Dysregulated genes and miRNAs in the apoptosis pathway in colorectal cancer patients

Apoptosis is genetically regulated and involves intrinsic and extrinsic pathways. We examined 133 genes within these pathways to identify whether they are expressed differently in colorectal carcinoma (CRC) and normal tissue (N?=?217) and if they are associated with similar differential miRNA expression. Gene expression data (RNA-Seq) and miRNA expression data (Agilent Human miRNA Microarray V19.0) were generated. We focused on dysregulated genes with a fold change (FC) of >?1.50 or <?0.67, that were significant after adjustment for multiple comparisons. miRNA:mRNA seed-region matches were determined. Twenty-three genes were significantly downregulated (FC?<?0.67) and 18 were significantly upregulated (FC?>?1.50). Of these 41 genes, 11 were significantly associated with miRNA differential expression. BIRC5 had the greatest number of miRNA associations (14) and the most miRNAs with a seed-region match (10). Four of these matches, miR-145-5p, miR-150-5p, miR-195-5p, and miR-650, had a negative beta coefficient. CSF2RB was associated with ten total miRNAs (five with a seed-region match, and one miRNA, miR-92a-3p, with a negative beta coefficient). Of the three miRNAs associated with CTSS, miR-20b-5p, and miR-501-3p, had a seed-region match and a negative beta coefficient between miRNA:mRNA pairs. Several miRNAs that were associated with dysregulated gene expression, seed-region matches, and negative beta coefficients also were associated with CRC-specific survival. Our data suggest that miRNAs could influence several apoptosis-related genes. BIRC5, CTSS, and CSF2R all had seed-region matches with miRNAs that would favor apoptosis. Our study identifies several miRNA associated with apoptosis-related genes, that if validated, could be important therapeutic targets.     

Genome-wide identification of novel microRNAs from genome sequences using computational approach in the mudskipper (<Emphasis Type="Italic">Boleophthalmus pectinirostris</Emphasis>)

MicroRNAs (miRNAs), approximately 22 nucleotides (nt) long, are small, non-coding RNA molecules with important regulatory functions in gene expression. They are mostly conserved among the organisms and this conservation makes them a good source for the identification of novel miRNAs by computational genomic homology. The miRNA repertoire of a major aquaculture species, Boleophthalmus pectinirostris, has been unknown until recently. Currently, the B. pectinirostris whole-genome sequences have been completed, making it more convenient for us to focus on computational prediction for novel miRNA homologs. Following a range of strict filtering criteria, a total of 62 potential miRNAs were identified for the first time; they belong to 39 different miRNA families. All these miRNAs were observed in the stem portion of the stable stem–loop structures. The minimum free energy (MFE) of the predicted miRNAs ranged from ?21.6 to ?62.7 kcal/mol with an average of ?39.2 kcal/mol. The A + U ranged from 32.5 to 69.1% with an average value of 52.2%. The phylogenetic analysis of predicted miRNAs revealed that miR-23a-3p, miR-184-3p, miR-214-5p, and miR-338-3p from B. pectinirostris are evolutionary highly conserved showing more similarity with other fish species. To verify the predicted miRNAs, selected miRNAs representing 16 of the 39 families were confirmed by stem–loop RT-PCR, indicating that the computational approach that we used to identify the miRNAs is a highly efficient and affordable alternative method. Taken together, these findings provide a reference point for further research on miRNAs identification in fish species, meanwhile, our study also will be useful for further insight into biological functions of miRNAs and improved understanding of genome in B. pectinirostris.     

<Emphasis Type="Italic">miR-3075</Emphasis> Inhibited the Migration of Schwann Cells by Targeting Cntn2

Peripheral nerve injury is a complex biological process that involves the expression changes of various coding and non-coding RNAs. Previously, a number of novel miRNAs that were dysregulated in rat sciatic nerve stumps after peripheral nerve injury were identified and functionally annotated by Solexa sequencing. In the current study, we studied one of these identified novel miRNAs, miR-3075, in depth. Results of transwell-based cell migration assay showed that increased expression of miR-3075 suppressed the migration rate of Schwann cells while decreased expression of miR-3075 elevated the migration rate of Schwann cells, demonstrating that miR-3075 inhibited Schwann cell migration. Results of BrdU cell proliferation assay showed that neither miR-3075 mimic nor miR-3075 inhibitor would affect Schwann cell proliferation. We further studied candidate target genes of miR-3075 by using bioinformatic tools and analyzing gene expression patterns and found that miR-3075 might target contactin 2 (Cntn2). Previous study showed that Cntn2 regulated cell migration and myelination. Our current observation suggested that the biological effects of miR-3075 on Schwann cell phenotype might by through the negative regulation of Cntn2. Overall, our study revealed the function of a novel miRNA, miR-3075, and expanded our current understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.     

