3.1 PTX up-regulated miR-221-3p, which directly regulated MDM2 and P53 in PTX-sensitive NSCLC strain (A549), and was lowly expressed in PTX-resistant A549 cells (A549/Taxol) cells
First, miR-221-3p expression was detected via qPCR after PTX treatment in A549 cells and A549/Taxol cells to explore the relationship between miR-221-3p and PTX resistance. After 10 µM PTX administration for 48 h, A549 cells were noticed to have cell shrinkage and cell size reduction, whereas the morphological changes of A549/Taxol cells were not evidently observed (Fig. 1A). MTT assay results showed that PTX inhibited NSCLC cell proliferation in a dose-dependent manner. A549/Taxol cells (IC50 value = 55.47 µM) showed evident PTX resistance compared with A549 cells (IC50 value = 17.18 µM) (Fig. 1B). After treatment of PTX, the miR-221-3p expression was significantly increased in A549 cells (Fig. 1C, P = 0.036), but was extremely low in A549/Taxol cells at only 1/3 of that in A549 cells (Fig. 1C, P = 0.045). No significant change was found in the miR-221-3p expression in A549/Taxol cells after PTX treatment (Fig. 1C, P = 0.070).
Second, MDM2 and P53 levels were analyzed by Western blot to explore the molecular mechanisms of miR-221-3p, and dual-luciferase reporter assay was conducted to verify the interaction between miR-221-3p and MDM2. As shown in Fig. 1D, TargetScanHuman 7.2 was employed first to predict that MDM2 is the putative target of miR-221-3p. According to Western blot analysis, A549 cells had significantly decreased MDM2 protein expression (P = 0.004, Figs. 1E and 1F), but was significantly increased P53 protein expression (P < 0.001, Figs. 1E and 1G) after PTX administration. The MDM2 protein expression in A549/Taxol cells was almost three times higher than that in A549 cells (P < 0.001, Figs. 1E and 1F), whereas the P53 protein expression was low at approximately 1/8 of that in A549 cells (P < 0.001, Figs. 1E and 1G). No significant was found in the MDM2 and P53 expression in A549/Taxol cells after PTX treatment. MDM2 was sixfold higher than that in A549 + PTX group (P < 0.001, Figs. 1E and 1F), whereas P53 was approximately 1/25 of that in A549 + PTX group (P < 0.001, Figs. 1E and 1G). A luciferase reporter plasmid-containing wild-type or mutant MDM2 was used to observe the effects after transfection (Fig. 1H), and the luciferase activity was measured 48 h after transfection. When miR-221-3p was co-transfected with MDM2-WT, the relative luciferase activity was significantly lower than that in the control group (P = 0.027, Fig. 1I) or the miR-NC group (P = 0.026, Fig. 1I). However, when miR-221-3p was co-transfected with MDM2-MT, no observed significance was found (Fig. 1I). These results indicate that miR-221-3p interacts with MDM2.
3.2 Knockdown or overexpression of miR-221-3p regulated the expression of MDM2 and P53 in A549 cell line
First, miR-221-3p expression in A549 cells was down-regulated or up-regulated via the transfection of inhibitor-miR-221-3p or mimic-miR-221-3p, and the expression changes of MDM2 and P53 were analyzed by qPCR and Western blot to further validate the miR-221-3p/MDM2/P53 pathway. As shown in Fig. 2A, inhibitor-miR-221-3p was designed to specifically target the binding sites in miR-221-3p. QPCR results in Fig. 2B showed that inhibitor-miR-221-3p and mimic-miR-221-3p transfections significantly down- and up-regulated the expression of miR-221-3p to 1/20 (P < 0.001) and 500 times (P < 0.001) of that in the negative control group, respectively. Therefore, transfection was successful. As shown in Fig. 2C, the expression of MDM2 in A549 cells was up-regulated to 1.6 times of that in negative control group (P < 0.001) after inhibitor-miR-221-3p transfection, and mimic-miR-221-3p transfection significantly down-regulated the expression of MDM2 to 1/3 of that in negative control group (P = 0.0017). As shown in Fig. 2D, inhibitor-miR-221-3p transfection up- and down-regulated MDM2 (P = 0.008, Fig. 2E) and P53 protein expression (P = 0.040, Fig. 2F), respectively, whereas mimic-miR-221-3p transfection down- and up-regulated MDM2 expression (P = 0.022, Fig. 2E) and P53 expressions (P = 0.016, Fig. 2F), respectively. The above results indicated that miR-221-3p was negatively and positively correlated with MDM2 and P53, respectively, which was consistent with the results shown in Figs. 1H and 1I.
