An integrated analysis of dysregulated SCD1 in human cancers and functional verification of miR-181a-5p/SCD1 axis in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC), one of the most lethal cancers, has become a global health issue. Stearoyl-coA desaturase 1 (SCD1) has been demonstrated to play a crucial role in human cancers. However, pan-cancer analysis has revealed little evidence to date. In the current study, we systematically inspected the expression patterns and potential clinical outcomes of SCD1 in multiple human cancers. SCD1 was dysregulated in several types of cancers, and its aberrant expression acted as a diagnostic biomarker, indicating that SCD1 may play a role in tumorigenesis. We used ESCC as an example to demonstrate that SCD1 was dramatically upregulated in tumor tissues of ESCC and was associated with clinicopathological characteristics in ESCC patients. Furthermore, Kaplan-Meier analysis showed that high SCD1 expression was correlated with poor progression-free survival (PFS) and disease-free survival (DFS) in ESCC patients. The protein-protein interaction (PPI) network and module analysis by PINA database and Gephi were performed to identify the hub targets. Meanwhile, the functional annotation analysis of these hubs was constructed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Functionally, the gain-of-function of SCD1 in ESCC cells promoted cell proliferation, migration, and invasion; in contrast, loss-of-function of SCD1 in ESCC cells had opposite effects. Bioinformatic, QPCR, Western blotting and luciferase assays indicated that SCD1 was a direct target of miR-181a-5p in ESCC cells. In addition, gain-of-function of miR-181a-5p in ESCC cells reduced the cell growth, migratory, and invasive abilities. Conversely, inhibition of miR-181a-5p expression by its inhibitor in ESCC cells had opposite biological effects. Importantly, reinforced SCD1 in miR-181a-5p mimic ESCC transfectants reversed miR-181a-5p mimic-prevented malignant phenotypes of ESCC cells. Taken together, these results indicate that SCD1 expression influences tumor progression in a variety of cancers, and the miR-181a-5p/SCD1 axis may be a potential therapeutic target for ESCC treatment.

Keywords: ESCC; MiR-181a-5p; Stearoyl-CoA desaturase.

Graphical abstract

Fig. 1

SCD1 acted as a diagnostic factor in many human cancers. (A and B) SCD1 expression in normal tissues (from GTEx database) and in single cells (single-cell types database from HPA website) were analyzed by radar diagrams. (C and D) The SCD1 expression level in unpaired adjacent normal and tumor tissues from TCGA with GTEx databases are shown. T: tumor; N: normal. * p < 0.05.

Fig. 2

The diagnostic feature of SCD1 was estimated by ROC analysis. The aberrant SCD1 expression with upregulation in 13 cancers from TCGA and GTEx databases was analyzed by ROC curve. AUC > 0.9 was considered a high diagnostic value, 0.9 ≥ AUC > 0.7 was median, and 0.7 ≥ AUC > 0.5 was low.

Fig. 3

The genetic alteration and DNA methylation of SCD1 in human cancers. (A) The genetic alteration frequency of SCD1 from cBioPortal database is depicted by histograms. (B) The map of correlations between SCD1 and CNAs is presented. (C) The SCD1 promoter methylation in normal and tumor tissues were determined based on the UALCAN database. T: tumor; N: normal. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 4

Protein expression, protein-protein interaction, and functional enrichment assay of SCD1 in multiple human cancers. (A) The SCD1 protein expression in normal and tumor tissues using CPTAC samples from UALCAN were presented. (B) Construction of PPI network based on Gephi software. The bubble plots of GO enrichment such as (C) biological process, (D) cellular components, and (E) molecular function were performed by DAVID tool. MIS-H: microsatellite instability high; MIS-L: microsatellite instability low. T: tumor; N: normal. **, p < 0.01; ***, p < 0.001.

Fig. 5

The clinical features and outcomes of SCD1 expression in esophageal cancer. The correlation between SCD1 expression and pathologic characteristics such as (A) sample types, (B) cancer stages, (C) histology types, (D) patients’ race, (E) gender, and (F) weight in esophageal cancer were determined from the UACAN database. (G and H) Kaplan-Meier survival curves of the association between SCD1 expression and survival probability, including DFS and PFS in esophageal cancer from the cBioportal database were analyzed. T: tumor; N: normal. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 6

SCD1 expression modulates cell proliferation in ESCC. (A) The protein levels of SCD1 in SCD1-overexpressing KYSE270 and TE11 cells were determined by Western blotting assay. (B and C) The CCK-8 and colony formation assays were adopted for assessment of cell growth in gain-of-function of SCD1 in ESCC cells. (D) The mRNA and protein levels of SCD1 were evaluated in loss-of-function of SCD1 in ESCC cells. (E and F) The CCK-8 and colony formation assays were performed to assess the cell growth ability in loss-of-function of SCD1 in ESCC cells. * p < 0.05, *** p < 0.001.

Fig. 7

The effect of SCD1 on migration, and invasion in ESCC cells. (A and B) The migratory abilities of KYSE270 and TE11 cells with SCD1 overexpression were detected by wound healing assay. (C and D) The migratory abilities of KYSE270 and TE11 cells with SCD1 knockdown were examined by wound healing assay. (E and F) Transwell assays were used to verify the effects of gain- and loss-of-function of SCD1 on migration and invasion of ESCC cells. (G) The mRNA levels of N-cadherin, vimentin, and E-cadherin were examined in SCD1-overexpressing and SCD1-depleted ESCC cells by QPCR. Scale bar: 100 µm; ** p < 0.01, *** p < 0.001.

Fig. 8

miR-181a-5p directly regulates SCD1 in ESCC cells. (A) The Venn diagram showing target miRNAs of SCD1, as predicted from five miRNA databases. The bioinformatics analysis predicted that the 3′UTR sequence of SCD1 was complementary to the sequence of miR-181a-5p. The binding sequences of SCD1 were mutated (in red color). (B and D) The mRNA and protein expression patterns of SCD1 were assessed in ESCC cells transfected with miR-181a-5p mimics or mimic control by QPCR and Western blotting assays. (C) Kaplan-Meier survival curve of miR-181a-5p in patients with ESCC is shown. (E and F) The mRNA and protein expression patterns of SCD1 were assessed in ESCC cells transfected with miR-181a-5p inhibitor or inhibitor control by QPCR and Western blotting assays. (G) The luciferase report assay revealed that SCD1 was a direct target of miR-181a-5p. * p < 0.05, *** p < 0.001.

Fig. 9

miR-181a-5p/SCD1 axis regulates ESCC progression. (A and C) The cell growth and motility abilities were assessed in ESCC cells transfected with miR-181a-5p mimic or mimic control. (B and D). The cell growth and motility abilities of KYSE270 and TE11 were assessed in cells transfected with miR-181a-5p inhibitor or inhibitor control. (E to G) The CCK8 and Transwell assays with or without Matrigel were used to evaluate the growth and motility effects of miR-181a-5p mimic/ESCC transfectants with SCD1 overexpression. * p < 0.05, ** p < 0.01, *** p < 0.001.