Bone is the main site of metastasis from prostate cancer; therefore, it is important to investigate the microRNAs (miRNAs) and mRNA associated with bone metastases from prostate cancer. Since an appropriate mechanical environment is important in the growth of bone, in the present study, the miRNA, mRNA, and long non-coding RNA (lncRNA) profiles of mechanically strained osteoblasts treated with conditioned medium (CM) from PC-3 prostate cancer cells were studied. MC3T3-E1 osteoblastic cells were treated with the CM of PC-3 prostate cancer cells and were simultaneously stimulated with a mechanical tensile strain of 2,500 µε at 0.5 Hz; the osteoblastic differentiation of the MC3T3-E1 cells was then assessed. In addition, the differential expression levels of mRNA, miRNA and lncRNA in MC3T3-E1 cells treated with the CM of PC-3 cells were screened, and some of the miRNAs and mRNAs were verified by reverse transcription-quantitative PCR (RT-qPCR). The signal molecules and signaling pathways associated with osteogenic differentiation were predicted by bioinformatics analysis. The CM of PC-3 prostate cancer cells suppressed osteoblastic differentiation of MC3T3-E1 cells. A total of seven upregulated miRNAs and 12 downregulated miRNAs were selected by sequencing and further verified using RT-qPCR, and related differentially expressed genes (11 upregulated and 12 downregulated genes) were also selected by sequencing and further verified using RT-qPCR; subsequently, according to the enrichment of differentially expressed genes in signaling pathways, nine signaling pathways involved in osteogenic differentiation were screened out. Furthermore, a functional mRNA-miRNA-lncRNA regulatory network was constructed. The differentially expressed miRNAs, mRNAs and lncRNAs may provide a novel signature in bone metastases of prostate cancer. Notably, some of the signaling pathways and related genes may be associated with pathological osteogenic differentiation caused by bone metastasis of prostate cancer.
Bone is associated with electrolyte balance, energy metabolism and mechanical competence; it performs these functions through a continuous homeostatic balance between modeling and remodeling processes that is carried out mainly by osteoblasts (OBs) and osteoclasts (OCs), which act via bone formation and resorption, respectively (
Bone metastasis of prostate cancer leads to abnormal osteoblastic differentiation, and causes fatal osteolytic and osteoblastic abrasions, which results in a poor quality of life in individuals with prostate cancer (
The growth of bone requires an appropriate mechanical environment, osteoblasts, important functional cells in bone, are usually stimulated by a mechanically loaded, and there is a dynamic balance between bone resorption and bone formation (
In the present study, the MC3T3-E1 osteoblastic cells were treated with the CM of prostate cancer cells and physiological mechanical tensile strain (2,500 µε at 0.5 Hz) was simultaneously applied to the cells using a four-point bending device. Subsequently, the osteogenic differentiation of cells under the conditions of pathology (CM and physiological mechanical strain) and physiology (fresh medium and physiological mechanical strain) were detected. The differentially expressed mRNAs and miRNAs in the pathology group were detected and verified by high-throughput RNA sequencing and reverse transcription-quantitative PCR (RT-qPCR). Finally, bioinformatics technology was used to screen and predict the signal molecules and signaling pathways related to bone metastasis.
PC-3 prostate cancer cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in an incubator containing 5% CO2 at 37˚C. PC-3 cells (1x106 ) were grown in a 25-cm2 culture flask in cell culture medium until 60-70% confluent. After two washes with phosphate-buffered saline (PBS), cells were incubated in DMEM containing 1% FBS for 48 h, and the crude CM (CCM) was then collected, filtered through a 0.2-µm sterilizing filter and stored at -80˚C. The PC-3 CM contained 20% PC-3 CCM, and 80% DMEM containing 10% FBS (FBS; Gibco; Thermo Fisher Scientific, Inc.) culture medium.
