LINC00514 promotes gastric cancer cell growth and EMT progression via miR-204-3p/KRAS

Long noncoding RNAs (LncRNAs) participate in tumor development and tumorigenesis. However, the mechanism, function and expression of LINC00514 in GC remain unknown. We showed that LINC00514 was upregulated in GC specimens compared with nontumor specimens. Overexpression of LINC00514 induced cell growth and EMT progression in GC cells. By using bioinformatics prediction, we found that miR-204-3p contained binding sequences for LINC00514. Luciferase reporter analysis noted that miR-204-3p overexpression decreased the luciferase expression under LINC00514-wild-type and KRAS-wild-type reporters but not that under mutant reporter. Ectopic LINC00514 expression decreased miR-204-3p expression. miR-204-3p expression was decreased in GC specimens compared with nontumor specimens and that LINC00514 was negatively correlated with miR-204-3p in GC specimens. Furthermore, KRAS was identified as a target gene for miR-204-3p according to TargetScan. Elevated miR-204-3p expression inhibited KRAS expression in HGC-27 cells, and ectopic expression of LINC00514 enhanced KRAS expression. Elevated LINC00514 expression enhanced cell growth and EMT progression by sponging KRAS. Our data indicated that LINC00514 may act as an oncogene and therapeutic target for GC.

thyroid tumor cell migration, growth and invasion. Yu et al. [29] illustrated that LINC00514 enhanced osteosarcoma development by sponging miR-708/ URGCP. Mi et al. [30] also noted that SP1-influenced LINC00514 overexpression induced metastasis and growth by modulating miR-708 in osteosarcoma. Until now, the roles of LINC00514 in gastric tumor are undefined and need to be studied.
We found that LINC00514 expression was upregulated in GC specimens compared with nontumor specimens. Overexpression of LINC00514 induced cell growth and EMT progression in GC cells.

MATERIALS AND METHODS
GC specimens and paired control nontumor samples were collected from 40 cases of GC at our department. Samples were immediately stored and snap-frozen in liquid nitrogen. Our protocol was approved by the Clinical Ethics Committee of Ningxia Medical University. HGC-27 and SGC-7901 cells were obtained from ATCC (Invitrogen, USA) and plated in DMEM supplemented with streptomycin, FBS and penicillin. PcNDA-LINC00514 and miR-204-3p mimic and control vectors were obtained from GenePharma (Shanghai, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen, USA).

CCK-8 analysis
Cells were plated in a 96-well microplate, and cell proliferation was assessed by CCK-8 kit (Dojindo). The growth rate was detected at 0, 1, 2 and 3 days posttransfection, and the absorbance at 450 nM was detected with a microtiter reader.

Dual Luciferase Reporter
The mutated and wild-type putative miR-204-3p targets on LINC00514 and the KRAS 3'UTR were then cloned into the pGL3 expression vector (Invitrogen). Cells were cultured in 24-well dishes and transfected with scramble or miR-204-3p mimic and wt LINC00514 and KRAS 3'UTR or mutated LINC00514 and KRAS 3'UTR vectors. After 48 hours, luciferase values were measured with Dual-Luciferase System (Promega, USA).

Statistical analysis
Data are indicated as the means ± SD, and statistical assays were utilized by SPSS. Statistical significance was detected using Student's t-test. Statistical significance was measured at P<0.05.

LINC00514 was overexpressed in GC specimens
First, the level of LINC00514 was detected using qRT-PCR in 40 pairs of GC specimens and paired control nontumor specimens. As illustrated in Figure 1A, 1B, the expression of LINC00514 in 40 pairs of GC specimens and paired control nontumor specimens is indicated. LINC00514 was overexpressed in 29 cases (29/40, 72.5%) compared to paired control nontumor specimens ( Figure 1C). LINC00514 expression was upregulated in GC specimens compared with nontumor specimens ( Figure 1D).

Overexpression of LINC00514 induced cell growth and EMT progression in GC cells
The  AGING LINC00514 enhanced CDK2 expression in both SGC-7901 and HGC-27 cells ( Figure 3C). Overexpression of LINC00514 induced cell growth in both SGC-7901 and HGC-27 cells ( Figure 3D). Ectopic expression of LINC00514 inhibited expression of E-cadherin and increased vimentin and N-cadherin expression ( Figure 3E).

Elevated expression of LINC00514 enhanced cell growth and EMT progression by sponging KRAS
To further understand the functional roles of the LINC00514/KRAS axis in GC development, we used KRAS siRNA vectors for experiments. The level of KRAS was downregulated in HGC-27 cells after transfection with si-KRAS vectors ( Figure 5A).  Figure  5D). Downregulation of KRAS promoted E-cadherin expression and decreased N-cadherin and vimentin expression ( Figure 5E).

