MicroRNA-mRNA functional pairs for cisplatin resistance in ovarian cancer cells.
ABSTRACT: Ovarian cancer is the leading cause of death in women worldwide. Cisplatin is the core of first-line chemotherapy for patients with advanced ovarian cancer. Many patients eventually become resistant to cisplatin, diminishing its therapeutic effect. MicroRNAs (miRNAs) have critical functions in diverse biological processes. Using miRNA profiling and polymerase chain reaction validation, we identified a panel of differentially expressed miRNAs and their potential targets in cisplatin-resistant SKOV3/DDP ovarian cancer cells relative to cisplatin-sensitive SKOV3 parental cells. More specifically, our results revealed significant changes in the expression of 13 of 663 miRNAs analyzed, including 11 that were up-regulated and 2 that were down-regulated in SKOV3/DDP cells with or without cisplatin treatment compared with SKOV3 cells with or without cisplatin treatment. miRNA array and mRNA array data were further analyzed using Ingenuity Pathway Analysis software. Bioinformatics analysis suggests that the genes ANKRD17, SMC1A, SUMO1, GTF2H1, and TP73, which are involved in DNA damage signaling pathways, are potential targets of miRNAs in promoting cisplatin resistance. This study highlights candidate miRNA-mRNA interactions that may contribute to cisplatin resistance in ovarian cancer.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs, 8-23 nucleotides in length, which regulate gene expression at the post-transcriptional level. The present study was performed to analyze the association between microRNA-21 and cisplatin resistance in epithelial ovarian cancer (EOC) SKOV3 and SKOV3/DDP cells. In this experiment, the resistance of SKOV3 and SKOV3/DDP cells to cisplatin was evaluated using the MTT assay. Reverse transcription-quantitative polymerase chain reaction analysis was used to assess miRNA-21 levels and phosphatase and tensin homolog (PTEN) mRNA levels. Western blotting was used to assess PTEN protein levels. miRNA-21 mimics or inhibitors were transfected into SKOV3 and SKOV3/DDP cells. Prior to transfection, higher expression levels of miRNA-21 were observed in SKOV3/DDP cells compared with SKOV3 cells. Following transfection with miRNA-21 mimics, SKOV3 cells demonstrated increased sensitivity to cisplatin compared with negative control cells. Following transfection with the miRNA-21 inhibitor, SKOV3/DDP cells demonstrated decreased sensitivity to cisplatin compared with negative control cells. Furthermore, PTEN mRNA expression levels in SKOV3 cells transfected with miRNA-21 mimics was significantly lower compared with negative control cells. These results suggested that miRNA-21 may regulate cisplatin resistance by negatively targeting PTEN in EOC.
Project description:Cisplatin (DDP) resistance in patients suffering from ovarian cancer is a considerable hurdle to successful treatment. The present study aimed to identify a possible long non‑coding RNA (lncRNA)‑microRNA (miRNA)‑mRNA axis participating in ovarian cancer DDP‑resistance based on the critical roles of non‑coding RNAs, including lncRNAs and miRNAs, in carcinogenesis. According to online data and experimental results, lncRNA HAND2‑AS1 expression was significantly downregulated within ovarian carcinoma, especially within recurrent and DDP‑resistant ovarian carcinoma. The expression of HAND2‑AS1 was also shown to be markedly inhibited in SKOV3/DDP (DDP) cells with resistance to DDP. In SKOV3/DDP cells, HAND2‑AS1 overexpression inhibited cell viability and promoted cell apoptosis upon DDP treatment through the Bcl‑2/caspase‑3 apoptotic signaling. It was hypothesized that PTEN mRNA expression was also markedly inhibited in SKOV3/DDP ovarian cancer cells, while HAND2‑AS1 overexpression rescued PTEN proteins and blocked PI3K/AKT signaling activation. Moreover, miR‑106a was found to bind directly to PTEN 3' UTR and HAND2‑AS1. Upon DDP treatment, miR‑106a overexpression in SKOV3/DDP cells promoted cell viability. It inhibited cell apoptosis through the Bcl‑2/caspase‑3 apoptotic signaling pathway and downregulated the protein levels of PTEN and upregulated PI3K/AKT signaling activity. Furthermore, miR‑106a overexpression partially reversed the effect of HAND2‑AS1 overexpression upon PTEN proteins and SKOV3/DDP cell proliferation upon DDP treatment. In conclusion, a lncRNA HAND2‑AS1/miR‑106a/PTEN axis that re‑sensitizes DDP‑resistant SKOV3/DDP cells to DDP treatment has been established.
