TP53-Activated lncRNA GHRLOS Regulates Cell Proliferation, Invasion, and Apoptosis of Non-Small Cell Lung Cancer by Modulating the miR-346/APC Axis

Abstract

Non-small cell lung cancer (NSCLC) is the main type of lung cancer with high mortality worldwide. To improve NSCLC therapy, the exploration of molecular mechanisms involved in NSCLC progression and identification of their potential therapy targeting is important. Long noncoding RNAs (lncRNAs) have shown important roles in regulating various tumors progression, including NSCLC. We found lncRNA GHRLOS was decreased in NSCLC cell lines and tissues which correlated with poor prognosis of NSCLC patients. However, the role and underlying mechanisms of lncRNA GHRLOS in NSCLC progression remains elusive. The expression of lncRNA GHRLOS was examined in NSCLC cell lines and biopsy specimens of patients with NSCLC by quantitative real time polymerase chain reaction (qRT-PCR). The effects of GHRLOS on proliferation, invasion and apoptosis of NSCLC cells were determined by both in vitro and in vivo experiments. The interaction between GHRLOS and TP53 was determined by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) combined with qRT-PCR analysis. RNA immunoprecipitation (RIP) was conducted to validate the binding between GHRLOS and microRNA-346 (miR-346). Dual-luciferase reporter assays were also carried out to reveal the interaction between miR-346 and the 3' untranslated region (3'UTR) of adenomatous polyposis coli (APC) mRNA.Our data demonstrated that overexpression of lncRNA GHRLOS suppressed cancer cell proliferation and invasion as well as promoted cell apoptosis by regulating the expression of CDK2, PCNA, E-cadherin, N-cadherin, Bax, and Bcl-2 in NSCLC cells. Moreover, lncRNA GHRLOS was upregulated by the binding of TP53 to the GHRLOS promoter. The binding target of lncRNA GHRLOS was identified to be miR-346. Impressively, overexpression of miR-346 promoted cell proliferation and invasion, as well as inhibited cell apoptosis, however, these effects can be blocked by overexpression of lncRNA GHRLOS both in vitro and in vivo. In summary, this study reveals lncRNA GHRLOS, upregulated by TP53, acts as a molecule sponge of miR-346 to cooperatively modulates expression of APC, a miR-346 target, and potentially inhibits NSCLC progression via TP53/lncRNA GHRLOS/miR-346/APC axis, which represents a novel pathway that could be useful in targeted therapy against NSCLC.

Keywords: APC; NSCLC; TP53; lncRNA GHRLOS; miR-346.

Figure 1

LncRNA GHRLOS expression decreased in lung tumor tissues and is clinically significant. (A) H&E staining of clinical lung tissue samples, including tumor tissues and non-tumor tissues (adjacent tissues). (B) qRT-PCR analysis of lncRNA GHRLOS expression in lung tissues, &p< 0.01 vs. non-tumor. (C) qRT-PCR analysis of lncRNA GHRLOS expression in human normal pulmonary epithelial cell line (BEAS-2B) and NSCLC cell lines, &p< 0.01 vs. BEAS-2B. (D) Kaplan–Meier analysis of overall survival in NSCLC patients based on lncRNA GHRLOS expression.