The MC3T3-E1 mouse pre-osteoblastic cell line (provided by the Institute of Basic Medicine of Peking Union Medical College, Beijing, China), at the third passage, was seeded into mechanical loading dishes, which were reformed from cell culture dishes (Nunc; Thermo Fisher Scientific, Inc.) in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.), mechanical loading dishes were reformed from cell culture dishes (Nunc; Thermo Fisher Scientific, Inc.) according to the previously described method (
At 100% confluence, the medium was replaced with the PC-3 CM in the pathological group and with DMEM in the physiological group. All of the MC3T3-E1 cells were subjected to a mechanical tensile strain of 2,500 µε at 0.5 Hz, 1 h/day for 3 days. The mechanical strain was generated using a specially designed four-point bending device (provided by The Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin, China), as previously described (
After treatment with CM and mechanical stimulation, the MC3T3-E1 cells were lysed by brief sonication at 20 kHz for 1 min on ice in radioimmunoprecipitation (RIPA) lysis buffer (CW Biosciences). The protein concentration of the cell lysates was measured using the Bicinchoninic Acid (BCA) Protein Assay kit (CW Biosciences). ALP activity in the lysates was subsequently measured with an alkaline phosphatase assay kit (cat. no. A059-2-1; Nanjing Jiancheng Bioengineering Institute) using the p-nitrophenyl phosphate method, according to the manufacturer's instructions.
Following treatment, the cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at 37˚C. The fixed cells were incubated with the ALP staining kit (cat. no. D001-2-1; Nanjing Jiancheng Bioengineering Institute) and Alizarin red-S staining kit (cat. no. C0148; Beyotime Institute of Biotechnology) to reveal the calcium deposition of cells, according to the manufacturer's instructions. Cells were then observed under an optical microscope.
Following treatment, the protein extracts of the cells were harvested in RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) and protein concentration in the cell lysates was quantified using the BCA Protein Assay kit. Proteins (15 µg) were then separated by SDS-PAGE on 10% gels and transferred to nitrocellulose membranes, which were blocked with 2.5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) in Tris-buffered saline with 0.1% Tween-20 (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 h. Membranes were then incubated with the following primary antibodies at 4˚C for 15 h: Anti-RUNX-2 (1:200; cat. no. sc-390351) and anti-β-actin (1:1,000; cat. no. sc-8432) (both from Santa Cruz Biotechnology, Inc.). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) at 37˚C for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence detection reagent (Pierce; Thermo Fisher Scientific, Inc.). β-actin in the cell lysates was used as a loading control and data were normalized against the corresponding optical density of β-actin. ImageJ 1.48 software (National Institutes of Health) was used for semi-quantification of bands.
Total RNA was isolated from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA purity was assessed using the ND-1000 Nanodrop (NanoDrop; Thermo Fisher Scientific, Inc.). Each RNA sample had an A260:A280 ratio >1.8 and an A260:A230 ratio >2.0. RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies, Inc.) and each sample had the RNA integrity number >7.0. Briefly, RNA molecules were ligated with a 3' RNA adapter, followed by 5' adapter ligation, using a Truseq™ Small RNA sample prep Kit (cat. no. RS-200-0012; Illumina, Inc.). Subsequently, the adapter-ligated RNAs were reversed transcribed to cDNA and PCR amplified. cDNA was synthesized using an AmpliSeq cDNA Synthesis for Illumina kit (cat. no. 20022654; Illumina, Inc.), the thermocycling conditions used for reverse transcription were 12˚C for 15 min and 95˚C for 3 min. PCR amplification was performed using the Illumina® DNA PCR-Free Sequencing and Indexing Primer kit (cat. no. 20041797; Illumina, Inc.), the thermocycling conditions for PCR amplification were 94˚C for 180 sec, followed by 20 cycles of 98˚C for 20 sec, annealing at 56˚C for 15 sec and extension at 72˚C for 15 sec. Subsequently, the size of PCR products was selected by PAGE gel according to the instructions of the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (cat. no. E7330S; New England Biolabs, Inc.). The purified library products were evaluated using the Agilent 2200 TapeStation and the Qubit® 2.0 Fluorometer (Invitrogen; Thermo Fisher Scientific, Inc.), then diluted to 2 pM for sequencing. The type of sequencing performed was single-end from 3' ends and 50 bp was sequenced each time using HiSeq X Reagent Kits (cat. no. FC-501-2501; Illumina, Inc.).