Epithelial-specific histone modification of the <Emphasis Type="Italic">miR-96/182</Emphasis> locus targeting <Emphasis Type="Italic">AMAP1</Emphasis> mRNA predisposes p53 to suppress cell invasion in epithelial cells

Background

TP53 mutations in cancer cells often evoke cell invasiveness, whereas fibroblasts show invasiveness in the presence of intact TP53. AMAP1 (also called DDEF1 or ASAP1) is a downstream effector of ARF6 and is essential for the ARF6-driven cell-invasive phenotype. We found that AMAP1 levels are under the control of p53 (TP53 gene product) in epithelial cells but not in fibroblasts, and here addressed that molecular basis of the epithelial-specific function of p53 in suppressing invasiveness via targeting AMAP1.

Methods

Using MDA-MB-231 cells expressing wild-type and p53 mutants, we identified miRNAs in which their expression is controlled by normal-p53. Among them, we identified miRNAs that target AMAP1 mRNA, and analyzed their expression levels and epigenetic statuses in epithelial cells and nonepithelial cells.

Results

We found that normal-p53 suppresses AMAP1 mRNA in cancer cells and normal epithelial cells, and that more than 30 miRNAs are induced by normal-p53. Among them, miR-96 and miR-182 were found to target the 3′-untranslated region of AMAP1 mRNA. Fibroblasts did not express these miRNAs at detectable levels. The ENCODE dataset demonstrated that the promoter region of the miR-183-96-182 cistron is enriched with H3K27 acetylation in epithelial cells, whereas this locus is enriched with H3K27 trimethylation in fibroblasts and other non-epithelial cells. miRNAs, such as miR-423, which are under the control of p53 but not associated with AMAP1 mRNA, demonstrated similar histone modifications at their gene loci in epithelial cells and fibroblasts, and were expressed in these cells.

Conclusion

Histone modifications of certain miRNA loci, such as the miR-183-96-182 cistron, are different between epithelial cells and non-epithelial cells. Such epithelial-specific miRNA regulation appears to provide the molecular basis for the epithelial-specific function of p53 in suppressing ARF6-driven invasiveness.

     

MiRNA expression profile and miRNA–mRNA integrated analysis (MMIA) during podocyte differentiation

The podocyte is a prominent cell type, which encases the capillaries of glomerulus. Podocyte-selective deletion of Dicer or Drosha was reported to induce proteinuria and glomerulosclerosis, suggesting the essential role of microRNA (miRNA) in podocytes for renal function. However, no comprehensive miRNA expression or miRNA–mRNA integrated analysis (MMIA) can be found during podocyte differentiation. Herein, miRNA and mRNA microarrays are presented, which were carried out in differentiated and undifferentiated mouse podocyte cell lines (MPC5). A total of 50 abnormal miRNAs (26 down-regulated and 24 up-regulated) were identified in differentiated and undifferentiated podocytes. Using MMIA, 80 of the 743 mRNAs (>twofold change) were predicted for potential crosstalk with 30 miRNAs of the 50 abnormal miRNAs. In addition, the gene ontology of mRNAs and the pathway analysis of miRNAs revealed a new potential-regulated network during podocyte differentiation. The expressions of three remarkably changed miRNAs (miR-34c, miR-200a and miR-467e) and four mRNAs (Runx1t1, Atp2a2, Glrp1, and Mmp15), were randomly chosen for further validation by the quantitative real-time polymerase chain reaction, and their expression trends were consistent with the microarray data. Reference searching was also conducted to confirm our data and to find potential new molecules and miRNA-target pairs involved in the podocyte differentiation. The dual luciferase reporter assay for miR-200a/GLRX and let-7b/ARL4D confirmed the prediction of MMIA. The results of this study provide a detailed integration of mRNA and miRNA during podocyte differentiation. The molecular integration mode will open up new perspectives for a better understanding of the mechanism during podocyte differentiation.     

