Long noncoding RNA PCAT6 functions as an oncogene by binding to EZH2 and suppressing LATS2 in non-small-cell lung cancer

Abstract

Background: NSCLC (non-small-cell lung cancer) is the leading cause of cancer-related mortality worldwide. Both epigenetic and genetic changes contribute to the initiation, development and metastasis of NSCLC. Recently, accumulating data have begun to support the notion that long noncoding RNAs (lncRNAs) function as new crucial regulators of diverse biological processes, including proliferation, apoptosis and metastasis, and play crucial roles in tumorigenesis. Nevertheless, further study is warranted to comprehensively determine lncRNAs' functions and potential mechanism.

Methods: In this study, we performed a comprehensive analysis of the lncRNA expression profile of NSCLC using data from TCGA and Gene Expression Omnibus (GEO). PCAT6 expression level in a cohort of 60 pairs of NSCLC tissues using quantitative real-time PCR (qRT-PCR). Additionally, Loss-of-function assays and gain-of-function assays were used to assess the role of PCAT6 in promoting NSCLC progression. Tumor formation assay in a nude mouse model was performed to verity the role of PCAT6 in NSCLC in vivo. Meanwhile, RIP, ChIP, resue experiment and western blot assays were used to highlights the potential molecular mechanism of PCAT6 in NSCLC.

Findings: We identified that an oncogene, PCAT6, was upregulated in NSCLC, and this upregulation was verified in a cohort of 60 pairs of NSCLC tissues. Additionally, the expression level of PCAT6 was correlated with tumor size (P = .036), lymph node metastasis (P = .029) and TNM stage (P = .038). Loss-of-function and gain-of-function assays were used to assess the role of PCAT6 in promoting NSCLC progression. The results revealed that PCAT6 knockdown mitigated NSCLC cell growth by inducing G1-phase cell cycle arrest and apoptosis in vitro and in vivo. Whereas, PCAT6 overexpression could promoted tumor cell growth. Meanwhile, PCAT6 additionally promoted NSCLC cell migration and invasion. Furthermore, mechanistic investigation demonstrated that the oncogenic activity of PCAT6 is partially attributable to its repression of LATS2 via association with the epigenetic repressor EZH2 (Enhancer of zeste homolog 2). Overall, our study highlights the essential role of PCAT6 in NSCLC, suggesting that PCAT6 might be a potent therapeutic target for patients with NSCLC.

Keywords: EZH2; LATS2; Long noncoding RNA; Non-Small-Cell Lung Cancer; PCAT6.

Fig. 1

The expression profiles of lncRNAs in NSCLC tissues and normal tissues. (a) Heatmap of the differentially expressed lncRNAs expression in LUAD or LUSC and normal tissue samples was analyzed using the TCGA datasets. (b and c) Heatmap of the dysregulated lncRNAs in NSCLC was analyzed using the GSE19188, GSE18842 datasets. (d) Heatmap of the altered lncRNAs profiling (consistently altered at least two datasets, fold change) in TCGA-LUAD, TCGA-LUSC, GSE19188, GSE18842 datasets. (e) Venn diagram of altered lncRNAs in TCGA-LUAD, TCGA-LUSC, GSE19188, GSE18842 datasets.

Fig. 2

Higher PCAT6 expression levels in NSCLC and its clinical significance. (a) The PCAT6 expression levels in NSCLC tissues compared with normal tissues in TCGA-LUAD, TCGA-LUSC, GSE19188, GSE18842 datasets. (b) The PCAT6 expression level in 60 NSCLC tissues and corresponding adjacent non-tumor tissues was quantified by Quantitative real-time PCR analysis and normalized to GAPDH expression. Expression levels were shown as log2-fold change to match non-tumor tissues. Red column represented overexpression and Green column represented down-regulation. (c, d and e) The relationship between PCAT6 expression and clinicopathological parameters (such as maximum diameter, lymphatic metastasis and TNM stage) was shown. (f) Kaplan-Meier survival plots demonstrated that higher PCAT6 abundance correlated with a poor OS, using microarray data from 1926 lung cancer patients. Data are shown as the mean ± SD Based on at least three independent experiments. *P < .05, **P < .01.

