Up-regulation of miR-146a increases the sensitivity of non-small cell lung cancer to DDP by downregulating cyclin J

Cisplatin is the most widely used chemotherapy drugs for the treatment of lung cancer and other tumors [37]. Approximately 1,590,000 lung cancer patients succumb to the disease every year, 61% of which are primary drug resistance and 33% have an acquired drug resistance. Thus, an intense research underlying chemoresistance should be conducted to further establish better therapeutic approaches.

Currently, more and more studies focus on the research on oncogenes [38], epigenetic dysregulation [39] and abnormal expression of key genes (especially miRNAs) [10, 40] in drug resistance. Moreover, a series of miRNAs have been proposed as DDP resistance-associated genes according to miRNA microarray or RT-qPCR array profiling between A549 and A549/DDP cells [19, 41].

Most of studies have reported that miR-146a acts as an oncogene involved in tumor genesis and development [42], but other studies have demonstrated miR-146a functions as a tumor suppressor [43, 44]. Wu, C. et al. found that serum levels of miR-146a were potential biomarkers for the prediction of survival and response to chemotherapy in NSCLC [45]. Recently, direct targeting of EGFR by miR-146a was reported in castration-prostate cancer and HCC cells, leading to significant inhibition cell growth, colony formation, and migration in vitro [46, 47]. These evidences suggest that miR-146a plays an important role in the development and progression of cancer.

To our best knowledge, the role of miR-146a expression in DDP-resistent NSCLC has not been well documented. Interestingly, we found that miR-146a was down-regulated approximately 2-fold in A549/DDP cells, which attracts us to deeply explore the role of miR-146a in DDP-resistant NSCLC cells in vitro and vivo. Functional analysis indicated that miR-146a overexpression could sensitize NSCLC/DDP cells to DDP both in vitro and vivo by inducing G0/G1 phase arrest, inhibiting cell motility, and enhancing cell apoptosis. Therefore, we proposed that miR-146a might act as a chemosensitivity restorer to DDP in human NSCLC cells.

Further silico analysis showed that CCNJ was the target gene of miR-146a. The relevance between miR-146a and CCNJ was subsequently validated by luciferase reporter gene assay. As we know, CCNJ is a member of cyclin family protein that controls cell mitosis involved in oncogenesis and embryogenesis by forming CDK2/CCNJ complexes [48, 49]. Venturutti, L. et al. further suggest that the inhibition of CCNJ could repair the proliferation of breast carcinoma (BC) and gastric carcinoma (GC) cells in vitro and promote chemosensitive to trastuzumab and lapatinib in preclinical BC model [50]. A previous study also indicated that CCNJ could be a novel prognostic marker of HCC and acute leukemia (ALM) [51, 52]. To further investigate the effect of CCNJ in DDP-resistent NSCLC, both A549/DDP and SPC-A1/DPP cells were transfected with siCCNJ and then treated with DDP. As expected, we found that knockdown of CCNJ increased cell sensitivity to DDP by inducing cell cycle arrest and cell apoptosis. Notably, we found downregulation of CCNJ could enhance the gains of the sensitivity to DDP in miR-146a-overexpressing A549/DDP and SPC-A1/DDP cells.

There are several cell signaling molecules and pathways involved in drug resistance, including the ABC transporter subfamily B member 1 (ABCB1/MDR1/P-gp) [53, 54], ABC transporter subfamily C member 1 (ABCC1/MRP-1) [54], and lung resistance-related protein (LRP) [55]. Consistant with these reports, our results further demonstrated that miR-146a could downregulate drug-resistance-associated proteins (P-gp, MRP1 and LRP) and upregulate the expression of cleaved caspase-3 in vitro and vivo. However, we found P53, as a tumor suppressor, presented slightly downregulated in NSCLC/DDP cells after treated with miR-146a or siCCNJ, but no obvious change in tumor formed from A549/DDP cells stably transfected with miR-146a. These might be ascribed to cell growth status and different types of cells. Collectively, our results revealed that miR-146a overexpression could obviously increase the chemosensitivity of NSCLC/DDP cells to DDP by downregulating drug-resistance-associated proteins.