LncRNA AK023391 promotes tumorigenesis and invasion of gastric cancer through activation of the PI3K/Akt signaling pathway

Abstract

Background: Patients with gastric cancer commonly have a poor prognosis, owing to its invasiveness and distant metastasis. Recent studies have confirmed the pivotal role of long non-coding RNAs (lncRNAs) in tumorigenesis and the progression of malignant tumors, including gastric cancer. However, little is known about the molecular mechanism by which lncRNA AK023391 contributes to gastric cancer.

Methods: A lncRNA microarray was used to identify the differentially expressed lncRNA AK023391 in gastric cancer and adjacent normal tissues. In addition, RNA fluorescence in situ hybridization (FISH) was used to investigate the association between AK023391 expression and the clinicopathological characteristics and prognosis of patients with gastric cancer. Subsequently, a series of in vitro assays and a xenograft tumor model were used to observe the functions of lncRNA AK023391 in gastric cancer cells. A cancer pathway microarray, bioinformatic analysis, western blotting, and immunochemistry were carried out to verify the regulation of AK023391 and its downstream PI3K/Akt signaling pathway.

Results: Expression of lncRNA AK023391 was significantly upregulated in gastric cancer samples and cell lines in comparison to adjacent normal tissues, and was positively correlated with poor survival in patients with gastric cancer. The multivariate Cox regression model revealed that AK023391 expression acted as an independent prognostic factor for survival in patients with gastric cancer. Knockdown of AK023391 inhibited cell growth and invasion both in vitro and in vivo, and induced apoptosis and cell cycle arrest in gastric cancer cells, whereas its overexpression reversed these effects. Mechanistically, PI3K/Akt signaling mediated the NF-κB, FOXO3a, and p53 pathways. Moreover, downstream transcription factors, such as c-myb, cyclinB1/G2, and BCL-6 might be involved in AK023391-induced tumorigenesis in gastric cancer.

Conclusions: The novel oncogenic lncRNA AK023391 in gastric cancer exerts its effects through activation of the PI3K/Akt signaling pathway, and may act as a potential biomarker for survival in patients with gastric cancer.

Keywords: Akt; Gastric cancer; Invasion; PI3K; lncRNA AK023391.

Fig. 1

Expression of LncRNA AK023391 was upregulated in gastric cancer (GC). a LncRNA microarray analyses of five GC tissue samples and five corresponding adjacent normal tissues were performed. b LncRNA microarray identification of AK023391 and the most obvious changes in 20 upregulated lncRNAs and four downregulated lncRNAs. c qRT-PCR verification of the expression levels of 20 upregulated lncRNAs in GC tissues. d LncRNA expression profile and qRT-PCR analysis of the expression of AK023391 in GC tissues and adjacent normal tissues. T: tumor tissue; N: normal tissue

Fig. 2

Expression of lncRNA AK023391 was associated with poor survival in patients with gastric cancer (GC). a Fluorescence in situ hybridization(FISH) analysis of the expression and cellular localization of lncRNA AK023391 in 77 patients with GC and pair-matched normal tissues. b qRT-PCR analysis of AK023391 expression in different GC cell lines and GES-1. c Receiver operating characteristic (ROC) curve analysis of the cutoff value, sensitivity, specificity and area under the curve (AUC) of AK023391 in patients with GC. d Kaplan–Meier analysis of the correlation of AK023391 expression with survival in patients with GC, as well as early stage patients. *P < 0.05, **P < 0.01

Fig. 3

Knockdown of AK023391 inhibited cell proliferation and colony formation. a qRT-PCR analysis of the transfection efficiency of si-AK023391 in HGC-27, AGS, and SGC-7901 cells. b CCK-8 detection of cell proliferation activities of si-AK023391-transfected HGC-27, AGS, and SGC-7901 cells. c Analysis of cell colony formation capacity of si-AK023391-transfected HGC-27, AGS, and SGC-7901 cells. *P < 0.05, **P < 0.01

Fig. 4

Knockdown of AK023391 inhibited DNA synthesis of gastric cancer (GC) cells. The EdU assay was used to observe the effects of AK023391 knockdown on DNA synthesis of HGC-27, AGS, and SGC-7901 cells. *P < 0.05, **P < 0.01

Fig. 5

Knockdown of AK023391 inhibited migration and invasion of gastric cancer (GC) cells. a-b Cell migration abilities were respectively determined by the wound-healing assay and Transwell migration assay in si-AK023391-transfected HGC 27, AGS, and SGC-7901 cells. c The cell invasive potential was assessed by the Transwell invasion assay in si-AK023391-transfected HGC 27, AGS, and SGC-7901 cells. **P < 0.01

Fig. 6

Knockdown of AK023391 induced apoptosis and cell cycle arrest. a Flow cytometry was used to detect the proportion of apoptotic cells in si-AK023391-transfected HGC-27, AGS, and SGC-7901 cells. b Flow cytometry analysis of the cycle distribution of si-AK023391-transfected HGC-27, AGS, and SGC-7901 cells. **P < 0.01

Fig. 7

Overexpression of AK023391 promoted cell growth and invasion of gastric cancer (GC) cells. a qRT-PCR analysis of the transfection efficiency of AK023391 in MGC-803 and BGC-823 cells. b-d Cell proliferation activity and colony formation capability were estimated by CCK-8 and colony formation assays, respectively, in AK023391-transfected MGC-803 and BGC-823 cells. e-f Cell invasive potential was assessed by the Transwell invasion assay in AK023391-transfected MGC-803 and BGC-823 cells. **P < 0.01

Fig. 8

LncRNA AK023391 was involved in the regulation of the PI3K/Akt signaling pathway. a Pathway expression profile analysis of differentially expressed coding genes caused by si-AK23391 in AGS cells. b KEGG enrichment analysis of the 10 most important enriched pathways involved in the regulation of AK23391 in gastric cancer (GC) cells. c Gene ratio and enrichment score analysis revealed that PI3K/Akt and FOXO signaling pathways, respectively, had the most obvious enrichment among the altered pathways induced by AK023391 knockdown. d Cluster analysis of the differentially expressed genes involved in the PI3K/Akt and FOXO signaling pathways, induced by AK023391 knockdown. e-f Western blotting validation of the effects of AK023391 knockdown on the expression of PI3K/Akt, NF-κB, p53, and FOXO3a pathways, and their downstream transcription factors c-myb, cyclinB1/G2, and BCL-6 in HGC 27, AGS, and SGC-7901 cells

Fig. 9

Knockdown of AK023391 inhibited tumor growth in vivo. a Schematic representation of the SGC-7901 xenograft tumor size after 30 d of tumor growth in the si-AK023391 and negative control (NC)groups. b Tumor growth curve of the SGC-7901 xenograft tumor growth tendency in the si-AK023391 and NC groups. c Statistical comparison of the differences in tumor weight and volume between the si-AK023391 and NC groups. d IHC analysis of the expression levels of Ki-67, p-FOXO3a, p-PI3K, p-Akt, and p-NF-κB in xenograft tumor tissues treated with si-AK023391 and in the NC group (original magnification, ×200). e LncRNA AK023391 promoted GC tumorigenesis and invasion through activation of the PI3K/Akt pathway that further activated NF-κB, inactivated FOXO3a, upregulated c-myb, cyclinB1/G2, and BCL-6, and downregulated p53 expression