LINC00152 promotes pancreatic cancer cell proliferation, migration and invasion via targeting miR-150

Abstract

Pancreatic cancer (PC) is one of the top deaths causing cancers with low 5-year survival rate. Long non-coding RNAs (lncRNAs) are recognized as a crucial type of nonprotein-coding transcripts implicated in tumorigenesis. Emerging evidence has implied that LINC00152 exerts the potential oncogenic functions in various cancers. Nevertheless, the role of LINC00152 in PC remains elusive. In the present study, we found that LINC00152 was significantly up-regulated while miR-150 was down-regulated both in tissues and cell lines of PC, indicating their negative correlation in PC progression. Functionally, overexpression of LINC00152 promoted cell proliferation, migration and invasion, while LINC00152 knockdown reversed these effects. Mechanistic experiments reveal that miR-150 acted as a target of LINC00152 confirmed by luciferase reporter assay. Moreover, inhibition of miR-150 could markedly attenuate the suppression of cell proliferation, migration and invasion by knocking down LINC00152. Altogether, our findings concluded that LINC00152 facilitated PC progression through inhibiting miR-150 expression, indicating an innovative therapeutic target for PC.

Keywords: LINC00152; miR-150; pancreatic cancer.

Figure 1

Expression of LINC00152 and miR-150 in PC tissues and cell lines. A, B. The expression levels of LINC00152 and miR-150 in PC tissues (n=28) and adjacent normal tissues (n=28) by qRT-PCR assay. C. The Pearson correlation analysis between LINC00152 and miR-150 in PC tissues. D, E. The expression patterns of LINC00152 and miR-150 in five different PC cell lines, including BxPC3, Panc1, AsPC1, Sw1990, and HS-766T, as well as the normal pancreatic cell line HPDE6-C7 by qRT-PCR analysis. The mean ± SD in the graph presents the relative levels from three replications. *P < 0.05, **P < 0.01.

Figure 2

Effects of LINC00152 on cell proliferation, migration and invasion of PC cells. (A) The expression of LINC00152 in Panc1 and SW1990 cells, cells treated with pcDNA3.1 or pcDNA3.1-LINC00152, and cells treated with siNC and siLINC00152 was detected by qRT-PCR. (B-D) CCK-8, colony formation, EDU assays were used to determine the capacity of cell proliferation of the cells described for (A), respectively. (E) Cell migration by the four groups was detected by wound healing. (F) The transwell assays were used to determine cell migration and invasion ability of Panc1 cells transfected with pcDNA3.1 or pcDNA3.1-LINC00152, and SW1990 cells transfected with siNC or si-LINC00152. The mean ± SD in the graph presents the relative levels from three replications. *P < 0.05, **P < 0.01.

Figure 3

LINC00152 acts as a sponge for miR-150. A. HEK293T cells were co-transfected with luciferase reporter gene vector containing WT-LINC00152 or MUT-LINC00152 and miR-150 mimics or mimics-NC. B. The expression of miR-150 in Panc1 and SW1990 cells transfected with pcDNA3.1 or pcDNA3.1-LINC00152, siNC or si-LINC00152 was determined by qRT-PCR, respectively. C. The expression of miR-150 in Panc1 and SW1990 cells transfected with miR-150 mimics or mimics NC was measured by qRT-PCR. D. The expression of LINC00152 in Panc1 and SW1990 cells transfected with miR-150 mimics or mimics NC was measured by qRT-PCR. The mean ± SD in the graph presents the relative levels from three replications. *P < 0.05, **P < 0.01.

Figure 4

miR-150 suppresses cell proliferation, migration and invasion. A. qRT-PCR analysis of miR-150 in Panc1 and SW1990 cells transfected with mimic NC, miR-150 mimics or miR-150 inhibitor. B. The cell viability of Panc1 and SW1990 cells transfected with mimics NC, miR-150 mimics or miR-150 inhibitor was evaluated by CCK-8 assay at 24, 48, 72, and 96 hours. C. The effects of miR-150 in Panc1 and SW1990 cells on proliferation was determined using EDU assay. D. The cell migration and invasion ability of Panc1 and SW1990 cells transfected with mimics NC, miR-150 mimic or miR-150 inhibitor was evaluated by transwell assay. The mean ± SD in the graph presents the relative levels from three replications. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

miR-150 reverses the effect of LINC00152 on PC cells. A. The CCK-8 assay for Panc1 and SW1990 cells transfected with siNC + mimics NC, si-LINC00152 + mimics NC, and si-LINC00152 + miR-150 inhibitor at 24, 48, 72, and 96 hours. B. The cell proliferation by the three groups was detected by colony formation assay. C. The transwell assays of cell migration and invasion were performed in PANC-1 and SW1990 cells transfected with siNC + mimics NC, si-LINC00152 + mimics NC, and si-LINC00152 + miR-150 inhibitor. All results were presented as the mean ± SD and experiment were performed in triplicate. *P < 0.05, **P < 0.01. NC: negative control.

Figure 6

ZEB1 is a direct target of miR-150 in PC cells. A. Predicted miR-150 target sequence in the 3’-UTR of ZEB1 mRNA. B. miR-150 suppressed the luciferase activity of the wild-type ZEB1 3’-UTR expression vector but had no effect on the one with mutant ZEB1. C. The mRNA expression of ZEB1 in the PC cells after transfection with NC, miR-150 inhibitor, and miR-150 mimics, respectively. D. The protein level of ZEB1 in the PC cells after transfection with NC, miR-150 inhibitor, and miR-150 mimics, respectively (The original western blot images reference to Supplementary Figure 1). All results were presented as the mean ± SD and experiment were performed in triplicate. *P < 0.05, **P < 0.01. NC: negative control.

Figure 7

LINC00152 promoted PC tumor growth by sponging miR-150 in mice. A. Representative images of xenograft were shown after transfecting with si-NC or si-LINC00152. B, C. Tumor growth was monitored by analyzing tumor volume and weight. D. HE and immunohistochemical staining were performed to analyze the Ki-67, ZEB1, and E-cadherin expressions in xenografts. E. The expressions of LINC00152 and miR-150 in xenografts by qRT-PCR. The mean ± SD in the graph presents the relative levels from three replications. *P < 0.05, **P < 0.01.