Regulation Effect of miR-34a Expression on Radiosensitivity of Lung Adenocarcinoma Cells by Targeting Bcl-2 and CDK4/6 Signaling Pathways

Objective: Radiotherapy has been widely used to treat lung cancer. However, non-small lung cancer cells are insensitive to radiation, diminishing their radiotherapy effects. Although the radiosensitivity of the non-small lung cancer cells was reported to be enhanced through regulating miR-34a, the regulation effects of miR-34a expression on radiosensitivity of lung adenocarcinoma cells through target genes CDK4, CDK6, CyclinD1, and Bcl-2/Bax have not been systematically investigated. Methods: In this study, we investigated the effect of miR-34a expression on the Bcl-2, CDK4, and CDK6 pathways in lung adenocarcinoma cells, to provide new insights into the sensitization treatment of lung cancer. We first studied the effect of miR-34a expression on H1299 and A549 cell activity. Then to investigate the mechanisms of radiosensitivity, we focused on apoptosis, cell cycle, and target genes. Results: We find that overexpression of miR-34a in lung adenocarcinoma cells inhibits cell activity, and improves radiosensitivity. Specifically, overexpression of miR-34a suppresses the expression of target genes CDK4, CDK6, CyclinD1, and Bcl-2/Bax, which leads to cell cycle arrest and promotes apoptosis of lung adenocarcinoma cells. Conclusions: Overall, our results demonstrate that the overexpression of miR-34a enhances the radiosensitivity of lung adenocarcinoma cells, indicating that miR-34a is a sensitizer for lung adenocarcinoma radiotherapy.

Among all types of NSCLC, lung adenocarcinoma is found to be one of the most insensitive kinds to radiotherapy, which renders it an acritical research topic for future application of radiotherapy on lung cancer. Although a previous study has demonstrated that ectopic expression of miR-34a enhances radiosensitivity and promotes apoptosis by suppressing the LyGDI pathway in NSCLC cells [29], little is reported regarding the radiosensitivity of lung adenocarcinoma in Bcl-2 and CDK4, CDK6 pathway signaling. Therefore, we aimed to investigate the effect of the expression of miR-34a on the Bcl-2, CDK4, and CDK6 pathways in lung adenocarcinoma cells, to provide new insights into the sensitization treatment of lung cancer.

Transfection
Pre-miR-34a was transiently transfected into H1299 cells, and anti-miR-34a was transiently transfected into A549 cells. Briefly, 15 μl of pre-miR-34a, anti-miR-34a, or negative control (ABI, USA) in 100 μl of serum-free RPMI1640 medium were mixed with 5 μl of siPORT transfection reagent (Invitrogen, USA) in 100 μl of the same medium, and allowed to stand at room temperature for 10 min. 2.3 ml of cell suspension was added to the mixture, and transfection efficiency could be detected after incubation for 8 -72 h.

Western Blot Analysis
Cells were harvested, lysed by cell lysate, and then transferred to NC membranes. The membranes were probed with a primary antibody (Nanjing Ba aode Biotechnology Company, China), followed by a peroxidase-conjugated secondary antibody (Beijing Zhongshan Golden Bridge Biotechnology Company, China). Immunoreactive proteins were visualized using the ECL Plus reagent (Beijing Pu lilai Gene Technology Company, China).

CCK-8 Cell Activity Assay
Cells were seeded in 96-well plates, every group with five wells, each well was added 100 μl of cell suspension, and the culture plate was placed in an incubator (37˚C, 5% CO 2 ) for 1 -4 h. Each well was added 10 μl of CCK-8 solution (Beijing Tongren Chemical Company, China), incubated for 1 -4 h at 37˚C, and measured with a microplate reader at absorbance values of 450 nm.

Apoptosis Analysis
Cells (1 × 10 5 ) were harvested at 24 h after transfection and 48 h after irradiation, and suspended in 500 μl of Binding Buffer, then stained with 5 μl of Annexin V-FITC and 5 μl of the PI (Nanjing KGI biotech companies, China), and kept in a dark place 15 min before being analyzed by flow cytometry.

Cell Cycle Analysis
Cells (1 × 10 5 ) were harvested at 24 h after transfection and 48 h after irradiation, and fixed with paraformaldehyde for 2 h. Cells were stained in 500 μl of the

Statistical Analysis
Data of the two comparing groups were analyzed by the Student's t-test using the SPSS, version 19.0 and values less than 0.05 were considered statistically significant.

