been improved and improved the patient’s quality of life over the past decades, the prognosis of HNSCC is still unsatisfying, with the 5-year survival rate remaining around . - Numerous studies thus far have shown that FOXD1 as a key role in the occurrence, development and malignant regulation of multitudinous human malignance and its abnormal up-regulation was obviously related to cell prolifera- tion, tumor metastasis and invasion as well as poor prognosis [5, 6]. - Abnormal overexpression of FOXD1 gene may be as an oncogene, via facilitating cell proliferation and transformation, promoting the process of EMT and inhibiting cell apoptosis [7–9]. - All of the above results indicate that FOXD1 is prob- ably a variety of new potential cancer-causing genes in human cancer. - Although FOXD1 is thought to be a key oncogene in tumorigenesis, whether it takes an pivotal part in the carcinogenesis of HNSCC remains unknown.. - Therefore, in the present study, we collected relevant HNSCC materials from the clinical specimens, TCGA and GEO databases and executed the current systematic analysis to study the potential molecular regulatory mechanisms and clinical significance of FOXD1 in HNSCC. - At the same time, the biological function of FOXD1 in HNSCC was further explored by combining small interference RNA and bioinformatics analysis techniques.. - A panel of HNSCC cell lines including Cal27, Fadu, SCC9, SCC25, HN4, HN6 and human immortalized oral mucosa keratinocytes (HOK) were used in this study.. - Bioinformatics analysis of HNSCC. - FOXD1 level and associated clinical data of HNSCC were downloaded from 2 publicly datasets: GEO (https://www.ncbi.nlm.nih.gov/gds/) and TCGA (https://. - FOXD1 correlated-gene chart (.tsv) was obtained from cBioPortal(https://www.cbioportal.org. - While as displayed in Figure1 A-D, data min- ing and questioning from GSE6631, GSE12452, GSE25099 and GSE30784 indicated that FOXD1 mRNA was significantly higher in HNSCC samples compared with the normal group, respectively. - These data indicate that FOXD1 high expression may be related to the ma- lignant regulation of HNSCC. - Analysis of FOXD1 gene expression in HNSCC clinical samples and its clinical significance. - Next, to further reveal the expression pattern of FOXD1 in HNSCC clinical specimens as well as its relationship with clinicopathological parameters, we semi- quantitatively detected FOXD1 protein expression pat- tern in 110 patients with primary HNSCC by immuno- histochemical staining. - 2, FOXD1 showed a positive staining mainly in nucleus in HNSCC paraffin sections, whereas weak/. - negative staining was identified in the normal oral mu- cosa and the stroma of HNSCC. - Based on our IHC- scoring system, FOXD1 expression in HNSCC/normal mucosa was classified. - Thus, the expression of FOXD1 protein can be classified as high (n = 61) or low expres- sion group (n = 49) in HNSCC clinical samples and negative (n = 8), high (n = 1) or low (n = 4) expression in normal HNSCC clinical specimens. - Therefore, these data confirmed that the FOXD1 protein was highly expressed in HNSCC (P <. - Aberrant overexpression of FOXD1 in HNSCC patients associated with poor outcome. - To explore prognostic significance of FOXD1 abundance in HNSCC patients, we analysis the relationship between its protein level and clinical prognostic. - 1 Overexpression of FOXD1 mRNA in five HNSCC datasets. - A-E: The mRNA levels of FOXD1 (normalized and log2-transformed) were compared between HNSCC samples and normal counterparts in GSE6631 (A), GSE12452 (B), GSE25099(C), GSE30784(D)and TCGA-HNSC(E) datasets. - To further evaluate the prognostic value of FOXD1 abundance in HNSCC, both univariate and multivariable survival analyses were executed by cox proportional hazards regression model. - FOXD1 knockdown inhibited migration and invasion, proliferation and facilitated apoptosis in HNSCC cells To explore the cancer-promoting effects in HNSCC, we first examined the FOXD1 abundance in several HNSC C cell lines and carried out the loss-of-function assay by siRNA modulated knockdown. - 4A, the expression of FOXD1 protein in all HNSCC cell lines was obviously higher than that in immortalized non-tumorigenic cells (HOK). - Two inde- pendent siRNAs targeting human FOXD1(siFOXD1–1, Table 1 Expression of FOXD1 and its associations with. - A, D: Representative negative staining of FOXD1 in normal oral epithelial (100× A, 200× D). - C, F: Representative high expression of FOXD1 in primary human HNSCC sample (100× C, 200× F). - siFOXD1–2) were brought in cal27 and Fadu cells to de- tect the changes of FOXD1 abundance and cell pheno- type. - 4B, the expression of FOXD1 protein decreased significantly after transfection with siRNA, which confirmed the effectiveness of our gene. - 3 High FOXD1 expression positively associates with reduced overall survival rates in HNSCC patients. - A-A, B: Overall survival (A) and disease- free survival (B) analyses of HNSCC patients with high or low expression of FOXD1 were estimated by IHC. - C, D: Overall survival (C) and disease- free survival (D) plot of FOXD1 in TCGA-HNSC patients downloaded from cBioPortal(https://www.cbioportal.org. - FOXD1 expression (high, low . - The migration and invasion ability of FOXD1 knockdown cells were also exam- ined by wound healing and Transwell experiments, respectively. - These results suggested that FOXD1 is involved in the regulation of the ma- lignant phenotype of HNSCC and may be a valuable potential therapeutic target.. - 4 FOXD1 knockdown inhibits cell migration and invasioncell proliferation, cell migration and invasion and triggers apoptosis in HNSCC cells.. - A: Endogenous FOXD1 protein expression was measured in