ZYG11A serves as an oncogene in non-small cell lung cancer and influences CCNE1 expression

Abstract

By analyzing The Cancer Genome Atlas (TCGA) database, we identified ZYG11A as a potential oncogene. We determined the expression of ZYG11A in NSCLC tissues and explored its clinical significance. And also evaluated the effects of ZYG11A on NSCLC cell proliferation, migration, and invasion both in vitro and in vivo. Our results show that ZYG11A is hyper-expressed in NSCLC tissues compared to adjacent normal tissues, and increased expression of ZYG11A is associated with a poor prognosis (HR: 2.489, 95%CI: 1.248-4.963, p = 0.010). ZYG11A knockdown induces cell cycle arrest and inhibits proliferation, migration, and invasion of NSCLC cells. ZYG11A knockdown also results in decreased expression of CCNE1. Over-expression of CCNE1 in cells with ZYG11A knockdown restores their oncogenic activities. Our data suggest that ZYG11A may serve as a novel oncogene promoting tumorigenicity of NSCLC cells by inducing cell cycle alterations and increasing CCNE1 expression.

Keywords: CCNE1; NSCLC; ZYG11A; bioinformatics; oncogene.

Figure 1. ZYG11A is highly expressed in NSCLC tissues

a. Venn Diagram for gene screening, TCGA datasets genes with fold change > 5 and tumor expression > 3 were included, and a list of 7 genes was obtained. b. Normal lung tissues do not express ZYG11A, but several lung tumor tissues are positive for ZYG11A. c. After analysis of 108 paired tissues, ZYG11A was found to be highly expressed in tumors (p<0.0001). d. ZYG11A is over-expressed in 93.7% (59 of 63) of the lung cancer tissues, with an average over-expression of 9.3-fold (P<0.001) as compared to paired normal tissues. e and f. ZYG11A over-expression is associated with greater T stage (p = 0.016) and TNM stage(p = 0.014). g and h. ZYG11A mRNA and protein are hyper-expressed in H1299 and SPC-A-1 cell lines.

Figure 2. Knockdown of ZYG11A alters NSCLC cell line proliferation, migration, invasion, and cell cycle stage in vitro

a. Two specific siRNAs (siRNA-1, siRNA-2) were designed and synthesized, and siRNA-1 had a better efficiency. b. Depletion of ZYG11A undermines both H1299 and SPC-A-1 cell lines' proliferation c. Colony numbers of H1299 and SPC-A-1 cells transfected with si-ZYG11A are less than those transfected with si-NC (p = 0.0015 and p = 0.0003). d. Migratory and invasion rates of H1299 or SPC-A-1 cells transfected with si-ZYG11A are decreased compared with NC group. e. H1299 cells transfected with si-ZYG11A display more arrest at G1 phase as compared with cells transfected with si-NC. f. si-ZYG11A impairs migration as compared with NC group (p<0.001).

Figure 3. Knockdown of ZYG11A retards tumor growth in vivo

a. Tumor nodules from mice injected with sh-ZYG11A cells are significantly smaller than those injected with NC cells. b. sh-ZYG11A tumors from both H1299 and SPC-A-1 cell lines have less dense PCNA staining. c. Compared with the NC group, the sh-ZYG11A group has reduced tumor size. d. Both mRNA and protein expression of CCNE1 are decreased in the sh-ZYG11A group compared with NC group, and ZYG11A expression is also decreased.

Figure 4. ZYG11A depletion influences CCNE1 expression

a. KEGG pathway enrichment analysis indicates genes co-expressed with ZYG11A are enriched for cell cycle pathways. b. CCNE1 mRNA expression is reduced after transfection with si-ZYG11A (p = 0.004), but the expression of CDKN1 (p21), CDKN2 (p27), and CCND1 were not altered. c. cBioPortal enrichment analysis indicated CCNE1 expression is positively related with ZYG11A expression (p<0.001). d. CCNE1 protein expression is decreased after transfection with si-ZYG11A, with no difference in expression of p21, p27, or CCND1.

Figure 5. Rescue of proliferation and migration by over-expression of CCNE1 in ZYG11A-depleted cells

a. ZYG11A-depleted H1299 cells transfected with a full-length human CCNE1 have increased expression of CCNE1. b. Depletion of ZYG11A undermines H1299 cell proliferation, but over-expressing CCNE1 recovers proliferation. c. H1299 cells transfected with sh-ZYG11A have a decreased migratory rate when compared with NC transfected cells, but CCNE1 over-expression restores migration.

Figure 6. Tissue microarray analysis

a, b, c, d. each present an example of normal, I, II, and III TNM stage, ZYG11A expression is associated with TNM stage. e. univariate survival analysis indicated that ZYG11A expression is associated with poorer prognosis p = 0.0022.