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Dendrosomal curcumin increases expression of the long non-coding RNA gene MEG3 via up-regulation of epi-miRs in hepatocellular cancer.

ABSTRACT

Background: Hepatocellular carcinoma is the fifth most common cancer worldwide, with poor prognosis and resistance to chemotherapy. This gives novel cancer treatment methods an overwhelming significance. Epigenetic therapy of cancer is useful in reversing some of the cancer defects because of reversibility of the epigenetic alterations. Non-protein coding transcripts are the major part of our transcriptome. MEC3 is a tumor suppressor long non-coding RNA being expressed in many normal tissues. Methylation of MEG3 promoter region elicits the decrease in its expression in hepatocellular cancer cells. Bioactive nutrients including curcumin offer great potential in altering DNA methylation status which is catalyzed via DNMT1, DNMT3A and 3B.

Purpose: Herein, we aimed to study RNA-based epigenetic effects of dendrosomal curcumin (DNC) on hepatocellular cancer (HCC).

Study design: To this end miRNA-dependent regulation of MEG3 expression under treatment with DNC was studied by evaluating the modulatory involvement of miR-29a for DNMT3A and 3B and miR-185 for DNMT1. Methods: We evaluated DNC entrance to HCC cells with the use of fluorescent characteristics of curcumin. Next we performed the MTT assay to evaluate DNC and dendrosome effects on HCC cell viability. The coding and non-coding genes expression analyses were done using quantitative-PCR.

Results: In result we found that the DNC dependent overexpression of miR-29a and miR-185 (P < 0.01) can down-regulate the expression of DNMT1, 3A and 3B (P < 0.05) and subsequently overexpresses MEG3 (P < 0.05).

Conclusion: DNC potentially can induce DNA hypomethylation and reexpression of silenced tumor suppressor genes in HCC. These data suggest that DNC could be an effective choice for epigenetic therapy of HCC.

Keywords:

Hepatocellular cancer

Epigenetic therapy

Dendrosomal curcumin

DNA methylation

Long non-coding RNA

miRNA

Introduction

Cancer is a hyperproliferative disorder that results from multiple genetic and epigenetic aberrations (Sawan et al. 2008). Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, with poor prognosis and resistance to chemotherapy. Activation of several oncogenes and the loss of several tumor suppressor genes induce liver tumorigenesis (Yao and Mishra 2009). Through transcriptome analyses using deep sequencing and tiling arrays it has been identified that a large portion of eukaryotic genomes are transcribed into non-protein coding RNAs, while only a small fraction has protein coding capacity (Amaral and Mattick 2008). Among the non-coding RNAs, miRNAs have been extensively studied in carcinogenesis. Their role in hepatocarcinogenesis has been revealed (Croce 2009). Although the repertoire of long non-coding RNAs (IncRNAs) remains to be elucidated, growing evidence suggests that IncRNAs have emerged as important regulators for diverse cellular function. These genes are frequently aberrantly expressed in various human cancers in both oncogenic and tumor suppressive pathways (Gibb et al. 2011). Studies to identify IncRNAs that have noticeable role in hepatocarcinogenesis led to identify several IncRNAs, among which maternally expressed gene 3 (MEG3) was down-regulated significantly compared with non-malignant hepatocytes via promoter hypermethylation. MEG3 has recently been shown to possess tumor suppressor activity. Expression of this IncRNA has been shown to suppress cell growth via p53 expression modulation and subsequently p53-mediated transactivation of downstream targets in colon and brain cancer cells (Zhang et al. 2010; Braconi et al. 2011). Numerous IncRNAs including HOTAIR contribute to bridge the limited number of polycomb proteins and the diverse tissue-specific genome modifications. HOTAIR is co-expressed with the HoxC genes, interacts with polycomb proteins and works in Trans to repress HoxD expression. HOTAIR gene was significantly overexpressed in tumor tissues from patients with HCC and breast cancer compared with non-tumor tissue. HOTAIR is an oncogenic IncRNA that has role in breast cancer and HCC metastasis (Geng et al. 2011). The potential role and involvement of MEG3 and HOTAIR with contradictory functions can be detected in hepatocarcinogenesis. The roles of ncRNAs lead us to this notion; they are top level regulators which correlate genetics and epigenetics. Epigenetic mechanisms include heritable and reversible changes in DNA methylation, histone modifications and altered miRNA expression without any change in the DNA sequence. The reversibility of the epigenetic alterations has emerged therapeutic potential of epigenetic treatments for improving cancer cells condition (Yoo and Jones 2006). Epigenetic regulation constitutes an important mechanism by which dietary components can selectively activate or inactivate gene expression (Meeran et al. 2010). Curcumin (diferuloylmethane) is the flavoring agent of turmeric powder (Curcuma longa) with various therapeutic properties especially antitumor activity and without any side effects on normal cells. This compound has recently been determined to induce epigenetic changes. However, to develop curcumin for clinical use as a regulator of epigenetic changes needs further investigation to improve its attributes. Insolubility of the compound in aqueous solutions, the main reason for its poor bioavailability, has limited its utilization as a therapeutic agent. To overcome this problem and increase the solubility of curcumin, numerous approaches have been taken by liposomes, micelles, adjuvants and phospholipid complexes (Anand et al. 2007; Bisht et al. 2007). Nevertheless, any perfect formulation has not been found yet. Here, we improved the solubility of curcumin thereby its anticancerous property through employing dendrosome nanoparticles. This nanocarrier is a neutral, amphipathic and biodegradable nano-material synthesized previously by our group (Sarbolouki et al. 2000;Sadeghizadeh et al. 2008). Its capability has been shown in boosting the solubility of curcumin and improving its uptake into cancer cells, afterwards inhibiting the proliferation of these cells in vitro and in vivo (Alizadeh et al. 2012;Babaei et al. 2012;Tahmasebi Mirgani et al. 2014). In present study we used the combination of this nanocarrier and curcumin called dendrosomal curcumin (DNC) toward effective curcumin entrance to HCC and maximize epigenetic activity of curcumin treatment. Previous studies by our group have revealed that dendrosomal nanocarrier and DNC have not any cytotoxic effect on normal cells (Babaei et al. 2012; Tahmasebi Mirgani et al. 2014).