a panel of HNSCC cell lines as compared to normal human oral keratinocytes (HOK).. - 5A) and performing gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and the data suggests that FOXD1-positive-corrlated genes enriched in numerous cancer related biofunc- tion and pathways such as cell-substrate junction and focal adhesion(Fig. - In conclusion, combined with cell assay in vitro and bioinformatics results extremely support the idea that FOXD1 is an newly oncogene in HNSCC.. - Previous studies have shown that FOXD1 may be an oncogene related to tumor develop- ment and act as a potential diagnostic biomarker and. - 5 The biofunction of FOXD1 correlation genes. - A: Schematic diagram of the numbers of FOXD1-associated-genes with Spearman ’ s correlation >. - Herein, this study revealed the FOXD1 expression pattern in HNSC C, uncovered its prognostic and clinicopathological sig- nificance and also revealed its the oncogenic roles of FOXD1 by the loss-of function method and bio- informatics analysis. - In particular, dys- regulation of the FOX family have been proved to be involved in nearly all stages of HNSCC tumorigen- esis [23]. - Here, we found that FOXD1 is significantly ele- vated in most HNSCC specimens as proofed by ob- viously overexpressed FOXD1 mRNA in GEO and TCGA datasets as well as up-regulation of FOXD1 protein in our HNSCC patients cohort. - B: GO (upper panel) and KEGG (low panel) analysis for the genes in low- risk group.C-F: The most involved significant pathways via GSEA. - abnormal pattern of FOXD1 overexpression in HNSCC. - Previous findings have shown the clinical significance of FOXD1 abundance in various human cancers. - We further established overexpression pattern of FOXD1 and shown that its overexpression significantly associated with lymph node metastasis and clinical stage in HNSCC, while there was no significant correlation between FOXD1 expression and other clinicopathologic parameters.. - Moreover, up-regulation of FOXD1 was obviously as- sociated with the decrease of survival rate via Kaplan-Meier survival data analysis, thus suggesting that FOXD1 might be a valuable prognostic bio- marker for HNSCC. - Therefore, Multivariate survival analysis using cox proportional hazards regression model results confirmed that FOXD1 expression was an independent prognostic factor for HNSCC. - Accumulating evidence has revealed that FOXD1 is vitally participated in tumorigenesis by facilitating cell migration, invasion, proliferation, angiogenesis and inhibiting apoptosis [9, 26]. - Consistent with this, our in vitro loss-of-function experiment results indi- cate that FOXD1 knockdown leads to a diminished in proliferation, migration and invasion in HNSCC cells. - Additionally, bioinformatics analysis results fur- ther indicated that FOXD1 was involved significant pathways included KEGG Pathways in cancer, EMT, Focal adhesion, Apoptosis, TGF-beta signaling, Cell cycle etc. - Consistently, previous studies have re- vealed that FOXD1 facilitates cell proliferation and inhibits apoptosis by promoting the expression of PIK2 in colorectal cancer [24]. - Fengping Pan and his colleagues revealed that FOXD1 promoted CRC cell malignant phenotypes by activation of the ERK 1/2 signaling pathway [6]. - Of note, the inhibition of FOXD1 expression affected cell related malignant phenotypes such as cell prolif- eration, cell migration and invasiveness, cell apop- tosis and ultimately leads to tumor regression and reduction of tumor metastasis. - Taken together, com- bined with bio-informatics analysis and the results of our findings and others strongly favors the notion that FOXD1 probably functions as a hypothetical oncogene by facilitating cancer cell proliferation, mi- gration and invasion. - Suppression of FOXD1. - expression via genetic method might hold transla- tional promise in the treatment of HNSCC.. - In conclusion, our all results indicate that a subgroup of HNSCC patients who FOXD1 aberrantly up-regulation in an important and reveals its pro-oncogenic role in promoting the occurrence and development of HNSCC.. - However, there is still a large gap between the role of FOXD1 in the development of HNSCC and the effective treatment of FOXD1.. - The online version contains supplementary material available at https://doi.. - org/10.1186/s . - A-F: The mRNA levels of FOXD1 (normalized and log2-transformed) from TCGA-HNSCC datasets were compared between clinical subtypes: Clinical stage(I- IV, A), Patho- logical grade(I-III, B), HPV infection status(C), Tumor size(T1-T4, D), Cervical nodal metastasis(E), Distant metastasis(F). - https://doi.org/10.3322/caac.21492.. - https://doi.org/10.1038/s . - https://doi.org/10.3322/caac.21551.. - https://doi.org/10.3892/ijo.25.5.1495.. - https://doi.org/10.3892/or.2017.5344.. - https://doi.org CAN-15-2860.. - https://doi.org/10.1002/ijc.31799.. - CXCL5 induces tumor angiogenesis via enhancing the expression of FOXD1 mediated by the AKT/NF-kappaB pathway in colorectal cancer. - https://doi.org/10.1038/ncomms4197.. - https://doi.org/10.1016/j.bbrc . - https://doi.org/10.1016/j.molonc . - https://doi.org/10.1089/omi.2011.0118.. - Increased expression of FOXD1 is associated with cervical node metastasis and unfavorable prognosis in oral squamous cell carcinoma. - https://doi.org/10.1111/jop.13098.. - https://doi.org/10.3390/cells9010258.. - https://doi.org/10.1016/j.bbcan . - https://doi.org/10.3390/cancers11121897.. - https://doi.org/10.1038/. - https://doi.org/10.1038/s z.. - https://doi.org/10.1111/ajco.13451.. - https://doi.org/10.3233/CBM-1 91311.. - https://doi.org/10.1016/j.biopha . - https://doi.org/10.3390/cancers12092690.
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