miR-195b is required for proper cellular homeostasis in the elderly

doi:10.1038/s41598-024-51256-8

. 2024 Jan 8;14(1):810.

doi: 10.1038/s41598-024-51256-8.

miR-195b is required for proper cellular homeostasis in the elderly

Maria Del Mar Muñoz-Gallardo¹, Carlos Garcia-Padilla^{1

2}, Cristina Vicente-Garcia³, Jaime Carvajal³, Amelia Arenega^{1

4}, Diego Franco^{5

6}

Affiliations

¹ Cardiovascular Development Group, Department of Experimental Biology, University of Jaen, Jaen, Spain.
² Department of Anatomy, Embryology and Zoology, School of Medicine, University of Extremadura, Badajoz, Spain.
³ Andalusian Centre of Developmental Biology (CABD-CSIC-UPO-JA), Seville, Spain.
⁴ Fundación Medina, Granada, Spain.
⁵ Cardiovascular Development Group, Department of Experimental Biology, University of Jaen, Jaen, Spain. dfranco@ujaen.es.
⁶ Fundación Medina, Granada, Spain. dfranco@ujaen.es.

PMID: 38191655
PMCID: PMC10774362
DOI: 10.1038/s41598-024-51256-8

miR-195b is required for proper cellular homeostasis in the elderly

Maria Del Mar Muñoz-Gallardo et al. Sci Rep. 2024.

. 2024 Jan 8;14(1):810.

doi: 10.1038/s41598-024-51256-8.

Authors

Maria Del Mar Muñoz-Gallardo¹, Carlos Garcia-Padilla^{1

2}, Cristina Vicente-Garcia³, Jaime Carvajal³, Amelia Arenega^{1

4}, Diego Franco^{5

6}

Affiliations

¹ Cardiovascular Development Group, Department of Experimental Biology, University of Jaen, Jaen, Spain.
² Department of Anatomy, Embryology and Zoology, School of Medicine, University of Extremadura, Badajoz, Spain.
³ Andalusian Centre of Developmental Biology (CABD-CSIC-UPO-JA), Seville, Spain.
⁴ Fundación Medina, Granada, Spain.
⁵ Cardiovascular Development Group, Department of Experimental Biology, University of Jaen, Jaen, Spain. dfranco@ujaen.es.
⁶ Fundación Medina, Granada, Spain. dfranco@ujaen.es.

PMID: 38191655
PMCID: PMC10774362
DOI: 10.1038/s41598-024-51256-8

Abstract

Over the last decade we have witnessed an increasing number of studies revealing the functional role of non-coding RNAs in a multitude of biological processes, including cellular homeostasis, proliferation and differentiation. Impaired expression of non-coding RNAs can cause distinct pathological conditions, including herein those affecting the gastrointestinal and cardiorespiratory systems, respectively. miR-15/miR-16/miR-195 family members have been broadly implicated in multiple biological processes, including regulation of cell proliferation, apoptosis and metabolism within distinct tissues, such as heart, liver and lungs. While the functional contribution of miR-195a has been reported in multiple biological contexts, the role of miR-195b remains unexplored. In this study we dissected the functional role of miR-195b by generating CRISPR-Cas9 gene edited miR-195b deficient mice. Our results demonstrate that miR-195b is dispensable for embryonic development. miR-195b^-/- mice are fertile and displayed no gross anatomical and/or morphological defects. Mechanistically, cell cycle regulation, metabolism and oxidative stress markers are distinctly impaired in the heart, liver and lungs of aged mice, a condition that is not overtly observed at midlife. The lack of overt functional disarray during embryonic development and early adulthood might be due to temporal and tissue-specific compensatory mechanisms driven by selective upregulation miR-15/miR-16/miR-195 family members. Overall, our data demonstrated that miR-195b is dispensable for embryonic development and adulthood but is required for cellular homeostasis in the elderly.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Figure 1**
miR-195 expression analyses. In situ hybridization of miR-195 expression (panel A, E and F) and negative controls (B, C and D) in E9.5 (panels A, B) and E12.5 (panels C–F) mouse embryos. e′ and e′′ are close ups of panel (E). f′ and f′′ are close ups of panel (F). Sequence comparison of the pre-miRNAs of miR-15/miR-16/miR-195 family members (panel G), highlighting in red the seed sequence of each mature microRNAs. RT-qPCR analyses of miR-15/miR-16/miR-195 family members in E16 embryonic (panel H) and 6 months old adult (panel I) mouse tissues. Note that miR-195a, and to a lesser extend miR-195b, are widely expressed in both embryonic and adult tissues. n = 6 in all in situ hybridization experiments. n = 3 in all RT-qPCR experiments.

