LncRNA LINC00974 Upregulates CDK6 to Promote Cell Cycle Progression in Gastric Carcinoma

GC patients

In this study 66 GC patients (all were adenocarcinoma cases; gender: 36 men and 30 women; age: 32–67 years; mean age: 47.3 ± 5.0 years) were selected from the 122 GC patients admitted to Baotou Cancer Hospital between July 2010 and April 2014. This study passed the review board of Baotou Cancer Hospital before enrolling patients. Inclusion criteria: (1) new GC cases; (2) no therapies were initiated. Exclusion criteria: (1) recurrent GC; (2) history of malignancies; (3) other severe clinical disorders were observed. After admission, all the 66 GC patients were educated with the principle of this study and the potential publication of data from this study. All patients signed informed consent. According to the staging system established by AJCC, the 66 GC patients included 19, 20, and 27 cases at stages II–IV, respectively.

Follow-up

From the day of admission, survival conditions of all patients were monitored for 5 years. Follow-up was performed in a monthly manner through telephone. The patients died of other causes or lost before the end of follow-up were excluded from subsequent survival analysis.

GC tissues and cells

Before the initiation of therapies, nontumor gastric tissues and GC tissues were collected from each patient by performing fine needle gastric biopsy. The weight of each tissue sample ranged from 0.02 to 0.032 g. Based on histopathological examination results, GC tissues contained >95% cancer cells and nontumor tissues contained <5% cancer cells.

AGS human GC cell line (GAC, ATCC) was used. Cells were cultivated in a mixture of 90% F-12K Medium and 10% fetal bovine serum (FBS). Cell culture conditions were 37°C, 5% CO₂, and 95% humidity.

Transient transfections

LINC00974 and CDK6 expression vectors were constructed using pcDNA3.1 vector (Sangon, Shanghai, China). Negative control (NC) siRNA and LINC00974 siRNA were also from Sangon. To perform transfections, AGS cells were harvested at confluence of 70%–80%, followed by transfection of 10 nM vector (empty vector as NC group) or 35 nM siRNA (NC siRNA as NC group) into 6 × 10⁵ cells using Lipofectamine 2000 (Thermo Fisher Scientific). Cells were collected at 24 h post-transfection to perform the following experiments. Untransfected cells were control (C) cells.

RNA extractions and qPCR

Total RNAs in tissues (ground in liquid nitrogen) and AGS cells were extracted using RNAzol (Sigma-Aldrich). After digestion with DNase I, total RNAs were subjected to reverse transcriptions using AMV Reverse Transcriptase (Sangon). The synthesized cDNA and SYBR^® Green master mix (Takara, Japan) were used to prepare qPCR mixtures with GAPDH as endogenous control to measure the expression levels of LINC00974 and CDK6 mRNA.

Cell cycle assay

AGS cells were collected 24 h after transfection and trypsinization was performed. Pre-cold PBS was used to wash cells and ethanol (75%) was then used to incubate with the cells at 4°C for 4 h. After washing with pre-cold PBS again, cells were stained with BD Pharmingen™ PI/RNase for 30 min. Finally, cells were separated by flow cytometer and 10⁵ events were included in each experiment.

Cell proliferation assay

At 24 h after transfection, AGS cells were collected and mixed with the mixture of 90% F-12K Medium and 10% FBS with a ratio of 4 × 10⁴ cells per 1 mL. Cells were mixed well with cell culture medium to prepare single cell suspensions. A 96 cell culture plate was used to cultivate cells with 0.1 mL per well at 37°C, followed by the addition of 10 μL CCK-8 solution (Sigma-Aldrich) at 4 h before the end of cell culture. After cell culture was terminated, OD values were measured at 450 nm.

Western blot

Total proteins in 5 × 10⁵ cells were extracted using RIPA solution (Thermo Fisher Scientific). Electrophoresis was performed using 10%-PAGE gel to separate different proteins after protein denaturing in boiling water for 5 min. Gel transfer was performed using PVDF membranes, and blocking was performed in PBS containing 5% FBS for 2 h at room temperature. Membranes were first blotted with GAPDH (1:900, ab9485; Abcam) or CDK6 (1:900, ab22349; Abcam) rabbit primary antibodies for 12 h at 4°C, followed by blotting with goat IgG-HRP (1:1500, MBS435036; MyBioSource) secondary antibody for 2 h at room temperature. Membranes were then incubated with ECL (Sigma-Aldrich) for 5 min at room temperature to develop signals. Data were processed using Image J v1.46 software.

