Long non-coding RNA LINC00665 gastric cancer tumorigenesis by regulation miR-149-3p/RNF2 axis

Abstract

Background: Recently, LINC00665 has been reported to be a pivotal regulator in kinds of malignancy, such as lung cancer and liver cancer. However, the functions and underlying mechanisms of LINC00665 in gastric cancer (GC) remain unclear.

Materials and methods: We recruited 49 paired GC tissue to explore LINC00665 expression by qRT-PCR. In vitro function assays were used to explore the roles of LINC00665 in GC progression. Moreover, the interaction among LINC00665, miR-149-3p and RNF2 was explored by bioinformatics analysis and luciferase reporter assay.

Results: In the present study, we found that LINC00665 expression was significantly elevated in GC tissues and cell lines. High LINC00665 expression was associated with TNM stage, histological grade, and poor prognosis of GC patients. Function assays showed that LINC00665 suppression significantly reduced GC cells viability and invasion ability in vitro. Mechanistic analysis showed that LINC00665 might serve as a ceRNA for miR-149-3p to regulate the expression of RNF2.

Conclusion: Our current study revealed the LINC00665/miR-149-3p/RNF2 axis was involved in GC progression, providing novel insights into the treatment for GC.

Keywords: LINC00665; RNF2; gastric cancer; miR-149-3p.

Figure 1

LINC00665 was upregulated in gastric cancer (GC). (A) The expression of LINC00665 in GC was determined by The Cancer Genome Atlas (TCGA) database. (B) The expression of LINC00665 in normal tissues explored by National Center for Biotechnology Information. (C) LINC00665 expression was significantly increased in GC tissues compared to normal tissues. (D, E) High LINC00665 expression was significantly associated with poor overall survival (D) and disease free survival rate (E) of GC patients. *P<0.05.

Figure 2

LINC00665 promoted gastric cancer (GC) cells proliferation and invasion. (A) Expression of LINC00665 in GC tissues was measured by qRT-PCR. (B) High LINC00665 expression was associated with advanced TNM stage. (C) High LINC00665 expression was associated with advanced histological grade. (D) Expression of LINC00665 in GC cell lines was measured by qRT-PCR. (E) sh-LINC00665 significantly reduced LINC00665 expression in AGS and MGC-803 cells. (F–I) Cell viabilities were measured in AGS and MGC-803 cells transfected with sh-LINC00665 or sh-NC by EdU assay (F, G) and colony formation assay (H, I). (J) Cell invasion was measured in AGS and MGC-803 cells transfected with sh-LINC00665 or sh-NC by Transwell assay. *P<0.05.

Figure 3

MiR-149-3p was a target of LINC00665. (A) Architecture of LINC00665 genomic region according to the University of California, Santa Cruz database. (B, C) Target sequences of miR-149-3p in LINC00665. (D) LINC00665 inhibition increased miR-149-3p expression in gastric cancer (GC) cells. (E, F) The interaction between LINC00665 and miR-149-3p was confirmed by luciferase reporter assay (E) and pull-down assay (F). (G) Expression of miR-149-3p in GC tissues was measured by qRT-PCR. (H, I) Low miR-149-3p expression was associated with poor relapse free survival (H) and overall survival (I) of GC patients. (J) miR-149-3p mimics reduced GC cells colony formation ability. *P<0.05.

Figure 4

LINC00665 regulated RNF2 by absorbing miR-149-3p. (A) miR-149-3p and its putative binding sequence sites in the RNF2 3′UTR. (B) miR-149-3p mimics reduced the luciferase activity of RNF2-Wt group, but not RNF2-Mut group. (C) miR-149-3p mimics reduced RNF2 protein expression in gastric cancer (GC) cells. (D) LINC00665 expression was positively associated with RNF2 expression in GC tissues. (E) RNF2 protein expression was suppressed in sh-LINC00665 group, while miR-149-3p inhibitors rescued the effects. (F) IHC determined RNF2 expression in GC tissues and normal tissues. (G) RNF2 expression in GC tissues was determined by GEPIA database. (H, I) High RNF2 expression was associated with poor relapse free survival (H) and overall survival (I) of GC patients. *P<0.05.

Figure 5

Schematic representation of LINC00665 in gastric cancer.