The Novel Role of LINC01235 in Metastasis of Gastric Cancer Cells by Inducing Epithelial-mesenchymal Transition

LncRNAs play a vital role in the tumorigenesis of gastric cancer (GC). The present study aims to explore the role of LINC01235 in clinical signicance, prognostic prediction and GC metastasis. We identied differentially expressed lncRNAs using stomach adenocarcinoma RNA-Seq data from The Cancer Genome Atlas (TCGA). The expression of LINC01235 in GC cell lines and tissues were conrmed by qRT-PCR assay. The overall survival analysis and univariate/ multivariate Cox regression analysis were performed to explore the prognostic value of LINC01235. In vitro assays were utilized to assess the effect of LINC01235 in cell migration and invasion. Western blotting measured the expression of EMT-induced proteins. This study determined that LINC01235 expression has a higher fold changes by analyzing TCGA RNA-Seq data. qRT-PCR assay conrmed that LINC01235 is signicantly over-expressed in GC cells and tissues. Additionally, the overall survival analysis showed that patients with a higher LINC01235 expression had a poorer prognosis than those with a lower LINC01235 expression. Univariate Cox regression analysis indicated that high LINC01235 expression is positively correlated with poor prognosis. Moreover, LINC01235 was an independent poor prognostic marker for GC in multivariate Cox analysis. In vitro assays suggested that LINC01235 knockdown suppresses GC cell migration and invasion. GSEA revealed that high LINC01235 expression is strongly enriched in EMT pathway. Western blotting results revealed that LINC01235 silencing decreases the expression of EMT-induced proteins.


Results
This study determined that LINC01235 expression has a higher fold changes by analyzing TCGA RNA-Seq data. qRT-PCR assay con rmed that LINC01235 is signi cantly over-expressed in GC cells and tissues. Additionally, the overall survival analysis showed that patients with a higher LINC01235 expression had a poorer prognosis than those with a lower LINC01235 expression. Univariate Cox regression analysis indicated that high LINC01235 expression is positively correlated with poor prognosis. Moreover, LINC01235 was an independent poor prognostic marker for GC in multivariate Cox analysis. In vitro assays suggested that LINC01235 knockdown suppresses GC cell migration and invasion. GSEA revealed that high LINC01235 expression is strongly enriched in EMT pathway. Western blotting results revealed that LINC01235 silencing decreases the expression of EMT-induced proteins.

Conclusion
LINC01235 could promote GC cell metastasis via EMT and function as a prognostic biomarker.

Background
Cancer remains a major public health issue with an estimated 18.1 million cases and 9.6 million cancer deaths in 2018 worldwide 1 . Gastric cancer (GC) is a common malignant digestive tract tumor with a high incidence and mortality and a poor prognosis 2, 3 . Even though GC incidence and mortality rates have been decreasing in the past few decades, there were still 1,033,701 new cases and 782,685 GC-related deaths around the world in 2018 4 . More than 40% of GC patients are diagnosed with metastatic diseases and their response rate to chemotherapy and targeted therapy is unsatisfactory 5,6 . Therefore, GC is a common contributor to the worldwide cancer burden as measured by disability-adjusted life years lost 7 .

