LncRNA CDKN2B-AS1 Promotes Cell Viability, Migration, and Invasion of Hepatocellular Carcinoma via Sponging miR-424-5p

Abstract

Objective: Hepatocellular carcinoma (HCC) results in high mortality and metastasis. In this study, the effects of long non-coding RNA (lncRNA) CDKN2B-AS1 on the progression of HCC were investigated.

Materials and methods: LncRNA CDKN2B-AS1 expression of HCC cancer and adjacent tissues, and HCC cells were detected. Subsequently, CDKN2B-AS1 was overexpressed and silenced in HCC cells to observe the effects of CDKN2B-AS1 on the cell viability, migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells by performing cell counting kit-8 (CCK-8), wound-healing, Transwell, and Western blot. The target gene of CDKN2B-AS1 was predicted and verified to be miR-424-5p, whose expression in HCC cells with up- or down-regulation of CDKN2B-AS1 expression was determined. Moreover, the effects of miR-424-5p on cell viability, migration, and invasion and EMT of HCC cells were investigated with miR-424-5p up-regulation or down-regulation, together with overexpression or silencing of CDKN2B-AS1.

Results: CDKN2B-AS1 expression was increased in HCC tissues and cells. Silencing of CDKN2B-AS1 suppressed cell viability, migration, invasion, and EMT, while overexpression of CDKN2B-AS1 produced the opposite results. Furthermore, CDKN2B-AS1 was predicted and verified to target miR-424-5p and was confirmed to negatively modulate miR-424-5p expression. Moreover, overexpression of miR-424-5p partially suppressed the previously high cell viability, migration, and invasion, and activated EMT resulted from up-regulation of CDKN2B-AS1, while silencing of miR-424-5p elevated the cellular processes inhibited by silencing the expression of CDKN2B-AS1.

Conclusion: The present study revealed that high-expressed CDKN2B-AS1 may associate with the progression of HCC by affecting the cell viability, migration, invasion, and EMT of HCC cells by negatively regulating miR-424-5p.

Keywords: hepatocellular carcinoma; invasion; lncRNA CDKN2B-AS1; miR-424-5p; migration.

Figure 1

CDKN2B-AS1 expression in HCC tissues and cells was detected by qRT-PCR. (A) The expression level of CDKN2B-AS1 in HCC and its adjacent tissues (n=30). **P<0.001, vs. Normal. (B) The expression level of CDKN2B-AS1 in THLE-2, Huh7, Hep3B, MHCC97H and Sk-Hep1 cells. **P<0.001, vs. THLE-2.

Figure 2

Transection efficiency and cell viability in Huh7 cells transfected with siCDKN2B-AS1 and MHCC97H cells transfected with CDKN2B-AS1, with the group divided into blank, siNC, siCDKN2B-AS1 or blank, NC, CDKN2B-AS1. (A, B) CDKN2B-AS1 expression in Huh7 and MHCC97H cells was determined by qRT-PCR, GAPDH was used as internal reference. (C, D) Cell viability in Huh7 and MHCC97H cells was determined by CC-8 assay. *P<0.05, **P<0.001, vs. Blank. ^#P<0.05, ^##P<0.001, vs. siNC or NC.

Figure 3

Cell migration and invasion capacities in Huh7 cells transfected with siCDKN2B-AS1 and MHCC97H cells transfected with CDKN2B-AS1, with the group divided into blank, siNC, siCDKN2B-AS1 or blank, NC, CDKN2B-AS1. (A–D) Determination of cell migration ability by wound-healing assay. (E–H) Determination of cell invasion ability by transwell assay. **P<0.001, vs. Blank. ^##P<0.001, vs. siNC or NC.

Figure 4

EMT related protein expression in Huh7 cells transfected with siCDKN2B-AS1 and MHCC97H cells transfected with CDKN2B-AS1, with the group divided into blank, siNC, siCDKN2B-AS1 or blank, NC, CDKN2B-AS1, GAPDH was used as internal reference. (A, B) Determination of E-Cadherin, N-Cadherin and Snail expression in Huh7 cells transfected with siCDKN2B-AS1 by Western blot. (C, D) Determination of E-Cadherin, N-Cadherin and Snail expression in MHCC97H cells transfected with CDKN2B-AS1 by Western blot. *P<0.05, **P<0.001, vs. Blank. ^#P<0.05, ^##P<0.001, vs. siNC or NC.

Figure 5

MiR-424-5p was the target gene of CDKN2B-AS1 and its expression in Huh7 cells transfected with siCDKN2B-AS1 and MHCC97H cells transfected with CDKN2B-AS1, with the group divided into blank, siNC, siCDKN2B-AS1 or blank, NC, CDKN2B-AS1. (A) The target gene of CDKN2B-AS1 was predicted by starBase. (B, C) The verification of CDKN2B-AS1 targeting miR-424-5p by dual-luciferase assay in Huh7 and MHCC97H cells. (D) The expression level of miR-424-5p in HCC and its adjacent tissues (n=30). (E) The correlations between CDKN2B-AS1 and miR-424-5p expression in HCC tissues. (F, G) The expression of miR-424-5p was measured by qRT-PCR, the internal reference was U6. **P<0.001, vs. Normal or Blank. ^##P<0.001, vs. siNC or NC.

Figure 6

Transection efficiency and cell viability in Huh7 cells and MHCC97H cells, with the group divided into MC, M, IC, I and siCDKN2B-AS1+I in Huh7 cells or MC, M, IC, I and CDKN2B-AS1+M in MHCC97H cells. (A, B) miR-424-5p expression in Huh7 and MHCC97H cells was determined by qRT-PCR, U6 was used as internal reference. (C, D) Cell viability in Huh7 and MHCC97H cells was determined by CCK-8 assay. **P<0.001, vs. MC. ^##P<0.001, vs. IC. ^{^^}P<0.001, vs. I or M.

Figure 7

Cell migration and invasion capacities in Huh7 cells and MHCC97H cells, with the group divided into MC, M, IC, I and siCDKN2B-AS1+I in Huh7 cells or MC, M, IC, I and CDKN2B-AS1+M in MHCC97H cells. (A–D) The migration ability of Huh7 and MHCC97H cells was determined by wound-healing assay. (E–H) The invasion ability of Huh 7 and MHCC97H cells was determined by transwell assay. **P<0.001, vs. MC. ^##P<0.001, vs. IC. ^{^^}P<0.001, vs. I or M.

Figure 8

EMT related protein expression in Huh7 cells and MHCC97H cells, with the group divided into MC, M, IC, I and siCDKN2B-AS1+I in Huh7 cells or MC, M, IC, I and CDKN2B-AS1+M in MHCC97H cells. GAPDH was used as internal reference. (A, B) Determination of E-Cadherin, N-Cadherin and Snail expression in Huh7 cells by Western blot. (C, D) Determination of E-Cadherin, N-Cadherin and Snail expression in MHCC97H cells by Western blot. **P<0.001, vs. MC. ^##P<0.001, vs. IC. ^{^^}P<0.001, vs. I or M.