Intramuscular injection of mechano growth factor E domain peptide regulated expression of memory-related <Emphasis Type="Italic">sod</Emphasis>, miR-134 and miR-125b-3p in rat hippocampus under simulated weightlessness

Objective

To investigate the expression of memory-related antioxidant genes and miRNAs under simulated weightlessness and the regulation of mechano growth factor (MGF) E domain, the peptide preventing nerve damage.

Results

Igf-iea and mgf mRNA levels, expression of antioxidant genes sod1 and sod2 and levels of miR-134 and miR-125b-3p increased in rat hippocampus after 14 days tail suspension to simulate weightlessness which was inhibited with intramuscular injection of E domain peptide. Therefore, administration of MGF E domain peptide could reverse increased expressions of memory-related igf-iea, mgf, sod1, sod2, miR-134 and miR-125b-3p in rat hippocampus under simulated weightlessness.

Conclusions

MGF may regulate the redox state and miRNA-targeted NR-CREB signaling, and intramuscular injection may be the alternative administration because of its safety, convenience and ability to pass through the blood brain barrier.

     

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets <Emphasis Type="Italic">MET</Emphasis> and <Emphasis Type="Italic">AXL</Emphasis> in triple-negative breast cancer

Background

Breast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.

Methods

Bioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.

Results

By applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line.

Conclusions

These findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

     

Deregulation of neuronal miRNAs induced by amyloid-β or TAU pathology

Background

Despite diverging levels of amyloid-β (Aβ) and TAU pathology, different mouse models, as well as sporadic AD patients show predictable patterns of episodic memory loss. MicroRNA (miRNA) deregulation is well established in AD brain but it is unclear whether Aβ or TAU pathology drives those alterations and whether miRNA changes contribute to cognitive decline.

Methods

miRNAseq was performed on cognitively intact (4 months) and impaired (10 months) male APPtg (APP^swe/PS1^L166P) and TAUtg (THY-Tau22) mice and their wild-type littermates (APPwt and TAUwt). We analyzed the hippocampi of 12 mice per experimental group (n =?96 in total), and employed a 2-way linear model to extract differentially expressed miRNAs. Results were confirmed by qPCR in a separate cohort of 4 M and 10 M APPtg and APPwt mice (n =?7–9 per group) and in human sporadic AD and non-demented control brain. Fluorescent in situ hybridization identified their cellular expression. Functional annotation of predicted targets was performed using GO enrichment. Behavior of wild-type mice was assessed after intracerebroventricular infusion of miRNA mimics.

Results

Six miRNAs (miR-10a-5p, miR-142a-5p, miR-146a-5p, miR-155-5p, miR-211-5p, miR-455-5p) are commonly upregulated between APPtg and TAUtg mice, and four of these (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) are altered in AD patients. All 6 miRNAs are strongly enriched in neurons. Upregulating these miRNAs in wild-type mice is however not causing AD-related cognitive disturbances.

Conclusion

Diverging AD-related neuropathologies induce common disturbances in the expression of neuronal miRNAs. 4 of these miRNAs are also upregulated in AD patients. Therefore these 4 miRNAs (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) appear part of a core pathological process in AD patients and APPtg and TAUtg mice. They are however not causing cognitive disturbances in wild-type mice. As some of these miRNA target AD relevant proteins, they may be, in contrast, part of a protective response in AD.

     

Plasma microRNAs are associated with acute exacerbation in idiopathic pulmonary fibrosis

Background

Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) has high short-term mortality with unknown causes. To predict this malignant condition in clinics is challenging. In this study, we aim to demonstrate whether there are miRNAs that differ between AE-IPF and stable IPF, which may be served as reliable biomarker for AE-IPF prediction.

Methods

Human fibrotic-associated miRNAs arrays were designed to detect miRNAs expression in plasma of 3 AE-IPF patients, 3 Stable-IPF (S-IPF) patients and 3 normal controls (NC). Differentially expressed miRNAs between AE-IPF and S-IPF patients were selected for further analyses. The validation studies were carried out in plasma of 12 AE-IPF patients, 45 S-IPF patients and 51 healthy control subjects. Signaling pathways and cellular processes interacted with validated miRNAs were predicted by DIANA-miRPath.

