lncRNA XIST regulates cell proliferation, migration and invasion via regulating miR-30b and RECK in nasopharyngeal carcinoma

Abstract

Long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) plays an essential role in the development and progress of nasopharyngeal carcinoma (NPC). MicroRNA-30b (miR-30b) has been confirmed to play an inhibitory role in various types of cancer. The molecular mechanisms underlying the lncRNA XIST-mediated regulation of the metastasis of NPC cells by miR-30b is not clear. qPCR and western blot analysis were used to detect the expression of XIST, miR-30b, and reversion inducing cysteine rich protein with kazal motifs (RECK) in NPC tissues and cell lines. The detection of luciferase reporter gene confirmed the relationship between lncRNA XIST, miR-30b and RECK. CCK-8 and Transwell assays were performed in order to detect the proliferation, migration and invasion of the NPC cells. The results of qPCR and western blotting indicated that the expression levels of lncRNA XIST and RECK were higher in the NPC tissues and cell lines than that of the control group, while the expression of miR-30b was lower. Knockdown of lncRNA XIST significantly inhibited cell proliferation, migration and invasion in the NPC cell lines. In addition, lncRNA XIST was found to negatively regulate the expression of miR-30b, resulting in the upregulation of RECK. Overexpression of RECK was found to reverse the inhibitory effect of lncRNA XIST knockdown or miR-30b on NPC cell metastasis. Our results showed that cell migration and invasion were inhibited by knockdown of lncRNA XIST, suggesting that the lncRNA XIST/miR-30b/RECK axis is involved in the development of NPC.

Keywords: RECK; long non-coding RNA XIST; miR-30b; nasopharyngeal carcinoma.

Figure 1.

Expression of lncRNA XIST was determined by qPCR in NPC and adjacent tissues and NPC SUNE1 and HK1 cells. (A) Expression of lncRNA XIST in NPC and adjacent normal tissues. (B) Expression of lncRNA XIST in NPC cells and NP69 normal cells. **P<0.01, compared to adjacent tissues or NP69 cells. lncRNA, long non-coding RNA; XIST, X-inactive specific transcript; NPC, nasopharyngeal carcinoma.

Figure 2.

lncRNA XIST is efficiently knocked down suppresses SUNE1 and HK1 cell proliferation and metastasis. (A) Expression of lncRNA XIST in NPC cells transfected with si-XIST or si-NC. (B) CCK-8 assay was performed to detect the effect of the knockdown of lncRNA XIST on cell proliferation. (C and D) Knockdown of lncRNA XIST suppressed NPC cell invasion. (×100 magnification). (E and F) Knockdown of lncRNA XIST suppressed NPC cell migration. (×100 magnification). **P<0.01, *P<0.05, compared to the si-NC group. lncRNA, long non-coding RNA; XIST, X-inactive specific transcript; NPC, nasopharyngeal carcinoma; NC, negative control.

Figure 3.

Expression of miR-30b was determined by qPCR in NPC and adjacent tissues and NPC SUNE1 and HK1 cells. (A) Expression of miR-30b in NPC and adjacent normal tissues. (B) Expression of miR-30b in NPC cells and NP69 normal cells. **P<0.01, compared to the adjacent tissues or NP69 cells. NPC, nasopharyngeal carcinoma.

Figure 4.

lncRNA XIST targets miR-30b and negatively regulates its expression (A) Putative binding sites between lncRNA XIST and miR-30b. (B) Expression of miR-30b is significantly increased by knockdown of XIST. (C) Expression of miR-30b after transfection with miR-30b mimic. (D and E) The luciferase activity was determined by dual-luciferase reporter gene assay in NPC SUNE1 and HK1 cells. (F) Correlation between lncRNA XIST and miR-30b relative expression. **P<0.01, *P<0.05, compared to the NC. XIST, X-inactive specific transcript; NPC, nasopharyngeal carcinoma; NC, negative control; WT, wild-type; MUT, mutated.

Figure 5.

lncRNA XIST knockdown suppresses NPC cell invasion and migration by targeting miR-30b. (A) Expression of miR-30b in NPC SUNE1 and HK1 cells transfected with si-XIST, si-NC, or si-XIST and miR-30b inhibitor. (B) The protein levels of EMT-related marker protein in SUNE1 and HK1 cells transfected with si-XIST, si-NC, or si-XIST and miR-30b inhibitor. E, E-cadherin; N, N-cadherin; V, vimentin. (C-F) SUNE1 and HK1 cell migration and invasion after transfection with si-XIST, si-NC, si-XIST and miR-30b inhibitor (×100 magnification). **P<0.01, *P<0.05, compared to the si-NC group. XIST, X-inactive specific transcript; NPC, nasopharyngeal carcinoma; NC, negative control; EMT, epithelial-to-mesenchymal transition.

Figure 6.

RECK is a direct miR-30b target in NPC cells. (A) Display of the RECK 3′UTR-WT or -MUT sequences with miR-30b sequences. (B and C) Luciferase activity of RECK 3′UTR-WT or -MUT in SUNE1 and HK1 cells after decreasing miR-30b. (D) RECK mRNA expression in NPC cells after transfection with the miR-30b mimic. (E) Protein levels of RECK in NPC cells after transfection with si-XIST, si-NC, si-XIST and miR-30b inhibitor. **P<0.01, *P<0.05, compared with the NC. RECK, reversion inducing cysteine rich protein with kazal motifs; XIST, X-inactive specific transcript; NPC, nasopharyngeal carcinoma; WT, wild-type; MUT, mutated; NC, negative control.