Inhibitors of micro-RNAs for use for preventing and/or attenuating skin ageing

Abstract

Identification and use of compounds which inhibit the expression or activity of micro-RNAs for preventing and/or attenuating ageing. An in vitro method for screening for candidate compounds for preventing and/or attenuating ageing of the skin including (a) bringing at least one test compound in contact with a sample of fibroblasts, (b) measuring the expression or the activity of at least one microRNA chosen from miR-134 and miR-152 in said fibroblasts, and (c) selecting the compounds for which an inhibition of at least 20%, preferably at least 30%, preferably at least 40% of the expression or an inhibition of at least 20%, preferably at least 30%, preferably at least 40% of the activity of at least one microRNA is measured in the fibroblasts treated in (a) compared with the untreated fibroblasts.

Claims

1. An in vitro method for screening for candidate compounds for attenuating ageing of the skin, comprising the following steps: (a) bringing at least one test compound in contact with at least one sample of fibroblasts; (b) measuring the expression or the activity of at least one microRNA chosen from miR-134 and miR-152 in said fibroblasts; (c) selecting the compounds for which an inhibition of at least 20% of the expression, or an inhibition of at least 20% of the activity, of said at least one microRNA is measured in the fibroblasts treated in step (a) compared with untreated fibroblasts.

2. The method according to claim 1, wherein step (b) is performed before and after step (a).

3. The method according to claim 1, wherein the test compounds are chosen from botanical extracts.

4. The method according to claim 1, wherein the inhibition of expression or activity of the microRNA measured in step (c) is at least 50%.

5. The method according to claim 1, wherein the inhibition of expression or activity of the microRNA measured in step (c) is at least 40%.

6. The method according to claim 1, wherein the inhibition of expression or activity of the microRNA measured in step (c) is at least 60%.

7. The method according to claim 1, wherein the at least one microRNA in step (b) is miR-134.

8. The method according to claim 1, wherein said fibroblasts are pre-senescent fibroblasts that have been obtained after 70 population doublings in classical culture conditions, wherein the classical culture conditions comprise culturing the fibroblasts in 106 medium added with LSGS growth supplements, and constantly keeping the fibroblasts in a subconfluent state.

9. The method according to claim 1, wherein said fibroblasts are pre-senescent fibroblasts.

10. The method according to claim 9, wherein the pre-senescent fibroblasts are obtained after 70 population doublings in classical culture conditions.

11. The method according to claim 10, wherein the classical culture conditions comprise culturing the fibroblasts in 106 medium added with LSGS growth supplements, and constantly keeping the fibroblasts in a subconfluent state.

12. An in vitro method for screening for candidate compounds for attenuating ageing of the skin, comprising the following steps: (a) preparing at least two samples of fibroblasts; (a) bringing at least one test compound into contact with at least one of said at least two samples of fibroblasts, while leaving at least one of said two samples of fibroblast untreated; then (b) measuring the expression or the activity of at least one microRNA chosen from miR-134 and miR-152 in said at least two samples of fibroblasts; and (c) selecting the compounds for which an inhibition of at least 20% of the expression, or an inhibition of at least 20% of the activity, of said at least one microRNA is measured in the at least one sample of fibroblasts brought into contact with the at least one test compound treated in step (a) compared with the sample of untreated fibroblasts.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following examples illustrate the invention without limiting the scope thereof. These examples are based on the figures listed below:

(2) FIG. 1

(3) Induction of Replicative Senescence in Human Dermal Fibroblasts.

(4) (A) Population doublings of primary human fibroblasts during 103 days of culture. After 70 population doublings the growth curve has a plateau, showing that cells stop dividing and are reaching the senescent state. (B) Western blots performed on protein extracts from human fibroblasts at passage 1(p1), p4, p8 and p16 showing the analysis of some senescence markers, such as sirt1 and p16. -Actin was used as loading control. (C) Human fibroblasts at p1, p4, p8 and p16 were subjected to a 3 h BrdU pulse, collected, propidium iodide (PI)-stained, and analyzed by flow cytometry. BrdU-positive cells are indicated as S-phase fluorescent population and are assesd by PI staining of DNA content of 2n or 4n (fixed to values of 250 and 400 in the plots). (D and E) SA--galactosidase staining and quantification by blue-cell/field of fibroblasts at p1, p4, p8 and p12.

(5) FIG. 2

(6) Expression of miR-134 in Fibroblasts Replicative Senescence.

(7) Relative quantification by Real Time PCR of miR-134 was performed on total RNAs collected from proliferating p1, pre-senescent p11 and senescent p16 fibroblasts.

