Effect of BRAF-mediated PI3K/Akt/mTOR pathway on biological characteristics and chemosensitivity of NSCLC A549/DDP cells

Abstract

The present study aimed to explore the biological characteristics of non-small cell lung cancer (NSCLC) cells and the mechanism of chemosensitivity through the role of the PI3K/Akt/mTOR signaling pathway mediated by BRAF gene silencing. Following cell transfection and grouping, an MTT assay detected the activity of NSCLC cells, a scratch wound test assessed the migration ability, flow cytometry using PI staining detected the cell cycle phase, TUNEL and flow cytometry through Annexin V-PI staining assessed the apoptosis, and colony formation was used to detect the sensitivity of lung cancer cells to cisplatin chemotherapy. Furthermore, the relative expression levels of BRAF, PTEN, PI3K, mTOR mRNA were assessed by RT-qPCR, and the protein expression levels of BRAF, PTEN, PI3K, phosphorylated (p)-PI3K, Akt, p-Akt, mTOR, p-mTOR, cisplatin resistance-related enzymes ERCC1 and BRCA1, apoptotic proteins Bax and Bcl-2 were assessed by western blotting. Compared with the control group and NC group, there were differences in decreased BRAF mRNA expression levels in the small interfering (si)BRAF group and siBRAF + IGF-1 group (both P<0.05). In addition, compared with the control group, the siBRAF, NVP-BEZ235 and siBRAF + NVP-BEZ235 groups had significant decreased cell viability at 2-6 days, decreased migration ability, shortened proportion of S-phase cells, increased proportion of G₁/G₀-phase cells, increased apoptosis rate, decreased number of colony-forming cells, decreased mRNA expression of PI3K, Akt and mTOR, increased PTEN mRNA expression, decreased protein expression levels of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, ERCC1, BRCA1 and Bcl-2, and increased protein expression levels of PTEN and Bax (all P<0.05); and more obvious trends were revealed in the siBRAF + NVP-BEZ235 group (all P<0.05); whereas opposite results were detected in the siBRAF + IGF-1 group when compared with the siBRAF group and NVP-BEZ235 group (all P<0.05). Silencing of BRAF gene expression to inhibit the activation of the PI3K/Akt/mTOR signaling pathway exerted a synergistic effect decreasing cell viability, inhibiting the cell cycle and migration, increasing the apoptosis rate, decreasing the number of colony-forming cells and increasing chemosensitivity of NSCLC. Activation of the PI3K/Akt/mTOR signaling pathway may reverse the role of silencing of BRAF gene expression, providing a potential approach for improving the chemosensitivity of NSCLC. The present study for the first time, to the best of our knowledge, clarified the possible mechanism of NSCLC cell biological characteristic changes and chemosensitivity from the perspective of BRAF gene silencing and PI3K/Akt/mTOR signaling pathway activation, providing a potential reference for suppressing tumor aggravation and improving the therapeutic outcomes of NSCLC at the genetic level.

Keywords: BRAF gene; PI3K/Akt/mTOR signaling pathway; biological characteristics; chemosensitivity; non-small cell lung cancer cells.

Figure 1.

Comparison of cell viability expressed as the OD value in each group after transfection by MTT colorimetric assay. The data were measured and expressed by mean ± standard deviation. The comparison between the two groups was performed by unpaired t-test, and the comparison between multiple groups was performed by one-way ANOVA. The cell experiment was repeated three times. *P<0.05, compared with the control group and NC group at the same time-point, revealing statistical difference; ^#P<0.05, compared with the siBRAF + NVP-BEZ235 group at the same time-point, revealing statistical difference. NC, negative control; si, small interfering.

Figure 2.

Comparison of cell migration ability expressed as the migrating number of cells and cell scratch distance in each group after transfection by scratch test. (A) Experimental images in each transfection group using the scratch test at 0 h (as the control) and 48 h respectively. (B) Comparison of the migration distance in each group. The data were measured and expressed by the mean ± standard deviation. The comparison between the two groups was performed by unpaired t-test, and the comparison between multiple groups was performed by one-way ANOVA. The cell experiment was repeated three times. *P<0.05, compared with the control group and NC group, revealing statistical difference; ^#P<0.05, compared with the siBRAF + NVP-BEZ235 group, showing statistical difference. NC, negative control; si, small interfering.

