miR-192-5p suppresses the progression of lung cancer bone metastasis by targeting TRIM44

Abstract

Lung cancer is the leading cause of cancer-related deaths worldwide, with 50-70% of patients suffering from bone metastasis. Accumulating evidence has demonstrated that miRNAs are involved in cell proliferation, migration, and invasion in malignancy, such as lung cancer bone metastasis. In the present study, we demonstrated that reduced miR-192-5p and increased TRIM44 levels were associated with the proliferation, migration and invasion of lung cancer. Furthermore, the potential functions of miR-192-5p were explored in A549 and NCI-H1299 cells. We found that miR-192-5p upregulation suppressed tumour behaviours in lung cancer cells. To further investigate whether miR-192-5p is associated with TRIM44, we used TargetScan software to predict the binding site between miR-192-5p and TRIM44. Luciferase activity assays were performed to verify this prediction. In addition, the significant role of miR-192-5p in negatively regulating TRIM44 expression was manifested by our research group. our results suggest that miR-192-5p inhibited the proliferation, migration and invasion of lung cancer through TRIM44.

Figure 1

Downregulation of miR-192-5P in human patient serum and lung cancer cell lines. (A) miR-192-5P expression in healthy volunteers and in patients with and without lung cancer bone metastasis. GAPDH mRNA was used as an internal control. (B) miR-192-5P expression was significantly lower in human lung cancer cells than in normal lung epithelial cells. GAPDH was used as an internal control. (C) TRIM44 expression was the highest in three tissue groups. GAPDH was used as an internal control. (D) Quantification of TRIM44 was normalized to GAPDH. Each bar represents the mean ± SEM. *P < 0.05, **P < 0.01, NS, non-significant. This study investigated serum from healthy volunteers (n = 130), lung cancer patients without bone metastasis (n = 68) and lung cancer patients with bone metastasis (n = 78).

Figure 2

miR-192-5p upregulation suppressed lung cancer cell growth. (A) A549 and NCI-H1299 cells were transfected with miR-192-5p antagomir, miR-NC, and miR-192-5P mimic. High miR-192-5p expression was examined by qPCR assay. (B) TRIM44 downregulation was detected by qPCR (B) and western blotting (C). Quantification of TRIM44 was normalized to GAPDH (D,E). MTT assays suggest that cell viability was enhanced upon miR-192-5p downregulation in (F) A549 and (G) NCI-H1299 cells. *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were performed at least three times.

Figure 3

miR-192-5P upregulation suppressed cell migration and invasion. (A) A549 and NCI-H1299 cells transfected with miR-192-5p antagomir, miR-NC, and miR-192-5P mimic were subjected to cell migration assays, and the cells were stained with a crystal violet solution. (B) The number of migrated cells was reduced by miR-192-5p. (C) Images of the invasion assays are shown, and (D) the invasive ability was weakened. NC, non-treated control group. Each bar represents the mean ± SEM. *P < 0.05, **P < 0.01. All experiments were performed at least three times with duplication.

Figure 4

TRIM44 is a direct target of miR-192-5p. (A) Potential targets of miR-192-5p were located in the 3′-UTR of TRIM44 through TargetScan Human. (B) A549 and (C) NCI-H1299 cells transfected with both miR-192-5p mimic and miR-192-5P antagomir in the presence of wild-type or mutant TRIM44 3′-UTR was detected though luciferase activity. (D) qPCR and western blotting (E) were performed to identify the mRNA and protein expression levels of TRIM44 in transfected cells. Quantification of TRIM44 was normalized to GAPDH. All experiments were performed at least three times with duplication within each individual experiment.

Figure 5

miR-192-5p negatively regulated TRIM44 expression in lung cancer cells. (A) A549 and NCI-H1299 cells were transfected with TRIM44 or plasmid vectors. A549 (B) and NCI-H1299 (C) cell viability was determined by MTT assay. (D) Representative images of A549 and NCI-H1299 cells showing cell migration various times after wounding. More cells migrated into the wound at 36 h in TRIM44 and miR-NC + TRIM44 cells than in miR-NC and miR-192-5p + TRIM44 cells. All experiments were performed at least three times.

Figure 6

Effect of miR-192-5P on tumour growth in vivo. A lung cancer bone metastasis model was established by injecting A549 cells (5 × 10⁶ cells) into the marrow cavity of nude mice (12 mice per group). (A) Representative images of femur tumours in nude mice. (B) Tumour size was measured using Vernier callipers once a week until the animals were sacrificed. (C) Tumour volume was measured at the last time point. (D) Representative H&E staining (100× magnification) and immunohistochemistry images for TRIM44 (40× magnification) in tumour sections from different groups. TRIM44 protein expression in tumour tissues was examined by western blotting (E) and quantified (F). Each bar represents the mean ± SEM. *P < 0.05.