Oridonin alters the expression profiles of MicroRNAs in BxPC-3 human pancreatic cancer cells

Background Oridonin, an ingredient used in traditional Chinese medicine, has been demonstrated to play an important role in antitumour effects, but the mechanism underlying its antitumour properties is still not clear. Methods To verify the anti-cancer effects of oridonin via a miRNA-dependent mechanism, comprehensive miRNA expression profiling of oridonin-treated BxPC-3 human pancreatic cancer cells was performed using a miRNA microarray assay based on Sanger miR-Base Release 20, followed by a validation using real-time PCR. MicroRNA target prediction and Gene Ontology and KEGG pathway analysis were performed to investigate possible pathways involved. Results The results showed that 105 miRNAs were significantly differentially expressed (signal reading >500, p ≤ 0.01, |Log2-value| ≥1) in oridonin-treated BxPC-3 human pancreatic cancer cells. Conclusions Our data indicates that oridonin inhibits BxPC-3 cells probably through regulating the expression of miRNAs. Interruption of miRNA profiling may provide new therapeutic methods for the clinical treatment of pancreatic cancer.


Background
Oridonin, a natural ent-kaurane diterpenoid compound, is isolated from the Chinese medicinal herb Rabdosia rubescens as well as other plants, such as Isodon trichocarpus and Isodon shikokianus. Oridonin has many physiological and pharmacological effects, including anti-inflammation, anti-bacterial and anti-tumour effects, and shows no obvious side effects when used for the treatment of various human diseases. Concerning anti-tumour effects, previous studies have reported that oridonin can induce cell growth inhibition, promote apoptosis and inhibit migration and invasion in many cancers [1][2][3]. Nevertheless, the mechanisms underlying the antitumour activity of oridonin have not been completely delineated.
MicroRNAs (miRNAs) are a novel class of non-coding RNAs with lengths of 17-25 nucleotides (nt) that can regulate gene expression in eukaryotic organisms by pairing with target mRNAs to repress translation or cause degradation of multiple target mRNAs [4]. Recent studies have shown that miRNAs play crucial roles in many biological processes, such as development, cell growth, differentiation, apodosis and even tumouriogenesis [5,6]. Furthermore, miRNAs can function both as tumour suppressors and oncogenes and might be a potential therapeutic target in cancer. Recent publications have shown that correcting abnormal miRNAs in tumours can inhibit the function of the target mRNA in vivo in a mouse model [7,8].
Traditional Chinese medicines have become a popular topic in relation to their potential anti-tumour properties. However, there are no available reports on oridonin which inhibits pancreatic cancer via miRNA regulation. In this study, we establish a sensitive microarray chip for miRNA expression profiling in BxPC-3 pancreatic cancer cells treated with oridonin to verify our hypothesis that oridonin alters the miRNA expression profile in pancreatic cancer, and we show that miRNAs have potential applications in the future clinical treatment of tumours.

Cell culture
The BxPC-3 human pancreatic cancer cell line was provided by the Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (ATCC® CRL1687™). The cells were cultured in RPMI 1640 (GIBCO, NY, United States) culture medium containing 10% foetal bovine serum (FBS, Gibco), 300 mg/L glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin in an incubator with 5% CO 2 at 37°C. Cells in logarithmic growth phase were seeded in 60 mm dishes at a density of 4 × 10 4 cell/cm 2 and incubated overnight. One group of these cells was subsequently treated with 87.8 μM oridonin (Gracia Chemical Technology Company, LTD, 98% purity, HPLC) dissolved in DMSO (final DMSO concentration in growth media is 0.1%), and another was used as a blank control group cultured in medium containing 0.1% DMSO for 24 hours. At least 3 independent experiments were performed.

