Expression and regulatory role of ultraconserved long non-coding RNA uc.77 in lung cancer

Abstract

Objective: To investigate the effect and molecular mechanism of ultra-conservative long non-coding RNA uc.77 in lung cancer. Methods: Lung cancer tissues and adjacent normal tissues were obtained from 61 patients with lung cancer who were diagnosed with lung cancer and underwent surgery from 2014 to 2016 in the General Hospital of the Southern Theater Command of the People's Liberation Army. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the uc.77 relative expressions in normal human bronchial epithelial cells 16HBE, lung cancer cell lines, and 61 pair lung cancer tissues. Uc.77 siRNA was transfected into lung cancer cells to interfere with the expression of uc.77, qRT-PCR was used to verify the interference effect, CCK8 method and clone formation experiment were used to detect cell proliferation ability, flow cytometry was used to detect apoptosis and cell cycle changes. H1299 cells transfected with uc.77 siRNA were injected into the subcutaneous right side of BALB/c nude mice to construct a tumor-bearing model for exploring the role of uc.77 on tumor growth. Western blot and qRT-PCR methods were used to detect the protein and mRNA expressions of p21. Results: The relative expression levels of uc.77 in lung cancer cell lines 95D, H1299, A549, H460, H446 and 16HBE-T were significantly higher than that of 16HBE cells (P<0.05). The uc.77 RNA expression levels of lung cancer tissues was significantly higher than that of the adjacent normal tissues (P<0.001). In addition, increased lncRNA uc.77 expression was significantly associated with big tumor size, lymph node metastasis and advanced TNM stage (P<0.05). After transfection with uc.77 siRNA, the expressions of uc.77 in H1299, 95-D and 16HBE-T cells were reduced (P<0.05), and the cell proliferation capacities were reduced at 48 hours and 72 hours (P<0.05). After transfection with uc.77 siRNA-1, the G(0)/G(1) phase cell ratio of H1299 siRNA-1 group [(71.86±3.46)%] was higher than those of H1299-control group [(47.62±5.48)%] and H1299 siRNA-NC group [(61.38±5.62)%, P<0.05], S phase cell ratio of H1299 siRNA-1 group [(14.99±3.61)%] was lower than those of H1299-control group [(34.95±7.05)%] and H1299 siRNA-NC group [(23.75±5.87)%, P<0.05], the apoptosis rate of H1299 siRNA-1 group [(4.90±1.80)%] was higher than those of H1299-control group [(3.30±0.80)%] and H1299 siRNA-NC group [(2.80±1.20)%, P<0.05], the colony formation rate of H1299 siRNA-1 group [(19.20±2.00)%] was lower than those of H1299 control group [(32.60±2.00)%] and H1299 siRNA-NC group [(34.40±1.00)%, P<0.05]. The results of the nude mice tumor formation experiment showed that the tumor volume of the H1299 siRNA-1 group was significantly lower than those of the H1299-control group and the H1299-negative control group (P<0.05), the average tumor weight of H1299 siRNA-1 group was significantly lower than those of H1299-control group and H1299-negative control group (P<0.05), tumor cell growth marker Ki-67 in the H1299 siRNA-1 group showed weak positive, and Ki-67 in the H1299-control group and H1299-negative control group showed positive. The result of qRT-PCR analysis showed that the mRNA expression level of p21 in H1299 siRNA-1 group (2.57±0.45) was higher than those in H1299 control group (1.00±0.00, P=0.001) and H1299 siRNA-NC group (1.52±0.37, P=0.009). The result of western blotting analysis also showed that the expression of p21 protein level in H1299 siRNA-1 group increased. Conclusions: The expression of ultraconserved long non-coding RNA uc.77 is elevated in lung cancer cell lines and lung cancer tissues. Silencing the expression of ultraconservative long noncoding RNA uc.77 can inhibit tumor growth, and blocking uc.77 expression may be a potential therapeutic target for lung cancer.