Figure 3.

Comparison of cell cycle phases in each group after transfection by flow cytometry using PI staining. (A) The distribution of the cell cycle in each phase in each group. (B) Histogram of the cell cycle distribution in each group expressed as the percentage (%). The data were measured and expressed as the mean ± standard deviation. The comparison between the two groups was performed by unpaired t-test, and the comparison between multiple groups was performed by one-way ANOVA. The cell experiment was repeated three times. *P<0.05, compared with the control group and NC group, revealing statistical difference; ^#P<0.05, compared with the siBRAF + NVP-BEZ235 group, showing statistical difference. NC, negative control; si, small interfering.

Figure 4.

Comparison of the cell apoptosis rate in each group after transfection by TUNEL assay. (A) The apoptosis of cancer cells in each group under light microscope (×200). (B) The apoptosis rate of cancer cells in each group expressed as the percentage of apoptotic cells (%). The data were measured and expressed as the mean ± standard deviation. The comparison between the two groups was performed by unpaired t-test, and the comparison between multiple groups was performed by one-way ANOVA. The cell experiment was repeated three times. *P<0.05, compared with the control group and NC group, revealing statistical difference; ^#P<0.05, compared with the siBRAF + NVP-BEZ235 group, showing statistical difference. NC, negative control; si, small interfering.

Figure 5.

Comparison of the cell apoptosis rate in each group after transfection by flow cytometry using Annexin V-PI staining. (A) The apoptosis of cancer cells in each group. (B) The apoptosis rate of cancer cells in each group expressed as the percentage (%). The data were measured and expressed by mean ± standard deviation. The comparison between the two groups was performed by unpaired t-test, and the comparison between multiple groups was performed by one-way ANOVA. The cell experiment was repeated three times. *P<0.05, comparison with the control group and NC group, revealing statistical difference; ^#P<0.05, comparison with siBRAF + NVP-BEZ235 group, showing statistical difference. NC, negative control; si, small interfering.

Figure 6.

Comparison of the colony-forming number of cells of each group after transfection. (A) The experimental images of colony formation in each group. (B) Histogram of the number of colony-forming cells in each group expressed as the (%). The data were measured and expressed as the mean ± standard deviation. The comparison between the two groups was performed by unpaired t-test, and the comparison between multiple groups was performed by one-way ANOVA. The cell experiment was repeated three times. *P<0.05, compared with the control group and NC group, revealing statistical difference; ^#P<0.05, compared with the siBRAF + NVP-BEZ235 group, showing statistical difference. NC, negative control; si, small interfering.

Figure 7.

Comparison of the mRNA expression levels of BRAF, PI3K, Akt, mTOR and PTEN in each group after transfection by reverse transcription-quantitative PCR. The data were measured and expressed as the mean ± standard deviation. The comparison between the two groups was performed by unpaired t-test, and the comparison between multiple groups was performed by one-way ANOVA. The cell experiment was repeated three times. *P<0.05, compared with the control group and NC group, revealing statistical difference; ^#P<0.05, compared with the siBRAF + NVP-BEZ235 group, showing statistical difference. NC, negative control; si, small interfering.

Figure 8.

Detection of the protein expression levels of BRAF, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, PTEN, ERCC1 and BRCA1, Bax, and Bcl-2 in each group after transfection by western blotting. (A) Western blotting bands and statistical analysis of protein expression including BRAF and PI3K/Akt/mTOR signaling pathway-related proteins. (B) Western blotting bands and statistical analysis of the protein expression including cisplatin resistance-related enzymes and apoptotic proteins. The data were measured and expressed as the mean ± standard deviation. The comparison between the two groups was performed by unpaired t-test, and the comparison between multiple groups was performed by one-way ANOVA. The cell experiment was repeated three times. *P<0.05, compared with the control group and NC group, revealing statistical difference; ^#P<0.05, compared with the siBRAF + NVP-BEZ235 group, showing statistical difference. p-, phosphorylated; NC, negative control; si, small interfering.