Materials and methods

Dendrosomal curcumin preparation

Curcumin was bought from Sigma-Aldrich Company, USA. Dendrosomal nanocarrier was a gift from Institute of Biochemistry and Biophysics, University of Tehran, Iran. Preparation of DNC, a combination of curcumin powder and liquid dendrosome nano-carrier, was performed using optimized protocol described in previous studies by our group (Babaei et al. 2012; Tahmasebi Mirgani et al. 2014).

Cell lines and culture condition

Two hepatoma cell lines including HepG2 and HuH-7 (from Pasteur Institute, Tehran, Iran) were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO[R],USA). Cells were supplemented with 10% fetal bovine serum (FBS; GIBCO[R], USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Life Technologies). All cells were grown at 37 [degrees]C in a humidified atmosphere of 5% carbon dioxide.

Cell viability test

Cell viability was measured by methylthiazol tetrazolium (MTT) assay according to the manufacturer's instructions (Sigma-Aldrich, USA). Briefly, identical numbers of cancerous cells in 200 [micro]l medium containing 10% FBS were seeded triplicate on 96-well plates and incubated overnight. These cells were subsequently treated 48 h (short time) and 120 h (long time) with various concentrations of DNC and dendrosome. Afterward, 20 [micro]l of 5 mg/ml MTT was added to each well and incubated for extra 4 h followed by addition of 200 [micro]l of dimethyl sulfoxide. The absorbance was determined at 570 nm by a 96-well plate reader (TECAN, Switzerland) then being proportional to cell viability. All values were compared with the matching controls. Cell viability was calculated as the percentage of cell viability of treated cells against control cells and the concentration at which cell growth was inhibited by 50% (inhibitory concentration: IC50 or lethal dose; LD50) determined by standard curve method (Malich et al. 1997). Each experiment was carried out in triplicate wells and repeated at least three times.

