Long non‑coding RNA HCG11 suppresses the malignant phenotype of non‑small cell lung cancer cells by targeting a miR‑875/SATB2 axis

Abstract

Long non‑coding RNAs (lncRNAs) are involved in the development and progression of a variety of diseases. However, the role of the lncRNA HLA complex group 11 (HCG11) in non‑small cell lung cancer (NSCLC) remains unclear. The present study showed that the expression levels of HCG11 were reduced in tumor tissues compared with adjacent normal tissues, and similar results were obtained in experiments using lung cancer cell lines. Additionally, patients with high HCG11 expression had an increased survival rate compared with patients with low HCG11 expression. Further studies have shown that overexpression of HCG11 inhibited NSCLC cell proliferation in vitro and in vivo. Interestingly, it was observed that HCG11 expression was negatively associated with the expression levels of oncogenic microRNA‑875 (miR‑875) in patient specimens. Specifically, HCG11 served as a sponge of miR‑875. Notably, it was determined that special AT‑rich sequence‑binding protein 2 (SATB2) was a direct target gene of miR‑875, and overexpression of miR‑875 largely abrogated the effects of HCG11 in NSCLC cells. In conclusion, HCG11 was shown to suppress the malignant properties of NSCLC cells by targeting a miR‑875/SATB2 axis, and may therefore be a promising target for the treatment of NSCLC.

Keywords: HLA complex group 11; long non‑coding RNA; microRNA‑875; non‑small cell lung cancer; special AT‑rich sequence‑binding protein 2.

Figure 1.

HCG11 expression is downregulated in NSCLC tissues and cell lines. (A) RT-qPCR analysis of the expression levels of HCG11 in NSCLC tissues and adjacent normal tissues obtained from 85 patients. (B) RT-qPCR analysis of the expression levels of HCG11 in NSCLC cell lines (A549, H1299, H446, H460 and H358) and 16HBE normal lung epithelial cells. (C) Overall survival rates of the 85 NSCLC patients undergoing surgery. Patients were divided into high (n=42) and low HCG11 expression group (n=43) based on the median expression levels. Data is shown as a Kaplan-Meier curve and was analyzed using a log-rank test. **P<0.01. NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR; HCG11, HLA complex group 11.

Figure 2.

HCG11 inhibits proliferation, invasion and migration, and induces apoptosis of non-small cell lung cancer cells in vitro. (A) Reverse transcription-quantitative PCR analysis of the expression levels of HCG11 in the Control, Vector and HCG11 groups. (B) MTT analysis of cell proliferation in the Control, Vector and HCG11 groups. (C) Flow cytometry analysis of cell apoptosis in the Control, Vector and HCG11 groups. (D) Western blot analysis of the protein expression of PCNA, cleaved-caspase-3 and cleaved-caspase-9 in the Control, Vector and HCG11 groups. (E and F) Transwell invasion assays showing (E) cell invasion and (F) migration in the Control, Vector and HCG11 groups. **P<0.01. HCG11, HLA complex group 11; PCNA, proliferation cell nuclear antigen; Vector, empty vector control.

Figure 3.

HCG11 suppresses NSCLC tumor growth in vivo. (A) Reverse transcription-quantitative PCR analysis of the expression levels of HCG11 in A549 cells after transduction with lentivirus carrying HCG11. (B-E) BALB/c nude mice were subcutaneously injected with A549 cells after transduction with a lentivirus carrying HCG11. (B) Tumor volume was measured once every 5 days. (C-E) A total of 30 days after implantation, all mice were euthanized, and tumor tissues were collected and weighed (n=8 mice per group). (F) H&E staining of the tumor tissue. (G) Immunohistochemical analysis of Ki-67 expression in tumor tissues. **P<0.01. H&E, hematoxylin and eosin; NSCLC, non-small cell lung cancer; Vector, empty vector control; HCG11, HLA complex group 11.

Figure 4.

HCG11 functions as a sponge of miR-875 in NSCLC cells. (A) Relative expression levels of miR-26a, miR-499a, miR-522, miR-224, miR-483, miR-214, miR-455, miR-534, miR-155, miR-105 and miR-875 in A549 cells following overexpression of HCG11. (B) Reverse transcription-quantitative PCR analysis of the expression levels of miR-875 in NSCLC tissues and adjacent normal tissues obtained from 85 patients. (C) Pearson correlation analysis of HCG11 and miR-875 expression in NSCLC tissues obtained from 85 patients. (D) Relative luciferase activity in 293T cells after co-transfection of WT or MUT HCG11 with miR-875-mimic or miR-NC. The specific binding site of miR-875 with HCG11 is shown in the top panel, based on StarBase. **P<0.01. NSCLC, non-small cell lung cancer; miR/miRNA, microRNA; NC, negative control; WT, wild-type; MUT, mutant; HCG11, HLA complex group 11.

Figure 5.

HCG11 inhibits the malignant phenotype of NSCLC cells by regulating a miR-875/SATB2 axis. (A) Relative luciferase activity in 293T cells after co-transfection of WT or MUT SATB2 with miR-875-mimic or miR-NC. The specific binding site of miR-875 with SATB2 mRNA is shown in the top panel, based on TargetScan. (B) Western blot analysis of the expression levels of SATB2 in the Control, Vector and HCG11 groups. (C) Transfection efficiency of miR-875-mimic, as determined by RT-qPCR. (D-I) A549 cells with overexpression of HCG11 were treated with miR-NC or miR-875-mimic. Subsequently, the expression levels of (D) miR-875 were measured by RT-qPCR, and (E) SATB2 protein expression was determined by western blotting. Finally, the (F) proliferation, (G) apoptosis, (H) invasion and (I) migration were analyzed by an MTT assay, flow cytometry or Transwell invasion and migration assays, respectively. **P<0.01. NSCLC, non-small cell lung cancer; miR/miRNA, microRNA; NC, negative control; WT, wild-type; MUT, mutant; RT-qPCR, reverse transcription-quantitative PCR; SATB2, special AT-rich sequence-binding protein 2; HCG11, HLA complex group 11 Vector, empty vector control.