Long non-coding RNA, CHRF, predicts poor prognosis of lung adenocarcinoma and promotes cell proliferation and migration

Abstract

Research has demonstrated that long non-coding RNAs (lncRNAs) are crucial factors in carcinogenesis. LncRNA, cardiac hypertrophy-related factor (CHRF), has been demonstrated to act as an oncogene in a variety of types of tumor. However, its biological function in lung adenocarcinoma remains to be elucidated. The present study aimed to examine the level of CHRF expression in lung adenocarcinoma tissues and cell lines, and to analyze the association between CHRF and clinicopathological characteristics, as well prognosis of patients with lung adenocarcinoma. Loss-of-function assays were performed to determine the biological function of CHRF. The expression of CHRF was markedly upregulated in lung adenocarcinoma tissues and cell lines. Patients exhibiting upregulated CHRF also demonstrated advanced Tumor-Node-Metastasis stage, lymph node metastasis and larger tumor size compared with those exhibiting downregulated CHRF. Results of Cox proportional hazards regression analysis suggested that highly-expressed CHRF may be regarded as an independent prognostic factor of prognosis. In addition, loss-of-function assays indicated that downregulation of CHRF suppressed cell proliferation, migration and invasion, and induced cell cycle arrest and apoptosis. Western blotting revealed that the phosphoinositide-3-kinase/Akt signaling pathway activity is reduced in lung adenocarcinoma following the knockdown of CHRF. Together, these results indicate that lncRNA, CHRF, may serve a critical role in the development and progression of lung adenocarcinoma, and may act as a novel prognostic biomarker and therapeutic target in lung adenocarcinoma.

Keywords: cardiac hypertrophy-related factor; long non-coding RNA; lung adenocarcinoma; migration; proliferation.

Figure 1.

LncRNA CHRF expression is upregulated in lung adenocarcinoma tissues and cell lines. (A) The expression level of CHRF in LAD tissues and matched adjacent normal tissues. (B) The expression level of CHRF in LAD cell lines, SPC-A1, NCI-H441, NCI-H1975, and in normal BEAS-2B cells. Error bars represented the mean ± standard deviation of ≥3 independent experiments. **P<0.01 vs. BEAS-2 B group. LncRNA, long non-coding RNA; CHRF, cardiac hypertrophy-related factor; LAD, lung adenocarcinoma.

Figure 2.

Kaplan-Meier analysis of the association between CHRF expression level and overall survival probability of patients with lung adenocarcinoma (P<0.001). CHRF, cardiac hypertrophy-related factor.

Figure 3.

Knockdown of CHRF represses cell proliferation by affecting the cell cycle and apoptosis. (A) Reverse transcription-quantitative polymerase chain reaction was used to measure the expression level of CHRF in cells transfected with si-CHRF. (B) MTT assays revealed the effect of si-CHRF on cell proliferation of lung adenocarcinoma cells. (C) Flow cytometry was used to analyze the effect of si-CHRF on cell cycle. (D) Flow cytometry was used to analyze the effect of si-CHRF on apoptosis. Error bars represent the mean ± standard deviation of at ≥3 independent experiments. **P<0.01 vs. control group. CHRF, cardiac hypertrophy-related factor; si, small interfering RNA; NC, negative control.

Figure 4.

Knockdown of CHRF suppresses cell migration and invasion abilities of LAD cells. (A) Transwell assays demonstrated that knockdown of CHRF inhibited the migratory abilities of LAD cells. (B) Quantification of the transwell assay results showed that the invasive ability of LAD cells was suppressed by CHRF knockdown. n=200 µm. Error bars represented the mean ± standard deviation of ≥3 independent experiments. **P<0.01 vs. control group. CHRF, cardiac hypertrophy-related factor; LAD, lung adenocarcinoma; SI, small interfering RNA; NC, negative control.

Figure 5.

Knockdown of CHRF reduces the protein expression level of PI3K/Akt pathway in LAD. Western blotting revealed that knockdown of CHRF decreased the protein expression levels of p-PI3K and p-Akt in (A) SPCA-1 and (B) NCI-H441 cells. Error bars represented the mean ± standard deviation of ≥3 independent experiments. **P<0.01 vs. control group. CHRF, cardiac hypertrophy-related factor; PI3K, posphoinositide-3-kinase; LAD, lung adenocarcinoma; p-, phosphorylated; T-total; si, small interfering RNA; NC, negative control.