Correlation between LncRNA‐LINC00659 and clinical prognosis in gastric cancer and study on its biological mechanism

Abstract Non‐coding RNAs play important roles in tumorigenesis and tumour progression. In previous screening, lncRNA‐LINC00659 (LINC00659) is highly expressed in gastric cancer; however, its role in gastric cancer has not been illustrated yet. In this study, the expression of LINC00659 was detected in cancer tissues and paracancerous tissues of patients with gastric cancer. As a result, LINC00659 expression was increased in gastric cancer tissues, which was closely associated with tumour stage and lymph node metastasis, but was not correlated with age, gender and tissue differentiation. Survival curve analysis showed that patients with low expression of LINC00659 harboured higher overall survival. In vitro, the level of LINC00659 was increased in gastric cancer cells. Afterwards, the expression of LINC00659 was down‐regulated in SGC‐7901 and BGC‐823 cells by plasmid‐mediated si‐LINC00659 transfection. Consequently, the cell invasion ability was weakened, the cell cycle was inhibited, and cell viability was also suppressed. Luciferase reporter gene assay and RNA pull‐down assay showed that LINC00659 could bind to the transcription factor SUZ12, indicating that SUZ12 was a regulatory gene of LINC00659. The overexpression of SUZ12 could resist the roles of si‐LINC00659. In this study, we found that LINC00659 was highly expressed in gastric cancer, which might be related to the regulation of cell proliferation and promotion of cell invasion. Transcription factor, SUZ12, was a regulator of LINC00659. Additionally, LINC00659 could regulate cell cycle and invasion of gastric cancer by promoting the expression of SUZ12.


| BACKG ROU N D
can be used as tumour markers to diagnose gastric cancer. Previous studies have found that HOTAIR can be used as a critical marker for the diagnosis and prognosis of breast cancer. 7 SUMO1P3, a small ubiquitin-related LncRNA, is also closely associated with the clinicopathological characteristics of patients with gastric cancer. The expression of SUMO1P3 is significantly higher in gastric cancer than that of paracarcinoma tissue; additionally, SUMO1P3 can also be used as a marker to distinguish benign gastric ulcer, gastric polyp and gastric cancer. 8 In our previous screening, LINC00659 is highly expressed in gastric cancer; however, its role in gastric cancer and its possible correlation with pathological parameters have not been revealed. Therefore, in this study, we mainly investigate the correlation between LINC00659 and clinical and prognostic factors in patients with gastric cancer through clinical samples, and to reveal the mechanism of LINC00659 in metastasis and invasion in gastric cancer.

| Extraction of total RNA from tissues and detection of relative expression of LINC00695
A total of 50 mg cancer tissue/adjacent tissue was cut into piece by sterile surgical scissors, followed by addition of 600 μL of Trizol reagent (Invitrogen) to the centrifuge tube. The tissue was ground, homogenized for 2-3 minutes until no presence of solid mass. The homogenized sample was then transferred to a 1.5 mL centrifuge tube, followed by addition of 600 μL of Trizol reagent and subsequent incubation for 5 minutes at room temperature to separate the nucleic acid and the protein. Proper amount chloroform was added (according to the proportion that 0.2 mL of chloroform/1 mL of Trizol reagent), shaken for 15 seconds, incubated at room temperature for 2-3 minutes and centrifuged at 3000 g for 20 minutes. Centrifuged sample was divided into three layers, the upper layer contained RNA, which was transferred to RNase-free centrifuge tube, followed by addition of an equal volume of isopropanol to precipitate RNA and incubation at room temperature for 10 minutes. After centrifuging at 3000 g for 15 minutes and drying, 50 μL of RNA-free water was added dissolve RNA, followed by purity detection. In brief, UV spectrophotometer was used to measure the absorbance at 260 and 280 nm, and the ratio of OD260/OD280 was calculated. OD260/OD280 between 1.8 and 2.0 indicated qualified purity of RNA sample.
Reverse transcription of RNA: After preparing the reverse transcription system, the reverse transcription reaction was performed at 55°C for 30 minutes, and the reverse transcriptase was inactivated at 85°C for 5 minutes. Finally, PCR tube was placed on ice for 5 minutes to terminate the reaction, and the cDNA product was stored at −20°C for further use.

