A high-throughput screen identifies miRNA inhibitors regulating lung cancer cell survival and response to paclitaxel

Abstract

microRNAs (miRNAs) are small RNAs endogenously expressed in multiple organisms that regulate gene expression largely by decreasing levels of target messenger RNAs (mRNAs). Over the past few years, numerous studies have demonstrated critical roles for miRNAs in the pathogenesis of many cancers, including lung cancer. Cellular miRNA levels can be easily manipulated, showing the promise of developing miRNA-targeted oligos as next-generation therapeutic agents. In a comprehensive effort to identify novel miRNA-based therapeutic agents for lung cancer treatment, we combined a high-throughput screening platform with a library of chemically synthesized miRNA inhibitors to systematically identify miRNA inhibitors that reduce lung cancer cell survival and those that sensitize cells to paclitaxel. By screening three lung cancer cell lines with different genetic backgrounds, we identified miRNA inhibitors that potentially have a universal cytotoxic effect on lung cancer cells and miRNA inhibitors that sensitize cells to paclitaxel treatment, suggesting the potential of developing these miRNA inhibitors as therapeutic agents for lung cancer. We then focused on characterizing the inhibitors of three miRNAs (miR-133a/b, miR-361-3p, and miR-346) that have the most potent effect on cell survival. We demonstrated that two of the miRNA inhibitors (miR-133a/b and miR-361-3p) decrease cell survival by activating caspase-3/7-dependent apoptotic pathways and inducing cell cycle arrest in S phase. Future studies are certainly needed to define the mechanisms by which the identified miRNA inhibitors regulate cell survival and drug response, and to explore the potential of translating the current findings into clinical applications.

MiR‐145, miR‐133a and miR‐133b inhibit proliferation, migration, invasion and cell cycle progression via targeting transcription factor Sp1 in gastric cancer

“…Reportedly, miR‐133a and miR‐133b could inhibit cell proliferation, migration and invasion in bladder cancer [27] and prostate cancer [28] by targeting the epidermal growth factor receptor (EGFR). A high‐throughput screen identified that miR‐133a and miR‐133b could decrease lung cancer cell survival by activating caspase‐3/7‐dependent apoptotic pathways and inducing cell cycle arrest in S phase [29]. Suppressive role of miR‐133a could also be found in esophageal squamous cell carcinoma [30], ovarian cancer [31], colorectal cancer [32] and so on.…”

miR-137 inhibits the proliferation of human non-small cell lung cancer cells by targeting SRC3

“…The mutated putative miR-137 binding site in the SRC3 3'-UTR was generated using a Site-Directed Gene Mutagenesis kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. NSCLC cells were seeded at a density of ~1x10 5 cells/well into 24-well plates and cultured for 24 h prior to transfection. Co-transfections with 500 ng pGL3-Basic firefly luciferase reporter and 100 nM miR-137 mimic or scramble mimic into cells using Lipofectamine 2000 for 4 h at 37˚C was performed.…”

“…Changes to the expression of microRNAs (miRNAs/miRs) in tumors are a focus being studied at present and it has been demonstrated that the dysregulation of miRNAs is involved in the malignancy of lung cancer (4,5). miRNAs are endogenous non-coding 19-23 nucleotide long RNA molecules that are located on chromosome 1p22.…”

RNAi screening comes of age: improved techniques and complementary approaches

“…Several such libraries have been developed and commercialized, enabling functional high-throughput, unbiased screens to be performed. This format has facilitated the identification of miRNAs that contribute to a variety of diseases and physiological responses, including viral infection 95 , breast cancer 96,97 and drug treatment responses 98,99 . A study involving both miRNA mimics and siRNA screening using the same assay, identified miRNAs involved in cisplatin resistance, as well as the kinases targeted by these miRNAs 100 .…”