miR-944 inhibits lung adenocarcinoma tumorigenesis by targeting STAT1 interaction

Abstract

Lung adenocarcinoma (LAC) is a leading cause of cancer-associated mortalities, particularly in developed countries. The aberrant expression of microRNAs (miRNAs) has been proven to regulate numerous diseases in the past two decades. miRNAs have been identified in almost all human cancer types. In the present study, the role of miR-944 in LAC proliferation was examined. It was identified that miR-944 was downregulated in LAC tissues and cells, and miR-944 overexpression inhibited A549 and H1299 cell proliferation, as determined by the Cell Counting Kit-8 and colony formation assay. Signal transducer and activator of transcription 1 (STAT1) was upregulated in LAC tissues and cells. Kaplan-Meier analysis demonstrated that the 5-year overall survival in patients with high STAT1 levels was significantly reduced, compared with patients with negative and low STAT1 expression. STAT1 was the direct target of miR-944. Additionally, a miR-944 mimic inhibited A549 cell growth in vitro. Collectively, these data demonstrate that miR-944 serves a pivotal role in LAC tumor growth by targeting STAT1. The data obtained indicated that miR-944 may be a novel biomarker and could result in potential therapies for LAC.

Keywords: lung adenocarcinoma; microRNA; proliferation; signal transducer and activator of transcription 1.

Figure 1.

miR-944 expression is reduced in LAC tissues and cell lines. (A) Relative levels of miR-944 expression were analyzed by reverse transcription-quantitative polymerase chain reaction in 25 pairs of patients with LAC. (B) The expression of miR-944 in LAC the cell lines A549, H1299, SK-Lu-1 and PC-9, and the human bronchial epithelial cell line 16HBE. Data presented as the mean ± standard deviation of three replicates. *P<0.05 vs. normal group; 16HBE group. LAC, lung adenocarcinoma; miR, microRNA.

Figure 2.

miR-944 inhibits cell proliferation. (A) Relative expression levels of miR-944 following transfection with miR-944 mimic or miR-NC in A549 and H1299 cells (B) A total of 2×10³ A549 and H1299 cells were plated per well in 96-well plates, and the Cell Counting Kit-8 assay was performed to detect cell proliferation absorbance at 450 nm at days 0, 2 and 4. (C) Colony formation assays were used to detect cell proliferation. *P<0.05 vs. miR-NC. NC, negative control; miR, microRNA.

Figure 3.

STAT1 is upregulated in LAC tissue and cells. (A) The STAT1 mRNA level was upregulated in human LAC tissues, compared with adjacent non-tumor tissues, as determined by RT-qPCR. *P<0.05 vs. adjacent non-tumor tissues. (B) The protein level of STAT1 is upregulated in human LAC tissues, compared with adjacent non-tumor tissues. *P<0.05 vs. adjacent non-tumor tissues. (C) Relative STAT1 mRNA expression levels in 16HBE and LAC cell lines at determined by RT-qPCR. *P<0.05 vs. 16HBE. (D) The Kaplan-Meier method was used to determine survival time. STAT1, signal transducer and activator of transcription 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; T, tumors; N, adjacent non-tumor tissues; LAC, lung adenocarcinoma.

Figure 4.

miR-944 directly downregulates the expression of STAT1. (A) The predicted targeting site of STAT1 3′-untranslated region combined with miR-944 is illustrated. (B) Luciferase assay of A549 cells. (C) The protein band mRNA levels and (D) quantification of STAT1 following transfection with miR-944 inhibitor and miR-NC, or miR-944 mimics and miR-NC. *P<0.05 vs. the miR-NC group. STAT1, signal transducer and activator of transcription 1; MUT, mutant; WT, wild-type; NC, negative control; miR, microRNA.

Figure 5.

miR-944 suppresses tumor growth in vivo by targeting STAT1. (A) Tumors formed in nude mice. A total of 5×10⁶ A549 cells were subcutaneously injected into nude mice (n=5). The mice were then sacrificed 21 day after injection. (B) Tumors were harvested, and images of representative tumors were obtained. (C) The miR-944 mimic transfection resulted in a decrease in final tumor weight. (D) The protein levels of STAT1 from tumor xenografts were measured by western blotting. (E) The mRNA levels of STAT1 in tumor xenografts were measured by reverse transcription quantitative polymerase chain reaction. A total of 30 mice were randomly divided into six groups to perform three independent experiments for each type of transfected cell. *P<0.05 vs. miR-NC. STAT1, signal transducer and activator of transcription 1; NC, negative control; miR, microRNA.