miRNA Expression Profile of Saliva in Subjects of Yang Deficiency Constitution and Yin Deficiency Constitution

Background/Aims: Based on the theory of constitution in Traditional Chinese Medicine (TCM), the Chinese Han population has been classified into nine constitutions. Of these, Yang deficiency constitution mainly exhibit cold intolerance while Yin deficiency constitution mainly exhibit heat intolerance. Some studies have been carried out to explore the modern genetic and biological basis of such constitution classification, but more remains to be done. MicroRNA (miRNA) serves as post-transcriptional regulators of gene expression and may play a role in the classification process. Here, we examined miRNA expression profile of saliva to further improve the comprehensiveness of constitution classification. Methods: Saliva was collected from Chinese Han individuals with Yang deficiency, Yin deficiency and Balanced constitutions (n=5 each), and miRNA expression profile was determined using the Human miRNA OneArray®v7. Based on 1.5 Fold change, means log2|Ratio|≥0.585 and P-value< 0.05, differentially expressed miRNA was screened. Target genes were predicted using DIANA-TarBasev7.0 and analysis of KEGG pathway was carried out using DIANA-mirPathv.3. Results: We found that 81 and 98 differentially expressed miRNAs were screened in Yang deficiency and Yin deficiency constitution, respectively. Among them, 16 miRNAs were identical and the others were unique. In addition, the target genes that are regulated by the unique miRNAs were significantly enriched in 27 and 20 signaling pathways in Yang deficiency and Yin deficiency constitution, respectively. Thyroid hormone signaling pathway is present in both constitutions. These unique miRNAs that regulated target genes of thyroid hormone signaling pathway may be associated with cold intolerance or heat intolerance. Conclusion: The results of our study show that Yang deficiency and Yin deficiency constitutions exhibit systematic differences in miRNA expression profile. Moreover, the distinct characteristics of TCM constitution may be explained, in part, by differentially expressed miRNAs

Glycerolipid biosynthesis in pea root plastids

Pea root plastids were isolated by differential centrifugation and resulting crude plastid fraction was purified by centrifugation through 10%(v/v) Percoll. Marker enzymes indicated that greater than 50% of the plastids were recovered essentially free from mitochondrial and endoplasmic reticulum contamination. The optimum in vitro conditions for glycerolipid biosynthesis from (U-$sp{14}$C) glycerol-3-phosphate have been determined. Total glycerolipid biosynthesis was approximately 15 nmole/hr/mg protein in the presence of 200 $mu$M glycerol-3-phosphate, 0.5 mM each of NADH and NADPH, 15 mM KH$sb2$CO$sb3$, 0.05 mM CoA, and 2 mM each of ATP and MgCl$sb2$, 100 mM Bis Tris Propane (pH 7.5) and incubated at the standard temperature of 25$sp circ$C. ATP, Coenzyme A and a divalent cation are absolutely required for glycerolipid biosynthesis, whereas reduced nucleotides and bicarbonate improve the synthesis to varying degrees. Dihydroxyacetone phosphate had little effect, while dithiothreitol, detergent and Mn$sp{2+}$ inhibited activity. Under the optimum conditions, isolated pea root plastids mainly synthesized approximately 15% phosphatidic acid, 16% phosphatidylcholine, 13% phosphatidylglycerol, 32% triacylglycerol. Galactolipid synthesis occurred only when UDP-galactose was supplied. Different concentrations of some cofactors resulted in alterations of glycerolipid distribution. Phospholipase A$sb2$ and Rhizopus lipase digestions of phospholipids and neutral lipids revealed that radioactive fatty acids were preferentially esterified to position sn 2 of each glycerolipid with generally 2-4 times as much radioactivity as position sn 1. Pea root plastids are composed of approximately 62% phospholipid, 24% neutral lipid and 14% glycolipid. Within these classes PG, TAG, and the galactolipids are the major components representing 24, 12, and 12% of the total plastid lipids

Oncomir miR-125b suppresses p14(ARF) to modulate p53-dependent and p53-independent apoptosis in prostate cancer.

MicroRNAs are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level and regulate complex patterns of gene expression. Our previous studies demonstrated that in human prostate cancer the miRNA miR-125b is highly expressed, leading to a negative regulation of some tumor suppressor genes. In this study, we further extend our studies by showing that miR-125b represses the protein product of the ink4a/ARF locus, p14(ARF), in two prostate cancer cell lines, LNCaP (wild type-p53) and 22Rv1 (both wild type and mutant p53), as well as in the PC-346C prostate cancer xenograft model that lentivirally overexpressed miR-125b. Our results highlight that miR-125b modulates the p53 network by hindering the down-regulation of Mdm2, thereby affecting p53 and its target genes p21 and Puma to a degree sufficient to inhibit apoptosis. Conversely, treatment of prostate cancer cells with an inhibitor of miR-125b (anti-miR-125b) resulted in increased expression of p14(ARF), decreased level of Mdm2, and induction of apoptosis. In addition, overexpression of miR-125b in p53-deficient PC3 cells induced down-regulation of p14(ARF), which leads to increased cell proliferation through a p53-independent manner. Thus, we conclude that miR-125b acts as an oncogene which regulates p14(ARF)/Mdm2 signaling, stimulating proliferation of prostate cancer cells through a p53-dependent or p53-independent function. This reinforces our belief that miR-125b has potential as a therapeutic target for the management of patients with metastatic prostate cancer

Schematic model of <i>miR-125b</i>-controlled oncopathway in CaP cells.

