Z2013 mir21通过pten和akt信号通过调控Kfb增殖和调亡

https://www.360docs.net/doc/a712221665.html, 561e

N

owadays, up to 4.5 percent of the general population suffers from hypertrophic scar-ring.1 Wounds, trauma, burns, surgical inci-sion, or disease can result in scar formation. To prevent infection, skin repairs itself from normal to scar tissue to close a wound.2 Keloid is an end of the full spectrum of scar that extends beyond the boundaries of the original wound. It can also spread to the surrounding skin by invasion. The clinical appearance of keloid is a raised growth, usually accompanied by pruritus and pain.3 Because the etiopathogenesis and mechanisms

of keloids are still not known, keloid therapy still leaves much to be desired.4 Treatment for keloid scars is dif?cult and frustrating, and the mecha-nisms underlying keloid formation are only par-tially understood.5

MicroRNAs (miRNAs) are small, highly con-served, 18- to 25-nucleotide-length, single-strand RNA molecules involved in the posttranscrip-tional regulation of gene expression. Because the functions of microRNAs are closely related to various aspects of cell physiology and biology, it is important to explore the molecular mechanism governing their expression levels. Our group has identi?ed differentially expressed miRNAs through an miRNA microarray between keloid tissue and normal skin tissue. MicroRNA-21 (miR-21) was revealed to have the highest fold Disclosure: The authors have no ?nancial interest to declare in relation to the content of this article.

Copyright ? 2014 by the American Society of Plastic Surgeons DOI: 10.1097/PRS.0000000000000577

Ying Liu, Ph.D.Xiaoxue Wang, M.D.Daping Yang, Ph.D.Zhibo Xiao, Ph.D.Xi Chen, Ph.D.

Harbin, People’s Republic of China

From the Departments of Plastic and Aesthetic and Gener-al Surgery, Second Af?liated Hospital of Harbin Medical

U niversity.Received for publication July 5, 2013; accepted January 28, 2014.

The ?rst two authors contributed equally to this article.MicroRNA-21 Affects Proliferation and

Apoptosis by Regulating Expression of PTEN in Human Keloid Fibroblasts

EXPERIMENTAL

Plastic and Reconstructive Surgery s /CTOBER

change (6.87-fold) among the 23 up-regulated miRNAs.6 Therefore, more work is needed to fur-ther explore the molecular mechanism between keloids and miR-21.

MicroRNA-21 is a speci?c miRNA that is over-expressed in many types of tumors, including stomach, prostate, head and neck, esophagus, glioblastoma, lung, colorectal, cholangiocarci-noma, breast, pancreatic cancer, and renal cell carcinoma.7–16It has been implicated in vari-ous processes involved in carcinogenesis such as inhibition of apoptosis, auxoaction of cell prolif-eration, and stimulation of tumor growth.12 Many genes under keen study have been con?rmed to be the target genes of miR-21.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene is a capital one. It was ?rst selected as a potential miR-21 target based on its mediating phenotypic characteris-tics of cancer cells17 and authenticated by several studies that followed.18,19 A large amount of data support the view that PTEN is a tumor suppressor gene. The phosphatidylinositol 3-kinase/PTEN/ AKT signaling pathway is involved in different cel-lular activities, including proliferation, migration, cell growth, cell survival, and tumorigenesis. It has been suggested that the constitutive activation of phosphatidylinositol 3-kinase/AKT signaling contributes to cancer formation because of con-current loss of function of the tumor suppressor molecule PTEN.

In this study, we ?rst found that PTEN was expressed at a low level in keloid tissues and nega-tively correlated with the level of miR-21. In addi-tion, we showed the in?uence of miR-21 on cell proliferation and apoptosis by modulating the expression of PTEN. We expect that these results could be explored as a gene therapy for the treat-ment of keloids.

PATIENTS AND METHODS Patient Samples

Keloid and normal skin tissue samples were obtained from 23 patients who underwent opera-tions from 2009 to 2012 at the Second Af?liated Hospital of Harbin Medical University. Informed consent was obtained from each patient recruited, and the study was approved by the hospital ethics committee. Keloid cells were obtained at surgi-cal release from six patients aged 18 to 37 years who had a nonpeduncular keloid on the main trunk, ear lobe, and upper arm of at least 1-year evolution, with clinical activity such as growth, hyperemia, pruritus, and pain. None of them had been treated previously.

Cell Culture

Primary cultures of ?broblasts from the sur-gical specimens were then established. Cells from passages 3 to 8 were used for experiments. Cells were maintained in Dulbecco’s Modi?ed Eagle Medium (Gibco BRL, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (Gibco) and 5 mM L-glutamine, 100 U/ml peni-cillin, and 100 mg/mL of streptomycin in a 5% carbon dioxide air incubator at 37°C. Cells were subcultured every 3 days using trypsin/ethyl-enediaminetetraacetic acid solution (saline containing 0.05% trypsin, 0.01 M sodium phos-phate, and 0.53 μM ethylenediaminetetraacetic acid, pH7.4).