| Cell transfection assay
Cell transfection was performed using the Lipofectamine RNAiMAX kit (Invitrogen). siRNA oligonucleotide si-LINC00659 and si-negative control of LINC00659 (as control) were transfected (Genepharma synthesis). The siRNA sequence of LINC00659 was Sense:

| Cell viability by CCK-8 assay
SGC-7901 and BGC-823 cells were divided into Control and si-LINC00659 groups and inoculated in 96-well plates. Three replicate wells were set, and blank medium was used as control. Cell viability was measured at 0, 3, 6, 12, 24, 36, 48 hours after transfection. In brief, 100 μL of medium/per well was replaced, followed by addition of 10 μL of CCK-8 reagent (Beyondtime Biotechnology Co., Ltd.).
After incubation for additional 2 hours, the absorbance was measured at 450 nm.

| Detection of cell invasion capacity by Transwell chamber
SGC-7901 and BGC-823 cells were transfected with siRNA.
Matrigel (BD) was melted overnight at 4°C for 24 hours.
Afterwards, 300 μL of serum-free medium was added to 60 μL Finally, the number of invading cells was observed and counted under microscope.

| Cell cycle detection
Cell cycle was detected using PI staining kit (BD) by FACS calibur.
Briefly, after siRNA transfection, SGC-7901 and BGC-823 cells were cultured for 24 hours, harvested with 0.25% trypsin and 0.02% EDTA, centrifuged at 1000 r/min to collect cells. The resuspended cells were washed with pre-cooled PBS, fixed in 70% ethanol at 4°C overnight. RNase was added at a final concentration of 50 μg/mL, and PI staining solution was added at 60 μg/mL after water bath.
Cell cycle was detected by flow cytometry after incubation in dark for 30 minutes.

| Detection of cell migration ability by wound healing assay
After siRNA transfection, SGC-7901 and BGC-823 cells were inoculated into 6-well plates. After cell confluency reached about 70%, s sterile pipette was used for scratching, and sterile cotton swab was utilized to wipe excess cells. Afterwards, culture medium was added for incubation, followed by observation of cell migration degree. Image Pro-Plus 6.0 software was used for quantitative analysis of migration rate.

| Colony formation assay
Colony formation assay was performed to assess the colony formation capacity. In brief, transfected cells were seeded in a 6-well plate (200-300 cells/per well) and incubated. Culture medium was changed every 2-3 days, and colony was formed for about two weeks. After discarding the medium, colonies were fixed with ethanol, stained with 1% crystal violet for 10 minutes. After rinsing with PBS, the number of colony formation was observed and quantitatively analysed.

| Detection of protein expression by Western blot
After siRNA transfection, cells were cultured for additional 24 hours. Then, cells were collected, washed twice with PBS and added with RIPA lysate (Beyondtime Biotechnology Co., Ltd.) and PMSF solution (Beyondtime Biotechnology Co., Ltd.) at a ratio of 100:1 for lysis on ice for 0.5 hour.
After centrifugation at 3000 g for 5 minutes, the precipitate was removed Image Pro-Plus 6.0 software was used to analyse optical density. GAPDH was used as the internal control, and results were shown as comparison of the optical density between target protein and internal control.

| RNA pull-down assay
Biotin-labelled RNAs were transcribed using Biotin RNA Labeling Mix showed that the expression of LINC00659 in the si-LINC00659 group was significantly lower than that of Control and si-Negative (P < .01). D-E, Cell viability by CCK-8 assay showed that the cell viability was increased within 3-48 h, which was significantly lower in the si-LINC00659 group than that of the Control group and the si-Negative group. Compared with Control group at the same time point, *P < .05; comparison with si-Negative group, #P < .05. F-H, Colony formation assay showed that the number of colony formed was significantly lower in the si-LINC00659 group than that in the Control and si-Negative groups (P < .01) RNA was reacted at 90°C for 2 minutes, then placed on ice for 2 minutes, followed by addition of RNA structure buffer (10 nmol/L Trist

