LINC00152 knock-down suppresses esophageal cancer by EGFR signaling pathway

2.1 Clinical sample

Thirty pairs of Non-tumor and esophageal cancer tissues were taken from esophageal cancer patients who received treatment in Nanjing University of Chinese Medicine from October 2015 to December 2016. All patients were primary esophageal carcinoma and un-treated before the operation. The tissues were resection and quickly stored in the 10% polyoxymethylene to fix until using.

2.2 In situ hybridization (ISH) assay

The tissues were embedded by paraffin and section as 5μm, sections dewaxing by xylene, after gradient alcohol hydration, using fresh 3% H₂O₂ to close and place at room temperature for 5-10 min; washing by distilled water (2 min×3 times); adding a drop fresh pepsin which was dilution by 30 g/l citric acid to incubate in the thermostat box for 10-30 min; ISH PBS was used to wash (5 min×3 times), washing by distilled water for 2 min; adding pre-hybrids as 20 μl/slice to place in the wet box to culture in the thermostat box (40°C) for 3h; adding pre-hybrids as 20 μl/slice, special cover film for in-situ hybridization and place in the wet box in thermostat box (40°C) overnight; Gently uncover the special cover film of the in situ hybridization (2×SSC buffer solution as 5 min×2 times; 0.5×SSC buffer solution for 15 min; 0.2×SSC buffer solution for 15 min); adding closed liquid as 30μl/slice to culture for 30 min at 37°C; getting rid of the excess liquid, adding 30μl/slice biotinylated rat digoxin to culture for 1 h at 37°C; ISH PBS was used to wash (5 min×4 times); adding SABC 30μl/slice to culture for 30 min at 37°C; washing by ISH PBS (5 min×4 times); adding fresh DAB to color, washing by water; Gradient alcohol dehydration, Xylene transparent, Neutral gum seal. Using image analyze software to analysis the IOD of different tissues.

2.3 RT-qPCR assay

Total RNA was extracted from clinical samples using Trizol reagent (Thermo Fisher Scientific) following the protocols of manufacturer. For the measurement of linc00152 mRNA level, RNA was reversely transcribed into cDNA using M-MLV Reverse Transcriptase (Thermo Fisher Scientific) together with random primers and then cDNA was used for qPCR detection by PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific) along with specific quantitative primers. The quantitative PCR primers were presented as follows: LINC00152 F: 5ʹ-GAAGGTGTCGGCAAGATC-3ʹ; R: 5ʹ-TCGGTGTCTGTCATATTCG-3ʹ. β-actin: F: 5ʹ-AAAGACCT-GTACGCCAACAC-3ʹ; R: 5ʹ-GTCATACTCCTGCTTGCTGAT-3ʹ.

2.4 Cell culture and grouping

The Eca 109 and Kyse 150 cells, two kinds of esophageal cancer cell lines purchased from ATCC (USA), were cultured by RPMI1640 medium (Sigma, USA) contained with 10% fetal bovine serum (FBS) (Sigma, USA) in incubator (37°C, 5% CO₂). When the cells reached 90% fluent, 0.25% trypsin was added to digest the cells and centrifuged to discard the supernatant. The suspension were added to cell culture medium and the incubated into the cell flask and future culture. Respectively dividing the Eca 109 and Kyse 150 cells into 3 groups: Normal control (NC) groups which were treated with non-treatment; Blank (BL) groups which were transfected with empty vector (GenScript (Nanjing) Co., Ltd., China) and si-lncRNA groups which were transfected with si-lncRNA LINC00152 (GenScript (Nanjing) Co., Ltd., China).

2.5 MTT assay

The Eca 109 or Kyse 150 cells of different groups were collected, the cell concentration of different groups were adjusted as 1×10⁵ cells/ml, the cells were inoculated in the 96-well plate, 200 μl cell suspension were added into every holes, placed into incubator at 37°C for 48 h, 20 μl of MTT solution (5mg/ml) was added into every well, cultured in the incubator at 37℃ for 4 h, after discarding the supernatant, adding the 150 μl DMSO solution, Oscillatory reaction for 10 min, The absorbance (A) value were measured at 490 nm. The cell proliferation rates were measured.

2.6 The cell apoptosis rate of different groups by flow cytometry

The Eca 109 or Kyse 150 cells of different groups which were treated by different methods for 48 h were collected and adjusted as 1×10⁵ cell/ml. Taking 1 ml cell suspension, added Annexin-V buffer solution, fully mixed, added the 5 μl Propidium iodide (PI) and Annexin-V-FITC, incubated avoid light for 20min, the cell apoptosis rate of different groups were measured by flow cytometry, the experiments were repeated for 3 times.

2.7 Cell cycle measurement by flow cytometry

The Eca 109 or Kyse 150 cells of different groups which were treated by different methods for 48 h were collected and adjustment as 1×10⁵ cell/ml. The cells were washed by pre-cool PBS twice, the pre-cool 70% ethanol was added to fix at 4℃ overnight, washed by PBS once. After this, the centrifugation collection fixed overnight, 500 μl PBS was added to suspension cell, adding 20 μl RNase A solution and 0.2% Triton X-100. It was then cultured in a 37℃ water-bath for 30 min; added 400 μl PI Staining solution, and mixed, Incubation under light conditions at 4℃ for 30 min. The cell cycle were measured at 488 nm by Flow Cytometry. The experiments were repeated for 3 times.

