Neuregulin1 acts as a suppressor in human lung adenocarcinoma via AKT and ERK1/2 pathway

Abstract

Background: Neuregulin1 (NRG1) is critical signaling protein that mediates the activation of downstream signaling pathways associated with malignancies. Multiple gene fusions related to NRG1 have been found in lung cancer. However, the underlying role NRG1 in lung cancer is yet unclear. Therefore, the present study investigated the biological functions on human lung adenocarcinoma (LUAD).

Methods: The expression of NRG1 was detected in LUAD tissues by Western blot (WB), quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The expression of NRG1 was upregulated by the addition of exogenous NRG1 and downregulated by small interfering RNA (siRNA), and the biological behaviors of LUAD cells were assessed: cell proliferation by MTT assay, cell cycle and apoptosis by flow cytometry analysis, and migration and invasion using Transwell system. Finally, the pathway underlying the cellular function was analyzed by WB.

Results: A lower expression of NRG1 was observed in LUAD cancer tissues (P<0.05). Moreover, the addition of exogenous NRG1 reduced the cell proliferation, migration, and invasion (P<0.001), while the downregulation of endogenous NRG1 promoted the three kinds of biological behaviors of LUAD cell lines (P<0.001); however, these manifestations did no effect on the distribution of cell cycle and apoptosis status (P>0.05). Furthermore, the deficiency of NRG1 reduced the expression of p-ERK1/2 and p-AKT at the protein level (P<0.001).

Conclusions: The current results suggested that NRG1 might be a suppressor in the development of LUAD, and its function was related to AKT and ERK1/2 pathway.

Keywords: AKT; Neuregulin1 (NRG1); biological behavior; inhibition; lung adenocarcinoma (LUAD).

Figure 1

NRG1 expression at the protein and RNA level in LUAD cancer and paracancerous tissues. (A) NRG1 protein bands obtained by WB from LUAD cancer and paracancer tissues; (B) the summary graph of NRG1 expression at the protein level; (C) the summary graph of NRG1 expression at the mRNA level. NRG1 was expressed in both LUAD tissues and the paired paracancerous tissues, and the expression was lower in the cancer tissues than that in the paracancerous tissues at the protein level (P=0.039) and mRNA level (P=0.024). NRG1, neuregulin1; LUAD, lung adenocarcinoma; WB, Western blot.

Figure 2

IHC staining for NRG1 in LUAD cancer tissues (200×). (A) Negative (−); (B) weak positive (+); (C) moderate positive (++); (D) strongly positive (+++); (E) negative control. The protein expressed in the nucleus, and in some cells, it was also found to express on the cell membranes and in the cytoplasm. IHC, immunohistochemistry; NRG1, neuregulin1; LUAD, lung adenocarcinoma.

Figure 3

Detection of cell transfection efficacy. (A) The transfection of fluorescent siRNA into cells was detected by immunofluorescence; (B) the expression of NRG1 was detected by gel electrophoresis assay after siRNA was transfected into the cells; (C) the transfection efficacy was detected by qRT-PCR in A549 cells; (D) the transfection efficacy was detected by qRT-PCR in H1975 cells. The transfection efficiency was 70.3% in A549 and 73.7% in H1975 cells. The two transfections were effective. NRG1, neuregulin1; siRNA, small interfering RNA; qRT-PCR, quantitative real-time polymerase chain reaction; NC, non-specific control.

Figure 4

Effects of NRG1 on the proliferation of A549 and H1975 cells. (A) The effects of seven different Re-NRG1 concentrations on the proliferation of A549 cell line at four time points; (B) the effect of exogenous NRG1 on the proliferation of H1975 cells; (C) the effect of NRG1 gene knockdown on the proliferation of A549 cells; (D) the effect of NRG1 gene knockdown on the proliferation of H1975 cells. Furthermore, NRG1 played an inhibitory role in the proliferation of A549 and H1975 cells at same doses when reacted for 72 h (P<0.001). The downregulated NRG1 expression stimulated the proliferation of A549 and H1975 cells (P<0.001). NRG1, neuregulin1; OD, optical density; NC, non-specific control.

Figure 5

Effects of NRG1 on the cell cycle of A549 and H1975 cells. (A) The effect of upregulated NRG1 expression on the cell cycle of A549 cells; (B) the effect of exposure to Re-NRG1 on the cell cycle of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell cycle of A549 cells; (D) the effect of downregulated NRG1 expression on the cell cycle of H1975 cells. These results showed that the upregulated NRG1 expression did not affect the cell cycle of these two cell lines (P=0.753, 0.599). Moreover, the downregulated NRG1 expression by gene knockdown did not alter the cell cycle of A549 and H1975 cells (P=0.532, 0.500). NRG1, neuregulin1; siRNA, small interfering RNA; NC, non-specific control.

Figure 6

Effects of NRG1 on the cell apoptosis status of A549 and H1975 cells. (A) The effect of upregulated NRG1 expression on the cell apoptosis of A549 cells; (B) the effect of exposure in Re-NRG1 on the cell apoptosis of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell apoptosis of A549 cells; (D) the effect of downregulated NRG1 expression on the cell apoptosis in H1975 cells. The upregulated NRG1 expression did not affect the cell apoptosis of these two cell lines (P=0.753, 0.858). Moreover, the downregulated NRG1 expression by knockdown of the gene could not alter the cell apoptosis status of A549 and H1975 cells (P=0.831, 0.332). NRG1, neuregulin1; siRNA, small interfering RNA; NC, non-specific control.

Figure 7

Effects of NRG1 on the cell migration ability of A549 and H1975 cells. (A) The role of different doses of exogenous Re-NRG1 on the migration of A549 cells; (B) the role of different doses of exogenous Re-NRG1 on the migration of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell migration of A549 cells; (D) the effect of downregulated NRG1 expression on the cell migration of H1975 cells. These effects displayed an inhibitory effect of NRG1 on the cell migration (P<0.001). In addition, a robust inhibition occurred at high Re-NRG1 concentration. NRG1, neuregulin1; NC, non-specific control.

Figure 8

Effect of NRG1 on the cell invasion ability of A549 and H1975 cells. (A) The role of different doses of exogenous Re-NRG1 on the cell invasion of A549 cells; (B) the role of different doses of exogenous Re-NRG1 on the cell invasion of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell invasion of A549 cells; (D) the effect of downregulated NRG1 expression on the cell invasion of H1975 cells. These effects displayed an inhibitory effect of NRG1 on the cell invasion (P<0.001). In addition, a strong inhibition occurred at high Re-NRG1 concentration. NRG1, neuregulin1; NC, non-specific control.

Figure 9

Expression of AKT and EKT1/2 pathway proteins after upregulated NRG1 by exposure of the cells to exogenous Re-NRG1 and downregulated NRG1 effectuated by gene knockdown. The expression of t-AKT and t-ERK1/2 was not altered in these experimental conditions. However, the expression of p-AKT and p-ERK1/2 was reduced in the case of downregulated NRG1. Consecutively, the expression of the two phosphorylated proteins increased after NRG1 upregulation. +, ++, ++++ indicated that the concentration of exogenous Re-NRG1 was 0, 300, and 1,200 ng/mL, respectively; *, **, *** referred to the control, NC, and siRNA-NRG1 groups, respectively. NRG1, neuregulin1; NC, non-specific control.