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. 2020 Mar 3;4:5.
doi: 10.1038/s41698-020-0110-5. eCollection 2020.

Annotation and Cluster Analysis of Long Noncoding RNA Linked to Male Sex and Estrogen in Cancers

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Free PMC article

Annotation and Cluster Analysis of Long Noncoding RNA Linked to Male Sex and Estrogen in Cancers

Shouping Liu et al. NPJ Precis Oncol. .
Free PMC article

Abstract

The sex difference in cancer occurrence is a consistent finding in cancer epidemiology. Several solid tumors, including lung cancer, colorectal cancer, hepatic carcinoma, and renal carcinoma, are generally more common in males. Although sexual dimorphism is attributed to hormonal or behavioral differences, evidence for the function of lncRNA is lacking in sex-specific cancers. We show here that LINC00263 is one of the most dysregulated lncRNAs in lung adenocarcinomas and is upregulated in lung adenocarcinoma, colorectal cancer, and renal carcinoma, especially in male patients compared to females. LINC00263 functions as an oncogene by promoting translocation of p65 into the nucleus to activate the NF-κB-signaling pathway through interaction with IKKα in the cytoplasm. The expression of LINC00263 is strongly correlated with ESR1, and it is decreased after treatment with estrogen. Ligand-activated ER could inhibit the function of LINC00263 by inhibiting NF-κB from cytoplasmic translocation into the nucleus. The inhibitory effect of estrogen on LINC00263 indicates its differential expression in male and female patients. Our findings indicate that LINC00263 is linked to male sex and estrogen as an oncogene, and these findings might help in the exploration of the mechanisms of differential gene regulation in sex-specific cancers.

Keywords: Cancer genomics; Computational biology and bioinformatics.

Conflict of interest statement

Competing interestsThe authors declare no competing interests. This manuscript has been read and approved by all authors and is not under consideration for publication elsewhere.

