Long noncoding RNA Z38 promotes cell proliferation and metastasis and inhibits cell apoptosis in human gastric cancer

Abstract

Gastric cancer is one of the leading causes of cancer-associated mortality and has a high tendency to metastasize, making it a priority to develop novel diagnostic and treatment methods at the early stages. The present study investigated the role of a newly-discovered long non-coding RNA, Z38, in gastric cancer cell proliferation, metastasis and apoptosis. It was observed that Z38 was upregulated in tissues from patients with gastric cancer as well as in cultured gastric cancer cells. Knockdown of Z38 decreased the cell proliferative rate, as evidenced by colony formation assays and cell proliferation assays. In addition, Transwell assays and wound-healing assays demonstrated that depletion of Z38 significantly inhibited cell migration and invasion in AGS and MKN74 cells. Furthermore, a cell apoptosis assay and measurement of relative activities of related caspases revealed that depletion of Z38 increased cell apoptosis by promoting the activities of caspase-3 and caspase-9, but not that of caspase-8. Finally, western blot analysis further demonstrated the role of Z38 in the apoptosis of AGS and MKN74 cells. These results suggested that Z38 promotes cell proliferation and metastasis, and inhibits cell apoptosis in gastric cancer. Z38 may represent a novel therapeutic target for the treatment of gastric cancer in clinic.

Keywords: Z38; apoptosis; gastric cancer; metastasis; proliferation.

Figure 1.

Long non-coding RNA Z38 was overexpressed in human gastric cancer tissues and cells. (A) A total of 100 patients with gastric cancer were included and the expression of Z38 in the tumor tissues and their adjacent non-cancerous tissues was investigated. ***P<0.0001, Cancer vs. Non-cancer. (B) Five gastric cancer cell lines KATO III, MKN45, SGC-7901, AGS and MKN74 and a control cell line 293T were involved to investigate the expression of Z38 using reverse transcription-quantitative polymerase chain reaction analysis. *P<0.05 vs. 293T cells.

Figure 2.

Knockdown of Z38 in AGS and MKN74 cells inhibited cell proliferation. (A) Two specific siRNAs against Z38 were transfected into AGS and MKN74 cells and the relative transcript levels of Z38 were examined. (B) Colony formation assay was performed on siZ38 (short for siZ38-2) transfection in AGS and MKN74 cells. *P<0.05, vs. Control in AGS cells; ^#P<0.05, vs. Control in MKN74 cells. (C) The cell proliferation rate was investigated when AGS cells were transfected with siZ38 or siNC. (D) The cell proliferation rate was investigated when MKN74 cells were transfected with siZ38 or control siNC. *P<0.05, vs. Control. siRNA, small interfering RNA; siNC, negative control siRNA.

Figure 3.

Knockdown of Z38 in AGS and MKN74 cells inhibited cell metastasis. (A) Cell migration assays were performed when AGS and MKN74 cells were transfected with siZ38 for 72 h. (B) Cell invasion assays were performed when AGS and MKN74 cells were treated with siZ38 for 72 h. *P<0.05 vs. Control in AGS cells; ^#P<0.05 vs. Control in MKN74 cells. (C) Representative images of wound-healing assays. (D) Following random selection of 5 fields for each group of cells, the wound closure rate was quantified in siZ38-transfected AGS and MKN74 cells. *P<0.05 vs. Control in AGS cells; ^#P<0.05 vs. Control in MKN74 cells. siZ38, Z38 small interfering RNA; siNC, negative control small interfering RNA.

Figure 4.

Knockdown of Z38 in AGS and MKN74 cells increased the rate of cell apoptosis. (A) Cell apoptotic rates were examined upon siZ38 transfection in AGS and MKN74 cells. (B) The relative activities of caspase-3 were examined upon siZ38 transfection in AGS and MKN74 cells. (C) The relative activities of caspase-8 were examined upon siZ38 transfection in AGS and MKN74 cells. (D) The relative activities of caspase-9 were examined upon siZ38 transfection in AGS and MKN74 cells. *P<0.05 vs. Control in AGS cells; ^#P<0.05 vs. Control in MKN74 cells. siZ38, Z38 small interfering RNA; siRC, negative control small interfering RNA.

Figure 5.

Knockdown of Z38 in AGS and MKN74 cells increased the protein expression levels of caspase-3 and caspase-9. AGS and MNK74 cells were treated with siZ38 for 72 h, and western blot analysis was performed to reveal the protein expression levels of caspase-3 and caspase-9. GAPDH was included as an internal control. Two bands of cl-caspase-3 were detected, but the top bands were specific to cl-caspase-3, the molecular weight of which is 17KD, and the lower one was also from cl-caspase-3, which has a smaller molecular weight (12KD). siZ38, Z38 small interfering RNA; cl-caspase, cleaved-caspase.