**Figure 2**
Histological and molecular characterization of miR-195b deficient liver. Hepatic histological sections of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− stained with H&E and picrosirius red, corresponding to 30 days and 420 days postnatal livers (panel A). Observe that no gross histological differences are observed, including no significant differences in collagen deposition. RT-qPCR analyses of miR-15/miR-16/miR-195 family members in livers corresponding to miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice, respectively, at different postnatal stages (i.e. 30, 60, 240 and 420 days postnatal) (panels B–G). Observe that miR-195b expression is severely downregulated in miR-195b^+/− livers and almost completely absent in miR-195b^−/− livers at all stages analyzed, while the rest of the miR-15/miR-16/miR-195 family members display no overt significant up-regulation in miR-195b^+/− and miR-195b^−/− livers in most stages analyzed, with the exception of a significant upregulation in the liver of miR-195b^+/− at 240 days postnatal, but not for miR-195b^−/−, at any of the stages analyzed. qRT-PCR analyses of *Foxa2, Hnfβ1, Prox1, Pparγ, Fatp, Acaca, Serbf1* and *Gpam* in livers of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice at two distinct postnatal stages, i.e. 240 days and 420 days (panel H–I). Observe that a global up-regulation of these hepatic developmental and metabolic markers is observed in miR-195b^+/− at 240 days postnatal, but not for miR-195b^−/− while at elderly stages, i.e. 420 days, a severe down-regulation of these markers is observed at both miR-195b^+/− and miR-195b^−/− conditions, with the exception of *Serbf1* and *Foxa2* that are mildly and significantly upregulated in miR-195b^−/− but not in miR-195b^+/− livers, respectively. RT-qPCR analyses of ROS markers in livers of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice at four distinct postnatal stages, i.e. 30, 60, 240 and 420 days (panel J). Observe that expression levels of *Sod1, Sod2, Gpx* and *Grs* are progressively increased in miR-195b^−/− livers with age, while *Prx* genes are severely downregulated at early postnatal stages in miR-195b^+/− and miR-195b^−/− mice, while at later postnatal stages, there are no significant differences (60 and 240 days) or they become significantly up-regulated (420 days). Mitochondria distribution assessed by Mitotracker labelling (panels M and N) and quantified (panel K) in cell cultures transfected with anti-miR-195 (panel N) as compared to controls (panel M). Note that anti-miR-195 leads to significant upregulation of the mitochondria labelling. Cell oxidative capacity was measured by CellROX labelling (panels O–P) and quantified (panel L) in cell cultures transfected with anti-miR-195 (panel P) as compared to controls (panel O). Observe an increase in cellular oxidative capacity after miR-195 inhibition (panel L). Normalized qPCR data were graphically plotted as heatmaps using Morpheus software (https://software.broadinstitute.org/morpheus/, accessed on 3 March 2020). All experiments were performed in at least three distinct biological samples. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

**Figure 3**
Cell cycle and senescence analyses in miR-195b deficient liver. RT-qPCR analyses of cell cycle markers such as *ccnd1*, *ccnd2*, *ccnd3* (panel A), *cdk4* and *cdk6* (panel B) in livers of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice at four distinct postnatal stages, i.e. 30, 60, 240 and 420 days. Note that these cell cycle markers are increased at early postnatal stages in miR-195b^+/− and miR-195b^−/− mice but are severely downregulated at late postnatal stages. Immunohistochemical analyses of ki67 expression on hepatic histological sections of miR-195b^+/+ (panel C), miR-195b^+/− (panel E) and miR-195b^−/− (panel D) mice at 60 days old and their quantitative analyses (panel F) demonstrated increased and decreased proliferation in miR-195b^+/− and miR-195b^−/− mice, respectively, in line with RT-qPCR analyses. Luciferase assays of *ccnd1* 3′UTR (panel J), *ccnd2* 3′UTR (panel K) and *ccnd3* 3′UTR (panel L) demonstrating significant downregulation after miR-195 overexpression in 3T3 fibroblasts. SA-β-galactosidase positive cells in cell cultures of 3T3 fibroblasts (panel M) and MEVEC endocardial cells (panel N) transfected with anti-miR-195, β-galactosidase expression vector or treated with 100 mM and 300 nM of H₂0₂. Observe the distinct behavior of anti-miR-195 administration in 3T3 fibroblasts (panel M) as compared to MEVEC endocardial cells (panel N). SA-β-galactosidase positive cells on hepatic histological sections of miR-195b^+/+ (panel O), miR-195b^+/− (panel Q) and miR-195b^−/− (panel P) mice and their quantitative analyses (panel R) at 240 days, demonstrating increased senescence in miR-195b^−/− (panel P) mice. SA-β-galactosidase positive cells on hepatic histological sections of miR-195b^+/+ (panel S), miR-195b^+/− (panel U) and miR-195b^−/− (panel T) mice and their quantitative analyses (panel V) at 420 days, demonstrating increased senescence on miR-195b^−/− (panel T) mice. RT-qPCR analyses of ER stress markers such as *Stim1, Atf6* and *Ire1* (panels W–X) in livers of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice at two distinct postnatal stages, i.e. 60 (panel W) and 420 days (panel X). Luciferase assays of *Stim1* 3′UTR (panel Y) and *Atf6* 3′UTR (panel Z) demonstrating significant downregulation after miR-195 overexpression in 3T3 fibroblasts. All experiments were performed in at least three distinct biological samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