Restoration of miR-34a Expression on H1299 Cell Activity
As shown in Figure 1  Since miR-34a showed obviously reduced expression in H1299 cells, we restored miR-34a expression in H1299 to observe cell proliferation. Pre-miR-34a or negative control miRNA (NC) was transfected into H1299 cells, and transfection efficiency was detected by the flow cytometry, which was 93%. Real-time PCR detection results showed that the expression of miR-34a in the pre-miR-34a transfection group was significantly increased (P < 0.01) up to 17 times (shown in Figure 1(b)), when compared with the negative control transfection group.
Therefore, these results demonstrate a significant increase in miR-34a expression in miR-34a transfection.
Cell activity of H1299 cells was detected by the CCK-8 kit, which was shown in Figure 1(c). As expected, H1299 cells transfected with pre-miR-34a showed dose-dependent growth inhibition after different doses of 60 Co γ-ray irradiation.
These results suggest that restoration of miR-34a expression enhanced the radiosensitivity of H1299 cells.

Effect of miR-34a Up-Regulation on Apoptosis and Cell Cycle of H1299
To further investigate the mechanism of miR-34a on radiosensitivity of H1299 cells, we performed cell apoptosis and cell cycle analyses, and the results were shown in Figure 1(d) and Table 1.
When compared to NC transfected group, the cell apoptosis rate in pre-miR-34a transfected group increased significantly. Table 1 showed that, compared to NC transfected group, G0/G1 arrested and S phase decreased in pre-miR-34a transfected group. Since CDK4, CDK6, and CyclinD1 are proved to be cell cycle-related target genes of miR-34a, and Bcl-2 and Bax are apoptosis-related target genes, we focused on CDK4, CDK6, CyclinD1, Bcl-2, and Bax, as targets of miR-34a. As shown in the results of real-time PCR, transcript levels of CDK4, CDK6, and Bcl-2/Bax in the pre-miR-34a transfected group were significantly lower than those for the NC transfected group. The results were further confirmed by western blot analysis. Protein levels of Bcl-2, CDK4, and CDK6 in the pre-miR-34a transfected group were significantly lower than those for the NC transfected group. These results indicated that miR-34a suppressed its target genes expression, enhanced radiation-induced apoptosis and cell cycle arrest (Figure 2(a) and Figure 2(b)).

Effect of Inhibition of miR-34a Expression on Radiosensitivity of A549 Cells
In order to verify the impacts of miR-34a expression on H1299 cells, we inhibited the expression of miR-34a in A549 cells to observe the cell proliferation of A549. Anti-miR-34a was transfected into A549 cells to observe cell activity, cell cycle, and apoptosis. When compared to the NC transfected group, cell activity in anti-miR-34a transfected group was significantly increased after irradiation (Figure 3(a)), and the difference was a dose-dependent increase.   As shown in Figure 3(b), the rate of cell apoptosis in the anti-miR-34a transfected group was significantly lower. Table 2 showed that the percentage of the G0/G1 phase in anti-miR-34a transfected cells significantly reduced, and the S phase increased significantly. The target gene (CDK6, CyclinD1, and Bcl-2/Bax) expressions of A549 cells increased significantly in anti-miR-34a transfected cells, as shown in Figures 3C and 3D. These results suggested that down-regulation of the miR-34a expression can reduce the radiosensitivity of A549 cells, which was mutual verification with the results of H1299 cells.

