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Abstract
BackgroundMelanoma 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.MethodsIn 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.ResultsMiR-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.ConclusionOur findings suggest LNA-anti-miR-21 can be potentially used as an anticancer agent for the treatment of melanoma.
Highlights
Melanoma is a cancer that has a high mortality rate in the absence of targeted therapy
We aimed to evaluate the effects of miR-21 inhibition in the B16F10 melanoma cell line in vitro and C57BL/6 mice in vivo
After ensuring the efficiency of transfection, changes in miR-21 expression in untreated cells and cells transfected with LNA scrambled and LNA-anti-miR-21, using 2−ΔΔCT method by real-time reverse transcriptase (RT)-PCR miRNA
Summary
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. According to the American Cancer Society, approximately 100,350 new melanomas (about 60,190 men and 40,160 women) are predicted in the United States by 2020. The average age of patients with melanoma is 65 years, its incidence is still high among individuals under 30 years of age; it is recognized as one of the most prevalent cancers in the young, women [1]. Despite significant advances in treatment, malignant melanoma (MM) has a poor prognosis [2]. Drug resistance is usually related to the abnormal expression of apoptosis molecules, including FLIP, Bcl-2, BclXL, MCL-1, P53, Bax, and FADD [3]
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