(8) FIG. 3

(9) Expression of miR-152 in Fibroblasts Replicative Senescence.

(10) Relative quantification by Real Time PCR of miR-134 was performed on total RNAs collected from proliferating p1, pre-senescent p11 and senescent p16 fibroblasts.

(11) FIG. 4

(12) MiR-134 and miR-152 Reduce Proliferation in Fibroblasts Upon Transfection in Proliferating/Young Fibroblasts.

(13) (A) Ninety-six hours after transfection of fibroblasts with a negative control, miR-134 or miR-152 sequences, cells were subjected to a 3 h BrdU pulse, collected, PI-stained, and analyzed by flow cytometry as described in FIG. 1. (B) Quantification of BrdU positive fibroblasts after transfecion with negative control, miR-134 and miR-152 sequences.

(14) FIG. 5

(15) MiR-134 and miR-152 Induce Senescence in Fibroblasts Upon Transfection in Proliferating/Young Fibroblasts.

(16) (A and B) SA--galactosidase staining and quantification by blue-cell/field of fibroblasts ninety-six hours after transfection with negative control, miR-134 and miR-152 sequences.

(17) FIG. 6

(18) ITGA9 is a Putative Target of miR-134 and miR-152 and Decreases in Fibroblast Replicative Senescence.

(19) (A) Relative quantification by Real Time PCR of ITGA9 mRNA level at p1 and p16. (B) Predicted miR-134 and miR-152 target sites on ITGA9 3UTR were identified by TargetScan 6.1 software. (C) Western blots performed on protein extracts from fibroblasts at p1 and p16 showing the protein level of ITGA9. -Actin was used as loading control.

(20) FIG. 7

(21) ITGA9 mRNA is Downregulated Upon miR-134 and miR-152 Transfection in Fibroblasts.

(22) Relative quantification by Real Time PCR of ITGA9 mRNA level of fibroblasts ninety-six hours after transfection with negative control, miR-134 and miR-152 sequences.

(23) FIG. 8

(24) ITGA5 is a Putative Target of miR-152 and Decreases in Fibroblasts Replicative Senescence.

(25) (A) Relative quantification by Real Time PCR of ITGA5 mRNA level at p1 and p16. (B) Predicted miR-152 target sites on ITGA 3UTR were identified by TargetScan 6.1 software. (C) Western blots performed on protein extracts from fibroblasts at p1 and p16 showing the protein level of ITGA5. -Actin was used as loading control.

(26) FIG. 9

(27) ITGA5 is Downregulated Upon miR-152 Transfection in Fibroblasts.

(28) (A) Relative quantification by Real Time PCR of ITGA5 mRNA level of fibroblasts ninety-six hours after transfection with negative control and miR-152 sequences. (B) Western blots performed on protein extracts from fibroblasts ninety-six hours after transfection with negative control, miR-152 and anti-miR 152 sequences showing the protein level of ITGA5. -Actin was used as loading control.

(29) FIG. 10

(30) Inhibition of miR-134 Using Specific AntagomiR in Fibroblasts.

(31) Relative quantification by Real Time PCR of miR-134 level ninety-six hours after transfection of fibroblasts with negative control and anti-miR-134 sequences.

(32) FIG. 11

(33) Inhibition of miR-152 Using Specific AntagomiR in Fibroblasts.

(34) Relative quantification by Real Time PCR of miR-152 level ninety-six hours after transfection of fibroblasts with negative control and anti-miR-152 sequences.

(35) FIG. 12

(36) Inhibition of miR-134 by Compound 4 and 5.

(37) Relative quantification by Real Time PCR of miR-134 level forty-eight hours after treatment of fibroblasts with negative control and compounds 4 or 5 at different concentrations.

(38) FIG. 13

(39) Inhibition of miR-152 by Compounds 2, 4 and 8.

(40) Relative quantification by Real Time PCR of miR-152 level forty-eight hours after treatment of fibroblasts with negative control and compounds 2, 4 or 8 at different concentrations.

EXAMPLE 1

Material and Methods

(41) Cell Culture and Transfection

(42) Neonatal Human Primary Dermal Fibroblasts (HDFn, Cascade, Invitrogen, Carlsbad, Calif., USA) were cultured in 106 medium added with LSGS growth supplements (Cascade). Cells were passaged usually once a week, at each passage the harvested cells number and seeded cell number were recorded in order to calculate the population doublings occurring between passages and the population doubling time. At each passage different aliquots of the cells were harvested to extract in triplicate RNA and proteins and an aliquot was submitted to senescence activated -galactosidase staining in order to assay the senescent or non-senescent state of the cells.

