MiR-221-3p-Mediated Downregulation of MDM2 Reverses the Paclitaxel Resistance of Non-Small Cell Lung Cancer in Vitro and in Vivo

Background: MicroRNAs (miRNAs) are involved in the initiation and development of cancer, and participate in drug resistance. Paclitaxel (PTX) is rst-line chemotherapy drug for advanced non-small cell lung cancer (NSCLC). The abnormal miRNA expression in NSCLC and its association with chemotherapy drug resistance remains largely unknown. The study aimed to investigate the aberrant expression of miR-221-3p in NSCLC and to elucidate its molecular mechanisms in relation to PTX resistance. Methods: Quantitative polymerase chain reaction (qPCR) was used to examine miRNA and messenger RNA (mRNA) in NSCLC tissues and cell lines. The roles of miR-221-3p in NSCLC progression and drug resistance were assessed by Western blot analysis, colony formation assay, and CCK-8. Dual-luciferase reporter assay was conducted to evaluate the interaction between miR-221-3p and MDM2. Xenograft tumor models were established by subcutaneously injecting PTX-resistant A549 cells (A549/Taxol) to assess the effect of miR-221-3p on tumor growth. Data regarding miRNAs and target proteins from 20 NSCLC tissues and paired non-cancerous matched tissues were also obtained for correlation analysis. Results: PTX increased miR-221-3p expression, which regulated MDM2/P53 expression in the PTX-sensitive NSCLC strain (A549). Meanwhile, miR-221-3p was rarely expressed and not interfered by PTX in A549/Taxol. Dual luciferase reporter assay conrmed that miR-221-3p specically binds to MDM2 mRNA. MiR-221-3p down-regulation reduced the sensitivity of A549 cells to PTX, whereas its up-regulation partially reversed the A549/Taxol cells resistance to PTX and increased the chemosensitivity of A549/Taxol cells to PTX in xenograft models. QPCR analysis revealed that miR-221-3p expression increased, whereas the MDM2 level decreased in NSCLC tumor tissues. Furthermore, the result of Western bolt analysis showed that P53 was lowly expressed in tumor tissues with MDM2 overexpression. Conclusions: MiR-221-3p overexpression could regulate MDM2/p53 signaling pathway to reverse the PTX resistance of NSCLC and induce apoptosis in vitro and vivo.


Introduction
Lung cancer (LC) is one of the most common causes of cancer-related death worldwide [1][2][3], and ranked second and rst among the new cancer cases and cancer-related mortality in 2018, respectively [4,5].
Non-small cell LC (NSCLC) accounts for 80% of all LC cases, with a 5-year survival rate of approximately 10-15% [6,7]. For patients with advanced NSCLC who do not receive a molecular targeted therapy or immune checkpoint therapy, the standard rst-line treatment remains cytotoxic chemotherapy [8].
Paclitaxel (PTX) is an important rst-line treatment of advanced NSCLC [9], and interferes with cell division by promoting microtubule polymerization and promotes apoptosis [10]. However, other anti-tumor mechanisms for PTX remain undiscovered. One shortcoming of PTX is the emergence of drug resistance [11], but the underlying molecular mechanism of PTX resistance is still under investigation.
MicroRNAs (miRNAs) are a class of non-coding RNAs (ncRNAs) that are 18-25 nucleotides long and typically cause messenger RNA (mRNA) degradation or translation inhibition by directly binding to the 3' untranslated region (3' UTR) of their target mRNA [12]. MiRNA is involved in various of cellular processes, including cell survival, proliferation, differentiation, apoptosis, and autophagy, through negative regulation of its targets [13]. The mechanism of miR-221-3p in tumor progression is complex. In 2012, miR-221 was reported to be an oncogene or tumor suppressor depending on the tumor system [14]. MiR-221 overexpression in the cancer stroma is associated with malignant potential in colorectal cancer [15]. Oral squamous cell carcinoma cells enhance resistance to doxorubicin by up-regulating miR-221 [16].
However, miR-221 can inhibit the growth of erythroleukemia cells via kit receptor down-modulation [17].
In this study, we investigated the aberrant expression of miR-221-3p in NSCLC and elucidated the molecular mechanisms underlying its in uence on apoptosis and induction of PTX resistance. Our ndings provide novel viewpoints for the anti-tumor mechanisms of PTX and the molecular mechanism of PTX resistance in NSCLC.

MTT assay
The cells were seeded into a 96-well plate (Corning) at a density of 5 × 10 3 cells/well in 200 ul culture medium. After treatment, the cells were incubated in 20 ul DMEM/F12 containing 0.5 mg/ml MTT at 37℃ for 4 h. Afterward, the supernatant was removed, and the cells were lysed in 150 ul dimethyl sulfoxide (DMSO) for 10 min at 37℃. Optical density (OD) values were detected at 490 nm. The obtained values were presented as folds of the control group.