<p>In CaP cancer cells, p14^ARF facilitates apoptosis in a p53-dependent (<i>left</i>) and p53-independent (<i>right</i>) manner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061064#pone.0061064-Muer1" target="_blank">[30]</a>. Since <i>miR-125b</i> directly targets p14^ARF and other pro-apoptotic molecules, deregulation of <i>miR-125b</i> can modulate proliferation and apoptosis in both p53-positive and p53-deficient CaP cells. Black arrows represent upregulated molecules and white arrows represent downregulated molecules. Broken arrow indicates undefined upregulation of Bak1 activity by p14^ARF.</p

<i>MiR-125b</i> regulates the p53 network.

<p><i>A</i>) Western blot analysis of Mdm2 and p53 in miR-125bm-treated LNCaP (<i>top</i>) and 22R<i>v</i>1 cells (<i>bottom</i>). Cells were transfected with 50 nM of miR-125bm or miR-negative control (miR-NC) for 72 hrs. Equal amounts of protein (50 µg) were used to detect the expression levels of Mdm2, p53, p21 and Puma. <i>B</i>) Western blot analysis of p14^ARF, Mdm2 and p53 in <i>p14^ARF</i> siRNA (sip14)-treated LNCaP (<i>top</i>) and 22R<i>v</i>1 cells (<i>bottom</i>). Cells were treated with sip14 and the cellular levels of p14^ARF, p53 and Mdm2 were analyzed. β-actin was used as a loading control. <i>C</i>) Co-immunoprecipitation analysis of protein interaction between p14^ARF and Mdm2 in 22R<i>v</i>1 cells. Cells were transfected with miR-125bm and 1.0 mg protein was immunoprecipitated with anti-p14^ARF antibody or the rabbit IgG. The resultant immunecomplexes were used to detect the level of Mdm2 by Western blot analysis using anti-Mdm2 antibody. Input: 50 µg protein from total cell lysate. IP: immunoprecipitation. IB: immunoblotting.</p

WST-1 proliferation assay of LNCaP cells (<i>A</i>) and 22R<i>v</i>1 cells (<i>B</i>).

<p>Cells were transfected with 50 nM of miR-125bm or 50 nM of miRNA negative control (miR-NC) for 5 days. Cell proliferation was measured by WST-1 assay. <i>p14^ARF</i> siRNA (sip14) was used as a control. The results are expressed as proliferation relative to that of miR-NC-treated cells, and shown as mean ± SD (n = 4).</p

<i>MiR-125b</i> down-regulates p14^ARF in CaP cells.

<p><i>A</i>) Western blot analysis of expression levels of p14^ARF in LNCaP (<i>top</i>) and 22Rv1 cells (<i>bottom</i>). Cells grown in 10% FBS media were transfected with 50 nM of miR-125bm or anti-<i>miR-125b</i> (anti-125b) for 72 hrs or treated with 5.0 nM of R1881 androgen for 48 hrs. Then, 50 µg of protein per sample was analyzed. Both miR-negative control (miR-NC) and anti-miR negative control (anti-NC) were used as controls, and β-actin was used as a loading control. <i>B</i>) Western blot analysis of expression levels of p14^ARF, mdm2 and p53 in lenti-<i>miR-125b</i>-overexpressed PC-346C xenograft tumor. Both untreated xenograft (untreat.) and lenti-miRNA control vector-infected PC-346C xenograft (vector) were used as controls. In both <i>A</i> and <i>B</i>, the numbers under the gels are the average fold changes of p14^ARF protein from three independent gels relative to the corresponding controls. Fold changes were calculated by scanning the p14^ARF bands and normalizing for β-actin bands. <i>C</i>) Luciferase assay of <i>miR-125b</i> binding to the 3′-UTR of <i>p14^ARF</i> mRNA in LNCaP cells. The assay was repeated three times with each assay being performed in three wells and similar results were obtained each time. The representative results are shown as a mean ±SD (n = 3).</p

Inactivation of <i>miR-125b</i> induces apoptosis in p53-positive CaP cells.

<p><i>A</i>) Detection of SMAC and activated caspase 3 (Cas-3) in LNCaP (<i>left</i>) and 22R<i>v</i>1 (<i>right</i>) cells. Cells were transfected with 50 nM miR-125bm or 50 nM anti-<i>miR-125b</i> (anti-125b) for 5 days, and the levels of SMAC and Cas-3 were measured by Western blot analysis. β-actin was used as loading control. The numbers under the gels are the average fold changes of SMAC and Cas-3 from three independent gels relative to the corresponding controls. <i>B</i>) Detection of anti-<i>miR-125b</i>-induced apoptosis in 22R<i>v</i>1 cells. Cells were transfected using 50 nM anti-<i>miR-125b</i> for 72 hrs and apoptotic cell death was detected using TUNEL assay. The green nuclear fluorescence indicates the apoptotic cleavage of nuclear DNA (<i>left</i>). For quantitation of apoptotic cell death, 400 cells were counted and apoptosis is expressed as % apoptosis (apoptotic cells/400×100%). Quantitative analysis was performed three times and result was expressed as mean ± SE (n = 3) (<i>right</i>). Cells treated with irradiation (IR, 6 Gy) were used as a positive control. <i>C</i>) TUNEL assay of apoptotic death of 22R<i>v</i>1 cells that were treated with anti-<i>miR-125b</i> followed by <i>p14^ARF</i> antisense (sip14). Result was expressed as mean ± SE (n = 3). <i>D</i>) Western blot analyses of p14^ARF, p53 and Bak1 levels in 22R<i>v</i>1 cells. <i>Left</i>: 22R<i>v</i>1 cells were transfected with anti-<i>miR-125</i>; <i>right</i>: anti-<i>miR-125</i>-transfected 22R<i>v</i>1 cells were treated with sip14. Both anti-miR-NC (anti-NC) and scramble siRNA were used as controls.</p

ERα is an RNA-binding protein sustaining tumor cell survival and drug resistance

Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response