(43) Human primary fibroblasts were transfected with human pre-miR 134, anti-miR-134, pre-miR-152, anti-miR-152 and scramble sequence as negative control (Ambion, Tex., USA) using the Lipofecatmine RNAimax transfection reagent (Invitrogen) according to manufacturer protocols. 24 hrs after transfection, the medium was removed and replaced with fresh medium.

(44) 400.000 human primary fibroblasts were plated in 106 medium with LSGS growth supplements and treated with test compounds. After 24 and 48 hours cells were collected and mRNA extracted following standard procedures. Real time PCR were performed as described below.

(45) RNA Extraction and Real Time PCR Analysis

(46) Total RNA from cells was isolated using mirVana mirRNA Isolation Kit (Ambion) by following the manufacturer's protocol for total RNA extraction. Total RNA was quantified using a NanoDrop Spectophotometer (Thermo Scientific, Delaware, USA) and RNA quality was controlled on an agarose gel. For microRNA detection, RNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription kit and qRT-PCR was performed with TaqMan universal master mix (Applied Biosystem) and specific primers for miR-134 and miR-152. U18 was used as an internal control (Applied Biosystem). The expression of each gene and miR was defined from the threshold cycle (Ct), and relative expression levels were calculated by using the 2-Ct method after normalization with reference to the expression of the housekeeping gene U18.

(47) Senescence-Associated -Galactosidase Staining

(48) Cells were grown in 6-well culture plates, washed with PBS, and fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 minutes. After another washing step with PBS, cells were incubated with -galactosidase staining solution (150 mmol/L NaCl, 2 mmol/L MgCl.sub.2, 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, 40 mmol/L citric acid, 12 mmol/L sodium phosphate, pH 6.0, containing 1 mg/mL 5-bromo-4-chloro-3-indolyl--Dgalactoside [X-gal]) for 24 hours at 37 C. The reaction was stopped by replacing the staining solution with 70% glycerol.

(49) Cell Proliferation and Cell Cycle Analysis

(50) Methods used to evaluate cell proliferation are generally based on incorporation of thymidine analogues such as 3H thymidine or bromodeoxyuridine (BrdU) during DNA synthesis. The Click-iT EdU flow cytometry assay kit is a novel alternative to BrdU assay (Molecular Probes, Eugene, Oreg., USA). This method replaces antibody-based detection of the nucleoside analogue, BrdU, with EdU (5-ethynyl-2-deoxyuridine), which is a nucleoside analogue of thymidine that is incorporated into DNA during active DNA synthesis. Briefly, cells were incubated with EdU for 4 hrs. After incubation, samples were fixed, permeabilized and stained according to the manufacturer's protocol. Cell cycle was analysed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, Calif., USA). Fifteen thousand events were evaluated using the Cell Quest (BD) and Modfit LT (Verity Software; BD) programs.

(51) Bioinformatics

(52) Analysis of miR-134 and miR-152 target sites on ITGA9 3UTR were performed using the TargetScan 5.1 software available at http://www.targetscan.org/

(53) Western Blotting

(54) Total cell extracts were resolved on a SDS polyacrylamide gel, blotted on a Hybond P PVDF membrane (G&E Healthcare, UK). Membranes were blocked with PBST 5% non fat dry milk, incubated with primary antibodies for 2 h at room temperature, washed and hybridized for 1 h at room temperature using the appropriate horseradish peroxidase-conjugated secondary antibody (rabbit and mouse, BioRad, Hercules, Calif., USA). Detection was performed with the ECL chemiluminescence kit (Perkin Elmer, Waltham, Mass., USA). anti-Sirt1 (Abcam; dilution 1:500), anti-actin (Sigma, St Louis, Minn., USA; dilution 1:5000), anti-p16 (Santa Cruz Biotechnology, California, USA; dilution 1:1000), anti-ITGA9 (Sigma, St Louis, Minn., USA; dilution 1:400) were used.

(55) Results

(56) 1Establishment of a Model for Replicative Senescence in Primary Human Fibroblasts.

(57) Human primary fibroblasts were cultured and serially passaged until the reached the senescent state. FIG. 1A is showing the population doublings of primary human fibroblasts during 103 days of culture. After 70 population (passage 16-p16) doublings the growth curve has a plateau, showing that cells stop dividing and are reaching the senescent state. Cells were collected at p1, p4, p8 and p16 to perform western blot analysis p16/INK4a and Sirt-1. Diminished expression of Sirt-1 and increasing levels of p16/INK4a show that cells are entering into the replicative senescent state. -actin protein levels are reported as a loading control (FIGS. 1A and 1B). Percentage of proliferating cells is diminishing during senescence as shown by the percentage of BrdU positive primary human fibroblasts at different passages (FIG. 1C). Primary human fibroblasts undergo replicative senescence after the 16 passage at 70 population doublings. Photomicrographs of SA--galactosidase staining in primary human fibroblasts at different passages show the expression of the senescent marker in older cells. Cells expressing SA--galactosidase show a deep blue staining (FIG. 1D), staining quantification is shown in FIG. 1E.