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from the tissue sam-ples using RNApure Tissue Kit (CW BioTech, Bei-jing, China). Quantitative real-time polymerase chain reaction for PTEN was performed with Prime Script RT reagent Kit (Takara Bio, Shiga, Japan). The quantitative real-time polymerase chain reac-tions were carried out using the SYBR green PCR master mix (Takara) and the miRcute miRNA quantitative real-time polymerase chain reaction detection kit (Tiangen, Beijing, China) with a Multiplex Quantitative PCR System (Applied Bio-systems, Foster City, Calif.), and β-actin was used as an internal standard. Primers were designed for quantitative real-time polymerase chain reac-tion from Primer Express software (Applied Biosystems). The primer sequences used were as follows: PTEN, 5′-TTTTGAAGACCATAA CCCACCA-3′ (forward) and 5′-ATCATTACACCA GTTCGTC C CT-3′(reverse); β-actin, 5′-AGAAGGA-GATCACTGCCCTGGCACC-3′(forward) and 5′-CCTGCTTGCTGATCCACATCTGCTG-3′(reverse); miR-21, 5′-GCGGTAGCTTATCAGACT-GATGTTGA-3′(forward); and U6, 5′-ACACG-CAAATTCGTGAAGCGT TCC-3′(forward). The polymerase chain reaction cycling conditions were as follows: PTEN, 5 minutes at 94°C, 40 cycles of 30 seconds at 94°C, 30 seconds at 54°C, and 30 seconds at 72°C, and ?nally 5 minutes at 72°C; and miR-21, 2 minutes at 94°C, 40 cycles of 20 seconds at 94°C, and 34 seconds at 51°C. Melt-ing curve analysis was conducted to determine the speci?city of the reaction. Each sample was tested in triplicate. The miR-21 cDNA was used for

562e

6OLUME .UMBER s MI2 2EGULATES +ELOID &IBROBLASTS

563e

quantitative real-time polymerase chain reaction analysis of miR-21 expression levels. U6 was used as an endogenous control. Relative expression level changes were calculated according to 2–?Ct [?Ct= Ct (miR-21)-Ct(U6)] method as described previously.20,21

Transfection of the miRNA Inhibitor or Mimics

An miR-21 inhibitor was designed according to the mature miR-21 sequence. The sequence of miR-21 antisense oligonucleotides with 2′O -methyl modi?cation and ?uorescently labeled was 5′-UCAACAUCAGUCUGAUAAGCUA-3′, and the negative control sequence was 5′-CAGUA-CUUUUGUGUAGUACAA-3′. The sequences were purchased from Genepharm Biotechnol-ogy (Shanghai, People’s Republic of China) and veri?ed by sequencing. Human keloid ?broblasts were transfected with an miR-21 inhibitor (50 nM) using X-tremeGENE siRNA TRansfection Reagent (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Cells were harvested after 24, 48, and 72 hours to test the transfec-tion ef?ciency. All procedures were performed in triplicate. MicroRNA-21 mimic and a scramble control were purchased (Genepharm; BioTech, Shanghai, China and transfected into cells similar to miR-21 inhibitor. The sequence of miR-21 mim-ics was 5′-UAGCUUAUCAGACUGAUGUUGA-3′ and that of the scramble control was 5′-UUCUC-CGAACGUGUCACGUTT-3′. Cells were infected and subjected to further experiments.

Measurement of DNA Synthesis

We measured DNA synthesis by using the Cell-Light EdU Apollo567 In Vitro Imaging Kit (Ribo Bio Co., Guangzhou, China) according to the manufacturer’s protocol. Adherent ?broblasts in a 96-well plate (Nest Scienti?c, Rahway, N.J.) plated at a density of 5 × 103 cells/well were trans-fected for 24 hours and incubated with 5-ethynyl-2′-deoxyuridine for 2 hours.22 Cells were washed twice with phosphate-buffered saline and then ?xed with 4% paraformaldehyde in phosphate-buffered saline for 30 minutes at room tempera-ture. After washing with phosphate-buffered saline followed by incubation with 2 mg/ml amino acetic acid for 5 minutes, the cells were permeabilized with 0.5% Triton X-100 (Sigma, St. Louis, Mo.) in phosphate-buffered saline for 10 minutes. The cells were then incubated with freshly made Apollo reaction cocktail for 30 minutes at room temper-ature under light protection. After that, nuclei were stained with Hoechst 33342. The stained

cells were examined with a ?uorescence micro-scope and photographed. From the photographs of each cell sample, the ratio of red (5-ethynyl-2′-deoxyuridine–positive) versus blue (nuclear staining) ?uorescent cells was determined as the percentage of 5-ethynyl-2′-deoxyuridine–positive cells. Five ?elds of vision were observed for each group with a coverslip under high-power lens ran-domly, counting arid scoring cells, and the sum of the scores was divided by the cell number,Cell Proliferation Assay