2.8 Western blot (WB) assay

The Eca 109 or Kyse 150 cells of different groups which were logarithmic growth phase was collection. 3 ml Trizol cell lysate was added into the cells, incubated on ice for 10 min, transfer to the centrifuge tube, after centrifugation, the supernatant was transferred into the EP tube. Detecting protein concentration according to the BCA protein concentration kit. Mixed the protein with sample buffer and boiled for 5 min at 100°C, added into SDS-PAGE electrophoresis gel, 50 μl/well. After electrophoresis, proteins were transferred to PVDF (Millipore, Bedford, MA, USA) membrane at 4°C. After blocking, the membrane was incubated with primary antibodies: EGFR (1:800) (Abcam, USA), PI3K (1:800) (Abcam, USA), AKT (1:800) (Abcam, USA), P21 (1:800) (Abcam, USA) and GAPDH (1:1000) (Abcam, USA). Followed by incubation with secondary antibody (1:1000) (Abcam, USA). EGFR, PI3K, AKT and P21 gray value of protein bands were measured by Ge-1 Pro Analyzer (ver 3.0) software.

2.9 Statistical analysis

The data of this study were analyzed by SPSS 19.0 software, the data were expressed as Mean±SD (standard deviation). T-test was used to compare the differences between groups. The one-way ANOVA was used to analyze the differences between groups. The LSD-t test was used for multiple comparisons between groups, the value was bilateral probability, and the test level was α=0.05. P<0.05 indicated that the difference was statistically significant.

Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors' institutional review board or equivalent committee.

3.1 LINC00152 expression in different tissues

Compared with no-tumor tissues, the LINC00152 expression was significantly increased in esophageal cancer tissues (P＜0.001). By RT-qPCR, the result was shown that LINC00152 mRNA expression of esophageal cancer tissues were significantly up-regulated compared with that of non-tumor tissues. Depending on these results, we inferred that LINC00152 might be an oncology factor in esophageal cancer occur and development. The relative data was shown in Figure 1.

Figure 1

The LINC00152 expression of different tissues by ISH and RT-qPCR (×100)

***:P＜0.001, compared with no-tumor tissues

3.2 LINC 00152 affects cell proliferation rate

To investigate the effect of the lncRNA LINC00152 on the esophageal cancer proliferation, the cells proliferation rates we measured by MTT assay. The relative results found that the lncRNA LINC00152 knockdown (si-lncRNA groups) suppressed cells proliferation compared with that of NC groups in Eca 109 (P<0.05,69.69±5.65 vs.127.33±5.02, Figure 2A) and Kyse 150 (P<0.05,71.50±4.48 vs.130.58±6.00, Figure 2B). The relative data were shown in Figure 2.

Figure 2

The cell proliferation rate of difference groups in Eca 109 and Kyse 150 cells. A. The Eca 109 cell proliferation rate of difference groups by MTT assay.

NC: Normal control group; BL: Blank group; si-lncRNA: si-lncRNA LINC00152 group

***: P<0.05, compared with NC group. B. The Kyse 150 cell proliferation rate of difference groups by MTT assay. ***: P<0.05, compared with NC group.

3.3 The cell apoptosis rates of different groups

To evaluate the effects of lncRNA LINC00152 in esophageal cancer apoptosis, we measured the cell apoptosis rates of difference groups by flow cytometry. Compared with NC groups, the cell apoptosis rates of si-lncRNA groups, which were knocked-down the lncRNA LINC00152, were significantly enhanced in Eca 109 (P<0.05, Figure 3A) and Kyse 150 (P<0.05, Figure 3B). The relative data were shown in Figure 3.

Figure 3

Cell apoptosis measurement by flow cytometry in Eca 109 and Kyse 150 cells.

NC: Normal control group; BL: Blank group; si-lncRNA: si-lncRNA LINC00152 group

A. The Eca 109 cell apoptosis rate of difference groups by flow cytometry. ***: P<0.05, compared with NC group. B. The Kyse 150 cell apoptosis rate of difference groups by flow cytometry. ***: P<0.05, compared with NC group.

3.4 The cell cycle by flow cytometry

To investigate the effects of lncRNA LINC00152 in esophageal cancer cell cycle, the cell cycle were evaluated by flow cytometry in different groups. Compared with NC groups, the G1 phase rate of si-lncRNA groups which the lncRNA LINC00152 were down-regulated were significantly increased in Eca 109 (P<0.05, Figure 4A) and Kyse 150 (P<0.05, Figure 4B). The relative data were shown in Figure 4.

Figure 4

The cell cycle rates of difference groups in Eca 109 and Kyse 150 cells.

NC: Normal control group; BL: Blank group; si-lncRNA: si-lncRNA LINC00152 group

A. The Eca 109 cell cycle rates of difference groups by flow cytometry. ***: P<0.05, compared with NC group. B. The Kyse 150 cell cycle rates of difference groups by flow cytometry. ***: P<0.05, compared with NC group.

3.5 LINC00152 affects relative protein expression

The EGFR, PI3K and AKT proteins expressions of si-ln-cRNA groups were significantly suppressed compare with those proteins expression of NC groups in Eca 109 (P<0.05, Figure 5A) and Kyse 150 (P<0.05, Figure 5B). Meanwhile, the P21 protein expression of si-lncRNA group was significantly increased compared with that of NC groups in Eca 109 (P<0.05, Figure 5A) and Kyse 150 (P<0.05, Figure 5B).

Figure 5

The relative proteins expressions of difference groups in Eca 109 and Kyse 150 cells.

NC: Normal control group; BL: Blank group; si-lncRNA: si-lncRNA LINC00152 group

A. The relative proteins expressions of difference groups in Eca 109 by WB assay. ***: P<0.05, compared with NC group. B. The relative proteins expressions of difference groups in Kyse 150 by WB assay. ***: P<0.05, compared with NC group.