**Figure 4**
Histological and molecular characterization of miR-195b deficient heart. Cardiac histological sections of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− stained with H&E and picrosirius red, corresponding to 30 days and 420 days postnatal livers (panel A). Observe that no gross histological differences are observed, including no significant differences in collagen deposition. RT-qPCR analyses of miR-15/miR-16/miR-195 family members in hearts corresponding to miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice, respectively, at different postnatal stages (i.e. 30, 60, 240 and 420 days postnatal) (panels B–F). Observe that several miR-15/miR-16/miR-195 family members display a significant upregulation in miR-195b^+/− and miR-195b^−/− hearts in elderly stages analyzed, particularly miR-15a_5p and miR-15b_5p. RT-qPCR analyses of cardiac developmental markers such as *Mef2c, Gata4, Nkx2.5, Tnnt2* and *Tbx5* in hearts of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice at four distinct postnatal stages, i.e. 30 days to 420 days (panel G). Observe that a global upregulation of these cardiac developmental markers is observed in miR-195b^+/− and miR-195b^−/− hearts from 60 days postnatal onwards, respectively. SA-β-galactosidase positive cells on cardiac histological sections of miR-195b^+/+ (panel H), miR-195b^+/− (panel I) and miR-195b^−/− (panel J) mice and their quantitative analyses (panel K) at 240 days, demonstrating no increased senescence on miR-195b^−/− (panel J) mice. SA-β-galactosidase positive cells on cardiac histological sections of miR-195b^+/+ (panel L), miR-195b^+/− (panel M) and miR-195b^−/− (panel N) mice and their quantitative analyses (panel O) at 420 days, similarly demonstrating no increased senescence on miR-195b^−/− (panel N) mice. qRT-PCR analyses of ROS markers in hearts of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice at four distinct postnatal stages, i.e. 30, 60, 240 and 420 days (panel P). Observe that expression of ROS markers are similarly increased in miR-195b^+/− and miR-195b^−/− hearts with age. All experiments were performed in at least three distinct biological samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

**Figure 5**
Histological and molecular characterization of miR-195b deficient lung. Lung histological sections of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− stained with H&E and picrosirius red, corresponding to 30 days and 420 days postnatal lungs (panel A). Observe that no gross histological differences are observed, including no significant differences in collagen deposition. RT-qPCR analyses of miR-15/miR-16/miR-195 family members in lungs corresponding to miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice, respectively, at different postnatal stages (i.e. 30, 60, 240 and 420 days postnatal) (panels B–F). Observe that several miR-15/miR-16/miR-195 family members display a significant up-regulation in miR-195b^+/− and miR-195b^−/− lungs in elderly stages analyzed, particularly miR-195a_5p, miR-15b_5p and miR-16_5p. RT-qPCR analyses of ROS markers in lungs of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice at four distinct postnatal stages, i.e. 30, 60, 240 and 420 days (panels G–H). Observe that expression of ROS markers are moderately increased in miR-195b^+/− and miR-195b^−/− hearts with age. SA-β-galactosidase positive cells on lung histological sections of miR-195b^+/+ (panel I), miR-195b^+/− (panel J) and miR-195b^−/− (panel K) mice and their quantitative analyses (panel L) at 240 days, demonstrating increased senescence on miR-195b^+/− (panel J) and miR-195b^−/− (panel K) mice. SA-β-galactosidase positive cells on lung histological sections of miR-195b^+/+ (panel M), miR-195b^+/− (panel N) and miR-195b^−/− (panel O) mice and their quantitative analyses (panel P) at 420 days, similarly demonstrating no increased senescence on miR-195b^−/− (panel P) mice. RT-qPCR analyses of cell cycle markers such as *ccnd1, ccnd2, ccnd3, cdk4* and *cdk6* (panel Q) in lungs of miR-195b^+/+, miR-195b^+/− and miR-195b^−/− mice at four distinct postnatal stages, i.e. 30, 60, 240 and 420 days. Note that *ccnd2* cycle marker is increased only at early postnatal stages in miR-195b^+/− and miR-195b^−/− mice while most of them are severely downregulated at late postnatal stages (60–420 days postnatal). All experiments were performed in at least three distinct biological samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