Page 3/26
The Human Genome Project found that protein-coding genes account for only 1% of the human genome, while the remaining genes have no protein-coding function 8 . The non-coding transcripts conclude housekeeping noncoding RNAs (for example: ribosomal, transfer, and small nuclear RNAs) and regulatory noncoding RNAs (for example: microRNAs and small interfering RNAs) 9 . Long non-coding RNAs (lncRNAs) with sequences > 200 nucleotides in length have no protein-coding function and play vital biological roles in human cancers 10 . LncRNAs can be classi ed into ve categories: sense, antisense, bidirectional, intronic, and intergenic 9 . The various molecular roles of lncRNAs vary for their different types.
Increasing evidence has indicated that lncRNAs are misregulated and participate in the development and progress of various cancers, including GC. For example, lncRNA HOTAIR promotes GC cell proliferation, migration, and invasion by sponging miR-331-3p and regulating HER2 expression 11 . Ectopic expression of HOTAIR also contributes to paclitaxel and doxorubicin resistance of GC cells by suppressing miR-217 expression 12 . Our previous study found that the DLX6-AS1/miR-204-5p/OCT1 positive feedback axis induces GC progression via EMT 13 . However, many vital biological roles of novel lncRNAs in GC need to be explored and veri ed.
The present study characterized stomach adenocarcinoma RNA-Seq data from The Cancer Genome Atlas (TCGA) and identi ed differentially expressed lncRNAs. LINC01235 expression was found to be of vital clinical signi cance. Further functional experiments con rmed that upregulated LINC01235 facilitates GC cell metastasis via EMT and may serve as a prognostic biomarker.

Patients and samples
A total of 36 paired GC and adjacent non-cancerous tissues (> 5 cm from cancer tissue) used in the study were obtained from the Fourth A liated Hospital of China Medical University. All samples were con rmed by histopathological examination and were stored at -80 °C immediately after surgical resection. The study was authorized by the Research Ethics Committee of the Fourth A liated Hospital of China Medical University and informed consent was obtained from all patients.
Rna Isolation And Quantitative Real-time Polymerase Chain Reaction (qrt-pcr) Total RNA samples were extracted from tissues and cell lines using RNAios Plus kits (TaKaRa Bio Technology, Dalian, China). RNA reverse transcription and quantitative PCR were performed with the ABI 7500 system (Applied Biosystems) using lnRcute lncRNA First-Strand cDNA Synthesis kit and SYBR Green qPCR kit (TIANGEN, Beijing, China). The primers used for qRT-PCR were listed in Table 1. Relative gene expression was determined using the 2 −ΔΔCt method.

Cell Transfection
Small interfering RNAs (siRNAs) designed and synthesized for decreasing gene expression by RiboBio were siRNA-LINC01235 #1, #2, #3, and negative control siRNA-NC. The siRNA sequences are shown in Table 1

Data Source And Bioinformatics Analysis
The RNA-Seq data and clinical information for GC were obtained from the UCSC Xena platform 14 , which stores various datasets from TCGA. The lncRNA expression pro les were selected and used for screening differentially expressed lncRNAs with a limma package 15 in R software. Relative LINC01235 expression values in GC and normal tissues were also collected from three microarray datasets (GSE65801, GSE51575, and GSE63089) and the GEPIA platform 16 . LINC01235 coding potential was calculated and predicted using CPC 2.0 project 17 . Gene set enrichment analysis (GSEA) was carried out to explore potential molecular functions that can be in uenced by the LINC01235 expression level 18

Statistical analysis
The SPSS software V19.0 and GraphPad Prism 7.0 were used for statistical analysis. LINC01235 expression levels between groups were analyzed by paired or unpaired test. Overall survival analysis was performed using the Kaplan-Meier method. The univariate and multivariate Cox regression analyses were used to test the effects of variables on survival. Pearson correlation analysis was utilized for calculating the expression correlation between genes. P-values < 0.05 were considered statistically signi cant.

LINC01235 is up-regulated in GC
The lncRNA expression pro les, including 375 GC tissues and 32 adjacent normal tissues, were utilized for identifying differentially expressed lncRNAs. According to the cut-off criterion of |FC| > 2.0 (fold change) and adjusted P-value < 0.05, there are 1537 up-regulated and 329 down-regulated lncRNAs in GC compared to adjacent normal tissues (Fig. 1A). LINC01235 had a log2FC value of 3.1 and was signi cantly up-regulated in GC tissues from the TCGA data (Fig. 1B). The retrieved microarray datasets from the Gene Expression Omnibus (GEO) repository showed that LINC01235 is signi cantly up-regulated in GC tissues compared to corresponding normal tissues (Fig. 1C). In addition, qRT-PCR was used to con rm the aberrant expression of LINC01235. In line with the above results, LINC01235 was frequently overexpressed in GC tissues and cell lines (Fig. 1D, E). LINC01235 was found to have a biased expression in multiple human organs, including brain, thyroid, esophagus, breast, stomach, and colon ( Fig. 2A). LINC01235 coding capacity was also investigated. The prediction indicated that LINC01235 has a coding probability of 0.058001 and belongs to noncoding genes (Fig. 2B).