DISCUSSION
LncRNAs have drawn increasing attention due to their roles in the development and progression of tumors. Many references have noted that lncRNAs play critical roles in GC development, indicating new insight into GC pathogenesis. For instance, HNF1A-AS1 induced cell angiogenesis, metastasis, invasion and lymphangiogenesis by sponging the miR-30b-3p/PI3K/ AKT axis in GC [31]. Liang et al. [32] noted that LINC00691 overexpression increased the invasion and growth of GC cells through JAK/STAT signaling. Zhou et al. [33] proved that BCAR4 increased cell growth and inhibited cell apoptosis by modulating MAPK/ERK in GC. Dai et al. [34] indicated that UCA1 enhanced cisplatin resistance by inducing the PI3K/AKT signaling pathway and recruiting EZH2 in GC. Recently, Li et al. [28] noted that knockdown of LINC00514 suppressed papillary thyroid tumor cell migration, growth and invasion. Yu et al. [29] illustrated that LINC00514 enhanced osteosarcoma development by sponging miR-708/URGCP. Mi et al. [30] also noted that SP1-influenced LINC00514 overexpression induced metastasis and growth by modulating miR-708 in osteosarcoma. However, the role of LINC00514 in GC is still unknown and needs to be studied. We illustrated that LINC00514 expression was upregulated in GC specimens compared with nontumor specimens. Overexpression of LINC00514 induced cell growth and EMT progression in GC cells.
LncRNAs play a ceRNA role in sponging miRNAs and their target genes to regulate cell functions [35,36]. For example, Wang et al. [37] illustrated that PVT1 induced GC cell migration by sponging miR-30a/Snail. Li et al. [38] noted that IGF2-AS enhanced GC cell invasion, growth and migration by regulating miR-937/EZH2. Deng et al. [39] showed that DLGAP1-AS1 induced GC progression by sponging miR-628-5p/AEG-1. Liu et al. [40] noted that SNHG1 induced GC cell EMT progression via modulation of the DCLK1/miR-15b/Notch1 axis. Recently, Li et al. [28] found that LINC00514 suppressed thyroid tumors by sponging the CDC23/miR-204-3p axis. By using bioinformatics prediction, miR-204-3p contained binding sequences for LINC00514. Luciferase reporter analysis illustrated that overexpression of miR-204-3p decreased the luciferase expression under LINC00514wild-type and KRAS-wild-type reporters but not that under the mutant reporter. Ectopic expression of LINC00514 decreased miR-204-3p expression. We illustrated that miR-204-3p expression was downregulated in GC specimens compared with nontumor specimens and that LINC00514 expression was negatively correlated with miR-204-3p expression in GC specimens. Previous studies have shown that miR-204 plays important roles in gastric cancer development. For example, Zhang et al. showed that miR-204-5p inhibited tumor metastasis by modulating CXCR4 and CXCL12 in gastric cancer [41]. Furthermore, another study indicated that miR-204-5p suppressed gastric cancer cell growth by inhibiting RAB22A and USP47 [42]. Furthermore, KRAS was identified as a potential target gene for miR-204-3p according to TargetScan bioinformatics prediction. Elevated expression of miR-204-3p inhibited KRAS expression in HGC-27 cells, and ectopic expression of LINC00514 enhanced KRAS expression. Elevated expression of LINC00514 enhanced cell growth and EMT progression by sponging KRAS. Previous studies have shown that KRAS plays critical roles in gastric tumor development. However, the underlying mechanisms are still unclear. Our results suggested that the ability of LINC00514 to modulate KRAS may provide the mechanism of posttranscriptional regulation of KRAS.
In summary, LINC00514 was overexpressed in GC specimens, and elevated expression of LINC00514 enhanced cell growth and EMT progression by sponging the miR-204-3p/KRAS axis. Our data indicated that LINC00514 may act as an oncogene and therapeutic target for GC.

Editorial note
& This corresponding author has a verified history of publications using a personal email address for correspondence.

AUTHOR CONTRIBUTIONS
Ling Yuan, Jiaxin Li, Yi Yang, Yan Chen conducted experiments and collected data and Ling Yuan, Yang Bu, Mengyi Ye, Xiongjie Mao, Tingting Ma, Yi Nan analyzed data, Ling Yuan, Yi Nan write and revised this manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.

FUNDING
This study was supported by Ningxia high school Top Discipline construction (Traditional Chinese Medicine Discipline, No. NXYLXK-2017A06) funded project.