DNA isolation, bisulfite conversion and methylation analysis

DNA was extracted using AccuPrep[R] Genomic DNA Extraction Kit (Bioneer, Korea) and treated with bisulfite using EZ DNA Methylation Gold' (Zymoresearch, Orange, CA, USA). Bisulfite converted genomic DNA was amplified using Hot Start Master Mix (Ampliqon, Denmark). Bisulfite-treated DNA was then used for bisulfite sequencing PCR (BSP) (Fraga and Esteller 2002). BSP was performed using Hot Start Master Mix (Ampliqon, Denmark) and PCR products were sequenced by Macrogen Company, Korea. Touchdown MSP was performed using Hot Start Master Mix (Ampliqon, Denmark) and PCR products were identified by ethidium bromide staining after 2% agarose gel electrophoresis. All primers used for MSP and BSP are available in Table 1.

RNA extraction, reverse transcription and Real-Time quantitative reverse transcription-PCR (Real-Time qRT-PCR)

Total RNA was extracted using TRIzol[R] reagent (Life Technologies) followed by DNase I (Thermo Fisher Scientific, Waltham, MA, USA) digestion according to the manufacturer's instructions. Complementary DNA was synthesized by PrimeScript[TM] RT reagent kit (Takara Bio Inc., Shiga, Japan). The list of primers for specific genes and housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are indicated in Table 2. Real-time PCR was performed using the SYBR[R] Premix Ex Taq[TM] II (Takara Bio Inc., Shiga, Japan). Relative gene expression was calculated as [2.sup.-[DELTA][DELTA]Ct].

RNA extraction, reverse transcription and semi-quantitative reverse transcription PCR (sqRT-PCR)

Analysis of gene expression of MEG3, DNMT1, DNMT3A, DNMT3B and GAPDH in time dependent manner was done in 12 h, 24 h and 48 h under treatment with DNC. RNA was extracted using TRIzof reagent (Life Technologies) followed by DNase I (Thermo Fisher Scientific, Waltham, MA, USA) digestion according to the manufacturer's protocols. Complementary DNA was synthesized by PrimeScript[TM] RT reagent kit (Takara Bio Inc., Shiga, Japan). PCR was done using Master Mix (Ampliqon, Denmark) and PCR products were identified by ethidium bromide staining after 2% agarose gel electrophoresis. Quantification of bands was done with ImageJ 1.45s software (National Institutes of Health, USA).

MiRNA expression analysis

RNA was extracted using TRIzol[R] reagent (Life Technologies) followed by DNase 1 (Thermo Fisher Scientific, Waltham, MA, USA) digestion according to the manufacturer's protocols. MiR mature transcripts have short lengths without poly A tail so cDNA synthesis was done using poly A polymerase for extension of 3' of miRNA mature transcripts. Then specific reverse primer and reverse transcriptase enzyme were used according to the manufacturer's construction, MiR-Amp kit (Pars Genome, Tehran, Iran) for synthesis of complementary DNA. Real time PCR was performed using 5x HOT FIREPOL[R] Eva Green[R] qPCR Mix plus ROX (Solis Bio Dyne, Tartu, Estonia) kit and PCR was run according to its protocol. The expression of miR-29a, miR-185 and U6 (reference gene) was analyzed using universal and specific primer sets MiR-Amp kit (Pars Genome, Tehran, Iran).

Statistical analysis

Statistics were presented in Prism[R] 5 software (GraphPad Software, Inc., La Jolla, CA, USA) or Microsoft Excel and analyzed using one-way analysis of variance followed by Newman-Keuls multiple comparison test or Student's t-test. Differences among groups were stated to be statistically significant when P < 0.05.

Results

Uptake of DNC

The absorbance spectrum of DNC using innate fluorescent characteristic of curcumin to HuH-7 and HepG2 cells could be viewed by light and UV microscopy in Fig. la-h.

Effects of curcumin on cancer cell lines

We determined the toxicity of curcumin on HepG2 and HuH-7 cell lines. The cytotoxic effect of DNC on cancerous cells could be seen microscopically as vesicular bodies in DNC treated cells in Fig. 2b, c, e and f compared to control ones in Fig. 2a and d. Also as Fig. 3a-d shows, DNC significantly suppresses proliferation of cancerous cell lines in a dose- and time-dependent manner (P < 0.01). The IC50 value of DNC for HepG2 cell line within 48 h was about 23 [micro]M which relatively reduced to 8 [micro]M in 120 h. The IC50 values of DNC for HuH-7 cells were; 21 [micro]M for 48 h and 11 [micro]M for 120 h. Furthermore, dendrosomal nanocarrier did not show any side effect on cell lines even at 30 [micro]M, which in turn confirms the safety of this nanoparticle on human cell lines. According to the IC50 results, for both cell lines we adjusted a concentration; 18 [micro]M, for 48 h and fewer times and 8 [micro]M for 120 h.