Western blot analysis
Western blot analysis was performed using standard procedures. Brie y, total protein was extracted and isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene uoride (PVDF) membrane. To block non-speci cally bound, the membrane was incubated with 5% skim milk powder for 1 h at room temperature. Membranes were then incubated with primary antibody against MDM2 or P53 (1:1000) followed by horseradish peroxidase (HRP) labeled secondary antibody and detected by chemiluminescence. Ananti-GAPDH antibody (1:1000) was used as a protein loading control.

Transfection
A549 cells were seeded into 6-well plates, incubated overnight, and transfected with inhibitor-miR-221-3p or inhibitor-miR-NC. A549/Taxol cells were transfected with the mimic-miR-221-3p or miR-NC under the same conditions. The sequences used for transfection were listed in Table S2. Lipofectamine®3000 was used as a transfection reagent according to the manufacturer's recommendations. After 48 h of incubation, cells were used for functional analysis.

Colony formation assay
Transfected A549 or A549/Taxol cells were seeded in 6-well plates at 5 × 10 3 cells per well. After incubation for 36 h at 37℃ in a 5% CO 2 humidi ed incubator, the cells were incubated with medium supplemented with PTX (2 µM) and cultured at 37℃ in a 5% CO 2 humidi ed incubator for 7 days. After colony formation was observed, the medium was removed. The cells were washed twice with phosphate buffered saline (PBS), xed with 4% formaldehyde for 10 min, and stained with 5% crystal violet for 10 min. The stained cell area ratio was calculated by randomly photographing 15 elds per well under a 10 × microscope. Finally, after dissolving crystal violet with 10% glacial acetic acid, optical density (OD) values were detected at 595 nm. The obtained values were presented as folds of the control group.

CCK8 assay
After 48 h of transfection in 96-well plates, the freshly prepared medium contained PTX at a nal concentration of 10 µM. The medium was added to the wells with 7 replicate wells per set. After 48 h of incubation, cell viability was measured using CCK-8 kit according to the manufacturer's instructions. The absorbance at 450 nm was measured using NanoDrop ND-1000 spectrophotometer.

Xenograft assay
All experimental protocols were approved by the Animal Ethics Committee of Soochow University. Animal experiments were carried out following the ARRIVE guidelines [17] and the EU Directive 2010/63/EU for animal experiments [18]. A total of 15 male BALB/c nude mice (4 weeks old) weighing 20.35 ± 1.0 g were fed a pellet diet and housed under controlled environment with a temperature of 24 ± 2˚C and air humidity of 60 ± 2%. For the drug-resistant xenograft model, A549/Taxol cells were subcutaneously injected into the armpits of nude mice (1 × 10 6 cells per animal). From the 10th day after cell injection, the engraftment of tumor was con rmed and the baseline tumor size was evaluated. The xenograft-bearing mouse models were randomized into 3 groups, 1) mice were intraperitoneally injected with PTX (15 ug per gram each time, bid) and intratumorally injected with agomir-221-3p (2 OD each time, qd); 2) mice were intraperitoneally injected with PTX and intratumorally injected with PBS; 3) mice were intratumorally and intraperitoneally injected with PBS. Tumor formation was monitored by measuring the length (L) and width (W) with calipers every 2 days, and the volumes were calculated using the following formula: (L × W × W)/2. All mice were sacri ced 10 days after rst PTX injection, and the tumors were neatly excised. Tumor tissues were then subjected to total RNA isolation for qPCR analysis.

Statistical analysis
All statistical analyses were performed using SPSS 22.0 software (IBM) and Graph pad Prism 5.0.
Differences between NSCLC tumor and adjacent non-cancerous tissues were analyzed using the Chisquare test. The correlations between miR-221-3p expression levels and clinicopathological factors were further analyzed by one-way ANOVA. The correlations between miR-221-3p expression, and MDM2 expression were explored by Pearson correlation analysis. P < 0.05 was considered statistically signi cant. 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 (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 signi cantly 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 signi cance was found (Fig. 1I). These results indicate that miR-221-3p interacts with MDM2. 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 elds per well under the 10 × magni cation. 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 signi cantly 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 signi cantly 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.

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 signi cantly 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 signi cantly 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.

