Apoptosis of SPC-A1 cells induced by Cisplatin.
SPC-A1 cells were treated with 2.5 µg/ml cisplatin for different periods of time for evaluating the effect of cisplatin on SPC-A1 apoptosis. Flow cytometry was used to detect the apoptosis rate at 12 h, 24 h, 48 h and 72 h after treatment with or without cisplatin. Comparing with those of paired untreated control groups, the morphology of SPC-A1 cells were changed (Fig. 1). Moreover, the apoptosis rates of the SPC-A1 cells cultured for 12, 24, 48 and 72 h with cisplatin were significantly greater (Figs. 2 and 3). With prolonged exposure with cisplatin, these apoptosis rates increased more markedly. It is suggested that cisplatin can induce apoptosis of SPC-A1 cells in a time-dependent manner.
MiR-27a expression increased in SPC-A1 cells cultured supernatants in a time-dependent manner with cisplatin treatment.
MiR-27a expression levels in the culture supernatants of SPC-A1 cells treated with cisplatin were detected by qRT-PCR. We revealed that the miR-27a levels in the SPC-A1 cultured supernatants after being treated with cisplatin a concentration of 2.5 µg/mL for 72 h were more four-fold higher than those in the untreated control group (Fig. 4a). MiR-27a expression levels in cisplatin (2.5 µg/mL) group increased gradually at 12 h, 24 h, 48 h and 72 h, and the difference between any time period of two groups was significant (P < 0.05). When 2.5 µg/mL cisplatin was used for treatment for different periods of time (12–72 h), the levels of miR-27a in supernatants increased with time dependence (Fig. 4a).
MiR-27a expression increased in SPC-A1 cells in a time-dependent manner with cisplatin treatment.
MiR-27a expression levels in SPC-A1 cells treated with cisplatin were detected by qRT-PCR. The results showed that the expression level of miR-27a in SPC-A1 cells treated with 2.5 µg/mL cisplatin for 72 hours was more 8 times higher than that in untreated control cells(Fig. 4b). Cells miR-27a expression levels in cisplatin (2.5 µg/mL) group increased between 12 h and 24 h, and miR-27a expression levels between the two group were significantly different (P < 0.05). However, there was no significant change in 24 h, 48 h and 72 h. This result showed that cells miR-27a expression levels in cisplatin (2.5 µg/mL) group entered into the platform after 24 h (Fig. 4b).
Correlation between prognosis and the change of miR-27a expression levels before and after chemotherapy in NSCLC patients.
As it demonstrated that, accompanying the apoptosis of SPC-A1 cells, miR-27a expression levels increased in SPC-A1 culture supernatants with time dependence after cisplatin treatment. Therefore, it is necessary to assess the prognostic value of in NSCLC patients who receiving first-line chemotherapy. MiR-27a expression levels of 52 patients receiving first-line chemotherapy in serum were monitored before and after chemotherapy. The results showed that the changes include 24 patients increasing and 28 patients decreasing. The follow-up completed until March 2013. The outcome of the patients includes 26 patients PR, 26 patients NR (include 10 patients SD and 16 patients PD). The detail information is shown in Table 1. The chi-square test showed significantly elevation of miR-27a after chemotherapy was more common in patients with PR compared with the patients who achieved NR (61.5% vs 30.8%, P = 0.026). This result found that there was a certain correlation between serum miR-27a levels increasing after chemotherapy and the better outcome of NSCLC patient.
Correlation between prognosis and the change of miR-27a expression levels before and after chemotherapy in lung adenocarcinoma patients (n = 52).
miR-27a change after chemotherapy
NR(SD + PD)
|PR: partial response,NR: no response,SD:stable disease, PD: progressive disease.|
Correlation between miR-27a levels and NSCLC patients’ survival outcome.
At the end of follow-up, 52 patients who completed first-line chemotherapy were divided into two groups according to the increase or decrease of serum miR-27a after chemotherapy. The patients increased by 24 cases after chemotherapy and decreased by 28 cases. Kaplan-Meier survival analysis showed that after first-line chemotherapy, the average survival time of 24 patients with increasing serum miR-27a was 28.4 months, while the average survival time was 9 months for the decreasing 28 patients; The survival time in increasing group is significantly higher than the decreasing group, and the difference between the two is significant (P = 0.0051). As showed in Fig. 5, the result suggested that miR-27a decrease patients had poor outcome compared with the increase patients.
Dynamic changes in serum miR-27a levels during chemotherapy.
The dynamic changes of serum mir-27a in 4 patients with NSCLC after different chemotherapy cycles were monitored. As showed in Fig. 6, patient A received 3 cycles of first-line drug therapy. Computed tomography (CT) showed that the tumor was significantly reduced, while tumor markers decreased and serum miR-27a levels increased gradually. At the end of follow-up, the patient survived and showed PR. Patient B received 3 cycles of first-line chemotherapy, but due to the disease progression of multiple metastases throughout the body, 10 months later, the relative content of serum miR-27a continued to decrease during chemotherapy. After patient C ended the first-line drug chemotherapy for 1 cycle, the progress of the disease was changed to second-line drug chemotherapy. After 3 cycles of second-line chemotherapy, the relative content of serum miR-27a continued to increase. By the end of follow-up, the patient's conditions show PR. Patient D received 2 cycles of first-line drug chemotherapy, the condition was stable, and was allowed to be discharged. After 1 month, the disease progressed and the relative content of serum miR-27a was significantly decreased. Chemotherapy was used instead of second-line chemotherapy, and the patient died and survived for 11 months. The above 4 patients also demonstrated that miR-27a can be used as a tumor marker to guide individualized treatment efficacy during chemotherapy.