The Role And Possible Mechanisms Of MiR-135a/b In The Cisplatin Resistance Of Lung Cancer Cells

Background and Objective: Drug resistance constitutes a major obstacle tosuccessful chemotherapy in cancer patients. In many cases, chemotherapies failbecause of drug resistance of cancer cells either intrinsic or acquired after an initialround of treatment. It is believed that most of clinical observed drug resistance ofcancer patients belongs to acquired resistance, which is caused by long-termanti-cancer drug usage. The genetic disorders mainly lie in intrinsic drug resistance ofcancer cells, while acquired drug resistance of cancer cells is closely related toepigenetic abnormality of genes. Epigenetics abnormality does not involve DNAsequence changes, which through DNA methylation, histone modifications and thenon-coding RNA regulation, playing an important role in modulating drug resistanceof cancer cells. Cisplatin (CDDP) is a commonly used chemotherapeutic agent fornon-small-cell lung cancer (NSCLC). However, treatment with this agent ischaracterized by resistance, both acquired and intrinsic. microRNAs (miRNAs) asimportant aspects of the epigenetic, participate in a variety of biological processesthrough post-transcriptional regulation mechanism. Recent studies have revealed thatmiRNA can modify tumor resistance by target of drug efflux pump molecules, cellcycle proteins, apoptosis regulatory proteins and other pathway. The aim of this studywas to investigate whether miR-135a/b could modulate the drug resistance of thehuman lung cancer cell line A549/CDDP to cisplatin, and explore the mechanism ofmiR-135a/b on the CDDP sensitivity of A549/CDDP cells.Methods:(1) The total RNA of A549and A549/CDDP cell lines was isolated andexamined.(2) miR-135a/b expression was measured by quantitative real-time PCR. (3) MTT assay was used to evaluate the sensitivity of NSCLC cells transfected withmiR-135a/b mimic or inhibitor.(4) Potential mRNA targets of miR-21were predictedby TargetScan, miRBase and MiRanda software. MCL13’-untranslated region-basedluciferase reporter plasmids was constructed to testify the target gene of miR-135a/b.(5) Total cell protein extracted after72hours of transfecation. The expression ofMCL1protein in NSCLC cells transfected with the negative control or miRNAsmimics was detected by western blot.(6) Flow cytometry was used to detectCDDP-induced apoptosis.Results:(1) Real-time quantification of miR-135a/b by stem-loop RT-PCR showedthat miR-135a/b were downregulated in A549/CDDP cell line compared with A549cell line and the decreased fold changes were1.66±0.06and5.56±0.09,respectively.(P<0.05)(2) In A549/CDDP cells, MTT assay revealed that thosetransfected with miR-135a/b mimics exhibited greatly decreased resistance to CDDPcompared with the miRNA mimic control transfected cells, while in A549cells, thosetransfected with miR-135a/b inhibitors exhibited greatly enhanced resistance toCDDP compared with the miRNA inhibitor control–transfected cells.(3) Theluciferase activity of MCL13’-untranslated region-based reporter constructed inA549/CDDP cells suggested that MCL1was the direct target gene of miR-135a/b.(4)Western Blot demonstrated that the decreased expression of miR-135a/b inA549/CDDP cells was concurrent with the overexpression of MCL1protein,compared with the parental A549cells. While the upregulation of miR-135a/b inA549/CDDP cells was concurrent with the downregulation of MCL1protein.(5)Enforced miR-135a/b expression sensitized A549/CDDP cells to CDDP-inducedapoptosis.Conclusions:(1) miR-135a/b were downregulated in A549/CDDP cells comparedwith A549cells.(2) The over-expressed miR-135a/b sensitized A549/CDDP cells to cisplatin resistance and CDDP-induced apoptosis.(3) miR-135a/b could play a role inthe development of CDDP resistance in lung cancer cell line at least in part bymodulation of apoptosis via targeting MCL1.