Long Non-Coding RNA-NEAT1 Promotes Cell Migration and Invasion via Regulating miR-124/NF-κB Pathway in Cervical Cancer

Abstract

Background: This study aimed to investigate the regulatory role of lncRNA-NEAT1 on cervical cancer (CC) and the underlying molecular mechanisms.

Methods: The expression of lncRNA-NEAT1 and miR-124 was detected in CC tissues and cells (HeLa and SiHa cells) by qRT-RCR. The relation between lncRNA-NEAT1 expression and clinical parameters of CC patients was explored. The cell migration and invasion were detected by wound healing assay and transwell assay. The cell proliferation was detected by CCK-8 and anchorage-independent colony assay. The targeting relation between miR-124 and lncRNA-NEAT1 was predicted by TargetScan and identified by dual luciferase reporter gene and RNA pull-down assay. The expression of metastasis- (MMP-2 and MMP), EMT- (E-cadherin, N-cadherin and Vimentin), and NF-κB pathway-related factors (NF-κB p65, p-NF-κB p65 and IκBα) was detected by Western blot.

Results: The expression of lncRNA-NEAT1 was upregulated in CC tissues and cells and positively correlated with TNM stage and lymph node metastasis. Overexpression of lncRNA-NEAT1 promoted the proliferation, migration and invasion, influenced the expression of EMT markers, and activated NF-κB pathway in HeLa and SiHa cells. Silencing of lncRNA-NEAT1 exhibited opposite effects on HeLa and SiHa cells. LncRNA-NEAT1 could negatively regulate its target miR-124. MiR-124 reversed the effects of lncRNA-NEAT1 on the migration, invasion, EMT and NF-κB pathway of HeLa cells.

Conclusion: LncRNA-NEAT1 promoted the migration and invasion of CC cells via regulating miR-124/NF-κB pathway.

Keywords: cervical cancer; invasion; lncRNA-NEAT1; miR-124/NF-κB pathway; migration.

Figure 1

The expression of lncRNA-NEAT1 was upregulated in CC tissues and cells (HeLa and SiHa cells). (A) The expression of lncRNA-NEAT1 in CC tissues and adjacent tissues (N = 72) was detected by qRT-PCR. (B) The expression of lncRNA-NEAT1 in HaCaT, HeLa and SiHa cells was detected by qRT-PCR. Data were presented as mean ± standard deviation with three replicates. ***P < 0.001, vs. adjacent tissues (A) and HaCaT cells (B).

Figure 2

LncRNA-NEAT1 promoted the migration and invasion of HeLa and SiHa cells. (A) The expression of lncRNA-NEAT1 in transfected HeLa and SiHa cells was detected by qRT-PCR. (B) The migration ability of transfected HeLa and SiHa cells was detected by wound healing assay. (C) The migration ability of transfected HeLa and SiHa cells was detected by transwell assay. (D) The invasion ability of transfected HeLa and SiHa cells was detected by transwell assay. (E) The expression of MMP-2 and MMP-9 in transfected HeLa and SiHa cells was measured by Western blot. Data were presented as mean ± standard deviation with three replicates. ***P < 0.001, vs. BLANK group.

Figure 3

LncRNA-NEAT1 promoted the proliferation of HeLa and SiHa cells. (A) Cell viability of transfected HeLa and SiHa cells was detected by CCK-8 assay. (B) The colony formation of transfected HeLa and SiHa cells was detected by anchorage-independent colony assay. Data were presented as mean ± standard deviation with three replicates. *P < 0.05, **P < 0.01, ***P < 0.001, vs. BLANK group.

Figure 4

LncRNA-NEAT1 influenced EMT in HeLa and SiHa cells. (A) The expression of E-cadherin, N-cadherin and Vimentin in transfected HeLa cells was detected by Western blot. (B) The expression of E-cadherin, N-cadherin and Vimentin in transfected SiHa cells was detected by Western blot. Data were presented as mean ± standard deviation with three replicates. ***P < 0.001, vs. BLANK group.

Figure 5

LncRNA-NEAT1 activated NF-κB pathway in HeLa and SiHa cells. (A) The nuclear expression of NF-κB p65 in transfected HeLa and SiHa cells was detected by immunofluorescence. (B) The expression of NF-κB p65, p-NF-κB p65 and IκBα in transfected HeLa and SiHa cells was detected by Western blot. Data were presented as mean ± standard deviation with three replicates. **P < 0.01, ***P < 0.001, vs. BLANK group.

Figure 6

LncRNA-NEAT1 negatively regulated miR-124. (A) The expression of miR-124 in CC tissues and adjacent tissues was detected by qRT-PCR. (B) The expression of miR-124 in HaCaT, HeLa and SiHa cells was detected by qRT-PCR. (C) The correlation analysis between the expression of lncRNA-NEAT1 and miR-124 in CC tissues. (D) The binding target of miR-124 on lncRNA-NEAT1 was predicted by Target Scan. (E) The luciferase activity was detected by dual luciferase reporter gene assay. (F) The expression of miR-124 was detected by RNA pull-down assay. (G) The expression of miR-124 in transfected HeLa cells was detected by qRT-PCR. (H) The expression of miR-124 in transfected SiHa cells was detected by qRT-PCR. Data were presented as mean ± standard deviation with three replicates. **P < 0.01, ***P < 0.001, vs adjacent tissues (A), HaCaT cells (B), miR-NC group (E), Bio-NC group (F), and BLANK group (G and H).

Figure 7

MiR-124 reversed the effects of lncRNA-NEAT1 on the migration, invasion, EMT and NF-κB pathway in HeLa cells. (A) The migration and invasion ability of transfected HeLa cells were detected by wound healing and transwell assay. (B) The expression of MMP-2 and MMP-9 in transfected HeLa cells was detected by Western blot. (C) The expression of E-cadherin, N-cadherin and Vimentin in transfected HeLa cells was detected by Western blot. (D) The expression of NF-κB p65, p-NF-κB p65 and IκBα in transfected HeLa cells was detected by Western blot. Data were presented as mean ± standard deviation with three replicates. ***P < 0.001, vs pcDNA-NC + miR-NC group. ^&&&P < 0.001, vs. pcDNA-NC + miR-124 mimics and pcDNA- NEAT1 + miR-NC group.