Differential expression analysis was conducted using miRDeep2 (v2.0.0) software (
Briefly, the total RNA quality was assessed as in the previous subsection, rRNAs were removed from total RNA using Epicentre Ribo-Zero rRNA Removal Kit (Illumina, Inc.) and fragmented to ~200 bp. Subsequently, the purified RNAs were subjected to first strand and second strand cDNA synthesis according to the aforementioned method, followed by adaptor ligation and enrichment with a low-cycle according to instructions of the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (cat. no. E7420L; New England Biolabs, Inc.). The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit 2.0 Fluorometer, and the final library was diluted to 10 pM for sequencing. The type of sequencing was paired-end from 3' ends and 150 bp was sequenced each time using HiSeq X Reagent Kits.
The differential expression analysis was conducted using DEGseq 1.38.0 software (
RT-qPCR was performed to further validate selected differentially expressed mRNAs and miRNAs identified from small RNA sequencing and transcriptome sequencing.
Total RNA was isolated from cells using TRIzol reagent, according to the manufacturer's protocol. After total RNA was extracted from the cells, cDNA was synthesized using a Quant Script RT kit (Tiangen Biotech Co., Ltd.); the thermocycling conditions for RT were 12˚C for 15 min and 95˚C for 3 min. qPCR was performed to detect the mRNA expression levels using SYBR Green I PCR mix (CW Biosciences) on a Real-Time PCR system (7900; Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The amplification reaction included a denaturation step at 94˚C for 180 sec, followed by 40 cycles of 94˚C for 15 sec, annealing and extension at 60˚C for 30 sec. Each sample was analyzed in triplicate. The expression levels of mRNA were normalized to the internal control β-actin using the 2-ΔΔCq method (
The miRNA expression levels were assessed using RT-qPCR by Guangzhou Ribobio Co., Ltd. The miRNA Uni-Reverse Primers and miRNA Primers (specific primers) for RT-qPCR of miRNA were provided by Guangzhou Ribobio Co., Ltd. Poly(A) tailing. Total RNA was isolated from cells using TRIzol reagent, according to the manufacturer's protocol. After total RNA was extracted from the cells cDNA was synthesized by reverse transcription according to the aforementioned method and qPCR was successively performed using the miDETECT A Track miRNA qRT-PCR Starter Kit (Guangzhou Ribobio Co., Ltd.). The reactions were incubated in a 96-well optical plate at 95˚C for 20 sec, followed by 40 cycles of 10 sec at 95˚C, 20 sec at 60˚C and 10 sec at 70˚C. The miRNA expression levels were normalized to U6 snRNA. The miRNA primer sequences are shown in
According to the results of the differential expression analysis, the regulatory relationships between long non-coding RNA (lncRNA) and miRNA were predicted using miRanda (
Since miRNAs serve their biological roles through regulating the expression of target genes at the post-transcriptional level, target genes of the miRNAs were predicted using online software, including miRDB (
Experiments were performed in triplicate as a minimum, and data from RT-qPCR, the ALP activity assay and western blotting are presented as the mean ± standard deviation. To identify differentially expressed miRNAs and mRNAs among the groups and to compare differences between groups, unpaired Student's t-test was performed using SPSS 18.0 (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.
