Mir151 conventional knockout mice
9.3. The potential application of Mir151 knockout mice
The recent discovery of a miR-151-3p target gene ATP2A2 (19), which encodes for
a slow skeletal and cardiac muscle specific Ca2+ ATPase (SERCA2) has provide a new
insight into the understanding of MIR151A. SERCA2 is essential for Ca2+ uptake during
excitation–contraction coupling in cardiomyocytes. Impaired Ca2+ uptake resulting from
decreased expression and reduced activity of SERCA2a is a hallmark of heart failure
(34), and the efficacy of SERCA2a restoration has been proven in improving the disease
(35-37). Due to the important role of SERCA2 in both healthy and disease hearts, and
Mir151 depletion in whole body has no significant deleterious effects on organogenesis
and survival, it could be a future therapeutics.
150
Chapter 10. Conclusion and Prospective
In conclusion, our preliminary data obtained from N1F2 mice has revealed that
Mir151 is not essential to survival, the physiological effect of Mir151 deficiency is mild,
and has showed the inconsistent results of urethane-induced lung tumorigenesis
compared with N10F2 mice. Using the Mir151 conventional knockout mice with pure
C57BL/6J genetic background, increasing the sample size, and giving appropriate
stimulus to mice such as chronic hypoxia, carcinogen-, or oncogene-induced cancer
model may contribute to understand the physiological function and pathological role of
Mir151 gene in the future.
In addition to conventional knockout mice, we have also generated the Mir151
conditional knockout mice which can be used to specific deplete Mir151 in the use of
Cre recombinase driven by myocardial-specific promoter. It may provide an opportunity
to confirm whether the Mir151 regulates heart expressed SERCA2 in vivo and evaluate
the therapeutic potential of miR-151 in heart failure.
151
Bibliography
1. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001 Oct 26; 294(5543):
853-858.
2. Friedlander MR, Lizano E, Houben AJ, Bezdan D, Banez-Coronel M, Kudla G, et al. Evidence for the biogenesis of more than 1,000 novel human microRNAs.
Genome biology 2014; 15(4): R57.
3. Choudhury NR, de Lima Alves F, de Andres-Aguayo L, Graf T, Caceres JF, Rappsilber J, et al. Tissue-specific control of brain-enriched miR-7 biogenesis.
Genes & development 2013 Jan 1; 27(1): 24-38.
4. Kim VN. MicroRNA biogenesis: coordinated cropping and dicing. Nature reviews Molecular cell biology 2005 May; 6(5): 376-385.
5. Fabian MR, Sonenberg N, Filipowicz W. Regulation of mRNA translation and stability by microRNAs. Annual review of biochemistry 2010; 79: 351-379.
6. Subramanyam D, Blelloch R. From microRNAs to targets: pathway discovery in cell fate transitions. Current opinion in genetics & development 2011 Aug; 21(4):
498-503. research 2008 Oct 21; 1236: 185-193.
9. Gatt ME, Zhao JJ, Ebert MS, Zhang Y, Chu Z, Mani M, et al. MicroRNAs 15a/16-1 function as tumor suppressor genes in multiple myeloma. Blood 2010 Oct 20.
10. Ding J, Huang S, Wu S, Zhao Y, Liang L, Yan M, et al. Gain of miR-151 on
152
chromosome 8q24.3 facilitates tumour cell migration and spreading through downregulating RhoGDIA. Nature cell biology 2010 Apr; 12(4): 390-399.
11. Lips EH, van Eijk R, de Graaf EJ, Oosting J, de Miranda NF, Karsten T, et al.
Integrating chromosomal aberrations and gene expression profiles to dissect rectal tumorigenesis. BMC cancer 2008; 8: 314.
12. Luedde T. MicroRNA-151 and its hosting gene FAK (focal adhesion kinase) regulate tumor cell migration and spreading of hepatocellular carcinoma.
Hepatology 2010 Sep; 52(3): 1164-1166.
13. Uchida M, Tsukamoto Y, Uchida T, Ishikawa Y, Nagai T, Hijiya N, et al.
Genomic profiling of gastric carcinoma in situ and adenomas by array-based comparative genomic hybridization. The Journal of pathology 2010 May; 221(1):
96-105.