Project description:Drug resistance is an obstacle to the treatment of epithelial ovarian cancer. Recently, research has suggested that miRNAs (microRNAs) are involved in cancer development, and speculation has been made about their possible involvement in drug resistance. Thus, we attempted to identify selected miRNAs involved in the development of chemo-resistance in epithelial ovarian cancer. Using miRNA profiles of a panel of cisplatin-resistant (SKOV3/DDP) cells, we validated data using quantitative real time-PCR (QRT-PCR), and studied the effects of miR-429 on cancer cell chemo-sensitivity, using gain- and loss-of-function studies. Data show that SKOV3/DDP expressed less miR-429 compared with parental SKOV3 cells and lower miR-429 expression conferred shorter overall survival (OS) and less progression-free survival (PFS) than the patients with more miR-429 expression (P < 0.01). Upregulation of miR-429 increases cisplatin sensitivity in epithelial ovarian cancer cells. Studies have confirmed that the zinc finger E-box binding homeobox1 (ZEB1) is a direct and functional target of miR-429 and that over-expression of miR-429 reduces autophagy-related protein anti-ATG7, anti-LC3A/B (P < 0.05). Thus, overexpression of miR-429 may suppress ZEB1, and may be a potential sensitizer to cisplatin treatment that may have therapeutic implications.
Project description:<h4>Background</h4>Accumulating evidence has revealed that aberrant microRNA (miRNA) expression can affect the development of chemotherapy drug resistance by modulating the expression of relevant target proteins. Emerging evidence has demonstrated that miR-133a participates in the tumorigenesis of various cancers. However, whether miR-133a is associated with cisplatin resistance in ovarian cancer remains unclear.<h4>Objective</h4>To investigate the role of miR-133a in the development of cisplatin resistance in ovarian cancer.<h4>Methods</h4>MiR-133a expression in cisplatin-resistant ovarian cancer cell lines was assessed by reverse-transcription quantitative PCR (RT-qPCR). A cell counting kit-8 (CCK-8) assay was used to evaluate the viability of tumour cells treated with cisplatin in the presence or absence of miR-133a. A luciferase reporter assay was used to analyse the binding of miR-133a with the 3' untranslated region (3'UTR) of YES proto-oncogene 1 (YES1). The YES1 expression level was analysed using a dataset from the International Cancer Genome Consortium (ICGC) and assessed by RT-qPCR and western blotting in vitro. The roles and mechanisms of YES1 in cell functions were further probed via gain- and loss-of-function analysis.<h4>Results</h4>The expression of miR-133a was significantly decreased in cisplatin-resistant ovarian cancer cell lines (A2780-DDP and SKOV3-DDP), and the overexpression of the miR-133a mimic reduced cisplatin resistance in A2780-DDP and SKOV3-DDP cells. Treatment with the miR-133a inhibitor increased cisplatin sensitivity in normal A2780 and SKOV3 cells. MiR-133a binds the 3'UTR of YES1 and downregulates its expression. Bioinformatics analysis revealed that YES1 expression was upregulated in recurrent cisplatin-resistant ovarian cancer tissue, and in vitro experiments also verified its upregulation in cisplatin-resistant cell lines. Furthermore, we discovered that miR-133a downregulated the expression of YES1 and thus inhibited cell autophagy to reduce cisplatin resistance. Yes1 knockdown significantly suppressed the cisplatin resistance of ovarian cancer cells by inhibiting autophagy in vitro. Xenograft tumour implantation further demonstrated that Yes1 overexpression promoted ovarian tumour development and cisplatin resistance.<h4>Conclusions</h4>Our results suggest that the miR-133a/YES1 axis plays a critical role in cisplatin resistance in human ovarian cancer by regulating cell autophagy, which might serve as a promising therapeutic target for ovarian cancer chemotherapy treatment in the future.