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The KEGG enrichment analyses predicted certain signaling pathways were associated with osteoblastic differentiation, including ‘Wnt signaling pathway’, ‘MAPK signaling pathway’, ‘Calcium signaling pathway’, ‘Osteoclast differentiation’, ‘Pathways in cancer’, ‘TNF signaling pathway’, ‘HIF-1 signaling pathway’, ‘MicroRNAs in cancer’ and ‘Rheumatoid arthritis’ (
Based on the RT-qPCR results of miRNAs and mRNAs, and KEGG analysis, the complete targeting regulatory relationships of mRNAs, miRNAs and signaling pathways were listed. Seven differently expressed mRNAs, and associated miRNAs and signaling pathways were selected (
Bone is responsive to dynamic mechanical loading; a suitable dynamic mechanical loading maintains the dynamic balance of bone formation (
In the present study, ALP activity, the mRNA expression levels of Col I and OCN, calcium deposition and the protein expression levels of Runx-2 in MC3T3-E1 cells were all decreased in the pathology group, which was treated with the PC-3 CM. These results indicated that the PC-3 CM inhibited the osteogenic differentiation of MC3T3-E1 cells
Furthermore, bioinformatics analysis identified 23 miRNAs and 28 mRNAs from the differential expression profiles and verified them with RT-qPCR. Compared with in the physiological group, there were seven upregulated and 12 downregulated miRNAs, and 10 upregulated and 11 downregulated mRNAs, which were consistent with the results of next generation sequencing. The predicted target mRNAs of the differentially expressed miRNAs implied potential regulatory mechanisms of the miRNA targets in osteoblastic differentiation resulting from the treatment of PC-3 CM.
Moreover, the differentially expressed mRNAs and miRNA-predicted ‘target’ genes were matched to strengthen the significance of the selected genes and miRNAs, these genes and miRNAs mediated osteoblastic differentiation which was the result of treatment of PC-3 CM. In addition, nine potential pathways, which may be involved in bone metabolism during prostate cancer progression and bone metastasis, were revealed, as follows: ‘Wnt signaling pathway’, ‘MAPK signaling pathway’, ‘Calcium signaling pathway’, ‘Osteoclast differentiation’, ‘Pathways in cancer’, ‘TNF signaling pathway’, ‘HIF-1 signaling pathway’, ‘MicroRNAs in cancer’, ‘Rheumatoid arthritis’. Relevant literature have demonstrated that these nine pathways are all associated with cancer progression or osteoblastic differentiation (
At present, evidence has supported the ‘seed and soil’ model by Paget, which proposes a specific and strong interaction between metastatic prostate cancer cells and the bone microenvironment (
Currently, widely used cell lines for prostate cancer research include PC3, DU145 or LNCaP cells (
The present findings suggested that genetic interactions (miRNA-gene-signaling pathway) may be associated with the bone metastasis of prostate cancer. Molecular alterations in gene expression may therefore represent a novel signature in pathological osteoblastic differentiation of prostate cancer, which could be used to develop diagnostic and therapeutic strategies for patients with prostate cancer and pathological osteoblastic differentiation caused by bone metastasis. However, these target genes and miRNAs require further verification in subsequent studies, and the mechanism underlying the involvement of differentially expressed miRNAs and genes in the osteoblasts after bone metastasis of prostate cancer also need further investigation.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The sequencing datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus repository (
YG designed the study, analyzed the data and participated in the bioinformatics analysis. ZC performed all the RT-qPCR analyses at Guangzhou RiboBio Co., Ltd. (experimental equipment and most of the reagents were provided by Guangzhou RiboBio Co., Ltd.). ZXY performed ALP activity and Alizarin red-S staining. JHW performed western blotting. HY, BH and JTG performed the bioinformatics analysis. YG and ZC wrote and revised the manuscript. YG and ZC confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Osteoblastic differentiation of MC3T3-E1 cells treated with PC-3 CM and stimulated with mechanical strain. All of the MC3T3-E1 cells were stimulated with a mechanical tensile strain of 2,500 µε at 0.5 Hz. PC-3 CM decreased the (A) ALP activity, (B) mRNA expression levels of Col and OCN, (C) Runx2 protein expression levels and (D) the activity of ALP and Ca deposition of the MC3T3-E1 cells. *P<0.05 and **P<0.01 vs. control group (only stimulated with mechanical strain). ALP, alkaline phosphatase; Ca, calcium; CM, conditioned medium; Col I, collagen I; OCN, ostocalcin.