Figures

Fig. 1
Fig. 1. Bioinformatic analysis of lung ADC from the TCGA database.
a, b Heatmap of the differentially expressed genes in lung ADC a 612 differentially expressed lncRNAs with 489 upregulated and 123 downregulated. b The top five lncRNAs with highest expression. c Differentially expressed gene clustering and module screening based on gene expression patterns of 30 lung ADC samples. Gene dendrogram obtained by clustering the dissimilarity based on consensus Topological Overlap with the corresponding module colors indicated by the color row. The top is the gene dendrogram and the bottom is the gene modules with different colors. A total of 25 modules were identified. d, e Heatmap of the correlation coefficient expressed between modules. Red represents high adjacency (positive correlation) and blue represents low adjacency (negative correlation). f Relationships of consensus module eigengenes and different traits such as normal, tumor, male, female, alive, dead, tobacco, stage, and TNM. Each row in the table corresponds to a module, and each column to a trait. Numbers in the table report the correlations of the corresponding module eigengenes and traits, with the P-values printed below the correlations in parentheses. The table is color coded by correlation according to the color legend. The intensity and direction of correlations are indicated on the right side of the heatmap (red, positively correlated; green, negatively correlated). g Analogous scatter plots for the green module. The gene significance for a tumor (y-axis) is strongly correlated with module membership in the green module (x-axis) cor = 0.96, P < 1e−200. The red dot represents LINC00263 (GS = 0.930878, MM = 0.92419).
Fig. 2
Fig. 2. LINC00263 promotes cell growth, colony formation, invasion, and migration.
a The expression of LINC00263 increased in 535 lung ADC tumor tissues compared to 59 normal tissues in the TCGA database. b Kaplan–Meier curves for overall survival rates associated with LINC00263 expression in lung ADC (low expression numbers = 366, high expression numbers = 125) HR = 0.67(0.45–0.91), P = 0.013. c RT-qPCR shows an increased expression of LINC00263 in 36 paired lung ADC relative to corresponding normal lung tissue samples P < 0.0001. d RT-qPCR verified the expression of LINC00263 in 29 male tumor tissue and 7 female tumor tissue (P = 0.0481). e–g Comparison of the expression of LINC00263 between 232 male tumor tissues and 265 female tumor tissue (P = 0.015) e, normal tissue and tumor tissue in females (P = 0.087) f, normal tissue and tumor tissue in males (P = 0.012) g. h, i Kaplan–Meier curves for overall survival rates associated with LINC00263 expression in female (low expression numbers = 140, high expression numbers = 129) i and males (low expression number = 110, high expression number = 52). j, k The MTS assay was used to assess cell viability in A549 j and PC9 k cells stably overexpressing LINC00263. The sex of A549 and PC9 was male. l, m Colony-formation assay of A549 l and PC9 m cells stably overexpressing LINC00263. n, o The results of cell migration and invasion. A549 n cells and PC9 o cells stably overexpressed LINC00263 have more cells migrated and invaded through the basement membrane compared to empty vector cells. p–u Nude mice after the injection of A549 stably expressing the control vector or LINC00263 expression plasmids are shown. Tumor formation was monitored at the indicated times; images p, weights u were recorded v MTS assay to assess cell viability in H1299 cells after the knockdown of LINC00263. The sex of H1299 was male. w H1299 cells after the knockdown of LINC00263 have fewer cells migrated and invaded through the basement membrane. Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t-test: ns nonsignificant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 3
Fig. 3. GO enrichment, KEGG, and GSEA analysis results for differentially expressed genes after the overexpression of LINC00263.
a Heatmap of the differentially expressed genes after the overexpression of LINC00263 in A549 cells via RNA-seq, 58 mRNAs that were upregulated by two-fold (upregulated), and 26 mRNAs that were downregulated by two-fold. b GO-enrichment analysis results of differentially expressed genes with fold change>1. c KEGG-enrichment analysis of differentially expressed genes. d–f GSEA analysis of the whole transcriptome.
Fig. 4
Fig. 4. LINC00263 activates the NF-κB-signaling pathway through interaction with IKKα.
a RT-qPCR analysis of LINC00263 expression levels in different subcellular fractions in H1299 and H520 cells and LINC00263 mainly localized in cytoplasm. b The prediction of the interaction between LINC00263 and IKKα, IKKβ, IKKγ, P50, and P65. c RIP assays show the association between IKKα and LINC00263. d, e LINC00263 overexpression promoted P65 transfer into the nucleus. f–h The expression of related genes was measured with RT-qPCR in A549 e cells stably overexpressing LINC00263 and H1299. f Cells stably transfected with two distinct target shRNA vectors and control cells and tumors in nude mice h. i miRNA, LncRNA, RBP, TF interacting with LINC00263. j Mechanism of LINC00263 with miRNA and target genes. Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t-test: ns nonsignificant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 5
Fig. 5. The expression of LINC00263 is sex-specific in cancers.