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 noncancerous 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 signi cantly 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 signi cant 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

Discussion
This study focused to investigate the aberrant expression of miR-221-3p in NSCLC and to elucidate its molecular mechanisms that in uence apoptosis and induce PTX resistance. Our results indicate that PTX can regulate the expression of MDM2 and P53 via up-regulating the expression of miR-221-3p in A549 cells. Furthermore, miR-221-3p overexpression could reverse PTX resistance and induce apoptosis via targeting the MDM2/P53 signaling pathway in NSCLC cells. The miR-221-3p up-regulation could increase the chemosensitivity of A549/Taxol to PTX in xenograft models. Based on analysis of NSCLC tissues and paired non-cancer matching tissues, it was found that miR-221-3p was up-regulated in NSCLC tissues, and the low expression of miR-221-3p is associated with poor T staging. This topic provides a new direction for studying the role of microRNA in the development of NSCLC and a new perspective for the analysis of PTX resistance of NSCLC. In addition, miRNA mimics was explored as a new method to reverse PTX resistance.
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 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 signi cantly 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 signi cant 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 signi cant 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 signi cant 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.
PTX can promote the apoptosis of A549 cells by up-regulating the level of long-chain ncRNA MEG3 [20]. However, the ncRNA as a mechanism of paclitaxel drugs has been rarely reported. Association studies between PTX and ncRNA have often focused on PTX resistance. In terms of miRNAs, ursolic acid has recently been reported to reverse the chemical resistance of PTX to breast cancer cells by targeting miR-149-5p [21]. MiR-155-3p can also act as a tumor suppressor and reverse PTX resistance via the negative regulation of MYD88 in human breast cancer [22]. In 2015, PTX has been reported to cause the miRNA release, including miR-221-3p [23]. In our research, the expression of miR-221-3p was up-regulated by PTX in A549 cells; this founding could be a new mechanism of PTX. Regarding PTX resistence, our results indicated that the low expression of miR-221-3p was associated with NSCLC PTX resistance. To explore methods to reverse PTX resistance to NSCLC cells, we transfected inhibitor-and mimic-miR-221-3p. The results showed that the knockdown of miR-221-3p could reduce PTX sensitivity in A549 cell line whereas the overexpression of miR-221-3p could reverse PTX resistance in A549/Taxol cell line.
In our research, despite the cellular and molecular experiments, 20 of NSCLC tissue and paired noncancerous matched tissue samples were also collected through surgical resection. The level of miR-221-3p increased signi cantly in NSCLC tissues compared with that in paired non-cancerous matched tissues, which suggested that miR-221-3p could be used as a potential biomarker for NSCLC. The relationship between miRNA and NSCLC has been extensively studied. The recent report suggested that miR-621 was closely related to the pathological grade and poor prognosis of NSCLC. Furthermore, miR-621 can inhibit the malignant progression of NSCLC by modulating SIX4 expression [24]. MiRNA could also be used as a candidate biomarker for the early diagnosis of NSCLC. MiR-23a and miR-451 can be used as potential biomarkers for the early diagnosis of NSCLC, and their combined detection can be effective in diagnosis [25]. MiR-17 and miR-222 can also be considered as non-invasive biomarkers for detecting early LC development and metastasis in patients with NSCLC [26]. In addition, the role of serum miRNA as a prognostic factor in patients with advanced NSCLC and its association with tissue miRNA expression pro les have been studied [27]. MiR-221 is a carcinogenic risk factor and is highly expressed in NSCLC tissues [27], which was consisted with the results from our clinical samples.
Regarding the relationship between MDM2 protein and NSCLC tissues, MDM2 is highly expressed as a proto-oncogene in cancer tissues. MDM2 is signi cantly up-regulated in lung adenocarcinoma tissues compared with adjacent tissues [28]. Not only limited to LC, after the inoculation of primary bone tumors, MDM2 expression in the inoculated site has also increased signi cantly compared with that in the control after prolonged time [29]. However, MDM2 expression is not signi cantly different between cancer and paracancerous tissues in patients with NSCLC according to Western blot analysis results [30]. In our research, although the increased expression of MDM2 was evidently related to advanced T stage, the level of MDM2 expression in NSCLC tissues was signi cantly lower than that in paired non-cancerous matched tissues. The Oncomine database analysis indicated the low expression of MDM2 in different pathological subgroups. The above results showed that the expression of MDM2 in NSCLC remains uncertain. In addition, the contingency effect caused by the small sample size was also an interference factor.
Some limitations in our analysis deserve discussion. First, only 20 NSCLC tumor and adjacent noncancerous tissues were collected in this experiment. Limited sample size weakened the proof of the abnormal expression of miR-221-3p in NSCLC. Second, given the small sample size, many false positives in the chi-square test and the Student t-test were found, as shown in Tables S3-6.

Conclusion
MiR-221-3p was up-regulated by PTX in NSCLC cells, and the low expression of miR-221-3p may be associated with PTX resistance. MiR-221-3p overexpression could regulate MDM2/P53 signaling pathway to reverse PTX resistance of NSCLC and induce apoptosis in vitro and vivo.

Declarations
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