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SCIENCE CHINA Life Sciences, Volume 63 , Issue 5 : 724-736(2020) https://doi.org/10.1007/s11427-018-9515-1

Nuclear miR-665 aggravates heart failure via suppressing phosphatase and tensin homolog transcription

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  • ReceivedDec 5, 2018
  • AcceptedMar 12, 2019
  • PublishedOct 28, 2019

Abstract

Although numerous miRNAs have been discovered, their functions in the different subcellular organelles have remained obscure. In this study, we found that miR-665 was enriched in the nucleus of cardiomyocytes, and then investigated the underlying role of nuclear miR-665 in heart failure. RNA fluorescence in situ hybridization assays in human heart tissue sections and primary cardiomyocytes showed that miR-665 was localized in the nucleus of cardiomyocytes. Increased expression of nuclear miR-665 was observed not only in the cardiomyocytes isolated from the heart of mice treated in vivo by transverse aortic constriction (TAC), but also in phenylephrine (PE)-treated cultured cardiomyocytes in vitro. To further explore the role of miR-665 in heart failure, a type 9 recombinant adeno-associated virus (rAAV) system was employed to manipulate the expression of miR-665 in mice. Overexpression of miR-665 aggravated TAC-induced cardiac dysfunction, while down-expression of miR-665 showed opposite effects. Bioinformatic prediction and biological validation confirmed that the PTEN (phosphatase and tensin homolog) gene was one of the targets of miR-665 in the nucleus. Furthermore, restoring PTEN expression significantly eliminated the destructive effects of miR-665 over-expression in TAC-induced cardiac dysfunction. Our data showed that nuclear miR-665 aggravates heart failure via inhibiting PTEN expression, which provided a therapeutic approach for heart failure.


Funded by

grant from the National Natural Science Foundation of China(81822002,81630010,81790624,91839302,31771264,31800973)

data collection and analysis

decision to publish

or preparation of the manuscript.


Acknowledgment

We thank colleagues in the group of Dr. Daowen Wang for various technical help and stimulating discussion during the course of this investigation. This work was supported by grant from the National Natural Science Foundation of China (81822002, 81630010, 81790624, 91839302, 31771264 and 31800973). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Supplement

SUPPORTING INFORMATION

Figure S1ƒThe microRNA-Promoter Interaction Resource (microPIR) was used to search for PTEN promoter that could interact with miR-665.

Figure S2ƒThe levels of luciferase reporter mRNA were detected in 293T cells 36 h after transfection with indicated plasmids.

Figure S3ƒAgo2 protein levels in treated H9c2 cells detected by Western blotting.

Figure S4ƒNeonatal rat ventricular cells (NRVCs) were identified by immunofluorescence staining with α-actinin-2(ACTN2) antibody (ACTN2 is a specific marker for cardiomyocytes).

Table S1ƒList of PCR primers

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    miR-665 localizes in the nucleus of cardiomyocytes. A, miR-665 was abundant in cardiomyocytes of control human heart sections detected by FISH assay. B, miR-665 localized in the nucleus of cardiomyocytes in human heart sections detected by FISH assay. C, miR-665 localized in the nucleus of primary cardiomyocytes detected by FISH assay.

  • Figure 2

    Nuclear miR-665 in cardiomyocytes was increased during heart failure. A, miR-665 was increased in PE-treated H9c2 detected by real-time PCR. B, Cardiac miR-665 was increased in TAC-induced heart failure mice detected by real-time PCR. C, miR-665 was enriched in the heart compared with other organs detected by real-time PCR. D, miR-665 was increased in isolated primary cardiomyocytes from adult TAC-induced heart failure mice detected by real-time PCR (ACTN2 is a marker for cardiomyocytes). E, miR-665 was predominantly expressed in the nucleus in adult mouse cardiomyocytes detected by real-time PCR. F, miR-665 was increased in the nucleus of adult mouse cardiomyocytes from TAC-induced heart failure mice detected by real-time PCR. G, miR-665 was increased in the nucleus of treated H9c2 cells detected by real-time PCR. Data are representative of three experiments, n=3. Data are expressed as mean±SEM.

  • Figure 3

    miR-665 aggravates PE-induced hypertrophy in cultured cardiomyocytes. A, miR-665 aggravated PE-induced hypertrophy in H9c2 cells detected by FITC-phalloidin staining. B, miR-665 increased the ANP expression in H9c2 cells detected by real-time PCR. C, miR-665 increased the BNP expression in H9c2 cells detected by real-time PCR. D, miR-665 decreased the α-MHC expression in H9c2 cells detected by real-time PCR. E, miR-665 increased the β-MHC expression in H9c2 cells detected by real-time PCR. Data are representative of three experiments, n=3. Data are expressed as mean±SEM.

  • Figure 4

    miR-665 aggravates heart failure in TAC-induced heart failure mice. A, The cardiac expression of miR-665 in mice with different treatments was detected by real-time PCR. B, miR-665 aggravated the reduction of EF (%) and FS (%) in TAC-treated mice. C, miR-665 aggravated the reduction of dP/dtmax and dP/dtmin in TAC-treated mice. D, miR-665 increased heart weight to body weight ratio in TAC-treated mice. E, miR-665 increased cardiomyocyte size and collagen deposition in the heart from TAC-treated mice detected by H&E staining or picrosirius red staining. Data are representative of three experiments, n=8. Data are expressed as mean±SEM.

  • Figure 5

    miR-665 directly targets PTEN by interaction with the promoter. A, Human PTEN promoter contains two binding sites for miR-665. B, The sequence alignment between miR-665 and the promoter of PTEN. C, The predicted binding site-1 (–217/–211) of PTEN was highly conserved among species. D, The mRNA level of PTEN was reduced in H9c2 cells treated with miR-665 mimics. E, The protein level of PTEN was reduced in H9c2 cells treated with miR-665 mimics. F, The promoter regions of PTEN (including wild-type and the mutated sequence at predicted binding site-1 (–217/–211) or site-2 (–835/–827) of PTEN) were cloned into pGL3-report vector. G, Luciferase activity was detected in 293T cells 36 h after transfection with indicated plasmids with miR-con, miR-665 mimics. H, Ago2 protein was also expressed in the nucleus of primary cardiomyocytes detected by immunofluorescence staining. I, miR-665 overexpression in rat cardiomyocytes led to an increased binding of the PTEN promoter to Ago2. J, The decreased PTEN expression in miR-665-treated-H9c2 cells was abolished after treating with si-Ago2. Data are representative of three experiments, n=3. Data are expressed as mean±SEM.

  • Figure 6

    Restored PTEN expression eliminated the miR-665-induced cardiac dysfunction. A, Cardiac expression of miR-665 in mice with different treatments was detected by real-time PCR. B, Cardiac expression of PTEN in mice with different treatments was detected by real-time PCR. C, Restored PTEN eliminated miR-665-induced reduction of EF (%) and FS (%) in TAC-treated mice. D, Restored PTEN eliminated miR-665-induced reduction of dP/dtmax and dP/dtmin in TAC-treated mice. E, Restored PTEN eliminated miR-665-induced increase in heart size in heart weight to body weight ratio in TAC-treated mice. F, Restored PTEN eliminated miR-665-induced increase in cardiomyocyte size and collagen deposition in the heart from TAC-treated mice detected by H&E staining or picrosirius red staining. G, Nuclear miR-665 plays a crucial role in heart failure. Data are representative of three experiments, n=8. Data are expressed as mean±SEM.

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