Linc00467 promotes lung adenocarcinoma proliferation via sponging miR-20b-5p to activate CCND1 expression

Abstract

Background: Recently, numerous studies have demonstrated the emerging role of long non-coding RNAs (lncRNAs) in human cancers. Linc00467 is a newly defined lncRNA and was reported to promote cell survival in neuroblastoma. However, the function of linc00467 in lung cancer is still unclear.

Material and methods: We analyzed linc00467 expression and survival data derived from The Cancer Genome Altas lung adenocarcinoma (LUAD) dataset as well as in collected LUAD tissues. Then, we silenced linc00467 expression in two lung cancer cell lines using small interfering RNAs and explored the effect of linc00467 knockdown on cell growth in vitro and in vivo. Moreover, we revealed a novel target gene of linc00467 and elucidated the underlying competitive endogenous RNA regulatory mechanism in lung cancer cells.

Results: Our data suggested that linc00467 expression was elevated in LUAD tissues and correlated with overall survival of LUAD patients. Linc00467 knockdown resulted in reduced proliferation rate in lung cancer cells. Furthermore, we elucidated that linc00467 promoted CCND1 expression in lung cancer cells via functioning as a molecular sponge for miR-20b-5p.

Conclusion: Linc00467/miR-20b-5p/CCND1 signaling pathway may provide new insights into lung cancer treatment.

Keywords: CCND1; cell growth; linc00467; lung adenocarcinoma; miR-20b-5p.

Figure 1

Linc00467 was up-regulated in lung adenocarcinoma tissues and cell lines. (A) Linc00467 mRNA levels in lung adenocarcinoma tissues and normal lung tissues. Data were obtained from the TCGA database. (B) Kaplan–Meier plots of overall survival of lung adenocarcinoma patients, stratified by linc00467 expression. Data were obtained from the TCGA database. (C) Real-time PCR analysis of linc00467 expression normalized to GAPDH expression in collected lung adenocarcinoma tissues and matched normal tissues. **p<0.01. (D) Real-time PCR analysis of linc00467 expression normalized to GAPDH expression in H1299, H23, HCC827, and A549 lung cancer cells compared with IMR90 normal lung fibroblast cells. **p<0.01. Abbreviation: TCGA, The Cancer Genome Altas.

Figure 2

Linc00467 knockdown inhibited lung cancer cell growth. (A, B) Real-time PCR analysis of linc00467 expression normalized to GAPDH expression in H1299 (A) and A549 (B) cells transfected with NC-siRNA or siRNAs to linc00467 (si-linc00467-1, si-linc00467-2). **p<0.01. (C, D) Proliferation analysis of H1299 (C) and A549 (D) cells transfected with NC-siRNA or siRNAs to linc00467. **p<0.01. (E) EdU analysis of H1299 and A549 cells transfected with NC-siRNA or siRNAs to linc00467. **p<0.01. (F, G) Colony formation analysis of H1299 (F) and A549 (G) cells transfected with NC-siRNA or siRNAs to linc00467. Abbreviation: siRNA, small interfering RNA.

Figure 3

Linc00467 knockdown led to lung cancer cell G₀/G₁ arrest. (A, B) Apoptosis analysis of H1299 (A) and A549 (B) cells transfected with NC-siRNA or siRNA to linc00467. (C, D) Cell cycle analysis H1299 (C) and A549 (D) cells transfected with NC-siRNA or siRNAs to linc00467. (E, F) Real-time PCR analysis of CCND1, CDK4, and CDK6 mRNA levels normalized to GAPDH mRNA levels in H1299 (E) and A549 (F) cells transfected with NC-siRNA or siRNAs to linc00467. **p<0.01. (G, H) Western blot analysis of CCND1, pRB, CDK4, and CDK6 in H1299 (G) and A549 (H) cells transfected with NC-siRNA or siRNAs to linc00467. GAPDH was used as loading control. Abbreviation: siRNA, small interfering RNA.

Figure 4

Linc00467 knockdown inhibited tumor growth in vivo. (A) Tumor weight of xenograft tumors developed from NC control and linc00467 knockdown A549 cells. **p<0.01. Scale bar =1 cm. (B) Tumor volume of xenograft tumors developed from NC and linc00467 knockdown A549 cells on indicated days. **p<0.01. (C) Western blot analysis of CCND1 and pRB levels in xenograft tumors developed from NC control and linc00467 knockdown A549 cells. GAPDH was used as loading control. (D) Immunohistochemical staining of CCND1 in xenograft tumors from NC and si-linc00467 group mice. Abbreviation: NC, negative control.

Figure 5

Linc00467 acted as a sponge of miR-20b-5p in lung cancer cells. (A) Real-time PCR analysis of miR-20b-5p levels in H1299 and A549 cells transfected with NC-siRNA or siRNA to linc00467. **p<0.01. (B) Pearson correlation scatter plot of miR-20b-5p levels and linc00467 levels in human adenocarcinoma tissues. (C, D) Western blot analysis of CCND1 levels in H1299 (C) and A549 (D) cells transfected with miR-NC and miR-20b-5p mimics. GAPDH was used as loading control. (E, F) Luciferase reporter gene analysis of the effect of miR-20b-5p on wild type and mutant reporter vectors containing linc00467 and CCND1 3ʹUTR binding sites. **p<0.01. Abbreviations: NC, negative control; siRNA, small interfering RNA.