Heat map of differentially expressed miRNAs from MC3T3-E1 cells subjected to mechanical tensile strain and treated with PC-3 CM, as determined by RNA sequencing. Control group, stimulated with mechanical strain; PC-3 CM group, stimulated with mechanical strain and treated with PC-3 CM. The colors represent relative levels of miRNAs. CM, conditioned medium; miRNA/miR, microRNA.
Expression levels of differentially expressed miRNAs were verified using reverse transcription-quantitative PCR. Seven miRNAs were upregulated (miR-499-5p, miR-217-5p, miR-6412, miR-9-3p, miR-505-3p, miR-96-5p, miR-542-5p) and 12 were downregulated (miR-467h, miR-3473d, miR-466j, miR-497b miR-3963, miR-133a-3p, miR-219c-5p, miR-181a-2-3p, miR-1224-3p, miR-190b-5p, miR-470-5p, miR-33-5p), consistent with the results of sequencing. *P<0.05, **P<0.01 and ***P<0.001 vs. control. CM, conditioned medium; miRNA/miR, microRNA.
Heat map of differentially expressed mRNAs from MC3T3-E1 cells subjected to mechanical tensile strain and treated with PC-3 CM, as determined by RNA sequencing. Control group, stimulated with mechanical strain; PC-3 CM group, stimulated with mechanical strain and treated with PC-3 CM. The colors represent relative levels of mRNAs. CM, conditioned medium.
Expression levels of differentially expressed mRNAs were verified using reverse transcription-quantitative PCR. A total of 10 mRNAs were upregulated (Bc13, Pfkfb3, Mmp3, Bdkrb1, Nos2, Tnnc1, Il6, Timp1, Angpt4, Porcn) and 11 were downregulated (Angpt1, Lef1, Nfatc2, Tnc, Cdc25b, Nr4a1, Fos, Cdca8, Fosb, Kif23, Cdc25c), consistent with the results of sequencing. *P<0.05, **P<0.01 and ***P<0.001 vs. control. CM, conditioned medium.
Heat map of differentially expressed lncRNAs from MC3T3-E1 cells subjected to mechanical tensile strain and treated with PC-3 CM, as determined by RNA sequencing. Control group, stimulated with mechanical strain; PC-3 CM group, stimulated with mechanical strain and treated with PC-3 CM. The colors represent relative levels of lncRNAs. CM, conditioned medium; lncRNA, long non-coding RNA.
lncRNA-miRNA-mRNA ceRNA network based on sequencing. Red ovals represents miRNAs, green rhombuses represent mRNAs, blue ovals represent lncRNAs. lncRNA, long non-coding RNA; miRNA/miR, microRNA; ceRNA, competing endogenous RNA.
Primer sequence for all mRNAs.
