Exosome-mediated delivery of miR-30a sensitize cisplatin-resistant variant of oral squamous carcinoma cells via modulating Beclin1 and Bcl2

Abstract

Exosomes facilitate cross-talk amongst tumor cells, and thus also possess the potential to influence tumor-microenvironment and chemo-resistance. miRNAs, the important constituent of exosomes, are often dysregulated in cancer. They have been shown to play an essential role in tumor progression, metastasis, invasion, and resistance developed against different therapies. Acquisition of cisplatin-chemoresistance remains a major hurdle in the effective treatment of oral squamous cell carcinoma (OSCC). In this study, we demonstrate the importance of exosome-mediated miR-30a transfer in conferring cisplatin sensitivity in the otherwise resistant OSCC cells. Notably, miR-30a was found to be significantly reduced in exosomes isolated from the serum of OSCC patients, especially those having disease-recurrence, post cisplatin treatment. In conjunction with the findings in clinical samples, decreased miR-30a expression was observed in vitro in the cisplatin-resistant cultured OSCC cells compared to the cisplatin-sensitive cells. Besides, we identified Beclin1, an autophagy-related marker, as a target of miR-30a and found it to be overexpressed in cisplatin-resistant OSCC cells, thus indicating at its possible negative-regulation by miR30a. Exosomes from the cisplatin-resistant cells that have been transfected with miR-30a mimics, when delivered to the naïve cisplatin-resistant cells, caused not only the significant enhancements in miR-30a expression but also a concomitant decrease in Beclin1 and Bcl2 expression (autophagic and anti-apoptotic marker). More importantly, this together resulted in the sensitization of cisplatin-resistant cells. Thus, our study highlighted the role of exosomal-mediated miR-30a transfer in regaining sensitivity of the cisplatin-resistant OSCC cells via Beclin1 and Bcl2 regulation and hence suggests at its potential therapeutic role.

Keywords: autophagy; chemoresistance; exosomes; miRNA30a-5p; oral cancer.

Figure 1

(A) Binding position prediction of miR-30a with Beclin1 using TargetScan web-based tool. (B) Binding position prediction of miR-30a with Beclin1 using the DIANA microT-CDS tool. (C) Binding energy prediction of miR-30a with Beclin1 by RNAhybrid. (D) BECN1 luciferase activity in cis^Res cells co-transfected with either empty vector or pmirGLO-Becn1 vector having miR-30a target sequence and either NTC or miR30a mimics. Data are expressed as the mean +/– SD. ^*** P < 0.001, significant difference vs. control group (n = 3). Two independent experiments gave similar results.

Figure 2

(A) miR-30a expression in clinical samples was quantified by qRT-PCR and normalized to U6 as a housekeeping gene. miR-30a expression profiling in cis^Res and cis^Sens oral cancer cells at the (B) total cellular and (C) exosomal level. (D) Western blot analysis for Beclin1 expression in cis^Res and cis^Sens oral cancer cells. (E) Densitometry of Beclin 1 Western blot normalized to actin as the loading control. (F) Beclin 1 mRNA expression as analyzed by qRT-PCR. 18S was used as a housekeeping gene.

Figure 3. cis^Res oral cancer cells were transfected with either miR-30a mimics or NC (negative control) and after 24 h, the following effects were analyzed.

cis^Sens cells were employed for the comparison. (A) miR-30a expression as measured by qRT-PCR. (B) Beclin1 protein expression as determined by the Western blot analysis. (C) Densitometry analysis of Beclin1 Western blot was carried out by normalization to β-actin employed as the loading control. Following miR30a restoration, cells were treated with different concentrations of cisplatin. Cell viability was measured by MTT assay after (D) 24 h and (E) 48 h. Error bars represent the mean ± SD of three independent experiments. ^* p < 0.05, ^*** p < 0.001.

Figure 4. cis^Res oral cancer cells were transfected with either miR-30a mimics or NC (negative control) and after 24 h, the following effects were analyzed.

cis^Sens cells were employed for the comparison. (A) Bcl2 protein expression by Western blot. (B) Densitometry analysis of Bcl2 Western blot normalized to β-actin as the loading control. (C) Mimics or NC transfected cells were treated with or without cisplatin and caspase-3 expression was analyzed by Western blot analysis. (D) Cells were treated as described in (C) and after 72 h of cisplatin treatment, cells were collected and stained with FITC-conjugated annexin-V and PI and analyzed by flow cytometry. (E) Graphical representation of the percentage of apoptotic cells as analyzed by FACS analysis. Error bars represent the mean ± SD of three independent experiments. ^* p < 0.05, ^*** p < 0.001.

Figure 5. The effects of cisplatin treatment were analyzed on the migration of cis^Res oral cancer cells transfected with either miR-30a mimics or NC (non-target control) by wound assay.

cis^Sens cells were employed for the comparison. (A–D) Representative images of wound closure taken at 0 and 24 h after the scratch was made and cisplatin treatment was given. (E) quantification of wound closure as analyzed using Image J. Data are expressed as mean ± SD. ^*** p < 0.001.

Figure 6. Exosomes were isolated from cis^Res cells transfected with either NC or miR-30a mimics and were used to treat other naïve cis^Res cells.

cis^Sens, cis^Res, and cis^{Res-miR-30a mimic} cells were employed as controls and for comparison. (A) The expression of CD9 in isolated exosomes from different cells was determined by Western blotting. (B) Beclin1 protein expression as determined by western bot. (C) Densitometry analysis of Beclin1 Western blot normalized to β-actin as a loading control. (D) Following exosomes treatment, cells were exposed to different concentrations of cisplatin (μM), as indicated) for 24 h. Cell viability was measured by the MTT assay. (E) For determining apoptosis, after cisplatin treatment, cells were harvested, stained with FITC-conjugated annexin-V and PI and analyzed by flow cytometry. (F) Graphical representation of the percentage of apoptotic cells as analyzed by FACS analysis. (G) BCl2 protein expression as determined by Western blot. (H) Densitometry analysis of BCl2 Western blot normalized to β-actin as a loading control. (I) miR-30a expression levels as determined by real-time PCR. Error bars represent the mean ± SD of three independent experiments. ^*** p < 0.001.

Figure 7. Diagram illustrating the role of exosome-mediated miR30a delivery in a reversal of cisplatin resistance in OSCC cells.