Figure 2

LncRNA GHRLOS inhibited NSCLC cell growth and invasion, and increased apoptosis. (A) qRT-PCR determination on lentivirus-induced overexpression of lncRNA GHRLOS at 48 h in A549 and NCI-H460 cells, &p< 0.01 vs. Lv-vector. (B) CCK-8 assay on the effect of lncRNA GHRLOS on cancer cell proliferation after lentivirus infection at the indicated time points, &p< 0.01 vs. Lv-vector. (C, D) Colony formation assay on the effect of lncRNA GHRLOS on growth of cancer cells after 14 days of culture, &p< 0.01 vs. Lv-vector. (E) qRT-PCR analysis on the expression of cell growth-associated genes, PCNA and CDK2, in A549 and NCI-H460 cells after overexpressed lncRNA GHRLOS for 48 h, &p< 0.01 vs. Lv-vector. (F, G) Trans-well assay analysis on the effect of lncRNA GHRLOS on the invasion of cancer cells, &p< 0.01 vs. Lv-vector. (H) qRT-PCR analysis on cell adhesion-related genes, including E-cadherin and N-cadherin, in A549 and NCI-H460 cells after infection for 48 h, &p< 0.01 vs. Lv-vector. (I, J) Annexin V-FITC/PI analysis on the effect of lncRNA GHRLOS on cancer cell apoptosis, &p< 0.01 vs. Lv-vector. (K) qRT-PCR analysis on the expression of cell apoptosis related genes, Bax and Bcl-2, in NSCLC cells infected by lentivirus for 48 h, &p< 0.01 vs. Lv-vector.

Figure 3

The TP53 activated lncRNA GHRLOS transcription in NSCLC cells. (A) The predicted binding motif of TP53 in the human lncRNA GHRLOS promoter based on analysis using JASPAR. (B, C) qRT-PCR analysis of TP53 expression after knockdown or overexpression of TP53 for 48 h in NSCLC cells, &p< 0.01 vs. Lv-sh-NC or Lv-vector. (D, E) qRT-PCR analysis on lncRNA GHRLOS expression after knockdown or overexpression of TP53 for 48 h in NSCLC cells, &p< 0.01 vs. Lv-sh-NC or Lv-vector. (F, G) ChIP assays evaluated TP53 binding sites, &p< 0.01 vs. anti-IgG. (H) Schematic diagram of plasmids used for luciferase reporter assays. (I, J) Dual luciferase reporter assays examined luciferase activity, &p< 0.01 vs. Lv-vector. (K) Colony formation assay on the growth of cancer cells infected with indicated lentivirus for 48 h followed by 14 days of culture, &p< 0.01 vs. Lv-vector, Фp< 0.01 vs. Lv-TP53. (L) Transwell assays on invasion of cancer cells 48 h after infection, &p< 0.01 vs. Lv-vector, Фp< 0.01 vs. Lv-TP53. (M) Annexin V-FITC/PI analysis on cancer cell apoptosis after infection for 48 h, &p< 0.01 vs. Lv-vector, Фp< 0.01 vs. Lv-TP53.

Figure 4

LncRNA GHRLOS is a molecular sponge of miR-346. (A) qRT-PCR analysis of subcellular localization of lncRNA GHRLOS. GAPDH was used as an internal cytoplasmic control, and U6 served as an internal nuclear control. (B) qRT-PCR analysis of miR-346 in clinical NSCLC tissues, &p< 0.01 vs. non-tumor. (C) The predicted binding site between lncRNA GHRLOS and miR-346 was obtained. (D) qRT-PCR analysis on the expression of lncRNA GHRLOS and miR-346 after anti-Ago2-mediated RIP assays in A549 and NCI-H460 cells, &p< 0.01 vs. input. (E) Dual luciferase reporter assay on the interaction between lncRNA GHRLOS and miR-346 after infection with Mock, miR-346 mimic, or miR-346 inhibitor for 48 h, &p< 0.01 vs. Lv-Mock. (F) qRT-PCR analysis of miR-346 after indicated infection for 48 h in A549 and NCI-H460 cells, &p< 0.01 vs. Lv-vector.