RNA isolation and miRNA microarray
After 24 hours of treatment, total RNA (containing small RNAs) was extracted using the TRIzol LS reagent (Invitrogen Life Technologies) following the manufacturer's protocol. The microarray assay (μParafloTM MicroRNA Microarray Assay) was performed by a service provider (LC Sciences), including quality control, labelling, chip hybridisation, signal amplification image acquisition and microarray data analysis. Hybridisation was performed overnight on a μParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies) [9]. On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://www.mirbase.org/) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection

MiRNA expression was altered in BxPC-3 cells treated with oridonin
To study the responses of miRNAs to oridonin, microarray analysis of miRNA expression in BxPC-3 cells treated with oridonin was compared with the expression of miRNAs in DMSO treated cells. Only miRNAs showing significant expression among the oridonin treatments and their controls are reported ( Table 1). As shown in Table 1, 105 reporters presented a strong response (signal reading >500, p ≤ 0.01, |Log2-value b | ≥1) and significant regulation. Among these 105 miRNAs, 49 miRNAs were significantly down-regulated, whereas 56 were significantly up-regulated by oridonin. Among them, there are many new miRNAs whose function has been scarcely described in the literature. Previous studies related to miRNA expression in human pancreatic cancer are collected and summarised in Table 2 for comparison and discussion. Results showed that the expression of some miRNAs was changed dramatically after treatment with oridonin, as shown in Table 3 (20 miRNAs, including miR-205, miR-10b, miR-125b, miR-200b, miR-132, miR-320, miR-185, miR-424-5p, and miR-17-5p), which indicated that oridonin may influence BxPC-3 pancreatic cancer cells through regulating miRNAs, though verifying this hypothesis will require further investigation.

Target prediction and GO and KEGG pathway analyses
It has been demonstrated that one miRNA could target more than one gene, whereas some genes were targets of more than one miRNA. To predict the target mRNAs of the differentially expressed miRNAs, we performed target prediction for the differentially expressed miRNAs identified in the BxPC-3 cells using three different types of online software: TargetScan, PicTar and miRanda. The intersection of three software's predictions was taken as the finally potential target genes.
GO and KEGG pathway analyses were performed on the target genes of the significantly differentially expressed miRNAs. Figure 1 Distribution of GO categories for the predicted target genes of differentially expressed miRNAs identified in BxPC-3 cells treated with oridonin. The left vertical axis represents the percent of genes, the right vertical axis represents the number of genes and the horizontal axis represents the GO category, including biological processes, cellular components and molecular functions. Figure 1. The results revealed that the significantly enriched predicted target genes were involved mainly in the following categories: biological processes (e.g., signal transduction, regulation of transcription, DNA-dependent and multicellular organismal development), cellular components (e.g., cytoplasm, nucleus, membrane, integral to membrane and plasma membrane) and molecular functions (protein binding, metal ion binding and zinc ion binding).

The enriched GO annotations are shown in
The KEGG pathway annotations of all of the target genes of the significantly differentially expressed miR-NAs are shown in Table 4 (P ≥ 0.05). KEGG is a major public database of biological pathways, and significant enrichment in KEGG categories can identify differentially expressed genes involved in the main biochemical metabolic pathways and signal transduction pathways. The results presented in Table 4 revealed that the influence of BxPC-3 pancreatic cancer cells by oridonin may  (Figure 2), which correlated well with the microarray results in Table 1.