mRNA | Primer sequences, 5'-3' |
---|---|
Col I | F: GGTATGCTTGATCTGTATCT |
R: TCTTCTGAGTTTGGTGATACG | |
OCN | F: AGTCTGACAAAGCCTTCA |
R: AAGCAGGGTTAAGCTCACA | |
Nr4a1 | F: TGGTGAAGGAAGTTGTGCGT |
R: ATCAAGGTCTCTGGGCGTTG | |
Porcn | F: CTACGAGCCTACGAGAGTGCT |
R: CTGTGGCCTCAGACAGAAAGC | |
Angpt1 | F: ATCCCGACTTGAAATACAACTGC |
R: CTGGATGATGAATGTCTGACGAG | |
Lef1 | F: GCCACCGATGAGATGATCCC |
R: TTGATGTCGGCTAAGTCGCC | |
Fos | F: CGGGTTTCAACGCCGACTA |
R: TGGCACTAGAGACGGACAGAT | |
Cdca8 | F: AAAAGCGAAAGGTAATCGAGGT |
R: TGCAGATCGAAGATTCTTATGGC | |
Bcl3 | F: CCGGAGGCCCTTTACTACCA |
R: GGAGTAGGGGTGAGTAGGCAG | |
Fosb | F: CCTCCGCCGAGTCTCAGTA |
R: CCTGGCATGTCATAAGGGTCA | |
Bdkrb1 | F: CCCCTCCCAACATCACCTC |
R: GGACAGGACTAAAAGGTTCCCC | |
Osmr | F: GCATCCCGAAGCGAAGTCTT |
R: GGGCTGGGACAGTCCATTCTA | |
Podn | F: GACTGTCCCCGAGATTGTGC |
R: CAGGTCGCCTGGAAACTCA | |
Prc1 | F: AACTCACCTCCGGGAAATATGG |
R: GGATATGCTTTTGAGCAGCCT | |
Pfkfb3 | F: CAACTCCCCAACCGTGATTGT |
R: TGAGGTAGCGAGTCAGCTTCT | |
Nos2 | F: GTTCTCAGCCCAACAATACAAGA |
R: GTGGACGGGTCGATGTCAC | |
Tnnc1 | F: GCGGTAGAACAGTTGACAGAG |
R: GACAAGAAACTCATCGAAGTCCA | |
Slc6a2 | F: TTCTGGCGCGAATGAATCC |
R: AGTAGATCGGCGGTTTTGCAG | |
Nfatc2 | F: TCATCCAACAACAGACTGCCC |
R: GGGAGGGAGGTCCTGAAAACT | |
Tnc | F: TTTGCCCTCACTCCCGAAG |
R: AGGGTCATGTTTAGCCCACTC | |
Car9 | F: GGTTAGAGGATCTATCGACTCCC |
R: GGTGCCTCCATAGCTCCAA | |
Timp1 | F: CGAGACCACCTTATACCAGCG |
R: ATGACTGGGGTGTAGGCGTA | |
Kif23 | F: ATGAAGTCAGCGAAGGCTAAGA |
R: GCGAACCCTACAGTACACCC | |
Angpt4 | F: CAGCCAGCTATGCTACTAGATGG |
R: CAGGCAAGTCCCTCTGGAG | |
Cdc25c | F: GTTTCAGCACCCAGTTTTAGGT |
R: AGAATGCTTAGGTTTGCCGAG | |
Mmp3 | F: GGCCTGGAACAGTCTTGGC |
R: TGTCCATCGTTCATCATCGTCA | |
Prc | F: GAGTCAGTCACAACAGATGC |
R: TGCACAAGATACACCTTCATCC | |
β-actin | F: CACCATTGGCAATGAGCGGTTC |
R: AGGTCTTTGCGGATGTCCACGT |
F, forward; R, reverse.
Primer sequences of all miRNAs.
