LncRNA LINC00689 Promotes the Progression of Gastric Cancer Through Upregulation of ADAM9 by Sponging miR-526b-3p

Abstract

Introduction: Increasing studies have demonstrated that noncoding RNAs, including miRNAs and lncRNAs, have vital roles in mediating cancer progression. However, the expression features and biological functions of LINC00689 in gastric cancer (GC) remain largely unknown. This study was designed to investigate the functions of LINC00689, miR-526b-3p and ADAM9 as well as their interactions in GC.

Methods: Real time PCR(RT-PCR) was used to detect the expression of LINC0068, miR-526b-3p and ADAM9 in both GC tissues or cell lines. Gain- and loss- of functions of assays were conducted to verify the role of LINC0068, miR-526b-3p and ADAM9 in GC development. Cell proliferation were determined by CCK8 assay and transwell assay and scratch wound-healing assay were used to test cell invasion and migration. Further, the relationships between LINC00689 and miR-526b-3p, miR-526b-3p and ADAM9 were predicted by bioinformatics analysis and then proved by Luciferase reporter assay and RNA Immunoprecipitation(RIP) assay.

Results: We found that LINC00689 was upregulated in GC tissues and positively correlated with advanced tumor stage and tumor size, while miR-526b-3p was downregulated. Furthermore, gain- and loss-of-function experiments revealed that LINC00689 promoted the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of GC cells, while miR-526b-3p had the opposite effects. The underlying mechanisms indicated that LINC00689 functioned as a competing endogenous RNA (ceRNA) by sponging miR-526b-3p in GC cells. Further investigations confirmed that ADAM9 was a direct target of miR-526b-3p and positively modulated the progression of GC.

Conclusion: Our study suggests that LINC00689 functions as a novel oncogenic lncRNA in the development of GC by promoting ADAM9 expression through suppression of miR-526b-3p.

Keywords: ADAM9; LINC00689; competing endogenous RNA; gastric cancer; miR-526b-3p.

Figure 1

Expression of LINC00689 in GC tissues and cell lines. (A). The level of LINC00689 in human GC tissues and matched adjacent normal tissues was detected by RT-PCR (n = 41). ***p<0.001 vs the normal group. (B). The expression of LINC00689 was increased in patients with advanced TNM stage. (n= 16 in stage I, n=15 in stage II, n=10 in stage III). *represents p<0.05, ***represents p<0.001. (C and D). The correlation between LINC00689 expression and overall survival (C) or post progression survival (D) of GC patients through KM-plotter (http://kmplot.com/analysis/index.php?p=service). (E). The expression of LINC00689 was elevated in GC cells compared with the immortalized gastric epithelium cell line GES-1. *p<0.05, **P<0.01, ***p<0.001 vs the GES-1 group.

Figure 2

LINC00689 promoted the proliferation, migration and invasion of GC cells in vitro. MGC-803 and AGS cells were transfected with LINC00689-overexpressing plasmid, sh-LINC00689 or corresponding negative controls. (A and B). RT-PCR was used to detect the expression of LINC00689; (C and D). CCK8 assay was used to detect the proliferation of GC cells. (E). Cell invasion was detected by Transwell assay. (F). Scratch wound-healing assay was used to determine cell migration. (G). Western blot was used to detect the expression of the EMT-related markers E-cadherin and Vimentin. n=three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

miR-526b-3p is a target of LINC00689 in GC. (A). The base binding sites of miR-526b-3p and LINC00689 were predicted by StarBase (http://starbase.sysu.edu.cn/). (B). Dual luciferase reporter assay was conducted to verify the targeted relationship between miR-526b-3p and LINC00689, which showed that miR-526b-3p decreased the luciferase activity of the LINC00689 wild-type reporter construct (LINC00689-WT) but not the mutant type construct (LINC00689-MUT), N.S (no significance) represents p>0.05, **represents p < 0.01. (C and D). RIP assay was conducted to detect the binding between LINC00689 and Ago2 or miR-526b-3p and Ago2. (E). The expression level of miR-526b-3p was detected in GC tissues and matched adjacent normal tissues by RT-PCR (n=41). (F). Pearson’s regression analysis was used to analyze the relationship between miR-526b-3p and LINC00689. (G). Relative expression of miR-526b-3p in the GC cell lines and immortalized gastric epithelium cell line GES-1, *p < 0.05, ***p < 0.001 vs the GES-1 group. (H). miR-526b-3p expression was increased by inhibition of LINC00689 but inhibited upon overexpression of LINC00689, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