Gene expression analysis

Alterations in the expression of genes (MEG3, DNMT1, DNMT3A, DNMT3B, p53, and HOTAIR) after 48 h exposure to DNC (Fig. 4a-j, m and p) and miRNA genes (miR-29a and miR-185) after 24 h exposure to DNC (Fig. 4k, 1, n and o) in cancerous cells were evaluated by Real-Time qRT-PCR. Differently expressed genes and the amount of difference between treated and control cells are shown in Table 3.

Analysis of gene expression of main genes including MEG3, DNMT1, DNMT3A, DNMT3B and reference gene GAPDH in time dependent manner was done in 12 h, 24 h and 48 h under treatment of DNC in HuH-7 and HepG2 cell lines (Fig. S2). This data showed DNC in time dependent manner significantly, P < 0.05, alters expression of MEG3, DNMT1, DNMT3A, DNMT3B relative quantification to reference gene GAPDH.

MEG3 promoter hypermethylation

Bisulfite sequencing

MEG3 promoter was found to be methylated in cancerous cell lines whereas it was found to be unmethylated in normal cells. Hence, hepatocellular cancerous cell lines were used to study the potential of DNC to cause reversal of hypermethylation of MEG3 promoter. Reversal of promoter hypermethylation of MEG3 gene after treatment with DNC was comparatively evaluated with untreated cells using bisulfite sequencing. CpG sites were screened in two region selected according to the study by Zhao et al. (2005), and bioinformatics analysis using Methyl Primer Express Software (Applied Biosystems) and CpG Island Searcher, spanned by the core promoter and enhancer like region upper transcription initiation site. The sequences obtained were checked using Chromas software (Technelysium Pty Ltd.). Untreated cells had a methylated signature with all the CpG sites in two regions, whereas treatment with DNC led to hypomethylation of some CpG sites in enhancer like region in the fraction of the DNA templates extracted from treated HuH-7 cells (Fig. 5a) and HepG2 cells (Fig. 5b), which confirmed that DNC can affect CpG methylation condition. For quantitative and more accurate result, sequencing of clones of the whole promoter region would be helpful.

