Let-7a-3p overexpression increases chemosensitivity to carmustine and synergistically promotes autophagy and suppresses cell survival in U87MG glioblastoma cancer cells

Abstract

In terms of primary brain tumors, glioblastoma is one of the most aggressive and common brain tumors. The high resistance of glioblastoma to chemotherapy has made it vital to find alternative treatments and biological mechanisms to reduce the survival of cancer cells. Given that, the objective of the present research was to explore the potential of let-7a-3p when used in combination with carmustine in human glioblastoma cancer cells. Based on previous studies, the expression of let-7a is downregulated in the U87MG cell line. Let-7a-3p transfected into U87MG glioblastoma cells. Cell viability of the cells was assessed by MTT assay. The apoptotic induction in U87MG cancerous cells was determined through the utilization of DAPI and Annexin V/PI staining techniques. Moreover, the induction of autophagy and cell cycle arrest was evaluated by flow cytometry. Furthermore, cell migration was evaluated by the wound healing assay while colony formation assay was conducted to evaluate colony formation. Also, the expression of the relevant genes was evaluated using qRT-PCR. Transfection of let-7a-3p mimic in U87MG cells increased the expression of the miRNA and also increased the sensitivity of U87MG cells to carmustine. Let-7a-3p and carmustine induced sub-G1 and S phase cell cycle arrest, respectively. Combination treatment of let-7a-3p and carmustine synergistically increased arrested cells and induced apoptosis through regulating involved genes including P53, caspase-3, Bcl-2, and Bax. Combined treatment with let-7a-3p and carmustine also induced autophagy and increased the expression of the ATG5 and Beclin 1 (ATG6). Furthermore, let-7a-3p combined with carmustine inhibited cell migration via decreasing the expression of MMP-2. Moreover, the combination therapy decreased the ability of U87MG to form colonies through downregulating CD-44. In conclusion, our work suggests that combining let-7a-3p replacement therapy with carmustine treatment could be considered a promising strategy in treatment and can increase efficiency of glioblastoma chemotherapy.

Graphical Abstract

Summary of conducted assays in this research. After selection of let-7a-3p, this miRNA transfected into U87MG cells via electroporation. Optimum dose and time of the miRNA were evaluated by the qRT-PCR. In order to find IC50 of the carmustine, U87MG cells were treated with different doses of this chemotherapy drug and MTT assay conducted. Then cells were transfected with optimum doses of let-7a-3p and treated with IC50 of carmustine. In order to assess the combined effect of these two methods, apoptosis induction, cell cycle arrest, autophagy induction, cell migration, colony formation, and expression of the involved genes were evaluated.