Discussion
With the discovery of miRNAs, it has been shown that miRNAs can function as endogenous posttranscriptional gene regulators through binding to the 3′ untranslated region of target mRNAs, and emerging evidence suggests that miRNAs play an important role in regulating diverse biological processes. Abnormal expression of miRNAs is associated with many diseases, such as nervous system diseases, cardiovascular disease and cancer. Several studies have demonstrated that aberrant miRNA expression is involved in pancreatic cancer (Table 2). Pancreatic cancer is one of the most lethal malignancies, characterised by its highly metastatic potential, worst prognosis and strong resistance to chemotherapy and radiation therapy. The overall 5-year survival rate of pancreatic cancer is less than 5%. Chemotherapy and radiation therapy are the main therapeutic methods used to treat such cancers, however, these treatments produce deleterious side effects. Therefore, there is an urgent need to find safer treatments. Recently, traditional Chinese medicines have become a "hot spot" in relation to their potential anti-tumour properties, although the mechanisms of such anti-tumour effects are not clear. Some studies showed that the anti-cancer mechanisms of the active ingredients of traditional Chinese medicines may be associated with miRNAs, which can be treated as targets for cancer therapies [32][33][34]. Previous studies revealed that oridonin can cause cell cycle arrest, induce apoptosis and enhance the antitumour activity of gemcitabine in pancreatic cancer [35][36][37]. In this study, the miRNA expression was profiled in BxPC-3 human pancreatic cancer cells treated with oridonin. MicroRNA results showed that 105 miRNAs were significantly altered by oridonin treatment (Table 1). Among them, many have been reported to be associated with tumorigenesis or cancer progression. For instance, miR-424-5p (Table 3) is overexpressed in human pancreatic cancer. Down-regulation of miR-424-5p inhibits cell proliferation, migration and invasion and increases cell apoptosis in PANC-1 cells [22]. In addition, miR-17-5p, which is related to a poor prognosis, is overexpressed in pancreatic cancer [11]. Both miR-424-5p and miR-17-5p were found to be down-regulated by oridonin in our microarray data, implying that oridonin may inhibit pancreatic cancer cell proliferation, migration, invasion, and induce apoptosis by down-regulating miR-424-5p and miR-17-5p.
Four miRNAs (miR-409-3p, miR-103a-3p, miR-200b-3p and miR-107) were chosen to validate the microarray assay via quantitative real-time PCR. PCR results showed a well correlation with the microarray results, confirming the significant difference between oridonin treated and untreated cells. It has been reported that epigenetic silencing of miR-107 can regulate the expression of cyclin-dependent kinase 6 in pancreatic cancer [15]; while interfering miR-409-3p promotes tumour growth, the epithelial-to-mesenchymal transition (EMT) and bone metastasis [38]. miR-409-3p also suppresses the migration and invasion of bladder cancer T24 and 5,637 cells via targeting c-Met [39] and regulates cell proliferation and apoptosis by targeting PHF10 in SGC-7901 gastric cancer cells [40]. However, the effect of this miRNA on pancreatic cancer has rarely been described, similar to the situation for miR-103a-3p and miR-200b-3p. Based on the literature and our analysis on miRNA expression in cancer cells, we presume that these miRNAs likely play similar roles in pancreatic cancer, such as inhibiting cell proliferation, migration, invasion and inducing apoptosis. Thus, interruption of miRNA expression may be potential therapeutic targets for pancreatic cancer, although further studies are required to explore this possibility. Figure 2 qPCR validation of a subset of miRNA microarray data. The horizontal axis represents the miRNAs, and the vertical axis represents the expression of miRNAs. The black bar represents the control group, and the grey bar represents the oridonin group. The data are expressed as the mean ± standard deviation (SD). **Significantly different from the control (p < 0.01); *different from the control (p < 0.05).
For further investigation, Gene Ontology analysis and KEGG pathway annotation were applied. GO enrichment analysis showed that the mRNA clusters were significantly enriched for the categories that are essential for cell survival. A total of 31 enrichment pathways for predicted target genes were listed in Table 4. Among them, the top 5 signaling pathways were neuroactive ligand-receptor interactions, Pathways in cancer, MAPK, focal adhesion and calcium signalling pathways. The results showed that 176 predicted target genes are associated with neuroactive ligand-receptor interactions, 138 genes are associated with MAPK signaling pathways, while 118 genes are associated with calcium signalling pathways. Data from previous research suggest that oridonin can enhance the antitumour activity of gemcitabine in pancreatic cancer through the MAPK-p38 signalling pathway [36] and inhibit BxPC-3 cell growth through caspase signaling pathways [41], which verified the results of KEGG pathway annotation. In conclusion, The KEGG pathway annotation revealed that BxPC-3 pancreatic cancer cells may be influenced by oridonin through these pathways and provided new research directions.

Conclusion
In conclusion, the results of the present study provide new insights into the general mechanisms underlying the suppression of BxPC-3 cells by oridonin treatment and may provide new therapeutic methods for pancreatic cancer.