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Discussion

The modulation of expression of IncRNAs by other regulatory RNAs such as miRNAs through epigenetic regulation could be notable aspect of RNA regulatory network. Epi-miRNAs can contribute to the epigenetic landscape of cells by targeting specific epigenetic regulators such as DNA methyltransferases; the miR-29 family can directly target DNMT3A and 3B in lung, hepatoma and leukemia cancers, miR-185 directly controls the activity of DNMT1 in glioma and cervical cancers (Fabbri et al. 2007; Garzon et al. 2009; Braconi et al. 2011; Zhang et al. 2011; Xiang et al. 2013). Epigenetic related roles of miRNA including miR-29a and miR-185 in carcinogenesis and importance of IncRNAs such as MEC3 as potential downstream targets for RNA based epigenetic therapeutic intervention have been extended (Fabbri et al. 2007; Xiong et al. 2010; Braconi et al. 2011; Zhang et al. 2011). At present, epigenetic therapy refers to the use of agents with hypomethylating and histone deacetylating inhibitory activity. Bioactive nutrients including curcumin offer great potential in altering DNA methylation status with the intention to prevention and treatment of cancer. Curcumin affects the regulation of some histone deacetylases, histone acetyltransferases, miRNAs and DNMT1. Also there are some contradictory reports about its effects on DNMTs expression (Reuter et al. 2011). In this study miRNA-dependent regulation of MEG3 expression under treatment with DNC was assessed by evaluating the modulatory involvement of miR-29a for DNMT3A and 3B and miR-185 for DNMT1. As Fig. 5 shows DNA hypomethylation partly occurs under treatment with DNC in MEG3 promoter. We found that DNC dependent overexpression of miR-29a and miR-185 can down-regulate the expression of DNMT1,3A and 3B, and subsequently increases the expression of MEC3 in HCC (Fig. 4) through DNA hypomethyaltion. DNC uses the inter-relationship between two classes of ncRNAs, miRNAs and IncRNAs, and epigenetic regulation of gene expression in HCC for impeding with cancerous tumult. HCT116[p53.sup.+/+] cells transfected with MEC3 displayed significant enhancement in p53 protein levels. To gain insight into the features that transcription stimulation of MEG3 by DNC affects p53 in hepatoma cell lines, we assessed p53 expression at RNA level under DNC treatment. We found that p53 RNA levels in HepG2 cells (wild-type p53) do not display significant changes under DNC treatment but show significant decrease (P < 0.01) in HuH-7 cells with a different p53 status, mutated p53 (Fig. 4). p53 mutants through suppressing the expression of p53-target genes potentially augments drug resistance in cancer cells especially HCC (Vikhanskaya et al. 2007). DNC effects HuH-7 cells, shedding light that increase of p53 protein levels cannot occur following induction of MEG3 expression in mutated p53 cells, besides oncogenic mutant p53 was suppressed by DNC. So DNC potentially detracts drug resistance in HuH-7 cells. However we should do more studies to reveal it accurately. An imbalance between tumor suppressor genes and oncogenes expression is thought to be involved in molecular pathogenesis of HCC. Besides tumor suppressor IncRNA MEG3, we also assessed expression of oncogenic IncRNA EIOTA1R in the present study. HOTAIR gene was significantly over-expressed in HCC tissues compared with margin non-tumor tissues and associated with HCC progression and metastasis (Geng et al. 2011). DNC treatment decreased expression of HOTAIR significantly in HuH-7 cells but had no significant effect on its expression in HepG2 cells. Cheng et al. (2011) have explained high expression of EZH2 protein, a member of PRC2 complex in HuH-7 cell line. We also assessed the expression of HOTAIR in a glioblastoma cell line, U87MG (Fig. SI). DNC treatment had not any significant effect on its expression in U87MG, whereas there is not any report about oncogenic role of HOTAIR in' glioma up to now. Consistent with these findings, it could be realized that DNC targets HOTAIR in special circumstances, when it has oncogenic effects and the other side of equation, PRC2 members are markedly available. Eventually, DNC appears to interfere selectively with metastasis by means of repression of HOTAIR expression in HuH-7.

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Conclusion

Considering the above-mentioned findings, it seems that function and interaction of DNC with cell tactics in RNA-based epigenetic manner occur in a specific way. These results provide the evidence that DNC can induce DNA hypomethylation in part via up-regulation of epi-miRs, subsequently re-express silenced tumor suppressor genes including MEG3, and on the other hand DNC suppresses oncogenes including HOTAIR, targeting the histone modifying complexes, in HCC cells. These findings suggest that DNC potentially could be an effective choice for HCC epigenetic therapy.

Conflict of interest

The authors report no conflicts of interest in this work.

ARTICLE INFO

Article history:

Received 5 December 2014

Revised 30 January 2015

Accepted 25 May 2015

Abbreviations: DNC, dendrosomal curcumin; HCC, hepatocellular cancer; LncRNA, long non-coding RNA; MEG3, maternally expressed gene 3; HOTAIR, HOX transcript antisense RNA; DNMT1, DNA (cytosine-5-)-methyltransferase 1; DNMT3A, DNA (cytosine5-)-methyltransferase 3 alpha; DNMT3B, DNA (cytosine-5-)-methyltransferase 3 beta; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MiR-185, microRNA-185; MiR-29a, microRNA-29a; EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; PRC2, polycomb repressive complex 2; HoxD, homeobox D; HoxC, homeobox C; MTT, methylthiazol tetrazolium; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSP, bisulfite sequencing PCR; Real-Time qRT-PCR, Real-Time quantitative reverse transcription-PCR; SqRT-PCR, semi-quantitative reverse transcription PCR.

Acknowledgments

The authors gratefully acknowledge the Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran, and Deputy of Research, Ministry of Health and Medical Education, Tehran, Iran for supporting us in performing the project. This work is dedicated to the memory of Prof. Mohammad Nabi Sarbolouki who first constructed dendrosome and is presented to the scientific community with the hope of more advances in cancer therapy.

