LINC01127 promotes the development of ovarian tumors by regulating the cell cycle

Abstract

Background: Ovarian cancer is characterized by the high mortality rate and poor prognosis. Nevertheless, the oncogenesis mechanisms of ovarian cancer remain unclear. In our study, we focused on the potential role of lncRNA LINC01127 in the pathogenesis of ovarian cancer and its underlying mechanism.

Methods: LINC01127, which may participate in the development of ovarian cancer, was screened out by bioinformatics analysis. GSEA was used to analyze the function of LINC01127. QRT-PCR was used to analyze the LINC01127 level in 72 cases of ovarian cancer tissues and 53 cases of normal ovarian tissues. LINC01127 level in ovarian cancer cell lines was also determined by qRT-PCR. Subsequently, the selected ovarian tumor cells were transfected with LINC01127 siRNA by Lipofectamine 2000, followed by cell cycle detection using flow cytometry. The regulatory effects of LINC01127 on tumor growth and cell cycle in nude mice were verified by tumor formation assay. The mechanism of LINC01127 involving in cell cycle regulation was further explored by Western Blot.

Results: LINC01127 expression in ovarian cancer tissues was significantly higher than that in normal ovary tissues. The expression level of LINC01127 was negatively correlated with the prognosis of patients with ovarian cancer. GSEA analysis showed that LINC01127 was mainly enriched in the regulation of cell cycle. After transfection with LINC01127 siRNA, the proliferative abilities of SKOV3 and HO8910 cells were inhibited and cell cycle was arrested at G1/G0 phase. Tumorigenicity assay in nude mice showed that low expression of LINC01127 inhibited the growth of ovarian tumors. Further study found that LINC01127 knockdown upregulated expression levels of Cyclin D, Cyclin E and CDK4, but dramatically upregulated expression levels of P16 and P21. Meanwhile, the AKT and ERK pathways were inhibited by LINC01127 knockdown.

Conclusions: LINC01127 was up-regulated in ovarian cancer tissues. LINC01127 may be involved in the development of ovarian cancer by accelerating cell cycle progression through promoting the phosphorylation of ERK and AKT.

Keywords: AKT; ERK; LINC01127; Ovarian cancer; cell cycle.

Figure 1

LINC01127 is highly expressed in ovarian tumors and is negatively correlated with the prognosis. A. Hierarchical clustering analysis of lncRNAs that were differentially expressed (fold change >2; P<0.05) in ovarian tumors and normal tissues in GEO datasets. B. Co-upregulated and co-downregulated long non-coding RNAs in GEO datasets. C. Integration analysis of the 4 databases was performed using the Rank Prod method. D. Analysis of TCGA database showed that patients with high levels of LINC01127 expression showed worse prognosis (log-rank test). E. QRT-PCR of clinical samples showed that the expression levels of LINC01127 in ovarian tumor tissues were significantly higher than those in non-tumorous tissues. F. Ovarian tumor patients were divided into high expression group and low expression group according to the median of LINC01127 expression. G. LINC01127 expression was negatively correlated with the prognosis of patients with ovarian cancer. The mean values and SD were calculated from triplicates of a representative experiment, *P<0.05.

Figure 2

LINC01127 is associated with ovarian cancer cell cycle. A and B. GSEA analysis of integrated GSE18520, GSE38666, GSE40595 and GSE52037 through the InslicoMerging method indicated that the main regulatory biological process of LINC01127 was the cell cycle; C and D. GSEA analysis based on the TCGA database also indicated that the main regulatory biological process of LINC01127 was the cell cycle. The enrichment score (ES, green line) indicates the degree to which the gene set is over represented at the top or bottom of the ranked list of genes.

Figure 3

Interference with LINC01127 blocks the ovarian cancer cell cycle in G0/G1 phase. A. QRT-PCR analysis showed that LINC01127 was overexpressed in ovarian tumor cell lines compared with the normal cell lines; B. The efficiency of small interference RNAs was detected by sequences qRT-PCR; C and D. The cell cycle distribution of HO8910 and SKOV3cells after LINC01127 interference was analyzed by flow cytometry. The mean values and SD were calculated from triplicates of a representative experiment, *P<0.05.

Figure 4

LINC0027 regulates the ovarian cancer cell growth in vivo. A and B. sh-LINC01127 and empty vector were stably transfected into the ovarian cells and then injected to nude mice respectively. The subcutaneous xenograft tumor growth curve in nude mice was drawn according to the tumor volume recorded on the 6^th, 9^th, 12^th, 15^th, 18^th and 21^st days after subcutaneous injection. C. Tumor weights were represented. D. The expression level of LINC01127 in both groups were detected by qRT-PCR; E. The tumor sections of each group were stained with tumor proliferation markers Ki-67 and PCNA, cell cycle promoting proteins Cyclin D, Cyclin E and CDK4 and cell cycle inhibiting proteins P16 and P21 by Immunohistochemistry (magnification 400 ×). The mean values and SD were calculated from triplicates of a representative experiment, *P<0.05.

Figure 5

The regulatory mechanism of LINC01127 in ovarian cancer cell cycle. A. The expressions of Cyclin D, Cyclin E and CDK4 as well as P16 and P21 after LINC01127 interference by siRNA were detected by Western blot; B. The expressions of phosphorylated AKT and ERK as well as total Akt and ERK pathways were detected after LINC01127 interference by siRNA were detected by Western blot. The mean values and SD were calculated from triplicates of a representative experiment, *P<0.05.