14. Sayed D, Hong C, Chen IY, Lypowy J, Abdellatif M. MicroRNAs play an essential role in the development of cardiac hypertrophy. Circulation research 2007 Feb 16; 100(3): 416-424.
15. Fulci V, Colombo T, Chiaretti S, Messina M, Citarella F, Tavolaro S, et al.
Characterization of B- and T-lineage acute lymphoblastic leukemia by integrated analysis of MicroRNA and mRNA expression profiles. Genes, chromosomes &
cancer 2009 Dec; 48(12): 1069-1082.
16. Agirre X, Jimenez-Velasco A, San Jose-Eneriz E, Garate L, Bandres E, Cordeu L, et al. Down-regulation of hsa-miR-10a in chronic myeloid leukemia CD34+
cells increases USF2-mediated cell growth. Molecular cancer research : MCR 2008 Dec; 6(12): 1830-1840.
17. Barnabas N, Xu L, Savera A, Hou Z, Barrack ER. Chromosome 8 markers of metastatic prostate cancer in African American men: gain of the MIR151 gene and loss of the NKX3-1 gene. The Prostate 2011 Jun 1; 71(8): 857-871.
18. Liu Y, Cai H, Liu J, Fan H, Wang Z, Wang Q, et al. AmiR-151binding site polymorphism in the 3'-untranslated region of the cyclin E1 gene associated with nasopharyngeal carcinoma. Biochemical and biophysical research communications 2013 Feb 14.
153
19. Wei H, Li Z, Wang X, Wang J, Pang W, Yang G, et al. microRNA-151-3p regulates slow muscle gene expression by targeting ATP2a2 in skeletal muscle cells. Journal of cellular physiology 2015 May; 230(5): 1003-1012.
20. Liu P, Jenkins NA, Copeland NG. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome research 2003 Mar; 13(3): 476-484.
21. Yu IS, Lin SR, Huang CC, Tseng HY, Huang PH, Shi GY, et al. TXAS-deleted mice exhibit normal thrombopoiesis, defective hemostasis, and resistance to arachidonate-induced death. Blood 2004 Jul 1; 104(1): 135-142.
22. Jelkmann W. Regulation of erythropoietin production. The Journal of physiology 2011 Mar 15; 589(Pt 6): 1251-1258.
23. Stockmann C, Fandrey J. Hypoxia-induced erythropoietin production: a paradigm for oxygen-regulated gene expression. Clinical and experimental pharmacology & physiology 2006 Oct; 33(10): 968-979.
24. Eckardt KU, Kurtz A. Regulation of erythropoietin production. European journal of clinical investigation 2005 Dec; 35 Suppl 3: 13-19.
25. Pagel H, Jelkmann W, Weiss C. Erythropoietin production in the isolated perfused kidney. Biomedica biochimica acta 1990; 49(2-3): S271-274.
26. Pan X, Suzuki N, Hirano I, Yamazaki S, Minegishi N, Yamamoto M. Isolation and characterization of renal erythropoietin-producing cells from genetically produced anemia mice. PloS one 2011; 6(10): e25839.
27. Gruber M, Hu CJ, Johnson RS, Brown EJ, Keith B, Simon MC. Acute postnatal ablation of Hif-2alpha results in anemia. Proceedings of the National Academy of Sciences of the United States of America 2007 Feb 13; 104(7): 2301-2306.
28. Kapitsinou PP, Liu Q, Unger TL, Rha J, Davidoff O, Keith B, et al. Hepatic HIF-2 regulates erythropoietic responses to hypoxia in renal anemia. Blood 2010 Oct 21; 116(16): 3039-3048.
154
29. Percy MJ, Furlow PW, Lucas GS, Li X, Lappin TR, McMullin MF, et al. A gain-of-function mutation in the HIF2A gene in familial erythrocytosis. The New England journal of medicine 2008 Jan 10; 358(2): 162-168.
30. Bernhardt WM, Wiesener MS, Weidemann A, Schmitt R, Weichert W, Lechler P, et al. Involvement of hypoxia-inducible transcription factors in polycystic kidney disease. The American journal of pathology 2007 Mar; 170(3): 830-842.
31. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O'Rourke J, Mole DR, et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001 Oct 5; 107(1): 43-54.
32. Kellar A, Egan C, Morris D. Preclinical Murine Models for Lung Cancer:
Clinical Trial Applications. BioMed research international 2015; 2015: 621324.
