Overexpression of miR-758 inhibited proliferation, migration, invasion, and promoted apoptosis of non-small cell lung cancer cells by negatively regulating HMGB

Abstract

Non-small cell lung cancer (NSCLC) is one of the most fatal types of cancer with significant mortality and morbidity worldwide. MicroRNAs (miRs) have been confirmed to have positive functions in NSCLC. In the present study, we try to explore the role of miR-758 in proliferation, migration, invasion, and apoptosis of NSCLC cells by regulating high-mobility group box (HMGB) 3 (HMGB3.) NSCLC and adjacent tissues were collected. Reverse transcription quantitative PCR (RT-qPCR) was employed to detect expression of miR-758 and HMGB3 in NSCLC and adjacent tissues, in BEAS-2B cells and NSCLC cell lines. The targetted relationship between miR-758 and HMGB3 was identified by dual luciferase reporter gene assay. The effects of miR-758 on proliferation, migration, invasion, cell cycle, and apoptosis of A549 cells. MiR-758 expression was lower in NSCLC tissues, which was opposite to HMGB3 expression. The results also demonstrated that miR-758 can target HMGB3. The cells transfected with miR-758 mimic had decreased HMGB3 expression, proliferation, migration, and invasion, with more arrested cells in G₁ phase and increased apoptosis. Our results supported that the overexpression of miR-758 inhibits proliferation, migration, and invasion, and promotes apoptosis of NSCLC cells by negative regulating HMGB2. The present study may provide a novel target for NSCLC treatment.

Keywords: Apoptosis; HMGB3; Invasion; MicroRNA-758; Migration; Non-small cell lung cancer; Proliferation.

Figure 1. Lower expression of miR-758 was identified in NSCLC tissues

*, P<0.05 compared with the adjacent tissues. Measurement data were presented as mean ± S.D.; n=50; the comparisons between groups were conducted by the t test.

Figure 2. High HMGB3 expression while low miR-758 expression was observed in NSCLC tissues

(A) mRNA level of HMGB3 in NSCLC and adjacent tissues measured by RT-qPCR. (B) The gray value of HMGB3 protein band in NSCLC and adjacent tissues determined by Western blot analysis. (C) The protein level of HMGB3 in NSCLC and adjacent tissues; *, P<0.05 compared with the adjacent tissues; measurement data were presented as mean ± S.D.; n=50; the comparisons between groups were conducted by the t test.

Figure 3. Expression of miR-758 was lower and HMGB3 expression was higher in A549 cell line

(A) miR-758 expression in BEAS-2B, A549, H1650, H1975, and H292 cell lines detected by RT-qPCR. (B) mRNA level of HMGB3 in BEAS-2B, A549, H1650, H1975, and H292 cell lines detected by RT-qPCR. (C) The gray value of HMGB3 protein band in BEAS-2B, A549, H1650, H1975, and H292 cell lines. (D) The protein level of HMGB3 in BEAS-2B, A549, H1650, H1975, and H292 cell lines detected by Western blot analysis. *, P<0.05 compared with the BEAS-2B cell line; **, P<0.01 compared with the BEAS-2B cell line; ^#, P<0.05 compared with the A549 cell line. Measurement data were presented as mean ± S.D.; n=3; the comparisons amongst multiple groups were conducted by the one-way ANOVA.

Figure 4. MiR-758 targets HMGB3 gene

(A) MicroRNA.org website predicts that there is a binding site of miR-758 on HMGB3 mRNA 3′-UTR. (B) Dual luciferase reporter gene assay verified the target relationship between miR-758 and HMGB3. *, P<0.05 compared with the NC group. (C) Result of RT-qPCR showed that miR-758 regulated mRNA level of HMGB3. (D,E) Western blot analysis indicated that miR-758 affected HMGB3 protein level. *, P<0.05 compared with the control group; ^&, P<0.05 compared with the miR-758 mimic-NC group; ^#, P<0.05 compared with the miR-758 inhibitor-NC group. Measurement data were presented as mean ± S.D.; n=3; the comparisons amongst multiple groups were conducted by the one-way ANOVA.

Figure 5. Overexpression of miR-758 inhibited proliferation of NSCLC A549 cells in each group

The doubling time of A549 cells is typically 24–40 h. *, P<0.05 compared with the control group; ^&, P<0.05 compared with the miR-758 mimic-NC group; ^#, P<0.05 compared with the miR-758 inhibitor-NC group; measurement data were presented as mean ± S.D.; n=3; the comparisons amongst multiple groups were conducted by the one-way ANOVA.

Figure 6. Overexpression of miR-758 inhibited migration and invasion of A549 cells in each group

(A,C) Wound healing assay indicates that overexpression of miR-758 inhibits migration of A549 cells. (B,D), Transwell assay revealed overexpression of miR-758 suppresses invasion ability of A549 cells. *, P<0.05 compared with the control group; ^&, P<0.05 compared with the miR-758 mimic-NC group; ^#, P<0.05 compared with the miR-758 inhibitor-NC group. Measurement data were presented as mean ± S.D.; n=3; the comparisons amongst multiple groups were conducted by the one-way ANOVA.

Figure 7. Overexpression of miR-758 stimulated cell apoptosis of NSCLC

*, P<0.05 compared with the control group; ^&, P<0.05 compared with the miR-758 mimic-NC group; ^#, P<0.05 compared with the miR-758 inhibitor-NC group. Measurement data were presented as mean ± S.D.; n=3. The comparisons amongst multiple groups were conducted by the one-way ANOVA.