Project description:Exosomes carrying microRNAs (miRNAs) have been demonstrated to play critical roles in the regulation of development, growth and metastasis of cancer. Bioinformatic predictions identified differentially expressed SRY-box 9 (SOX9) in OC, and the regulatory miRNA miR-139-5p. Here, we aim to evaluate the function of exosomal miR-139-5p in the sensitivity of ovarian cancer (OC) cells to cis-diamminedichloroplatinum(II) (DDP). Expression pattern of miR-139-5p and SOX9 in ovarian cancer cells (SKOV3) and DDP-resistant cells (SKOV3/DDP) was identified using reverse transcription quantitative polymerase chain reaction and western blot analysis. The relationship between miR-139-5p and SOX9 was validated using a dual-luciferase reporter assay. SKOV3/DDP cell line was developed and introduced with miR-30a-5p mimic to analyse the effects of miR-30a-5p on resistance to DDP. The <i>in vitro</i> and <i>in vivo</i> effects of exosomal miR-30a-5p on resistance of SKOV3 cells to DDP were assessed in a co-culture system of exosomes and OC cells as well as in tumour-bearing nude mice. High expression of SOX9 and low expression of miR-30-5p were witnessed in OC. Furthermore, miR-30-5p, a downregulated miRNA in SKOV3/DDP cells, increased the rate of cell apoptosis and enhanced the sensitivity of SKOV3/DDP cells to DDP by targeting SOX9. Moreover, exosomes carrying miR-30a-5p were identified to sensitize SKOV3/DDP cells to DDP both <i>in vitro</i> and <i>in vivo</i>. These data together supported an important conclusion that DDP-resistant OC cell-derived exosomal miR-30a-5p enhanced cellular sensitivity to DDP, highlighting a potential strategy to overcome drug resistance.
Project description:Cisplatin resistance is a challenge in the treatment of epithelial ovarian cancer. Here, clinical data showed that the level of netrin-G1 (NTNG1) in cisplatin-resistant cancer was higher than that in cisplatin-sensitive cancer (2.2-fold, <i>p</i> = 0.005); patients with a high NTNG1 level in cancer tissues had shorter progression-free survival (11.0 vs. 25.0 months, <i>p</i> = 0.010) and platinum-free interval (5.0 vs. 20.0 months, <i>p</i> = 0.021) compared with patients with a low level. Category- or stage-adjusted analyses demonstrated that the association between the NTNG1 level and prognosis occurred in type II or FIGO III/IV cancer. The basal level of NTNG1 in SKOV3/DDP cells (a cisplatin-resistant subline) was higher than that in SKOV3 cells; therefore, NTNG1 was overexpressed in SKOV3 cells, or silenced in SKOV3/DDP cells. Knocking in NTNG1 reduced the action of cisplatin to decrease cell death and apoptosis of SKOV3 cells, accompanied by upregulation of p-AXL, p-Akt and RAD51; however, opposite effects were observed in SKOV3/DDP cells after knocking down NTNG1. Co-immunoprecipitation demonstrated that NTNG1 bound GAS6/AXL. Silencing NTNG1 enhanced cisplatin effects <i>in vivo</i>, decreasing tumor volume/mass. These data suggested that a high NTNG1 level can result in cisplatin resistance in ovarian cancer cells via the GAS6/AXL/Akt pathway and that NTNG1 may be a useful target to overcome resistance.
Project description:Our previous study manifested that lanthanum chloride (LaCl<sub>3</sub>) can enhance the anticancer ability of cisplatin (DDP) in ovarian cancer cells. Here, ovarian cancer cells SKOV3 and SKOV3/DDP were subjected to DDP and LaCl<sub>3</sub>. Cell viability, apoptosis, DNA repair, and PI3K/Akt pathway were detected. LaCl<sub>3</sub> induced more cell death and apoptosis caused by DDP in two cell lines, accompanied by upregulation of Bax and Cleaved caspase 3 proteins, and downregulation of Bcl-2 protein. LaCl<sub>3</sub> also could decrease RAD51 protein by inactivation of the PI3K/Akt pathway. These data indicated that LaCl<sub>3</sub> could be a potential drug to modulate DDP resistance by inactivating of PI3K/Akt pathway and attenuating DNA repair in ovarian cancer.
