Therapeutic inhibition of microRNA-21 (miR-21) using locked-nucleic acid (LNA)-anti-miR and its effects on the biological behaviors of melanoma cancer cells in preclinical studies

Abstract

Background: Melanoma is a cancer that has a high mortality rate in the absence of targeted therapy. Conventional therapies such as surgery, chemotherapy, and radiotherapy are associated with poor prognosis. The expression of miR-21 appears to be of clinical importance, and the regulation of its expression appears to be an opportunity for treatment.

Methods: In this current study, we aimed to evaluate the effects of miR-21 inhibition in- vitro and in-vivo. In-vitro studies have investigated LNA-anti-miR-21 in mouse melanoma cells (B16F10), and in-vivo studies have proposed a model of melanoma in male C57BL/6 mice. To evaluate the anticancer effects of LNA-anti-miR-21, a QRT-PCR analysis was performed using the 2^-ΔΔCT method to determine the degree of inhibition of oncomiR-21. The MTT test, propidium iodide/AnnexinV in-vitro, and tumor volume measurement using the QRT-PCR test with the 2^-ΔΔCT method were used to estimate the inhibition of miR-21 and the expression of downstream genes including: SNAI1, Nestin (Nes), Oct-4, and NF-kB following miR-21 inhibition. Finally, immunohistochemistry was conducted for an in-vivo animal study.

Results: MiR-21 expression was inhibited by 80% after 24 h of B16F10 cell line transfection with LNA-anti-miR-21. The MTT test showed a significant reduction in the number of transfected cells with LNA-anti-miR-21. The transfected cells showed a significant increase in apoptosis in comparison with the control and scrambled LNA groups. According to our in vivo findings, anti-miR-21 could reduce tumor growth and volume in mice receiving intraperitoneal anti-miR after 9 days. The expression of the SNAI1gene was significantly reduced compared to the controls. Immunohistochemical analysis showed no change in CD133 and NF-kB markers.

Conclusion: Our findings suggest LNA-anti-miR-21 can be potentially used as an anticancer agent for the treatment of melanoma.

Keywords: Cutaneous melanoma; LNA-anti-miR-21; miR-21; miRNAs.

Fig. 1

Evaluation of transfection efficiency in B16F10 cells using a FAM-labeled scrambled LNA: Control compared with scrambled LNA transfected cells, a Fluorescence microscopy detected green fluorescent cells. b Flow cytometry showed more than 80% transfected cells

Fig. 2

Evaluation of the expression of miR-21, 24 h after transfection using 2^−ΔΔCt method by real-time PCR. The control group was used as the reference for comparison with other groups

Fig. 3

LNA-anti-miR-21 caused a significant reduction in B16F10 cell viability compared to the scrambled and control groups (**P < 0.01)

Fig. 4

LNA-anti-miR-21 has a significant effect on apoptosis percentage in comparison with untreated and scrambled LNA cells (**P < 0.01). The Figure indicates the apoptosis profile following annexin V/PI staining; LNA-anti-miR-21 increased apoptosis. The histogram indicates the percentage of apoptotic cells

Fig. 5

Expression levels of miR-21 in both IP and IT performed 24 h following transfection, using 2^−ΔΔCt method by real-time PCR. The untreated group was used as the control for comparison with other groups

Fig. 6

Expression levels of Snai1, Nes, NF-kB, and NF-kB mRNA after 19 days of LNA-anti-miR-21 injection by 2^−ΔΔCt method using real-time PCR. The expression level of SNAI1 in cells treated with LNA-anti-miR-21 decreased significantly

Fig. 7

Effect of LNA-anti-miR-21 on tumor volume compared with the control group. The highest longitudinal and transverse diameters were determined to measure tumor volume (**P < 0.01 versus the controls). It seems that IP and IT LNA-anti-miR-21 injection has a greater effect on inhibiting tumor size

Fig. 8

a Immunohistochemical evaluation of CD133 and NF-kB. Images are tumor staining fields for each treatment. The low-power scale bar is 2 μm, and the high-power scale bar is 50 μm. b Quantification of melanoma tumors data compared to control (untreated cells). The levels of CD133 and NF-kB proteins reduced, though not significantly compared to the control cells *P < 0.05 and **P < 0.01