Figure 5

The interaction between lncRNA GHRLOS and miR-346 in cancer cell proliferation, invasion, and apoptosis. (A) Colony formation assay showed reciprocal suppression between lncRNA GHRLOS and miR-346 on the growth of transfected cancer cells after 14 days culture, &p< 0.01 vs. Lv-Mock+Lv-vector, #p< 0.01 vs. Lv-miR-346+Lv-GHRLOS. (B) Trans-well assays on invasion of lentivirus infected cancer cells, &p< 0.01 vs. Lv-Mock+Lv-vector, #p< 0.01 vs. Lv-miR-346+Lv-GHRLOS. (C) qRT-PCR on the expression of cell growth biomarkers, PCNA and CDK2, in A549 and NCI-H460 cells after infected with indicated lentivirus for 48 h, &p< 0.01 vs. Lv-Mock+Lv-vector, #p< 0.01 vs. Lv-miR-346+Lv-GHRLOS. (D) qRT-PCR on the expression of cell adhesion biomarkers, including E-cadherin and N-cadherin, in A549 and NCI-H460 cells, &p< 0.01 vs. Lv-Mock+Lv-vector, #p< 0.01 vs. Lv-miR-346+Lv-GHRLOS. (E) qRT-PCR analysis on the expression of cell apoptosis-associated genes, Bax and Bcl-2, in cancer cells after lentivirus infection for 48 h, &p< 0.01 vs. Lv-Mock+Lv-vector, #p< 0.01 vs. Lv-miR-346+Lv-GHRLOS. (F) Photo of tumors in nude mice after inoculation for 28 days. (G) The tumor volume of each group (n=6) measured at the indicated time points after inoculation of NCI-H460 cells. (H) The weight of tumors in nude mice after inoculation for 28 days, &p< 0.01 vs. Lv-Mock+Lv-vector, #p< 0.01 vs. Lv-miR-346+Lv-GHRLOS. (I) qRT-PCR analysis on the expression of PCNA, CDK2, E-cadherin, N-cadherin, Bcl-2, and Bax in xenografted tumors, &p< 0.01 vs. Lv-Mock+Lv-vector, #p< 0.01 vs. Lv-miR-346+Lv-GHRLOS.

Figure 6

APC was a target of miR-346 and regulated by interaction of lncRNA GHRLOS and miR-346. (A) Predicted binding site of miR-346 in the APC 3’UTR (STARBASE database). (B) qRT-PCR analysis on APC expression in NSCLC tissues, &p< 0.01 vs. non-tumor. (C) Dual luciferase reporter assay on luciferase activity of APC 3’UTR, &p< 0.01 vs. Lv-Mock+Lv-vector, #p< 0.01 vs. Lv-miR-346+Lv-GHRLOS. (D) qRT-PCR analysis on APC expression in cancer cells infected with lentivirus for 48 h, &p< 0.01 vs. Lv-Mock+Lv-vector, #p< 0.01 vs. Lv-miR-346+Lv-GHRLOS. (E) Western blot analysis on APC expression in cancer cells after lentivirus infection for 48 h.

Figure 7

Silencing APC blocked miR-346 knockdown- or lncRNA GHRLOS overexpression-controlled gene expression on biomarkers of cell proliferation, invasion, and apoptosis. (A, B) qRT-PCR analysis on gene expression after lentivirus infection for 48 h in A549 and NCI-H460 cells, &p< 0.01 vs.Lv-Mock+Lv-sh-NC, #p< 0.01 vs.Lv-miR-346+Lv-sh-APC. (C, D) qRT-PCR analysis on gene expression in NSCLC cells with lentivirus infection for 48 h, &p< 0.01 vs.Lv-Mock+Lv-sh-NC, #p< 0.01 vs.Lv-miR-346+Lv-sh-APC.

Figure 8

APC expression is modulated by overexpression or interference with TP53 expression. (A) qRT-PCR analysis on TP53 expression after overexpressed- or knockdown-TP53 for 48 h, &p< 0.01 vs. Lv-vector. (B) qRT-PCR analysis on APC, lncRNA GHRLOS, and miR-346 expression after indicated lentivirus infection for 48 hr in A549 and NCI-H460 cells, &p< 0.01 vs. Lv-vector. (C) Western blot analysis on APC expression in A549 and NCI-H460 cells with indicated infection. (D) Schematic presentation of molecular mechanism of lncRNA GHRLOS mediated cell proliferation, invasion and apoptosis in NSCLC.