33. Ma Y, Fiering S, Black C, Liu X, Yuan Z, Memoli VA, et al. Transgenic cyclin E triggers dysplasia and multiple pulmonary adenocarcinomas. Proceedings of the National Academy of Sciences of the United States of America 2007 Mar 6;
104(10): 4089-4094.
34. Meyer M, Schillinger W, Pieske B, Holubarsch C, Heilmann C, Posival H, et al.
Alterations of sarcoplasmic reticulum proteins in failing human dilated cardiomyopathy. Circulation 1995 Aug 15; 92(4): 778-784.
35. Kawase Y, Ly HQ, Prunier F, Lebeche D, Shi Y, Jin H, et al. Reversal of cardiac dysfunction after long-term expression of SERCA2a by gene transfer in a pre-clinical model of heart failure. Journal of the American College of Cardiology 2008 Mar 18; 51(11): 1112-1119.
36. Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B, et al.
Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure.
Circulation 2011 Jul 19; 124(3): 304-313.
37. Wahlquist C, Jeong D, Rojas-Munoz A, Kho C, Lee A, Mitsuyama S, et al.
Inhibition of miR-25 improves cardiac contractility in the failing heart. Nature 2014 Apr 24; 508(7497): 531-535.
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Figures
156
Figure 1. miR-151
Precursor structure and sequence of (A) human mir-151a and (B) mouse mir-151.
(C) Sequence of mature miR-151. blue: seed region, red: the difference between human and mouse miR-151-3p.
157
Figure 2. Knockout strategy of Mir151 gene.
Schematic strategy for introduction of the loxP sites flanked the region containing the mmu-mir-151 sequence. Mouse Mir151 gene is located in a large intron between exon 20 and exon 21 of Ptk2 gene. Two loxP sequences (indicated by black triangle), two Frt sequences, and a neomycin-resistant gene (indicated by green arrow) were inserted to the flanked region of Mir151 using a targeting vector. The wild type allele was replaced by the targeting allele through homologous recombination. The ES cells undergone neomycin selection could be transfected with either the Cre recombinase or the Flippase to delete the region between the two loxP or Frt sequences, respectively. The former became a Mir151 conventional knockout allele, while the latter became a conditional knockout allele.
158
Figure 3. Genotyping results.
(A) The primer design for genotyping. A multiplex PCR strategy using two forward primers (m151F2 and m151F3) and one reverse primer (m151R) was applied to detect wild type (WT) allele (393 bp) and knockout (KO) allele (494 bp) simultaneously. (B) Validation of the successful recombination was conducted by Southern blotting, the DNA fragment size of WT and KO allele was 10.8 kb and 9.3 kb, respectively. (C) Genotyping PCR was used as a routine method. The amplicon size of each genotype is as followed: Mir151+/+ 393 bp, Mir151-/- 494 bp, and Mir151+/- 393 and 494 bp.
159
Figure 4. Expression of Mir151 and Ptk2 (host gene).
Relative expression of 5p and 3p of mmu-mir-151 (A) in major organs of wild type mice (n=2) and (B) in heart tissue of each genotype (n=2) were detected by Taqman microRNA assays. (C) Ptk2 (Mir151 host gene) mRNA level was assessed in heart and thymus tissue, which expressed highest and lowest Mir151 level, by SYBR green qRT-PCR (n=2 for each genotype). snoRNA-202 and Gapdh gene were used as internal controls for miRNA and mRNA, respectively.
Bars represent the mean ± SD.
160
161
Figure 5. CBCs and DCs analysis
The CBCs and DCs of Mir151 wild type and knockout mice were monitored every month for more than one year. CBCs and DCs analysis were carried out with whole blood collected by puncture of the retro-orbital plexus of mice using capillary tubes with EDTA•K2 as anticoagulant. The (A) RBC count, (B) hemoglobin, and (C) hematocrit of knockout mice were increased from 4 to 6 months and then gradually decreased, while these in wild type mice remained stable though out the long-term observation. (Mir151+/+ n=8, Mir151-/- n=9) KO v.s WT * P ≤ 0.05, ** P ≤ 0.01 (ANOVA)
162
Figure 6. Epo mRNA level in mouse kidney and liver.