3.3 Knockdown and overexpression of miR-221-3p could reduce PTX sensitivity in A549 cell line and reverse PTX resistance in A549/Taxol cell line, respectively
To further analyze the association between miR-221-3p/MDM2/P53 pathway and PTX resistance, we down- and up- regulated miR-221-3p in A549 and A549/Taxol cells via the transfection of inhibitor- and mimic-miR-221-3p, respectively. Colony formation and CCK-8 assays were then employed to explore the changes of cell proliferation and cell viability in A549 cells and A549/Taxol cells after transfection. Figure 3A shows the cell proliferation ability of A549 and A549/Taxol cells after the transfection of inhibitor- and mimic-miR-221-3p via crystal violet staining, respectively. The stained cell area ratio was calculated in accordance with 15 random fields per well under the 10 × magnification. As shown in Fig. 3B, inhibitor-miR-221-3p transfection increased the proliferation of A549 cells (P = 0.025); however, this effect was not evident after PTX treatment (P = 0.191). Mimic-miR-221-3p transfection significantly inhibited the proliferation of A549/Taxol cells (P < 0.001) and increased the sensitivity of A549/Taxol cells to PTX (P < 0.001). After dissolving crystal violet with 10% glacial acetic acid, optical density values were detected at 595 nm by using the NanoDrop ND-1000 spectrophotometer. As shown in Fig. 3C, inhibitor-miR-221-3p transfection increased the cellular survival of A549 cells (P < 0.001); however, this effect was not evident after PTX treatment (P = 0.518). As shown in Fig. 3D, mimic-miR-221-3p transfection significantly inhibited cellular survival of A549/Taxol cells (P = 0.017) and increased the sensitivity of A549/Taxol cells to PTX (P = 0.004). The above results were consistent with the results shown in Fig. 3B. The cell viability of A549 and A549/Taxol cells treated with PTX after the transfection of inhibitor- and mimic-miR-221-3p was evaluated by CCK-8 assay (Fig. 3E). As shown in Fig. 3F, the absorbance at 450 nm of each group indicated that inhibitor- and mimic-miR-221-3p transfections could attenuate (P < 0.001) and strengthen (P < 0.001) the cell viability inhibition of PTX in A549 and A549/Taxol cell lines, respectively. The above results indicated that inhibitor- and mimic-miR-221-3p transfections could enhance PTX resistance in A549 cell line and reverse PTX resistance in A549/Taxol cell line, respectively.
3.4. MiR-221-3p overexpression could reverse PTX resistance in vivo
To determine the important role of miR-221-3p on PTX resistance in LC, we constructed drug-resistant xenograft by subcutaneously injecting A549/Taxol cells. As shown in Fig. 4A, the tumor size in the agomir-221-3p group was significantly smaller than those in the PTX group. The tumor size in the blank group was much larger than those in the PTX group (Fig. 4A). We also detected the miR-221-3p and MDM2 expression levels by qPCR analysis (Figs. 4C and 4D). We observed that the tumor growth was significantly suppressed by intratumorally injecting agomir-221-3p (Fig. 4B), with up-regulation of miR-221-3p expression (Fig. 4C) and down-regulation of MDM2 expression (Fig. 4D), which validated the effect of miR-221-3P/MDM2/P53 pathway on PTX resistance in vivo.
3.5 MiR-221-3p was up-regulated in NSCLC tissues and the low expression of miR-221-3p was correlated with advanced T stage
To further validate the role of the miR-221-3p/MDM2/P53 pathway in NSCLC, in addition to the above cellular and molecular experiments, we also collected 20 samples of NSCLC tumor and adjacent non-cancerous tissues through surgical resection from patients diagnosed between September 2018 and May 2019.
First, qPCR was used to measure the miR-221-3p and MDM2 expression levels in 20 paired NSCLC and paracancerous tissues (Figs. 5A and 5B). As shown in Fig. 5C, miR-221-3p was markedly up-regulated in NSCLC tissues compared with the control (P = 0.032). The expression of MDM2 in NSCLC group was significantly lower than that in paracancerous group (P = 0.003, Fig. 5D), which suggested a negative relationship between miR-221-3p and MDM2. However, no statistically significant correlation was found in 20 paired NSCLC and paracancerous tissues between miR-221-3p and MDM2 (R=-0.198, P = 0.221; Fig. 5E). Oncomine database was employed to verify the expression of MDM2 in NSCLC tissue, and the results indicated that the expression of MDM2 could be decreased in various pathological types of NSCLC (Fig. 5F).
Second, the correlation between miR-221-3p/MDM2 expression level and other clinicopathological parameters in patients with NSCLC was also analyzed. The mean value of miR-221-3p/MDM2 in NSCLC tissues was used as the threshold for distinguishing the high group and low groups.
Regarding miR-221-3p, the decreased expression of miR-221-3p was evidently related to advanced T stage (P = 0.005, Table S3; P = 0.008, Table S4). In summary, the expression of miR-221-3p in NSCLC tissues was significantly higher than that in paired non-cancerous matched tissues (P = 0.004, Table S4).
Regarding MDM2, the increased expression of MDM2 was evidently related to advanced T stage (P = 0.020, Table S5). In summary, the level of MDM2 expression in NSCLC tissues was significantly lower than that in paired non-cancerous matched tissues (P = 0.004, Table S6).
Third, based on the qPCR results in Fig. 5B, 20 paired NSCLC and paracancerous tissues were divided into 5 groups according to the sequence of MDM2 expression from low to high for Western blot experiments (Fig. 6A). The levels of MDM2 and P53 protein are shown in Figs. 6B and 6C, respectively. As shown in Fig. 6D, MDM2 was markedly up-regulated in NSCLC tissues compared with paracancerous tissues in MDM2 high-expression group (P = 0.046). However, no significant difference was found between NSCLC and paracancerous tissues in the 4 relatively low-MDM2 expression groups (P = 0.186, 0.131, 0.479, 0.470). As shown in Fig. 6E, P53 was markedly down-regulated in NSCLC tissues compared that in paracancerous tissues in two high-MDM2 expression groups (P = 0.013, 0.026 respectively). However, no significant difference was found between NSCLC and paracancerous tissues in the 3 relatively low-MDM2 expression groups (P = 0.201, 0.253, 0.514). A negative relationship between MDM2 and P53 was found in high-MDM2 expression group (R=-0.748, P = 0.033; Fig. 6F). No statistically significant correlation was found in another 4 relatively low-MDM2 expression groups between MDM2 and P53 (P = 0.474, 0.790, 0.741, 0.409). The above results indicated that MDM2 could be associated with the progression of NSCLC in the high-MDM2 expression group, which was negatively correlated with P53.