Results

According to the array analysis, 6 miRNAs showed differentiated expression between AE-IPF and S-IPF patients (P?<?0.05). In the validation studies, let-7d-5p was decreased in S-IPF and further decreased in AE-IPF, when compared to NC (0.0003?±?0.0002 vs 0.003?±?0.002, P?<?0.01 and 0.0007?±?0.0005 vs 0.003?±?0.002, P?<?0.01). While miR-25-3p was obviously decreased in S-IPF (0.0002?±?0.0001 vs 0.0003?±?0.0003, P?<?0.01) but significantly increased in AE-IP (0.0023?±?0.002 vs 0.0003?±?0.0003, P?<?0.01). In receiver-operator characteristic (ROC) curve analysis, the areas under the curve (AUCs) of miR-25-3p and let-7d-5p were 0.83 and 0.75, respectively. The sensitivity at fixed specificity of 90% was improved from 50% to 66.7% when the two miRNAs were combined. The functional prediction of miRNAs suggested that the loss of anti-fibrotic capacity and the gain of uncontrolled cell growth may be required in AE-IPF pathogenesis.

Conclusions

In conclusion, miR-25-3p and let-7d-5p in plasma were differentially expressed between AE-IPF and S-IPF. A combination of these two miRNAs may be a potential biomarker for AE-IPF from IPF.

     

<Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> Increases the Anti-apoptotic Micro RNA-21 and Decreases the Pro-inflammatory Micro RNA-155 in the LPS-Treated Human Endothelial Cells

Given the anti-inflammatory and protective role of probiotics in atherosclerosis and the regulatory role of micro RNA (miRNA) in endothelial cell (dys) functions, this study aimed to investigate the effect of Lactobacillus acidophilus (La) on cellular death and the expression of miRNA-21, 92a, 155, and 663 in human umbilical vein endothelial cell (HUVEC) induced by Escherichia coli lipopolysaccharide (Ec-LPS). LPS-treated and untreated HUVECs were cultured in the presence of different La conditions such as La-conditioned media (LaCM), La water extract (LaWE), La culture-filtered (LaFS) and unfiltered supernatants (LaUFS). After 24 h, apoptosis, necrosis and the levels of the mentioned miRNAs were measured using flow cytometry and real-time PCR methods, respectively. LaCM decreased apoptosis, necrosis and inflammatory miR-155 and conversely increased anti-apoptotic miR-21 in Ec-LPS-treated HUVECs. Association analysis revealed negative correlations between necrosis and the levels of miR-21, miR-92a, and miR-155. The beneficial effects of L. acidophilus on the ECs death and expression of atherosclerosis related miRNAs in these cells imply a new aspect of its regulation in cardiovascular diseases rather than previously described ones and suggest this probiotic bacterium as a candidate in the preventative therapy of atherosclerosis.     

Upregulation of p72 enhances malignant migration and invasion of glioma cells by repressing Beclin1 expression

p72 is the member of the DEAD-box RNA helicase family, which can unwind double-stranded RNA and is efficient for microRNA (miRNA, miR) processing. However, its specific role in glioma has not been elucidated. First, the expression of p72 in glioma cell lines and tissues was explored using Western blot. To explore the role of p72 on glioma progression, adenovirus inhibiting p72 was transfected into A172 and T98G cells. Cell autophagy was determined using GFPLC3 dots, and cell apoptosis was determined using flow cytometry. The effect of Beclin1 was explored using GFP-LC3 dots, flow cytometry, and colony formation. The possible miRNAs that target the 3′-untranslated region (3′-UTR) of Beclin1 were predicted using TargetScan. Dual luciferase reporter assay was applied to determine whether these miRNAs bind to the 3′-UTR of Beclin1. The expression of p72 was significantly increased in glioma cell lines and tissues. Autophagy-related protein Beclin1 was found to be significantly enhanced when p72 was inhibited. The accumulation of GFP-LC3 dots was significant in cells transfected with ad-sh-p72 compared with ad-con. Colony formation capacity and cell apoptosis were also found to be significantly decreased with p72 inhibition. Furthermore, upregulation of Beclin1 contributes to A172 cell autophagy, invasion, and apoptosis. Overexpression of p72 induces increased miR-34-5p and miR-5195-3p expression in A172 and T98G cells. Beclin1 was the target gene of miR-34-5p and miR-5195-3p. In conclusion, we found for the first time that overexpression of p72 decreased Beclin1 expression partially by increasing miR-34-5p and miR-5195-3p expression in A172 and T98G cells.     