Mir151 N1F2 wild type and knockout mice were sacrificed at the age of 3, 6, and 9 months and then extracted the total RNA from kidney and liver. (A) Renal Epo mRNA level in 3-, 6-, and 9-months-aged mice (n=4 per group at each time-point) and (B) liver Epo mRNA level in 6-months-aged mice (n=2) were determined by SYBR green qRT-PCR. Epo transcript was detected by SYBR green qRT-PCR, and Gapdh gene was used as an internal control. Gene expression was shown in relative to 3 months-aged wild type mice. Bars represent the mean ± SD. *** P ≤ 0.001 (ANOVA)
163
Figure 7. The hypoxia chamber for in vivo study.
With an oxygen sensor and continuous nitrogen supply, this system can reduce oxygen concentration in hypoxia chamber and maintained the oxygen level at 10±0.5%. The exhaust fan excludes the atmospheric air in the chamber, and the circulation fan makes uniform distribution of nitrogen and residual air in the chamber. In the control of timer, the exhaust fan turns around 30 seconds in every five minutes. Three capped cages can be put into the chamber simultaneously.
164
Figure 8. Chronic hypoxia induced the renal Epo mRNA level
N1F2 male mice (5 months old) were exposed to 10±0.5% O2 in the hypoxia chamber under the well control of oxygen sensor. After 0 and 6 hour exposure to chronic hypoxia, the mice were sacrificed and collected the kidney. Renal Epo mRNA was measured by SYBR green qRT-PCR and normalized by Gapdh. Bars represent the mean ± SD. (n=3 for each group) * P ≤ 0.05, ** P ≤ 0.01 (ANOVA)
165
Figure 9. Kidney section stained with hematoxylin and eosin.
(A) 5-month-old mice and (B) 9-month-old N1F2 mice did not revealed the increase of interstitial fibroblasts and renal fibrosis. (A) and (B) top ×50 ; bottom
×200. RC: renal corpuscle; PCT: proximal convoluted tubule; DCT: distal tubule; Red arrow: interstitial fibroblast
166
Figure 10. Expression of Hif-α target genes in the kidney
Total RNA was extracted from whole kidney lysate from N1F2 male mice (6 months). Expression of Hif-α target gene including (A) Epo, (B) Phd3, (C) Pgk, and (D) Vegf A was determined by SYBR green qRT-PCR. Gapdh gene was used as an internal control, and the gene expression in wild type mice was used as a calibrator. Bars represent the mean ± SD. (n=3 for each genotype) * P ≤ 0.05, **
P ≤ 0.01, *** P ≤ 0.001 (ANOVA)
167
Figure 11. CoCl2 treatment increased the expression of renal Epo mRNA.
9-month-aged N1F2 male mice were injected i.p with 60mg/kg of CoCl2 (n=7).
Control mice were injected with PBS (n=5). After 6 hour of injection, the mice were sacrificed and collected the kidney. Renal Epo mRNA was measured by SYBR green qRT-PCR and normalized by Gapdh.
Control CoCl
2168
Figure 12. Survival curve.
A cohort including 124 N1F2 mice (58 male and 66 female) were kept and monitored for more than two years. No difference in survival of these three genotype was found.
169
Figure 13. Examination of the lung in dead mice.
Lung cancer was spontaneously occurred in 23% homozygous and 33%
heterozygous N1F2 Mir151 knockout mice at their end stage. (A) The disease progression was subdivided according to the tumor size. [0: macro- and microscopic normal; 1: macroscopic normal, but tumor lesion can be observed in lung section under microscope; 2: nodule ≦ 2 (smaller than 1/4 lobe), normal part is still remained in the same lobe; 3: nodule > 2 or larger than 1/4 lobe, normal part is still remained in the same lobe; 4: Completely loss of normal tissue in the same lobe.] (B) Lung section stained with hematoxylin and eosin.
170
Figure 14. Urethane induction protocol for elder mice.
Mir151 N1F2 knockout mice (23 weeks old) as well as their littermate controls started to inject I.P. with 1 mg/g body weight urethane once weekly for 6 consecutive weeks. 20 weeks after the first urethane injection, mice were sacrificed, dissected their lungs, and examined the tumor size.
171
Figure 15. Lung cancer induced by urethane injection.