miRNA | miRBase accession no. | Primer sequence, 5'-3' |
---|---|---|
mmu-miR-190b-5p | MIMAT0004852 | TGATATGTTTGATATTGGGTT |
mmu-miR-296-5p | MIMAT0000374 | AGGGCCCCCCCTCAATCCTGT |
mmu-miR-33-5p | MIMAT0000667 | GTGCATTGTAGTTGCATTGCA |
mmu-miR-1224-3p | MIMAT0017231 | CCCCACCTCTTCTCTCCTCAG |
mmu-miR-3963 | MIMAT0019341 | TGTATCCCACTTCTGACAC |
mmu-miR-3473d | MIMAT0020632 | CCACTGAGCCACTTTCCAGCCCTT |
mmu-miR-219c-5p | MIMAT0029892 | GGACGTCCAGACGCAACTCTCG |
mmu-miR-505-3p | MIMAT0003513 | CGTCAACACTTGCTGGTTTTCT |
mmu-miR-467h | MIMAT0005855 | ATAAGTGTGTGCATGTATATGT |
mmu-miR-96-5p | MIMAT0000541 | TTTGGCACTAGCACATTTTTGCT |
mmu-miR-466m-3p | MIMAT0014883 | TACATACACACATACACACGCA |
mmu-miR-466j | MIMAT0005848 | TGTGTGCATGTGCATGTGTGTAA |
mmu-miR-542-5p | MIMAT0003171 | CTCGGGGATCATCATGTCACGA |
mmu-miR-6412 | MIMAT0025165 | TCGAAACCATCCTCAGCTACTA |
mmu-miR-181a-2-3p | MIMAT0005443 | ACCGACCGTTGACTGTACCTTG |
mmu-miR-133a-3p | MIMAT0000145 | TTTGGTCCCCTTCAACCAGCTG |
mmu-miR-470-5p | MIMAT0002111 | TTCTTGGACTGGCACTGGTGAGT |
mmu-miR-497b | MIMAT0031404 | CACCACAGTGTGGTTTGGACGTGG |
mmu-miR-7083-5p | MIMAT0028072 | TCGGGGCTGGACAAGCAGAGA |
mmu-miR-9-3p | MIMAT0000143 | ATAAAGCTAGATAACCGAAAGT |
mmu-miR-3061-3p | MIMAT0014829 | CTACCTTTGATAGTCCACTGCC |
mmu-miR-217-5p | MIMAT0000679 | TACTGCATCAGGAACTGACTGGA |
mmu-miR-499-5p | MIMAT0003482 | TTAAGACTTGCAGTGATGTTT |
Universal reverse primer | GTGCAGGGTCCGAGGT | |
U6 | F: CTCGCTTCGGCAGCACA | |
R: AACGCTTCACGAATTTGCGT |
miR/miRNA, microRNA; F, forward; R, reverse.
Enrichment analyses of KEGG pathway identified certain signaling pathways related to osteoblastic differentiation.
Target pathway | Target genes |
---|---|
‘Wnt signaling pathway’ | Nfatc2, Lef1, Porcn |
‘MAPK signaling pathway’ | Nr4a1, Dusp6, Cdc25b, Fos |
‘Calcium signaling pathway’ | Bdkrb1, Sphk1, Nos2, Tnnc1 |
‘Osteoclast differentiation’ | Nfatc2, Fosb, Fos |
‘Pathways in cancer’ | Bdkrb1, Il6, Nos2, Lef1, Fos |
‘TNF signaling pathway’ | Mmp3, Il6, Fos, Bcl3 |
‘HIF-1 signaling pathway’ | Angpt1, Angpt4, Timp1, Nos2, Pfkfb3, Il6 |
‘MicroRNAs in cancer’ | Tnc, Cdc25c, Kif23, Cdc25b |
‘Rheumatoid arthritis’ | Mmp3, Cxcl5, Angpt1, Il6, Fos |
Targeting regulatory relationships among mRNAs, miRNAs and signaling pathways based on the results of reverse transcription-quantitative PCR and Kyoto Encyclopedia of Genes and Genomes analysis.
miRNA | Gene | |||
---|---|---|---|---|
Up | Down | Up | Down | Signaling pathway |
miR-466j, miR-33-5p | Angpt1 | ‘HIF-1 signaling pathway’, ‘Rheumatoid arthritis’ | ||
miR-96-5p | miR-6412, miR-467h | Cdc25b | ‘MAPK signaling pathway’, ‘MicroRNAs in cancer’ | |
miR-505-3p | Lef1 | ‘Wnt signaling pathway’, ‘pathway in cancer’ | ||
miR-33-5p | miR-3473,d miR-1224-3p | Nfactd2 | ‘Wnt signaling pathway’, ‘Osteoclast differentiation’ | |
miR-219c-5p | miR-1224-3p, miR-542-5p | Tnc | ‘MicroRNAs in cancer’ | |
miR-466j | Bcl3 | ‘TNF signaling pathway’ | ||
miR-3473d, miR-217-5p, miR-133a-3p, miR-1224-3p | Pfkfb3 | ‘HIF-1 signaling pathway’ |