MicroRNA Biogenesis Pathway Gene Polymorphisms Are Associated with Breast Cancer Risk

MicroRNAs (miRNAs) play an important role as epigenetic regulators in cancer initiation and progression. One of the mechanisms of miRNA dysregulation is altered functioning of proteins involved in miRNA processing machinery. It has been suggested that single nucleotide polymorphisms (SNPs) within miRNA gene regions, miRNA target genes, and miRNA machinery genes may affect the miRNAs regulation. We selected 25 SNPs in the key genes of miRNA biosynthesis, including DROSHA/RNASEN, DGCR8, DICER1, XPO5, RAN, PIWIL1/HIWI, AGO1/EIF2C1, AGO2, GEMIN4, GEMIN3/DDX20, and DDX5, and investigated the association between these SNPs and the risk of breast cancer. The total number of breast cancer cases and cancer-free controls enrolled in the investigation were 778 (417 breast cancer patients and 361 healthy women). We found that rs11060845 and rs10773771 in the PIWIL1 gene, rs3809142/RAN, rs10719/DROSHA, rs1640299/DGCR8, rs563002/DDX20, rs595055/AGO1, and rs2740348/GEMIN4 were associated with breast cancer risk in Russians.     

MiR-1246 is upregulated and regulates lung cell apoptosis during heat stress in feedlot cattle

Globally, heat stress seriously threatens productivity of cattle. The objective of this study was to identify novel miRNAs that regulated heat stress in feedlot cattle. Experiment was conducted under heat stress and normal conditions. With profiling miRNAs of each feedlot cattle, our results showed the level of miR-1246 was significantly increased in these heat-stressed cattle (P <?0.05). Furthermore, by using bioinformatics analysis and luciferase reporter assays combined with qPCR and western blot, we found miR-1246 negatively regulated poly (C) binding protein 2 (PCBP2) and cAMP response element binding protein-like 2 (CREBL2) mRNA and protein levels through binding to the 3′-UTR region (P?<?0.05); further, it inhibited heat-induced apoptosis in lung cells. Finally, our results suggested that miR-1246 plays an important role in heat stress and it has the potential to be a novel modulation factor for heat stress in feedlot cattle.     

Regulatory roles of miR-155 and let-7b on the expression of inflammation-related genes in THP-1 cells: effects of fatty acids

The main aim of this investigation was to study the regulatory roles of let-7b and miR-155-3p on the expression of inflammation-associated genes in monocytes, macrophages, and lipopolysaccharide (LPS)-activated macrophages (AcM). A second goal was to analyze the potential modulatory roles of different fatty acids, including oleic, palmitic, eicosapentaenoic (EPA), and docosahexaenoic (DHA), on the expression of these miRNAs in the three cell types. This hypothesis was tested in human acute monocytic leukemia cells (THP-1), which were differentiated into macrophages with 2-O-tetradecanoylphorbol-13-acetate (TPA) and further activated with LPS for 24 h. Monocytes, macrophages, and AcM were transfected with a negative control, or mimics for miR-155-3p and miR-let-7b-5p. The expression of both miRNAs and some proinflammatory genes was analyzed by qRT-PCR. Interestingly, let-7b mimic reduced the expression of IL6 and TNF in monocytes, and SERPINE1 expression in LPS-activated macrophages. However, IL6, TNF, and SERPINE1 were upregulated in macrophages by let-7b mimic. IL6 expression was higher in the three types of cells after transfecting with miR-155-3p mimic. Similarly, expression of SERPINE1 was increased by miR-155-3p mimic in monocytes and macrophages. However, TLR4 was downregulated by miR-155-3p in monocytes and macrophages. Regarding the effects of the different fatty acids, oleic acid increased the expression of let-7b in macrophages and AcM and also increased the expression of miR-155 in monocytes when compared with DHA but not when compared with non-treated cells. Overall, these results suggest anti- and proinflammatory roles of let-7b and miR-155-3p in THP-1 cells, respectively, although these outcomes are strongly dependent on the cell type. Noteworthy, oleic acid might exert beneficial anti-inflammatory effects in immune cells (i.e., non-activated and LPS-activated macrophages) by upregulating the expression of let-7b.     