Statistical analysis

All data analyses were performed using mean values of the data of three biological replicates. Differences between GC and nontumor tissues were explored by paired t-test. Differences among multiple cell groups were analyzed by ANOVA (one-way) and Tukey test. Correlations were analyzed by linear regression. The 66 GC patients were grouped into high and low LINC00974/CDK6 level groups according to the median expression level. Survival curves were plotted using Kaplan–Meier plotter and compared by log-rank test. p < 0.05 was statistically significant.

LINC00974 and CDK6 mRNA were positively correlated in GC

Expression levels of LINC00974 and CDK6 mRNA in both nontumor and GC tissues were measured by qPCR and compared by paired t-test. Comparing with nontumor group, expression levels of LINC00974 (Fig. 1A) and CDK6 mRNA (Fig. 1B) were significantly higher in GC group (p < 0.05). Correlations between LINC00974 and CDK6 mRNA were analyzed by linear regression. It can be observed that expression level of LINC00974 was significantly and inversely correlated with that of CDK6 mRNA in GC tissues (Fig. 1C). However, the correlation was not significant in nontumor tissues (Fig. 1D).

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FIG. 1.

LINC00974 and CDK6 mRNA were positively correlated in GC. Expression levels of LINC00974 (A) and CDK6 mRNA (B) in both nontumor and GC tissues were measured by qPCR and compared by paired t-test. Correlations between LINC00974 and CDK6 mRNA in GC (C) and nontumor (D) tissues were analyzed by linear regression. All data were mean values of three biological replicates, *p < 0.05. GC, gastric cancer.

High levels of LINC00974 and CDK6 mRNA predicted poor survival

Survival curves of both high and low LINC00974/CDK6 level groups were plotted using Kaplan–Meier plotter and compared by log-rank test. It can be observed that survival rate of patients in high LINC00974 level group was significantly lower than that of patients in low LINC00974 level group (Fig. 2A). In addition, survival rate of patients in high CDK6 mRNA level group was also significantly lower than that of patients in low LINC00974 level group (Fig. 2B).

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FIG. 2.

High levels of LINC00974 and CDK6 mRNA predicted poor survival. Survival curves of both high and low LINC00974 (A)/CDK6 (B) level groups were plotted using Kaplan–Meier plotter and compared by log-rank test.

LINC00974 negatively regulated CDK6 in AGS cells

AGS cells were transfected with LINC00974 and CDK6 expression vectors as well as LINC00974 siRNA. Expression levels of LINC00974 and CDK6 mRNA were measured by qPCR. Comparing with two controls (C and NC), expression levels of LINC00974 and CDK6 mRNA were significantly altered at 24 h post-transfection (Fig. 3A, p < 0.05). Moreover, LINC00974 overexpression led to upregulated, whereas LINC00974 siRNA silencing led to downregulated CDK6 at both mRNA and protein levels (Fig. 3B, p < 0.05). However, CDK6 overexpression failed to affect LINC00974 expression.

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FIG. 3.

LINC00974 negatively regulated CDK6 in AGS cells. AGS cells were transfected with LINC00974 and CDK6 expression vectors as well as LINC00974 siRNA. Altered expression levels of LINC00974 and CDK6 mRNA were detected by qPCR at 24 h post-transfection (A). The effects of LINC00974 overexpression and siRNA silencing on CDK6 were analyzed by qPCR and Western blot (B), and the effects of CDK6 overexpression on LINC00974 expression were analyzed by qPCR (C). All data were mean values of three biological replicates, *p < 0.05.

LINC00974 positively regulated AGS cell cycle progression and proliferation through CDK6

The effects of LINC00974 and CDK6 overexpression as well as LINC00974 siRNA silencing on AGS cell progression and proliferation were analyzed by cell cycle assay and cell proliferation assay, respectively. Comparing with NC and C groups, LINC00974 overexpression promoted and siRNA silencing inhibited G1-S transition (Fig. 4A, p < 0.05; reflected by the percentage of cells at G1 and G2 phases) and cell proliferation (Fig. 4B, p < 0.05). In addition, CDK6 overexpression attenuated the effects of LINC00974 overexpression (p < 0.05).

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FIG. 4.

LINC00974 positively regulated AGS cell cycle progression and proliferation through CDK6. The effects of LINC00974 and CDK6 overexpression as well as LINC00974 siRNA silencing on AGS cell progression and proliferation were analyzed by cell cycle assay (A) and cell proliferation assay (B), respectively. All data were mean values of three biological replicates, *p < 0.05.