LncRNA AC093818.1 accelerates gastric cancer metastasis by epigenetically promoting PDK1 expression

Abstract

Gastric cancer (GC) is a highly prevalent type of metastatic tumor. The mechanisms underlying GC metastasis are poorly understood. Some long noncoding RNAs (lncRNAs) reportedly play key roles in regulating metastasis of GC. However, the biological roles of five natural antisense lncRNAs (AC093818.1, CTD-2541M15.1, BC047644, RP11-597M12.1, and RP11-40A13.1) in GC metastasis remain unclear. In this study, the expression of these lncRNAs was measured by quantitative reverse transcription-polymerase chain reaction. Migration and invasion were evaluated by wound-healing and the Transwell assay, respectively. Stable cells were injected into the tail veins of nude mice. Sections of collected lung and liver tissues were stained using hematoxylin and eosin. Protein expression was analyzed by western blot. RNA immunoprecipitation (RIP) assay was used to verify whether the STAT3 and SP1 transcription factors bound to AC093818.1 in GC cells. Expression levels of the five lncRNAs, especially AC093818.1, were significantly upregulated in metastatic GC tissues relative to those in nonmetastatic GC tissues. AC093818.1 expression was correlated with invasion, lymphatic metastasis, distal metastasis, and tumor-node-metastasis stage. AC093818.1 expression was highly sensitive and specific in the diagnosis of metastatic or nonmetastatic GC. AC093818.1 overexpression promoted GC migration and invasion in vitro and in vivo. AC093818.1 overexpression increased PDK1, p-AKT1, and p-mTOR expression levels. AC093818.1 silencing decreased these expressions. AC093818.1 bound to transcription factors STAT3 and SP1, and SP1 or STAT3 silencing could alleviated the effect of AC093818.1 overexpression. The data demonstrate that lncRNA AC093818.1 accelerates gastric cancer metastasis by epigenetically promoting PDK1 expression. LncRNA AC093818.1 may be a potential therapeutic target for metastatic GC.

Fig. 1. LncRNA AC093818.1 epression in GC tissues and it’s significance in GC metastasis.

a AC093818.1 expression measured in 20 nonmetastatic GC and 85 metastatic GC samples by qRT-PCR. b The performance of the AC093818.1-based model for the diagnosis of metastatic or nonmetastatic GC evaluated by ROC curve analysis.

Fig. 2. The effect of AC093818.1 knockdown or overexpression on migration and invasion of GC cells in vitro.

a, b Cell migration (a) and invasion (b) analyzed by the Transwell assay in sh-AC093818.1- and sh-NC-infected MGC803 cells, and ov-AC093818.1- and ov-NC-infected MKN28 cells. The amplification for the left representative images is ×200. c, d Cell migration analyzed by wound-healing assay in sh-AC093818.1- and sh-NC infected MGC803 (c), and ov-AC093818.1 and ov-NC-infected MKN28 cells (d). The amplification for the left representative images is ×100. e Expression of MMP-2, MMP-9, vimentin, and E-cadherin analyzed by western blot in sh-AC093818.1- and sh-NC-infected MGC803 cells and ov-AC093818.1- and ov-NC-infected MKN28 cells in vitro. ***P < 0.001.

Fig. 3. The effect of AC093818.1 knockdown or overexpression on distant metastasis of GC cells in vivo.

a Hematoxylin and eosin staining of sections prepared from lungs and livers of mice implanted with sh-AC093818.1- and sh-NC-infected MGC803 cells or ov-AC093818.1- and ov-NC-infected MKN28 cells. b, c Expression of MMP-2, MMP-9, vimentin, and E-cadherin in b lung and c liver analyzed by western blot in sh-AC093818.1- and sh-NC-infected MGC803 cells, and ov-AC093818.1- and ov-NC-infected MKN28 cells in vivo. *P < 0.05. **P < 0.01. ***P < 0.001.

Fig. 4. The effect of AC093818.1 on the expression of PDK1, AKT1, and mTOR.

a The effect of AC093818.1 on the mRNA expression of PDK1. b The effect of AC093818.1 on the protein expression of PDK1, AKT1, p- AKT1, mTOR and p- mTOR. **P < 0.01.

Fig. 5. AC093818.1 can bind to transcription factors STAT3 and SP1.

a, b RNA immunoprecipitation assay was carried out using sh-AC093818.1- and sh-NC-infected MGC803 cells, and ov-AC093818.1- and ov-NC-infected MKN28 cells. a AC093818.1 level was examined by qRT-PCR analysis and PCR products were resolved by 3% agarose gel electrophoresis. b The fold enrichment method (2^{−ΔΔCt[ChIP/NIS])} was used to calculated AC093818.1 level. **P < 0.01. c The protein levels of SP1 and STAT3 in RNA-binding proteins of labeled AC093818.1 enriched by RNA-protein pull-down assay using cell lysate of MGC803 and MKN28 cells. d AC093818.1 level can affect the transactivation effect of SP1 or STAT3 on PDK1 promotor. MGC803-sh-NC, MGC803-sh-AC093818.1, MKN28-ov-NC, MKN28-ov-AC093818.1 cells were transfected with luciferase reporter plasmid pGL3- PDK1 promotor along with control vector pRL-SV40, pcDNA-SP1, or pcDNA-STAT3. Cell lysates were prepared from cells after 48 h. Firefly luciferase activity was normalized with Renilla luciferase activity (F/R). ***P < 0.001.

Fig. 6. SP1 or STAT3 silencing inhibits the effect of AC093818.1 overexpression.

a SP1 or STAT3 level in MKN28-ov-AC093818.1 cells after transfected with siRNA targeting SP1 (si-SP1) or STAT3 (si-STAT3), or negative control siRNA (si-NC). b Cell migration and invasion analyzed by the Transwell assay in MKN28-ov-AC093818.1 cells after transfected with si-SP1, si-STAT3, or si-NC. The amplification for the left representative images is ×200. c Cell migration analyzed by wound-healing assay in MKN28-ov-AC093818.1 cells after transfected with si-SP1, si-STAT3, or si-NC. The amplification for the left representative images is ×100. d Expression of MMP-2, MMP-9, vimentin, and E-cadherin analyzed by western blot in MKN28-ov-AC093818.1 cells after transfected with si-SP1, si-STAT3, or si-NC. e PDK1 mRNA level in MKN28-ov-AC093818.1 cells after transfected with si-SP1, si-STAT3, or si-NC. f Expression of PDK1, AKT1, p- AKT1, mTOR and p-mTOR analyzed by western blot in MKN28-ov-AC093818.1 cells after transfected with si-SP1, si-STAT3, or si-NC. ***P < 0.001.