| The construction and detection method of mouse model
The animals were male nude mice of the BALB/C-nu/nu strain

| The expression of LINC00659 in gastric cancer and the correlation with clinicopathological characteristics and prognosis
The relative expression of LINC00659 in gastric cancer tissues was (0.53 ± 0.10), which was significantly higher than that in adjacent tissues (0.28 ± 0.08) (P < .01). In different tumour stages, LINC00659 was up-regulated with increasing tumour staging, indicating that LINC00659 was associated with tumour stage (shown in Figure 1A-C).
A total of 80 patients with gastric cancer were divided into LINC00659 high expression group and low expression group (N = 40 each) according to the cut-off value of 0.58 of LINC00659 relative expression.
The correlation analysis between LINC00659 expression and clinicopathological characteristics of gastric cancer patients showed that the expression level of LINC00659 was associated with tumour stage and lymph node metastasis, whereas was not associated with age, gender and histological differentiation. Multivariate regression analysis revealed that lymph node metastasis, tumour stage and LINC00659 expression level were independent prognostic predictors for patients with gastric cancer (shown in Tables 1 and 2). Survival curve analysis demonstrated that patients with low expression of LINC00659 had higher overall survival rate (shown in Figure 1D).

| The expression of LINC00659 in gastric cancer cells, determination of knock-down efficiency and effects on cell viability
The expression of LINC00659 was significantly higher in gastric cancer cell lines (SGC-7901 and BGC-823), compared with that in normal cells (GES-1) (P < .01). After siRNA silencing targeting LINC00659, the expression level of LINC00659 was significantly down-regulated in SGC-7901 and BGC-823 than that in Control group (P < .01). Colony formation assay showed that colony formation capacity was significantly weakened than Control group after LINC00659 silencing (P < .01). Cell viability assay by CCK-8 revealed that the cell viability was significantly down-regulated within 3-48 hours after LINC00659 silencing, compared with that in Control group (P < .01) (shown in Figure 2).

| The effects of LINC00659 on cell cycle, migration and invasion ability of gastric cancer cells
Transwell assay showed that the invasion ability was significantly down- Wound healing assay revealed that the cell migration ability was significantly decreased in the si-LINC00695 group than that in the Control group and the si-Negative group (P < .05) (shown in Figures 3 and 4).

| The regulatory roles of LINC00659 on cell cycle regulating protein and PI3K-AKT
In SGC-7901 and BGC-823 cells, the expression of key proteins in regulating cell cycle, p21, Cyclin E, Cyclin D and CDK2 was significantly down-regulated in the si-LINC00659 group than that in the Control and si-Negative groups (P < .05). Meanwhile, the PI3K-AKT signal was inhibited. The expression of key protein, PI3K, AKT, p-AKT and mTOR was significantly suppressed in the si-LINC00659 group, in comparison with Control group and si-Negative group (P < .05) (shown in Figure 5).

| Targeted regulation of transcription factor SUZ12 by LINC00659
In this study, we used co-expression analysis to find that LINC00659 and SUZ12 have regulatory relationship. At the same time, Western blot analysis showed that the expression of SUZ12 was significantly increased in SGC-7901 and BGC-823, and luciferase reporter assay revealed that the activity of SUZ12 was significantly enhanced after overexpression of LINC00659 in SGC-7901 and BGC-823. Therefore, LINC00659 could regulate the expression of transcription factor SUZ12 (shown in Figure 6).

