MiR-340 Reverses Cisplatin Resistance Of Hepatocellular Carcinoma Cell Lines By Targeting Nrf2-ARE Antioxidant Pathway

In recent years, chemotherapy outcome is usually poor because of drug resistance in cancer patients. It is an urgent concern for oncologist to investigate the mechanism and find a way to deal with chemoresistance. Failed chemotherapies are usually due to either intrinsic or acquired drug resistance in cancer patients and acquired resistance was observed in most cases. The intrinsic drug resistance is closely related to genetic disorders, while the epigenetic abnormality of genes mainly lies in acquired resistance. DNA methylation, histone modifications and the non-coding RNA modulation are involved in epigenetics abnormality, playing a crucial role in regulating drug resistance of cancer cells.The microRNAs (miRNAs) are a growing class of small endogenous non-coding RNA molecules, which post-transcriptionally modulate genes expression and acted as a critical regulator in diverse cellular processes, both physiologic and pathologic, including apoptosis, proliferation, development and differentiation. Previous studies have suggested that microRNAs could be served as a novel biomarker for diagnosis and prognosis in cancer patients. More importantly, recent works have underlined that changes in miRNAs levels might be involved in the evolution of drug resistance. Many studies have established that chemotherapy resistance could be reversed by knock-down or re-expression of specific miRNAs using precusors or mimics anti-sense oligonucleotides of miRNAs.Recently, there are few works on miRNAs related to chemotherapeutic drug resistance in hepatoma cells, Studies showed that miR-221 and miR-122 was overexpressed in drug resistant hepatoma cell lines, repression of these above miRNAs could restored the expression of PTEN and TIMP3, inhibited the AKT signaling pathway and sensitized hepatoma cells to chemotherapeutic drug-induced apoptosisFor further analysis of the possible role of miRNAs in the evolution of drug resistance in hepatoma cell line HepG2/CDDP, We conducted the following experiments:Objects and Methods1. High throughput miRNAs microarrays combined with qRT-PCR are used to detect and verify the difference of miRNA expression profiles between drug resistant hepatoma sublines, HepG2/CDDP and their parental cell line HepG2, computation softwares were used to explore the predieted accessible miRNAs which could bind to 3’UTR of Nrf2 mRNA. miR-340 was finally selected as our objective miRNA.2. To exogenously increase or decrease the expression of miR-340 in HepG2/CDDP or HepG2 by transient transfection of the corresponding miRNA mimics or inhibitors, respectively, validate the effect of the transfection on drug sensitivity for the resistant or parental hepatoma sublines through CCK8 assay, IC50 assay, and the apoptosis rate assay by cytoflowmetry.3. The relationship of miR-340 and Nrf2 was verified by dual-luciferase activity assay. the mechanism of miR-340 regulating drug resistance was elucidated by detection of Nrf2-related signaling using qRT-PCR and western blot analysisResults1.17 differentially expressed miRNA between HepG2/CDDP and HepG2 respectively were obtained through high throughput miRNA microarrays. Among them, there were 9 miRNAs significantly upregulated (change fold >=2.0) and 8 miRNAs downregulated (change fold <0.5 fold) in HepG2/CDDP sublines compared with HepG2, respectively. miR-340 was validated to be downregulated in drug resistant HepG2/CDDP cells by qRT-PCR, and it was found to be the most lowly downregulated miRNA (change fold>=0.07, P<0.01) in HepG2/CDDP sublines compared with HepG2 among the 8 downregulated miRNAs.2. HepG2/CDDP cells transfected with miR-340 mimics showed significantly increased apoptotic rate and decreased IC50 than that in the Nc group, wihle HepG2 cells transfected with miR-340 inhibitors showed significantly decreased apoptotic rate and increased IC50than that in the Nc group.3. It was validated by dual-luciferase activity assay that Nrf2 was the direct target genes of miR-340, Enforced miR-340 expression reduced Nrf2 level and Nrf2-relate gene expression.ConclusionsBased upon high-throughput screening of miRNA microarrays, we found a group of hepatoma CDDP resistance related miRNAs for the first time. Further, miR-340 may be the important molecule playing crucial role in the evolution of hepatoma CDDP resistance, miR-340 were significantly downregulated whereas Nrf2 was upregulated in HepG2/CDDP (cisplatin) cells, compared with parental HepG2 cells, miR-340 mimics could suppress Nrf2-dependent antioxidant pathway and enhancing the sensitivity of HepG2/CDDP cells to cisplatin. Collectively, the results first suggested that lower expression of miR-340 is involved in the development of CDDP resistance in hepatocellular carcinoma cell line, at least partly due to regulating Nrf2-dependent antioxidant pathway.