Project description:Advanced and chemotherapy-resistant ovarian cancer causes high mortality of ovarian cancer, and it is important to find safe and effective drugs to reduce the chemotherapeutic resistance of ovarian cancer. In our study, we attempted to clarify the resistance mechanisms of SKOV3/DDP cells <i>in vitro</i> and evaluated the sensitization to triptolide (TPL) <i>in vivo</i>. Our results indicated that the overexpression of AKT and p-AKT greatly enhanced the cisplatin (DDP) tolerance of SKOV3/DDP, and the combination of DDP+TPL had a significant tumour inhibition effect compared to DDP treatment (p<0.05), via reducing the expressions of p-PI3K, p-Akt, Survivin, VEGF and MMP-2, and the increase of Caspase-3. Collectively, these results suggest that the synergistic anticancer effect of TPL and DDP warrants their potential clinical applications in further.
Project description:The aim of the study was to explore the possible mechanisms that Guizhi Fuling Wan (GFW) enhances the sensitivity of the SKOV3/DDP ovarian cancer cells and the resistant xenograft tumours to cisplatin. Rat medicated sera containing GFW were prepared by administering GFW to rats, and the primary bioactive constituents of the sera were gallic acid, paeonol, and paeoniflorin analysed by HPLC/QqQ MS. Cell counting kit-8 analysis was shown that coincubation of the sera with cisplatin/paclitaxel enhanced significantly the cytotoxic effect of cisplatin or paclitaxel in SKOV3/DDP cells. The presence of the rat medicated sera containing GFW resulted in an increase in rhodamine 123 accumulation by flow cytometric assays and a decrease in the protein levels of P-gp, phosphorylation of AKT at Ser473, and mTOR in a dose-dependent manner in SKOV3/DDP cells by western blot analysis, but the sera had no effect on the protein levels of PI3K p110? and total AKT. The low dose of GFW enhanced the anticancer efficacy of cisplatin and paclitaxel treatment in resistant SKOV3/DDP xenograft tumours. GFW could sensitize cisplatin-resistant SKOV3/DDP cells by inhibiting the protein level and function of P-gp, which may be medicated through inactivation of the PI3K/AKT/mTOR pathway.
Project description:microRNAs (miRNAs) have regulatory roles in various cellular processes, including apoptosis. Recently, X-linked inhibitor of apoptosis protein (XIAP) has been reported to be dysregulated in epithelial ovarian cancer (EOC). However, the mechanism underlying this dysregulation is largely unknown.Using bioinformatics and a literature analysis, a panel of miRNAs dysregulated in EOC was chosen for further experimental confirmation from hundreds of miRNAs that were predicted to interact with the XIAP 3'UTR. A dual-luciferase reporter assay was employed to detect the interaction by cellular co-transfection of an miRNA expression vector and a reporter vector with the XIAP 3'UTR fused to a Renilla luciferase reporter. DAPI and TUNEL approaches were used to further determine the effects of an miR-137 mimic and inhibitor on cisplatin-induced apoptosis in ovarian cancer cells.We identified eight miRNAs by screening a panel of dysregulated miRNAs that may target the XIAP 3'UTR. The strongest inhibitory miRNA, miR-137, suppressed the activity of a luciferase reporter gene fused with the XIAP 3'UTR and decreased the levels of XIAP protein in SKOV3 ovarian cancer cells. Furthermore, forced expression of miR-137 increased cisplatin-induced apoptosis, and the depressed expression of miR-137 decreased cisplatin-induced apoptosis in SKOV3 and primary EOC cells. Consistently, the disruption of miR-137 via CRISPR/Cas9 inhibited apoptosis and upregulated XIAP in A2780 cells. Furthermore, the effect of miR-137 on apoptosis could be rescued by XIAP in SKOV3 cells. In addition, miR-137 expression is inversely correlated with the level of XIAP protein in both ovarian cancer tissues and cell lines.Our data suggest that multiple miRNAs can regulate XIAP via its 3'UTR. miR-137 can sensitise ovarian cancer cells to cisplatin-induced apoptosis, providing new insight into overcoming drug resistance in ovarian cancer.