miR-526b-3p inhibited the proliferation, migration, invasion and EMT process in GC cells in vitro. AGS and MGC-803 cells were transfected with miR-526b-3p mimics or inhibitors and the corresponding miRNA negative controls. (A). The expression of miR-526b-3p was detected by RT-PCR. (B and C). CCK8 assay was used to detect the proliferation of GC cells. (D). Cell invasion was detected by Transwell assay. (E). Scratch wound-healing assay was used to determine cell migration. (F). Western blot was used to detect the expression of the EMT-related markers E-cadherin and Vimentin. n=three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

ADAM9 is a direct target of miR-526b-3p in GC cells. (A). ADAM9 was a potential target of miR-526b-3p as predicted by miRmap (http://mirnamap.mbc.nctu.edu.tw/), PicTar(https://pictar.mdc-berlin.de/) and Targetscan (http://www.targetscan.org/vert_72/). (B). miR-526b-3p and its putative binding sequences in the 3’-UTR of ADAM9. (C). The relative luciferase activity in cells cotransfected with miR-526b-3p mimics and ADAM9-WT was decreased, while that in cells cotransfected with miR-526b-3p mimics and ADAM9-MT was not significantly changed. N.S (no significance) represents p>0.05, *** represents p<0.001. (D). Expression of ADAM9 in human GC tissues and matched adjacent normal tissues by Western blot (n = 4). (E). The expression level of ADAM9 was detected in GC tissues and matched adjacent normal tissues by RT-PCR (n=41). (F). Data of ADAM9 expression in Stomach adenocarcinoma was downloaded from GEPIA (http://gepia.cancer-pku.cn/). (G and H). Pearson’s regression analysis was used to analyze the relationship between ADAM9 and LINC00689 (G) or ADAM9 and miR-526b-3p (H). (I). LINC00689 overexpression increased the expression of ADAM9, and LINC00689 knockdown reduced the level of ADAM9. (J). miR-526b-3p overexpression reduced the expression of ADAM9, while miR-526b-3p knockdown had the opposite effect. N.S, **, and ***represents p >0.05, p<0.01 and p<0.001, respectively.

Figure 6

ADAM9 promotes the proliferation, migration and invasion of GC cells. (A and B). RT-PCR and Western blot were used to detect the expression of ADAM9 in MGC-803 cells that were transfected with si-ADAM9 or corresponding siRNA negative vector. (C). CCK8 assay was used to detect the proliferation of GC cells. (D). Cell invasion was detected by Transwell assay. (E). Scratch wound-healing assay was used to determine cell migration. (F). Western blot was used to detect the expression of the EMT-related markers E-cadherin and Vimentin. n=three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

LINC00689 increased ADAM9 expression by sponging miR-526b-3p. MGC-803 cells were transfected with miR-526b-3p mimics or LINC00689 overexpression plasmid. (A and B). Expression levels of LINC00689 and miR-526b-3p were detected by RT-PCR. (C). Western blot was conducted to detect the expression of ADAM9. (D). CCK8 assay was used to detect the proliferation of GC cells. (E). Cell invasion was detected by Transwell assay. (F). Scratch wound-healing assay was used to determine cell migration. (G). Western blot was used to detect the expression of the EMT-related markers E-cadherin and Vimentin. (H). A diagram of the mechanisms of LINC00689, miR-526b-3p and ADAM9 networks in regulating the progression of gastric cancer n=three independent experiments. NS p>0.05, *p < 0.05, **p < 0.01, ***p < 0.001.