(58) 2miR-134 Levels in Senescing Fibroblasts.

(59) At p1, p11 and p16, cells were collected to perform Real time PCR. Relative quantification of miR-134 levels between p1 and p16 shows significant increase in miR-134 expression in senescent fibroblasts: at p16, miR-134 expression is around 5.35 (0.08) higher than its expression at p1 (which is equal to 1.000.05), FIG. 2. Therefore, the fibroblasts senescent state, but not the fibroblasts proliferating state, is associated with an increase in miR-134 expression.

(60) 3miR-152 Levels in Senescing Fibroblasts.

(61) At p1, p11 and p16, cells were collected to perform Real time PCR. Relative quantification of miR-152 levels between p1 and p16 shows significant increase in miR-152 expression in senescent fibroblasts: at p16, miR-152 expression is around 3.37 (0.09) higher than its expression at p1 (which is equal to 1.000.07), FIG. 3. Therefore, the fibroblasts senescent state, but not the fibroblasts proliferating state, is associated with an increase in miR-152 expression.

(62) 4miR-134 and miR-152 Reduce Proliferation in Fibroblasts Upon Transfection in Proliferating/Young Fibroblasts.

(63) Ninety-six hours after transfection of human dermal fibroblasts with a negative control, miR-134 or miR-152 sequences, cells were subjected to a 3 h BrdU pulse, collected, PI-stained, and analyzed by flow cytometry as described in methods. The percentage of proliferating cells is diminishing upon miR-134 and miR-152 transfection as shown by the percentage of BrdU positive primary human fibroblasts transfected with negative control (26%), miR-134 (19%) and miR-152 (18%), FIGS. 4 A and 4B.

(64) 5miR-134 and miR-152 Induce Senescence in Fibroblasts Upon Transfection in Proliferating/Young Fibroblasts.

(65) Upon tranfection with miR-134 and miR-152, SA--galactosidase staining increased significantly in fibroblasts, demonstrating that these two miRNAs are sufficient per se to induce senescence (FIG. 5A). Quantification of blue-cell/field of human dermal fibroblasts, ninety-six hours after transfection, indicate that SA--galactosidase staining increases of 4-4.5 folds as compared with negative control (FIG. 5B).

(66) 6ITGA9 is a Target of Both miR-134 and miR-152.

(67) We have performed relative quantification by Real Time PCR of ITGA9 mRNA using cells at p1 and p16 (FIG. 6A), the data obtained clearly show that ITGA9 mRNA is strongly reduced in senescent fibroblasts (p16). Indeed, relative quantification by Real Time PCR of ITGA9 mRNA level of human dermal fibroblasts ninety-six hours after transfection indicate that ITGA9 is reduced 20% and 40%, respectively, upon miR-134 and miR-152 transfection. This is less evident at protein level (FIG. 6C) in the same experimental condition, probably for ITGA9 high protein stability. Using targetScan 6.1 software, we have identified on ITGA9 3UTR two predicted, for miR-134 and miR-152, target sites. ITGA9 mRNA is downregulated upon miR-134 and miR-152 transfection in fibroblasts as indicated in FIG. 7.

(68) 7ITGA5 is a Target of miR-152.

(69) We have performed relative quantification by Real Time PCR of ITGA5 mRNA using cells at p1 and p16 (FIG. 8A), the data obtained clearly show that ITGA5 mRNA is reduced in senescent fibroblasts (p16). Indeed, real Time PCR of ITGA5 mRNA level of human dermal fibroblasts ninety-six hours after transfection indicate that ITGA5 is reduced 30%, upon miR-152 transfection. This is also evident at protein level (FIG. 8C) in the same experimental condition. Using targetScan 6.1 software, we have identified on ITGA5 3UTR two predicted target sites for miR-152. Upon transfection, ITGA5 is downregulated by miR-152 in fibroblasts as shown by the relative quantification by Real Time PCR of ITGA5 mRNA level of human fibroblasts. Western blots performed on protein extracts from human fibroblasts after miR-152 and anti-miR-152 transfection showed a significant decrease of ITGA5. -Actin was used as loading control.

(70) 8Modulation of miR-134 by Synthetic Anti-miR-134.