| SUZ12 rescue assay verified the action mechanism of LINC00659 in SGC-7901
To further investigate that SUZ12 was a target protein of LINC00659, SGC-7901 cell line was transfected for SUZ12 overexpression.
In addition, Western blot was used to detect the levels of cell cycle regulatory proteins p21, Cyclin E, Cyclin D, and CDK2 and PI3K-AKT signal. As a result, SUZ12 overexpression led to significantly increased colony formation ability, migration capacity and invasion ability in si-LINC00659 + SUZ12 group, compared with those of si-LINC00659 group (P < .05). Meanwhile, the expression cell cycle regulatory protein was significantly higher in si-LINC00659 + SUZ12 group than that of si-LINC00659 group. And PI3K signal was activated. These results showed that SUZ12 overexpression could resist the effects caused by down-regulation of LINC00659 (shown in Figures 7 and 8).
F I G U R E 7 SUZ12 rescue assay on cell viability, migration and invasion ability of SGC-7901 cells. A, Cell viability by CCK-8 assay showed that cell viability was significantly decreased in si-LINC-00659 group than that in Control, whereas cell viability was significantly enhanced in si-LINC-00659 + SUZ12 group compared with that in si-LINC-00659 group. Compared with Control group, *P < .05; Comparison with si-LINC00659 group, #P < .05. B-C, Colony formation assay revealed that the number of colony formed was significantly lower in si-LINC00659 than that of Control group, whereas the number of colony formed was significantly higher in si-LINC-00659 + SUZ12 than that of si-LINC00659 group. Comparison between groups, P < .05. D-E, Transwell assay showed that the number of invaded cells was lower in si-LINC00659 group than that of Control group, whereas the number of invaded cells was significantly higher in si-LINC-00659 + SUZ12 group than that of si-LINC00659 group. Comparison between groups, P < .05. F-G, Wound healing assay showed lower migration ability in si-LINC00659 than that in control group, whereas cell migration ability was significantly higher in si-LINC-00659 + SUZ12 group than that si-LINC00659 group. Comparison between groups, P < .05

| Tumour size comparison and protein expression level
The tumour volume of mice after SGC-7901 cell inoculation was significantly higher than that of SGC-7901-LINC00659 −/− . After LINC00659 inhibition, the protein levels of SUZ12, p21, Cyclin E, Cyclin D and CDK2 in SGC-7901 tumour tissue were significantly higher than those of SGC-7901-LINC00659 −/− (shown in Figure 9). and MKN28 and inhibit cell proliferation. 11,12 In this study, we first validated that the expression of LINC00659 was increased in gastric cancer tissues. Additionally, the expression level of LINC00659 was associated with the stage and lymph node metastasis but was not correlated with age, gender or histological differentiation.

| D ISCUSS I ON
Multivariate regression analysis showed that lymph node metastasis, to S phase. In particular, the abnormal expression of CDK2 protein is detected in a variety of tumours, which is related to the proliferation of tumour cells. 16,17 The expression of Cyclins is significantly periodic and specific, and various signals and transcription factors Cyclin D, a well-studied protein, is an initial factor of cell cycle. Cells are progressed from G0 phase to G1 phase mediated by Cyclin D through G protein ras and mitotic activation protein kinase (MAPK).
Meanwhile, Cyclin D can also bind to CDK4 and CDK6 to shorten the G1 phase. The abnormal expression of Cyclin D in tumour cells can reduce the dependence of cell proliferation on mitogens, causing abnormal cell cycle and abnormal proliferation. 18,19 PI3K is a key signal in regulating cell proliferation/apoptosis. AKT, the downstream protein of PI3K, can be phosphorylated into p-AKT to further regulate cyclin. 20 PI3K can also transmit mitotic signals to p70S6K1 via AKT-mTOR. And PI3K inhibition can decrease the expression of Cyclin D1, causing G1 phase arrest. In addition, mTOR inhibitors could also lead to similar effects. 21 Therefore, these studies demonstrate that SUZ12 may play a role by promoting the expression of cyclins and by activating PI3K signals.

| CON CLUS IONS
In summary, here, we show that LINC00659 is involved in the carcinogenesis and progression of gastric cancer. The high expression of LINC00659 can promote the proliferation, metastasis and invasion of gastric cancer, which is related to the promotion of SUZ12 transcription factor expression. Moreover, SUZ12 further plays a role by promoting the expression of cell cycle regulatory proteins.

ACK N OWLED G EM ENTS
Thank the second hospital of JiaXing University for its help and Chenyang Han for its financial support.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
The study approved with Ethics Committee.

CO N S E NT FO R PU B LI C ATI O N
All authors approval published the article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data and material were availability.