(A) After open the chest of urethane-treated mice, the developing nodules can be observed on the lung surface. (B) Tumor size larger than 1 mm. (C) More than one nodule developed in one lobe. (C) Tumor size smaller than 1mm.
A B
C D
172
Figure 16. Quantification of tumor numbers induced by urethane in elder mice.
Tumor numbers were observed and counted under dissecting microscope. (WT n=5, Het n=6, and KO n=5) Het v.s WT, P=0.068 (Mann-Whitney test).
173
Figure 17. Urethane induction protocol for young mice.
Mir151 N10F2 knockout mice as well as their littermate controls started to inject I.P. at 6–8 weeks of age with 1 mg/g body weight urethane once weekly for 4 consecutive weeks. 36 weeks after the first urethane injection, mice were sacrificed, dissected their lungs, and examined the tumor size.
174
Figure 18. Quantification of tumor numbers induced by urethane in young mice.
Tumor numbers were subdivided according to the size: 0.5 mm (small), 0.8-1.2 mm (medium), and > 1.2 mm (large). Mir151 N10F2 knockout mice showed a significant decreased in small and medium size. (n=12 for each genotype) ** KO v.s WT, P ≤ 0.01; # KO v.s Het, P ≤ 0.05; $ Het v.s WT, P ≤ 0.05; $$ Het v.s WT, P
≤ 0.01 (Mann-Whitney test)
175
Tables
176
GenePrimer sequencePrimer nameSequence Gapdh151-AU5'-GAAAGAGTATCCCTGTGACCC-3' Forward5'-CTGGAGAAACCTGCCAAGTA-3'151-BD5'-AACTCTCCTGCTGTGAGTGG-3' Reverse5'-AAGAGTGGGAGTTGCTGTTG-3'151-CU5'-GACGCTTCATTCCAAGACGTC-3' Ptk2151-DD5'-TTCTGTGCTCACGAGGGTGAAC-3' Forward5'-AGGCGGCCCAGGTTTACT3'151-EU5'-AAGGACACAGAGACAACTTCC-3' Reverse5'-CACCTTCTCCTCCTCCAGGAT-3'151-FD5'-GTAGTATGGCCAACAGAAGAC-3' Epo151-GU5'-CGTGTCTATACGGAAAGGAG-3' Forward5'-CATCTGCGACAGTCGAGTTCTG-3'151-HD5'-AAGAGACAGTGGCAGTTGTG-3' Reverse5'-CACAACCCATCGTGACATTTTC-3'151-IU5'-CAGTGGAACTATTGAGCTCTC-3' Phd3151-JD5'-ATATGCAGCCTGAAACAGCTCC-3' Forward5'-TCGCTTCCTCCCGAACTCT-3'151-YU5'-TATGTGCAGAGCAGGAAGAAGC-3' Reverse5'-CAGAAACGAGGGTGGCTAACTT-3'151-ZD5'-AGTGCATGGTGGGACAATTGACC-3' Pgk Forward5'-GGAAGCGGGTCGTGATGA-3' Reverse5'-GCCTTGATCCTTTGGTTGTTTG-3'Primer nameSequence Vegf Am151F25'-TGGGACTGAGAGCTGAGAAAG-3' Forward5'-CCACGTCAGAGAGCAACATCA-3'm151F35'-TTCTTGTGGGAGTGTGTCAAGG-3' Reverse5'-TCATTCTCTCTATGTGCTGGCTTT-3'm151R5'-AGACGCTTCATTCCAAGACGTC-3' For targeting vector construct For genotyping
For SYBR green qRT-PCR
Table 1. Primer sequences
177
Table 2. N1F2 Pup number of each genotype at day 10 post birth
Mir151+/+ Mir151+/- Mir151-/- Total number
Expected 90 181 90 361
Observed 94 180 87 361
178
Table 3. Clinical biochemistry of Mir151 Conventional KO female mice *
Item Unit +/+ (n=4) -/- (n = 5) p-value
179
0 1 2 3 4 +/ +
140000+/ - *
120402-/ - *
201131 * Compared to WT mice, p<0.05 (non-parametric analysis)G ra d e
T a b le 4 . T h e 4 -s ta g e g ra d in g s y s te m
180
Appendixes
181
Appendix VI. Proposed model illustrating the expression, function and