Extracellular vesicles-mediated transfer of miR-208a/b exaggerate hypoxia/reoxygenation injury in cardiomyocytes by reducing QKI expression

Diabetes mellitus (DM) induces a variable degree of muscle sarcopenia, which may be related to protein degradation and to the expression of both E3 ubiquitin ligases and some specific microRNAs (miRNAs). The present study investigated the effect of diabetes and acute muscle contraction upon the TRIM63 and FBXO32 expression as well as the potential involvement of some miRNAs. Diabetes was induced by streptozotocin and studied after 30 days. Soleus muscles were harvested, stimulated to contract in vitro for twitch tension analysis (0.5 Hz), 30 min later for tetanic analysis (100 Hz), and 30 min later were frozen. TRIM63 and FBXO32 proteins were quantified by western blotting; Trim63 mRNA, Fbxo32 mRNA, miR-1-3p, miR-29a-3p, miR-29b-3p, miR-133a-3p, and miR-133b-3p were quantified by qPCR. Diabetes induced sarcopenia by decreasing (P < 0.05) muscle weight/tibia length index, maximum tetanic contraction and relaxation rates, and absolute twitch and tetanic forces (P < 0.05). Diabetes decreased (P < 0.05) the Trim63 and Fbxo32 mRNAs (30%) and respective proteins (60%), and increased (P < 0.01) the miR-29b-3p (2.5-fold). In muscle from diabetic rats, acute contractile stimulus increased TRIM63 protein, miR-1-3p, miR-29a-3p, and miR-133a/b-3p, but decreased miR-29b-3p (P < 0.05). Independent of the metabolic condition, after muscle contraction, both TRIM63 and FBXO32 proteins correlated significantly with miR-1-3p, miR-29a/b-3p, and miR-133a/b-3p. All diabetes-induced regulations were reversed by insulin treatment. Concluding, the results depict that muscle wasting in long-term insulinopenic condition may not be accompanied by increased proteolysis, pointing out the protein synthesis as an important modulator of muscle sarcopenia in DM.     

The use of miRNAs as reference genes for miRNA expression normalization during <Emphasis Type="Italic">Lilium</Emphasis> somatic embryogenesis by real-time reverse transcription PCR analysis

Expression profiling of miRNAs has the ability to reveal the essence of somatic embryogenesis (SE). qRT-PCR is one of the most commonly used techniques for dynamic miRNA detection but requires optimal reference genes for data reliability. This is the first report on reference gene validation for miRNA expression normalization in Lilium (Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor). In this study, seventeen miRNAs together with two snRNAs (U4, U6), one rRNA (5S rRNA) and three protein-coding genes (FP, ACT, GAPDH) were selected as reference candidates, and their expression stability was validated by qRT-PCR among eleven developing SE cultures in two lilies. Four normalization algorithms, including geNorm, BestKeeper, NormFinder and RefFinder, were also used to evaluate the stability of the reference candidates. For Lilium pumilum DC. Fisch., lpu-miR159a was the optimal reference gene during SE, followed by lpu-miR408b, while U6 was the least stable reference candidate. For Lilium davidii var. unicolor, FP presented greater stability than did half of the miRNA candidates, but the best reference gene was lda-miR162, followed by lda-miR159a. Further analysis of the expression level of miR156 and miR529 was used to evaluate the validity of the reference genes in both lilies. In general, miRNAs are superior to common protein-coding genes and snRNAs / rRNAs as reference genes for miRNA expression normalization during Lilium SE, and the most suitable reference miRNA is different between two species in the same Lilium genus. This is a pioneer study using suitable miRNAs as reference genes in Lilium and constitutes a small but essential step for the further exploration of miRNA function in Lilium, thus offering valuable references for other plants.