(71) Anti-miRNAs are miRNA inhibitors that specifically inhibit endogenous miRNAs. Anti-miRNAs are single stranded nucleic acids designed to specifically bind to and inhibit endogenous microRNA molecules. Anti-miRNAs have nucleic sequence complementary to the sequence of the target miRNA. These ready-to-use inhibitors can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs, and enable detailed study of miRNA biological effects. Use of the anti-miRNA enables miRNA functional analysis by downregulation of miRNA activity. Anti-miRNAs are commercially available; they can for example be obtained by Ambion or Applied Biosystems. Primary human fibroblasts were treated with an anti-miR-134 at different concentrations. After 96 hours cells were harvested for relative quantification of miR-134 levels using Real time PCR. The anti-miR-134 is significantly downregulating miR-134 levels with respect to the untreated cells; the expression of miR-134 in treated cells is only 0.1840.022 (whereas it is 1.000.01 for scramble) (FIG. 10).

(72) 9Modulation of miR-152 by Synthetic Anti-miR-152.

(73) Anti-miRNAs are miRNA inhibitors that specifically inhibit endogenous miRNAs. Anti-miRNAs are single stranded nucleic acids designed to specifically bind to and inhibit endogenous microRNA molecules. Anti-miRNAs have nucleic sequence complementary to the sequence of the target miRNA. These ready-to-use inhibitors can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs, and enable detailed study of miRNA biological effects. Use of the anti-miRNA enables miRNA functional analysis by downregulation of miRNA activity. Anti-miRNAs are commercially available; they can for example be obtained by Ambion or Applied Biosystems. Primary human fibroblasts were treated with an anti-miR-152 at different concentrations. After 96 hours cells were harvested for relative quantification of miR-152 levels using Real time PCR. The anti-miR-152 is significantly downregulating miR-152 levels with respect to the untreated cells; the expression of miR-152 in treated cells is only 0.3250.032 (whereas it is 1.000.01 for scramble) (FIG. 11).

(74) 10Inhibition of miR-134 by Compounds 4 and 5.

(75) Primary human fibroblasts were treated with compounds 4 and 5, respectively Epigallocatechine Gallate and Verbascoside, at the concentration indicated in FIG. 12. After 48 hours cells were harvested for relative quantification of miR-134 levels using Real time PCR. The compounds 4 and 5 are significantly downregulating miR-134 levels with respect to the untreated cells; the expression of miR-134 in treated cells is only 0.6030.03 and 0.7340.05 (whereas it is 1.000.01 for scramble) (FIG. 12).

(76) 11Inhibition of miR-152 by Compounds 2, 4 and 8.

(77) Primary human fibroblasts were treated with compounds 2, 4 and 8, respectively Catechine Hydrate, Epigallocatechine Gallate and Bois d'Ange PFA, at the concentration indicated in FIG. 13. After 48 hours cells were harvested for relative quantification of miR-152 levels using Real time PCR. The compounds 2, 4 and 8 are significantly downregulating miR-152 levels with respect to the untreated cells; the expression of miR-152 in treated cells is only 0.7690.03, 0.8040.05 and 0.3140.04 (whereas it is 1.000.01 for scramble) (FIG. 13).

EXAMPLE 2

Cosmetic Composition (O/W Serum)

(78) The following composition may be prepared in a classical manner for the man skilled in the art.

(79) The active agent is prepared as follows:

(80) The wood of Andira coriacea (i.e. Bois d'Ange) was extracted with ethanol, yielding 386 mg of crude extract after evaporation under reduced pressure. The crude extract was then purified by reverse phase preparative HPLC(C18 Varian Pursuit XRS, 250 mm41.1 mm5 nm) using a gradient of H2O+0.1% formic acid and MeOH (from H2O+FA/MeOH 70:30 to 60:40 in 17 min at 60 mL min-1) to afford pure Coatline A (12.3 mg) and Coatline B (40.2 mg).

(81) TABLE-US-00001 INCI name % (w/w) Water QSP 100.00 Chelating agent 0.05 pH balance 0.05 Preservatives 0.05 Glycol 3.25 AMMONIUM ACRYLOYLDIMETHYLTAURATE/VP 1.20 COPOLYMER ACRYLATES/C10-30 ALKYL ACRYLATE 0.20 CROSSPOLYMER GLYCERIN 3.00 GLYCERYLPOLYMETHACRYLATE 4.18 SODIUM ACETYLATED HYALURONATE 0.05 Oil 10.00 ALCOHOL 8.00 PERFUMES 0.30 Coatlines obtained as described above 0.05

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