Fig. 3

The effects of PCAT6 on NSCLC cell viability in vitro. (a) qRT-PCR analysis was performed to identify the expression level of PCAT6 in 7 NSCLC cell lines and human bronchial epithelial cell (HBE). The expression levels are normalized to HBEs. (b) The PCAT6 expression level in A549 and SK-MES-1 transfect with two discrete chemically synthesized siRNAs. (c) MTT assays were used to measure the growth curve of si-PCAT6–transfected A549 and SK-MES-1 cells. Values indicate the mean ± SD from three independent experiments. (d) Colony-forming assays were conducted to determine the proliferation of si-PCAT6-transfected A549 and SK-MES-1 cells. (e) The PCAT6 expression level in SPCA1 transfected with pcDNA3.1 PCAT6 and empty vector. (f) MTT assays were used to measure the growth curve of pcDNA3.1 PCAT6 and empty vector transfected in SPCA1. (g) Colony-forming assays were conducted to determine the proliferation of pcDNA3.1 PCAT6 and empty vector transfected in SPCA1. (h) Flow cytometry assays were performed to analyze the cell cycle progression when NSCLC cells transfected with PCAT6. * P < .05, **P < .01. N.S., not significant.

Fig. 4

The effects of PCAT6 on NSCLC cell apoptosis, cell migration and invasion in vitro. (a) The apoptosis of A549 and SK-MES-1S were analyzed by flow cytometry. LR, early apoptotic cells. UR, terminal apoptotic cells. (b and c) Transwell assays were used to determine the invasive ability of si-PCAT6 1#, si-PCAT6 2# or si-NC transfected A549 and SK-MES-1 cells. The cells on the lower chamber were stained and presented. Data represent the mean ± S.D. from three independent experiments. Scale bar represents 100 μm. *P < .05, **P < .01.

Fig. 5

The effects on tumor growth after PCAT6 downregulation in vivo. (a, b and c) The tumor growth curves were measured 4 days after the injection after injection of A549 cells stably transfected with shRNA PCAT6 1#, shRNA PCAT6 2# or empty vector and tumor volume was calculated once every 3 or 4 days. (d) Tumor weight when the tumors were harvested. (e) qRT-PCR analysis of PCAT6 expression level in tumor tissues formed from shRNA PCAT6 1#, shRNA PCAT6 2# or empty vector transfected A549 cells. (e) Representative images of HE staining and Ki-67 immunohistochemistry of the tumor. Up, H & E staining, Scale bar represents 50 μm; Down, immunostaining, Scale bar represents 20 μm. The data represent the mean ± S.D. from three independent experiments. *P < .05, **P < .01.

Fig. 6

PCAT6 co-expressed genes and downstream targets in NSCLC. (a) The co-expression genes of PCAT6. (b) GO pathway analysis for all genes with altered expressions in TCGA-LUAD, TCGA-LUSC datasets. (c) The interaction network of PCAT6. (d) Analysis of the relationship between PCAT6 expression and LATS2 mRNA level in TCGA-LUAD and TCGA-LUSC datasets.

Fig. 7

PCAT6 could recruit EZH2 to LATS2 promoter and represses LATS2 transcription. (a and b) The western blot analysis and qRT-PCR assay were conducted to detect the expression levels of LATS2 protein and mRNA in A549 and SK-MES-1 cells transfected with si-PCAT6 and si-NC. (c) PCAT6 expression levels in different subcellular fractions in A549 in A549 cell line. (d) RIP with rabbit monoclonal anti-EZH2 and preimmune IgG from A549 cell extracts. RNA levels in immunoprecipitates were detected by qPCR. Expression levels of PCAT6 RNA are presented as fold enrichment in EZH2 relative to IgG immunoprecipitates. (e) The expression level of LATS2 mRNA in A549 cells transfected with si-EZH2 and si-NC. (f) ChIP–qRT-PCR of EZH2 occupancy and H3K27me3 binding in the LATS2 promoter in A549 cells transfected with si-PCAT6 and si-NC. (g) MTT assays were used to measure the growth curve of si-NC, si-PCAT6 and si-PCAT6 + si-LATS2–transfected A549. (h) Colony-forming assays were conducted to determine the proliferation of si-NC, si-PCAT6 and si-PCAT6 + si-LATS2–transfected A549.The data represent the mean ± S.D. from three independent experiments. *P < .05, **P < .01.

Supplementary Fig. 1

(a) The expression level of downstream targets of PCAT6. (b) PCAT6 expression levels in different subcellular fractions in SK-MES-1. (c) The the western blot analysis were conducted to detect the expression levels of LATS2 protein in SPCA1 cells transfected with empty vector, pcDNA3.1-PCAT6 and pcDNA3.1-PCAT6 + si-EZH2. (d) The the western blot analysis were conducted to detect the expression levels of LATS2 protein in A549 cells transfected with si-NC, si-PCAT6 and si-PCAT6 + si-LATS2. *P < .05, **P < .01.