J Gastric Cancer.  2019 Dec;19(4):460-472. 10.5230/jgc.2019.19.e43.

LINC00703 Acts as a Tumor Suppressor via Regulating miR-181a/KLF6 Axis in Gastric Cancer

• Yang H
• Peng M
• Li Y
• Zhu R
• Li X
• Qian Z

• KMID: 2466034
• DOI: http://doi.org/10.5230/jgc.2019.19.e43

Abstract

PURPOSE
Long noncoding RNA 00703 (LINC00703) was found originating from a region downstream of Kruppel-like factor 6 (KLF6) gene, having 2 binding sites for miR-181a. Since KLF6 has been reported as a target of miR-181a in gastric cancer (GC), this study aims to investigate whether LINC00703 regulates the miR-181a/KLF6 axis and plays a functional role in GC pathogenesis.
MATERIALS AND METHODS
GC tissues, cell lines, and nude mice were included in this study. RNA binding protein immunoprecipitation (RIP) and pull-down assays were used to evaluate interaction between LINC00703 and miR-181a. Quantitative real-time polymerase chain reaction and western blot were applied for analysis of gene expression at the transcriptional and protein levels. A nude xenograft mouse model was used to determine LINC00703 function in vivo.
RESULTS
We revealed that LINC00703 competitively interacts with miR-181a to regulate KLF6. Overexpression of LINC00703 inhibited cell proliferation, migration/invasion, but promoted apoptosis in vitro, and arrested tumor growth in vivo. LINC00703 expression was found to be decreased in GC tissues, which was positively correlated with KLF6, but negatively with the miR-181a levels.
CONCLUSIONS
LINC00703 may have an anti-cancer function via modulation of the miR-181a/KLF6 axis. This study also provides a new potential diagnostic marker and therapeutic target for GC treatment.

Keyword

LincRNAs; KLF6; miRNA sponge; Gastric cancer

MeSH Terms

Animals
Apoptosis
Binding Sites
Blotting, Western
Cell Line
Cell Proliferation
Gene Expression
Heterografts
Immunoprecipitation
In Vitro Techniques
Mice
Mice, Nude
Real-Time Polymerase Chain Reaction
RNA, Long Noncoding
RNA-Binding Proteins
Stomach Neoplasms*
RNA, Long Noncoding
RNA-Binding Proteins

Figure

• 

  Fig. 1 LINC00703 interacts with miR-181a. (A) A schematic representation of LINC00703 location at chromosome 10 and 2 putative binding sites for miR-181a in LINC00703. (B) The luciferase reporter plasmid (KLF6 3′-UTR) was co-transfected with miR-181a and LINC00703-overexpressing plasmids into HEK-293T cells. The relative luciferase activity was measured 48h after transfection. (C) RIP with human monoclonal anti-Ago2, mouse preimmune IgG or 10% input from BGC-823 cell extracts. The qRT-PCR was performed to determine the RNA levels of miR-181a and LINC00703 in immunoprecipitates as compared to the input. (D) The expression of LINC00703 enriched by RNA pull-down assay. All data are presented as means±standard deviation (n=3, biological replicates). LINC00703 = long noncoding RNA 00703; KLF = Kruppel-like factor; UTR = untranslated region; RIP = RNA binding protein immunoprecipitation; IgG = immunoglobulin G; qRT-PCR = quantitative real-time polymerase chain reaction. *P<0.05, †P<0.01, and ‡P<0.001.

• 

  Fig. 2 LINC00703 regulates KLF6 expression. (A) The qRT-PCR analysis, demonstrating LINC00703 expression in several gastric cancer cell lines (MGC-803, SGC-7901, and BGC-823), compared with normal gastric epithelium cell line (GES-1). (B) LINC00703 expression analysis in BGC-823 cells, transfected with pcDNA3.1 or LINC00703-overexpressing vector (LINC00703). (C and D) Expression levels of miR-181a and KLF6 after 48 hours, following the transfection of pcDNA3.1 or LINC00703 in BGC-823 cells. (E) The KLF6 protein level in BGC-823 cells, transfected with miR-181a mimic, LINC00703 or both miR-181a mimic and LINC00703. All data are presented as means±standard deviation (n=5, biological replicates). LINC00703 = long noncoding RNA 00703; KLF = Kruppel-like factor; qRT-PCR = quantitative real-time polymerase chain reaction. *P<0.05 and †P<0.01.

• 

  Fig. 3 Functional analysis of LINC00703. BGC-823 cells were transfected with pcDNA3.1 or LINC00703 overexpressing vector (LINC00703). (A and B) Measurements of cell proliferation using MTT assay (A) and PCNA protein level (B). (C) Flow cytometry analysis of cell apoptotic rates. (D) Results of transwell assays reflecting the changes in cell migration and invasiveness (100×). All data are presented as means±standard deviation (n=3, biological replicates). LINC00703 = long noncoding RNA 00703; MTT = 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCNA = proliferating cell nuclear antigen. *P<0.05 and †P<0.01.

• 

  Fig. 4 Effect of LINC00703 on tumor growth in vivo. (A) Tumors were subcutaneously induced in nude mice. BGC-832 cells that were stably transfected with pcDNA3.1 or LINC00703 overexpressing vectors (LINC00703) were injected into the flanks of nude mice for 12 days. (B and C) Dynamics of tumor volume (A) and mice body weight (B) according to the measurements every 3 days. (D) The tumors were dissected and weighed at the end of experiment. (E) qRT-PCR analysis of the expression of LINC00703, miR-181a and KLF6 in resected tumor tissues from the nude mice. All data are presented as means±standard deviation (n=8, for each group). LINC00703 = long noncoding RNA 00703; qRT-PCR = quantitative real-time polymerase chain reaction; KLF = Kruppel-like factor. *P<0.05 and †P<0.01.

• 

  Fig. 5 LINC00703 correlates positively with KLF6 but negatively with miR-181a. qRT-PCR analysis of LINC00703 (A), KLF6 (B), and miR-181a (C) in 15 GC tissues and their adjacent normal tissue. Pearson correlation analysis was performed between LINC00703 and KLF6 (D), LINC00703 and miR-181a (E), miR-181a and KLF6 (F). All data are presented as means±standard deviation. LINC00703 = long noncoding RNA 00703; KLF = Kruppel-like factor; qRT-PCR = quantitative real-time polymerase chain reaction. *P<0.01.

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