LncRNA MALAT-1 promotes growth and metastasis of epithelial ovarian cancer via sponging microrna-22

Abstract

LncRNAs and miRNAs are emerging players in epithelial ovarian cancer (EOC). LncRNA MALAT-1 and miR-22 play vital roles in the onset and development of multiple cancers. Both of them are abnormally expressed in ovarian cancer, but the molecular basis for their involvement in EOC is unclear. In this study, we found MALAT-1 was up-regulated but miR-22 was down-regulated in EOC tissues and cell lines when compared to normal ovarian epithelial cell line IOSE80. Both of MALAT-1shRNA and miR-22 mimics inhibited ovarian cell proliferation, migration, and invasion, while simultaneously overexpressing MALAT-1 and miR-22 largely canceled out this inhibitory effect. Consistently, MALAT-1 silencing and miR-22 overexpression restrained tumor growth and metastasis to lungs in nude mice, which could be largely counteracted by co-overexpressing MALAT-1 and miR-22. Mechanistically, MALAT-1 targeted and sponged miR-22, counteracting its inhibitory effect on c-myc and c-myc-mediated epithelial-mesenchymal transition. Our findings for the first time demonstrated that MALAT-1 supports EOC progression through sponging miR-22, providing a novel insight into the role of MALAT-1 in ovarian cancer.

Keywords: Epithelial ovarian cancer; LncRNA MALAT-1; c-myc; miRNA-22.

Figure 1

MALAT-1 is up-regulated while miR-22 is down-regulated in epithelial ovarian cancer (EOC) tissues and cell lines. A: RNA levels of MALAT-1 and miR-22 indicated cell lines; B: And EOC or tumor-adjacent tissues (n=14) were determined by qPCR. Data are expressed as mean ± standard deviation (x̅ ± sd). *P<0.05 vs. IOSE80 cells, or tumor-adjacent normal tissues. MALAT-1: Metastasis-associated lung adenocarcinoma transcript-1; EOC: epithelial ovarian cancer.

Figure 2

miR-22 is a target of MALAT-1. A: The sequence comparison between wide-type or mutant MALAT-1 and miR-22; B: miR-22 and luciferase plasmids cloned with wild-type or mutant MALAT-1 were transfected into HEK293T cells, the luciferase activities in cell lysates were determined; C: RIP assay was performed to validate the interaction between MALAT1 and miR-22 in SKOV3 cells. Data are expressed as mean ± standard deviation (x̅ ± sd) and represent three replicated experiments *P<0.05. MALAT-1: Metastasis-associated lung adenocarcinoma transcript-1.

Figure 3

MALAT-1 promotes EOC cell proliferation, migration and invasion through suppressing miR-22. A: RNA levels of MALAT-1 and miR-22 in SKOV3 cells were examined 24 h after transfection of MALAT-1 overexpression vector, MALAT-1 shRNA, miR-22 mimics, and inhibitors using qPCR; B: The proliferative viability was assayed by MTT reagents; C: MALAT-1 shRNA, miR-22 mimics, or MALAT-1 and miR-22 mimics were introduced into SKOV3 cells, respectively. Migration capacities of these cells were assessed using scratching assay; D: Cell invasion assay was conducted using transwells and visualized using crystal violet staining. Data are expressed as mean ± standard deviation (x̅ ± sd) and represent three replicated experiments. *P<0.05 vs. the negative control (NC), **P<0.01 vs. the NC, and #P<0.05 vs. the miR-22 group. MALAT-1: Metastasis-associated lung adenocarcinoma transcript-1.

Figure 4

MALAT-1 enhanced epithelial-mesenchymal transition (EMT) of EOC cells via miR22/c-myc axis. A: MALAT-1 shRNA, miR-22 mimics, or MALAT-1 and miR-22 mimics were introduced into SKOV3 cells. The c-myc RNA levels were examined by qPCR; B: The c-myc protein was detected using immunoblots and then quantified by gray-scale analysis; C: The epithelial-mesenchymal transition markers, including E-cadherin, vimentin, snai1, were detected by immunoblots and then quantified by gray-scale analysis. Data are expressed as mean ± standard deviation (x̅ ± sd) and represent three replicated experiments. *P<0.05 vs. the negative control (NC), and #P<0.05 vs. the miR-22 group. MALAT-1: Metastasis-associated lung adenocarcinoma transcript-1.

Figure 5

MALAT-1 restrains ovarian cancer growth and metastasis in nude mice through suppressing miR-22. SKOV3 cells stably expressed MALAT-1 shRNA, miR-22, or MALAT-1 and miR-22 were established, respectively. These cell lines were inoculated subcutaneously into nude mice or intravenously injected into nude mouse tails (4 * 10⁶ cells/mouse). A: 4 weeks after inoculation, mice were sacrificed and the tumors were photographed; B: Tumor weights; C: Volumes in the 4 weeks were recorded. D: Expression levels of MALAT-1 and miR-22 in mouse tumor samples were determined by qPCR; F: For metastasis models, mouse lungs were photographed; E: Tumor nodules in lungs were counted. Data are mean ± standard deviation (x̅ ± sd). *P<0.05 vs. the negative control (NC), and #P<0.05 vs. the miR-22 group. MALAT-1: Metastasis-associated lung adenocarcinoma transcript-1.