Discussion
The key to tumor radiotherapy is reducing the radioresistance and improving radiosensitivity of tumors which are associated with miRNAs levels [39]. Among various miRNAs, miR-34a played an important role as predictive biomarker of tumor radiation [31]. In addition, the miR-34 family has been identified as a target of p53 and a potential tumor suppressor that plays a role downstream of the p53 pathway [13] [14] [15] [16] [17]. More than 50% of cancers, including NSCLC, have a p53-mutant and a low level of miR-34 expression [40]. In the present study, we also observed low levels of expression of miR-34a in lung adenocarcinoma cells, especially in p53-mutant cells H1299, which was consistent with previous studies. Our experimental results proved that miR-34a is an important molecule of lung adenocarcinoma cells.
Although ectopic expression of miRNAs has been frequently investigated for various types of cancer, the current cognition about the role of miRNAs on lung adenocarcinoma cells and corresponding mechanisms is still limited. Therefore, it is essential for promising the therapeutic effect of lung adenocarcinoma to identify the valuable miRNAs and targets. Previous studies have emphasized the key role of miR-34a as a radiosensitizer. Balca-Silva's study showed that overexpression of miR-34b in NSCLC cells increased radiosensitivity at low doses of radiation [41]. Liu et al. [2011] found that miR-34a played a key role in increasing radiosensitivity in different tissues by promoting cell apoptosis and decreasing cell viability [34]. Cortez et al. [2015] identified RAD51 as a new direct target of miR-34a to increase radiosensitivity in NSCLC cells [38]. Here, we found that overexpression of miR-34a inhibited cell activity of H1299. We further revealed that restoration of miR-34a enhanced the radiosensitivity of H1299 cells, suggesting that target genes of the miR-34a were sufficient to inhibit the cell activity of H1299. To investigate the mechanisms of radiosensitivity, we focused on apoptosis, cell cycle, and target genes. We profiled 5 genes from the cell cycle and apoptosis pathways after enforced overexpression of miR-34a in lung adenocarcinoma cell lines.
It is well known that exposure to ionizing radiation causes changes in the cell cycle. The changes at each stage of the cell cycle are a direct response to external injury factors. Among the various phases of the cell cycle, G1 is critical for regulating the cell cycle, providing enough time to promote damaged DNA repair. It has been previously shown that CyclinD1, CyclinE2, CDK4, and CDK6 are Journal of Cancer Therapy important molecules in the G1 checkpoint [42]. Moreover, CyclinD1, CyclinE2, CDK4, and CDK6 have been shown to be miR-34 target genes [42]. However, whether miR-34a could directly target CDK4 and CDK6 in lung adenocarcinoma is unclear. Our study showed that overexpression of miR-34a blocked in G0/G1 and G2 phases, and CDK4, CDK6, and CyclinD1 were all down-regulated in lung adenocarcinoma cells. These results indicated that miR-34 plays an important role in regulating the cell cycle, thereby enhancing the radiosensitivity of lung adenocarcinoma cells.
Apoptosis is a strictly controlled process of multiple genes, including the Bcl-2 family, caspase family, Bax, oncogenes such as C-myc, and tumor suppressor gene p53. Studies have shown that regulating the expression of the Bcl-2 family can inhibit cell proliferation and promote apoptosis [43] [44]. Bcl-2 expression is decreased by knockdown in brain glioblastoma and colon cancer cell lines, the apoptosis is enhanced after irradiation and the radio-protective role of miR-34a inhibitor is attenuated [34]. In different types of cancer, it has been reported that the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax is negatively regulated by miR-34a. Accordingly, our results showed that the overexpression of miR-34a increased apoptosis, inversely correlated with Bcl-2/Bax ratio. This is different from the effect of miR-34b on H1299 cell apoptosis and Bcl-2 level [41], suggesting the complex mechanism of the miR-34 family's influence on the radiosensitivity of tumor cells. Our results verified that miR-34a can promote cell apoptosis by regulating the expression of target genes, which is consistent with the previous results.
It is worth noting that the apoptosis rate of lung adenocarcinoma cells after irradiation increased, but the increase in apoptosis rate caused by overexpression of miR-34a was much smaller. The reason is that lung adenocarcinoma cells are not sensitive to γ-rays, and even larger doses of γ-rays cannot have a greater impact, indicating that lung adenocarcinoma cells have lower radiation sensitivity. Restoring the expression of miR-34a, the apoptosis rate of lung adenocarcinoma cells was significantly increased, which indicated that miR-34a can enhance the radiosensitivity of lung adenocarcinoma cells, promote cell apoptosis, and have a synergistic effect with the pro-apoptotic effect of γ-rays. Therefore, the combined effects of overexpression of miR-34a and γ-rays provide a new understanding of the treatment of lung adenocarcinoma.

Conclusion
In conclusion, our experimental study verified both positive and negative sides,

Authors' Contributions
All authors were involved in the design and implementation of the experiment.
Manuscripts have been reviewed and approved by all authors.