Clinical Signi cance Of Linc01235 In Gc
The aberrant expression of LINC01235 in patients with or without metastasis was investigated to explore its signi cance in metastasis. The results suggested that patients with metastasis have a higher LINC01235 expression level than those without metastasis (Fig. 3A). The GC patients with detailed survival time and status data were included in the Kaplan-Meier survival analysis. A total of 352 patients were sorted into high or low expression groups according to their LINC01235 median expression value.
The results showed that patients with a high LINC01235 expression have a poorer survival probability than those with a low LINC01235 expression (Fig. 3B). Univariate Cox regression analysis demonstrated that pathologic T, N, and M stages, tumor stage, and LINC01235 expression are signi cantly associated with patients' overall survival. Further multivariate Cox regression analysis indicated that pathologic M stage and LINC01235 expression can be independent prognostic factors in GC patients (Fig. 3C).

Linc01235 Knockdown Attenuates Gc Cell Migration And Invasion
SGC-7901 and MGC-803 cells were transfected with siRNA to silence LINC01235 expression and investigate the biological role of LINC01235. After a 24-h transfection, qRT-PCR con rmed the validity of the knockdown effect of the three siRNAs (Fig. 4A). Transwell experiments demonstrated that LINC01235 silencing signi cantly decreased the migration and invasion ability of SGC-7901 and MGC-803 cells (Fig. 4B).

Linc01235 Participates In Gc Cell Emt
GSEA was performed in order to explore potential biological processes involving LINC01235. According to the median value of LINC01235 expression, a total of 375 GC patients were classi ed into two categories (high vs. low). The ndings showed that high LINC01235 expression is signi cantly enriched in EMT, angiogenesis, and focal adhesions (Fig. 5A). Expression correlation between LINC01235 and EMTassociated genes was also analyzed. Pearson correlation analysis indicated that LINC01235 expression is negatively correlated with CDH1 (r = -0.27, P < 0.01) and positively correlated with VIM (r = 0.53, P < 0.01), SNAI2 (r = 0.49, P < 0.01), and CDH2 (r = 0.27, P < 0.01, Fig. 5B). Pearson correlation results for additional EMT-associated genes are shown in Supplementary Fig. 1. Furthermore, western blotting analysis demonstrated that silencing LINC01235 can increase E-cadherin (epithelial marker) expression and decrease N-cadherin, vimentin, and Slug (mesenchymal markers) expression (Fig. 5C). Thus, LIN01235 may promote GC cell migration and invasion via EMT.