a The expression of LINC00263 increased in 473 colon cancer tumor tissues compared to 41 normal tissue in the TCGA database (P < 0.0001). b Comparison of the expression of LINC00263 between 212 female tumor tissue and 235 male tumor tissues (P = 0.04). c–e Kaplan–Meier curves for overall survival rates associated with LINC00263 expression in colon cancers (low expression numbers = 140, high expression numbers = 129) c and females (low expression number = 28, high expression number = 47) d and males (low expression number = 32, high expression number = 58). f The expression of LINC00263 increased in 819 renal cell carcinoma tumor tissues compared to 113 normal tissue in the TCGA database (P < 0.0001). g Comparison of the expression of LINC00263 between 194 female tumor tissue and 396 male tumor tissues (P = 0.003). h–j Kaplan–Meier curves for overall survival rates associated with LINC00263 expression in renal cell carcinoma (low expression numbers = 418, high expression numbers = 399) h and females (low expression number = 155, high expression number = 107) i and males (low expression number = 298, high expression number = 257) j. k The expression of LINC00263 increased in 411 Hepatocellular carcinoma tumor tissues compared to 58 normal tissue in the TCGA database (P = 0.001). l–n Comparison of the expression of LINC00263 between normal tissues and tumor tissues in female (P = 0.08)(l) and in males (P = 0.006) m and between 120 female tumor tissues and 249 male tumor tissues (P = 0.38) n. o, p Kaplan–Meier curves for overall survival rates associated with LINC00263 expression in females (low expression number = 77, high expression number = 44) o and males (low expression number = 66, high expression number = 183) p. q The expression of LINC00263 increased in 499 prostate cancer tumor tissues compared to 52 normal tissue in the TCGA database (P = 0.005). r Kaplan–Meier curves for overall survival rates associated with LINC00263 expression in prostate cancer (low expression number = 247, high expression number = 248). s The expression of LINC00263 increased in 12 ovarian cancer normal tissues compared to 12 tumor tissue in the GEO database (GSE14407) (P = 0.01). t Kaplan–Meier curves for overall survival rates associated with LINC00263 expression in ovarian cancer (low expression number = 174, high expression number = 199). u The expression of LINC00263 increased in 1071 breast cancer tumor tissues compared to 98 normal tissue in the TCGA database (P = 0.007). v Comparison of the expression of LINC00263 between 1059 female tumor tissue and 12 male tumor tissues (P = 0.01). w Kaplan–Meier curves for overall survival rates associated with LINC00263 expression in breast cancer (low expression number = 354, high expression number = 723). Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t-test: ns nonsignificant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 6
Fig. 6. LINC00263 links with estrogen.
a Correlation between ESR1 mRNA and LINC00263 LncRNA levels in ovarian cancer. Data from GEO database (GSE14407). b Correlation between AR mRNA and LINC00263 LncRNA levels in prostate cancer. Data from TCGA database. c, d The expression of LINC00263 in MCF-7 cells after treatment with 100 nM estrogen (GSE119087) c or estradiol (GSE11352) d from the GEO database. e The expression of LINC00263 in LNCaP cells after AR gene knock down. Data from GEO database (GSE11428). f The expression of LINC00263 in LNCaP cells after DHT treatment. Data from the GEO database (GSE7868). g, h RT-qPCR analysis was conducted to detect the level of LINC00263 after treatment with E2 in MCF-7 cells g and A549 cells h. i The expression of LINC00263 in normal women during the menstrual cycle. Data from the GEO database (GSE6364). j The expression of LINC00263 in the vagina of postmenopausal women after treatment with E2. Data from the GEO database (GSE11622). k, l the expression of LINC00263 in ER negative k or ER positive l ovarian cells after treatment with E2. Data from the GEO database (GSE22600). m, n The expression of LINC00263 in tamoxifen-sensitive m and tamoxifen-resistant n after treatment with E2 or tamoxifen or E2 and tamoxifen. Data from the GEO database (GSE26459). Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t-test: ns nonsignificant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 7
Fig. 7. The xenograft model of A549-LINC00263 and schematic diagram predicting the role of LINC00263.
a–c Nude mice after the injection of A549 stably expressing the LINC00263 expression are shown. Tumor formation was monitored at the indicated times; images a, volume b and weights c were recorded (n = 8). Data are shown as the mean ± SEM; n ≥ 3 independent experiments, two-tailed Student’s t-test: ns nonsignificant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001. d LINC00263 could promote p65 translocation into the nucleus to activate the NF-κB pathway signaling through interaction with IKKα. Estrogen binding with estrogen receptor to inhibit p65 entry into the nucleus that can inhibit the function of LINC00263.

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References

    1. Dorak MT, Karpuzoglu E. Gender differences in cancer susceptibility: an inadequately addressed issue. Front. Genet. 2012;3:268. doi: 10.3389/fgene.2012.00268. - DOI - PMC - PubMed
    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA. 2015;65:5–29. - PubMed
    1. Ober C, Loisel DA, Gilad Y. Sex-specific genetic architecture of human disease. Nat. Rev. Genet. 2008;9:911–922. doi: 10.1038/nrg2415. - DOI - PMC - PubMed
    1. Wang Y, et al. Effect of luteinizing hormone-induced prohibitin and matrix metalloproteinases on ovarian epithelial tumor cell proliferation. Am. J. Cancer Res. 2015;5:114. - PMC - PubMed
    1. Mertens-Walker I, Baxter RC, Marsh DJ. Gonadotropin signalling in epithelial ovarian cancer. Cancer Lett. 2012;324:152–159. doi: 10.1016/j.canlet.2012.05.017. - DOI - PubMed

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