**Figure 6**
In vitro regulation of miR-15/miR-16/miR-195 family members. RT-qPCR analyses of miR-15/miR-16/miR-195 family members in HL1 cardiomyocytes, 3T3 fibroblasts, MEVEC endocardial and EPIC epicardial cells transfected with pre-miR-195 and anti-miR-195, respectively (panel A–F). RT-qPCR analyses of ROS markers in HL1 cardiomyocytes, 3T3 fibroblasts, MEVEC endocardial and EPIC epicardial cells transfected with pre-miR-195 and anti-miR-195, respectively (panel H–K). All experiments were performed in at least three distinct biological samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

**Figure 7**
miR-195b working model. Schematic representation of the distinct biological processes modulated by miR-195b. miR-195b deficiency leads to tissue-specific regulation of miR-15/miR-16/miR-195 family members, producing alterations in metabolic regulation, transcriptional control, cell proliferation, ROS homeostasis and ER stress that ultimately lead to cell senescence in the elderly.

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References

1. Hombach S, Kretz M. Non-coding RNAs: Classification, biology and functioning. Adv. Exp. Med. Biol. 2016;937:3–17. doi: 10.1007/978-3-319-42059-2_1. - DOI - PubMed
1. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 2014;15(8):509–524. doi: 10.1038/nrm3838.E. - DOI - PubMed
1. Bartel DP. Metazoan MicroRNAs. Cell. 2018;173(1):20–51. doi: 10.1016/j.cell.2018.03.006. - DOI - PMC - PubMed
1. van Solingen C, Bijkerk R, de Boer HC, Rabelink TJ, van Zonneveld AJ. The role of microRNA-126 in vascular homeostasis. Curr. Vasc. Pharmacol. 2015;13(3):341–351. doi: 10.2174/15701611113119990017. - DOI - PubMed
1. Aryal B, Singh AK, Rotllan N, Price N, Fernández-Hernando C. MicroRNAs and lipid metabolism. Curr. Opin. Lipidol. 2017;28(3):273–280. doi: 10.1097/MOL.0000000000000420. - DOI - PMC - PubMed

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[1] Hombach S, Kretz M. Non-coding RNAs: Classification, biology and functioning. Adv. Exp. Med. Biol. 2016;937:3–17. doi: 10.1007/978-3-319-42059-2_1. - DOI - PubMed

[2] Hombach S, Kretz M. Non-coding RNAs: Classification, biology and functioning. Adv. Exp. Med. Biol. 2016;937:3–17. doi: 10.1007/978-3-319-42059-2_1. - DOI - PubMed

[3] Ha M, Kim VN. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 2014;15(8):509–524. doi: 10.1038/nrm3838.E. - DOI - PubMed

[4] Ha M, Kim VN. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 2014;15(8):509–524. doi: 10.1038/nrm3838.E. - DOI - PubMed

[5] Bartel DP. Metazoan MicroRNAs. Cell. 2018;173(1):20–51. doi: 10.1016/j.cell.2018.03.006. - DOI - PMC - PubMed

[6] Bartel DP. Metazoan MicroRNAs. Cell. 2018;173(1):20–51. doi: 10.1016/j.cell.2018.03.006. - DOI - PMC - PubMed

[7] van Solingen C, Bijkerk R, de Boer HC, Rabelink TJ, van Zonneveld AJ. The role of microRNA-126 in vascular homeostasis. Curr. Vasc. Pharmacol. 2015;13(3):341–351. doi: 10.2174/15701611113119990017. - DOI - PubMed

[8] van Solingen C, Bijkerk R, de Boer HC, Rabelink TJ, van Zonneveld AJ. The role of microRNA-126 in vascular homeostasis. Curr. Vasc. Pharmacol. 2015;13(3):341–351. doi: 10.2174/15701611113119990017. - DOI - PubMed

[9] Aryal B, Singh AK, Rotllan N, Price N, Fernández-Hernando C. MicroRNAs and lipid metabolism. Curr. Opin. Lipidol. 2017;28(3):273–280. doi: 10.1097/MOL.0000000000000420. - DOI - PMC - PubMed

[10] Aryal B, Singh AK, Rotllan N, Price N, Fernández-Hernando C. MicroRNAs and lipid metabolism. Curr. Opin. Lipidol. 2017;28(3):273–280. doi: 10.1097/MOL.0000000000000420. - DOI - PMC - PubMed

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miR-195b is required for proper cellular homeostasis in the elderly

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miR-195b is required for proper cellular homeostasis in the elderly

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Abstract

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