Discussion
The higher incidence and mortality of GC are attributed to the fact that most patients are diagnosed at an advanced stage, thereby losing their opportunity for radical surgery. Research has shown that the veyear survival rate of patients with advanced GC is < 20% 20,21 . Increasing numbers of studies have found that lncRNAs can act as critical regulators in GC metastasis and tumor growth, potentially becoming promising targets for GC treatment [22][23][24] . LINC01235 is a large intergenic noncoding RNA that is located on human chromosome 9p23 and has three exons. RNA-Seq data from the Human Protein Atlas indicated that LINC01235 has a biased expression level in human tissues 25 . However, the role and function of LINC01235 in GC remain unclear.
The present study identi ed differentially expressed lncRNAs in GC and normal tissues by analyzing RNA-Seq data from the TCGA database. LINC01235 was found to have a log2FC value of 3.1 in cancer tissues compared to normal tissues. The GEO datasets were also explored. The results of three GC microarray datasets revealed that LINC01235 is signi cantly up-regulated in cancer tissues compared to paired normal tissues. Then, qRT-PCR results con rmed that LINC01235 is over-expressed in GC cell lines and tissues. All of these ndings support the aberrant expression of LINC01235 in GC. Therefore, LINC01235 may act as a novel proto-oncogene in the occurrence and progression of GC.
Furthermore, LINC01235 has a higher level in GC patients with metastasis than in those without. Kaplan-Meier survival analysis illustrated that patients with a high LINC01235 expression have a poorer survival probability than those with a low LINC01235 expression (P = 0.0017). Univariate Cox regression analysis indicated that high LINC01235 expression is positively correlated with poor prognosis (HR = 1.49, 95% CI [1.06-2.10], P = 0.021). Furthermore, LINC01235 is an independent poor prognostic marker for GC in multivariate Cox analysis (HR = 1.48, 95% CI [1.04-2.09], P = 0.029). To the best of our knowledge, no previous studies have investigated LINC01235 expression and biological role in GC. The function of LINC01235 in GC cells was explored using loss-of-function assay. The results revealed that LINC01235 silencing signi cantly decreases the migration and invasion capacity of GC cells. The above results indicated that LINC01235 is of vital clinical signi cance and may be a potential therapeutic target for metastatic GC.
Tumor metastasis is a multi-step process that includes migration and invasion of epithelial-derived tumor cells, blood circulation, evasion from blood vessels, and colonization of new microenvironments 26,27 .
GSEA was performed to explore potential biological processes involving LINC01235. LINC01235 expression was signi cantly enriched in EMT, angiogenesis, and focal adhesions. EMT, a biological process in which tumor cells lose their epithelial polarity and obtain mesenchymal characteristics, has been found to play a vital role in the initiation of metastasis 28,29 . Furthermore, expression correlation between LINC01235 and EMT-associated genes was analyzed. Pearson correlation analysis indicated that LINC01235 expression is negatively correlated with CDH1 (r = -0.27, P < 0.01) and positively correlated with VIM (r = 0.53, P < 0.01), SNAI2 (r = 0.49, P < 0.01), and CDH2 (r = 0.27, P < 0.01). Western blotting assays con rmed that LINC01235 silencing can increase E-cadherin expression and decrease Ncadherin, vimentin, and Slug expression. These ndings showed that LINC01235 may promote GC cell migration and invasion via EMT.

Conclusions
The present study found that lncRNA LINC01235 is up-regulated in GC cells and tissues, underscoring its vital clinical signi cance. Loss-of-function experiments demonstrated that upregulated LINC01235 promotes GC cell metastasis via EMT and may be a valuable prognostic biomarker and treatment target for metastatic GC. However, the in-depth mechanism by which LINC01235 facilitates GC cell migration and invasion requires more investigation in future studies.

Declarations
Ethics approval and consent to participate The study was authorized by the Research Ethics Committee of the Fourth A liated Hospital of China Medical University and informed consent was obtained from all patients.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
Dai DQ and Zhang C designed the study; Zhang C, Zhang CD and Liang Y performed data processing and analysis; Zhang C wrote the paper. All authors participated in revising the nal manuscript and approved it for publication.         Clinical signi cances of LINC01235 in GC. (A) Patients with metastasis have higher LINC01235 expression level than those without metastasis. (B) Overall survival analysis showed that patients with higher LINC01235 expression have poorer prognosis than those with lower LINC01235 expression (P = 0.0017). (C) Univariate and multivariate Cox analysis indicated that LINC01235 is an independent poor prognostic marker for GC. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3
Clinical signi cances of LINC01235 in GC. (A) Patients with metastasis have higher LINC01235 expression level than those without metastasis. (B) Overall survival analysis showed that patients with higher LINC01235 expression have poorer prognosis than those with lower LINC01235 expression (P = 0.0017). (C) Univariate and multivariate Cox analysis indicated that LINC01235 is an independent poor prognostic marker for GC. *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.