Supplementary Materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.05.071.

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Mina Zamani (a), Majid Sadeghizadeh (a),*, Mehrdad Behmanesh (3), Farhood Najafi (b)

(a) Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

(b) Department of Resin and Additives, Institute for Color Science and Technology, Tehran, Iran

* Corresponding author. Tel: +98 21 8288 4409; fax: +98 21 8800 7598.

E-mail address: sadeghma@modares.ac.ir(M.Sadeghizadeh).

http://dx.doi.org/10.1016/j.phymed.2015.05.071
Table 1
Primers sequence used for MSP and BSP.

                                                            Ampliqon
Primer                   Sequence                           length (bp)

BSP forward              5'-ATGTTTTTGTGGCGTTGTAGGGT-3'      256
BSP reverse (region 1)   5-CTAACCGAAACCAATCAACAACCAA-3'
BSP forward              5'-TTTTAGAGAAATGAGCGTATTGTAGT-3'   277
BSP reverse (region 2)   5-CCAAAAAAACAAAACCCATCCCAA-3'

Bp: base pair

BSP: bisulfite sequencing PCR

Table 2
Primers sequence used for q-PCR.

                                                     Ampliqon
Gene     Primer    Sequence                          length (bp)

DNMT1    Forward   5'-GAAGGAGCCCGTGGATG-3'           233
         Reverse   5'-GTTGATGTCTGCGTGGTAG-3'
DNMT3A   Forward   5-TACGCACCACCTCCAC-3'             no
         Reverse   5'-AGATGTCCTCAATGTTCC-3'
DNMT3B   Forward   5-CGACCTCACAGACGACAC-3'           170
         Reverse   5'-TTCCAAACTCCTTCCCATCC-3'
HOTAIR   Forward   5'-CCAAACAGAGTCCGTTCAGTG-3'       no
         Reverse   5-TACACAAGTAGCAGGGAAAGG-3'
GAPDH    Forward   5-GTGAACCATGAGAAGTATGACAAC-3'     123
         Reverse   5-CATGAGTCCTTCCACGATACC-3'
MEG3     Forward   5'-CGGCTGAAGAACTGCGGATGG-3'       198
         Reverse   5'-CGTGGCTGTCGAGGGATrTCG-3'
p53      Forward   5-TCCTCAGCATCTTATCCGAGTG-3'       265
         Reverse   5'-AGGACAGGCACAAACACGCACC-3'

bp: base pair.

Table 3
Relative levels of non-coding RNA and coding RNA, assessed by
quantitative Real-Time reverse transcription-polymerase chain
reaction. in dendrosomal curcumin treated samples relative to
the untreated control samples.

Gene      (HepG2) [2.sup.-       P value   Up-or down-
          [DELTA][DELTA]Ct]                regulation
          change of expression
          (mean factor)

DNMT1     0.081057               0.0419    (Down)
DNMT3A    0.012263               0.0088    (Down)
DNMT3B    0.821194               0.0303    (Down)
MEG3      5.553868059            0.0093    (Up)
miR-29a   1.544043               0.3678    (ns)
miR-185   2.174682               0.0071    (Up)
HOTAIR    0.943223               0.9646    (ns)
p53       0.948921               0.9921    (ns)

Gene      (HepG2) [2.sup.-       P value   Up-or down-
          [DELTA][DELTA]Ct]                regulation
          change of expression
          (mean factor)

DNMT1     0.010207               0.0223    (Down)
DNMT3A    0.014923               0.0344    (Down)
DNMT3B    0,464514               0.0378    (Down)
MEG3      4.29583                0.0210    (Up)
miR-29a   3.755633               0.0034    (Up)
miR-185   2.444983               0.0044    (Up)
HOTAIR    0.113602               0.0347    (Down)
p53       0.003273               0.0071    (Down)

NS, no statistically significant differences detected (P > 0.05).

MicroRNA genes were measured relative to U6 and other genes
relative to the housekeeping gene for glyceraldehyde 3-phosphate
dehydrogenase.

Student's t-test was used for statistical analysis.
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Author:Zamani, Mina; Sadeghizadeh, Majid; Behmanesh, Mehrdad; Najafi, Farhood
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Sep 15, 2015
Words:4508
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