Keloid cells were plated onto 96-well culture plates at 3 × 103 cells per well after 24 hours’ transfection; 3-4,5-dimethyl-2-thiazolyl)-2,5-di-phenyl-2-H-tetrazolium bromide(MTT) solution (5 mg/ml) was then added to each well on each day for 5 consecutive days (?ve wells per group each day). The cells were incubated for an addi-tional 4 hours, after which the supernatant was discarded and the formazan precipitate was dis-solved in dimethyl sulfoxide (100 μl); then, the absorbance was measured in an enzyme-linked immunosorbent assay reader (Thermo Molecu-lar Devices Co., Union City, N.J.) at 570 nm. The data are presented as the mean ± SD, which are derived from ?ve samples of three independent experiments.

Protein Isolation and Western Blot Analysis

Total protein extracts of the keloid and nor-mal skin samples and cells were prepared by homogenization in radioimmunoprecipitation assay (Beyotime BioTech, Jiangsu, China). Brie?y, for isolation of total protein fractions, cells (after 72 hours’ transfection) or tissue samples were collected, washed twice with ice-cold phosphate-buffered saline, and lysed using cell lysis buffer [20 mM Tris (pH 7.5), 150 mM sodium chloride, 1% Triton X-100, 2.5 mM sodium pyrophos-phate, 1 mM ethylenediaminetetraacetic acid, 1% sodium carbonate, 0.5 μg/ml leupeptin, and

Table 1. Flow Cytometric Analysis of Cell Apoptosis

Group Q1 (%)Q2 (%)Q3 (%)Q4 (%)miR-21 inhibitor 3.15 3.9478.914.72Negative control 6.38 2.684.27 6.75Scramble control 6.45 3.1784.06 6.32miR-21 mimic

4.99

1.49

84.06

2.68

Q1, dead cells; Q2, late apoptosis; Q3, normal cells; Q4, early apoptosis.

564e

Plastic and Reconstructive Surgery s /CTOBER

1 mM phenylmethanesulfonyl ?uoride]. The lysates were collected by scraping from the plates and then centrifuged at 12,000 rpm at 4°C for 15 minutes. Total protein samples (20 μg) were loaded onto a 12% of sodium dodecyl sulfate polyacrylamide gel for electrophoresis, and trans-ferred onto polyvinyl di?uoride transfer mem-branes (Millipore, Billerica, Mass.) at 0.8 mA/cm

2 for 70 minutes. Membranes were blocked at 37°C for 1 hour with blocking solution (1% bovine serum albumin in phosphate-buffered saline plus 0.05% Tween-20). The samples were incubated overnight at 4°C with primary anti-bodies for PTEN, AKT, and phosphorylated AKT (Cell Signaling Technology, Danvers, Mass.) and caspase

3 (Cell Signaling Technology) at a com-patible dilution in blocking solution. After three wash steps in Tris-buffered saline Tween-20 for 10 minutes each, membranes were incubated for 1 hour at room temperature with a horserad-ish peroxidase–conjugated anti-rabbit secondary antibody (ZhongShan JinQiao, Beijing, China) at a dilution of 1:10,000 in blocking solution. After washing three times for 30 minutes each time in Tris-buffered saline Tween-20, signals were visual-ized using an enhanced chemiluminescence kit (Beyotime BioTech).

Immuno?uorescence

Cells were seeded onto 24-well plates at a density of 4 × 104 cells/well. After 72 hours’ transfection, cells were ?xed with 2% formalde-hyde at 4°C for 30 minutes, and permeabilized

with 0.2% Triton X-100 (Sigma) for 15 minutes at room temperature. After three wash steps with phosphate-buffered saline, cells were blocked for 30 minutes with 5% bovine serum albumin in phosphate-buffered saline. The samples were incubated for 2 hours at room temperature with primary antibody at a 1:200 dilution (anticaspase 3 was rabbit polyclonal antibody). After being washed three times in phosphate-buffered saline, samples were incubated for 1 hour with 1 mg/ml ?uorescein isothiocyanate–labeled anti-rab-bit secondary antibody (ZhongShan JinQiao) in blocking solution. After three additional wash steps, the samples were visualized by ?uores-cence microscopy.

Flow Cytometric Analysis of Cell Apoptosis

The extent of apoptosis was measured by means of an annexin V–?uorescein isothiocya-nate/propidium iodide apoptosis detection kit (Beyotime BioTech) as described by the manu-facturer. After transfection with the miRNA inhibitor or mimics for 24 hours, cells were col-lected, washed twice with phosphate-buffered saline, gently resuspended in annexin V bind-ing buffer, and incubated with annexin V–?uo-rescein isothiocyanate/propidium iodide in the dark for 15 minutes and analyzed by ?ow cytom-etry using FloMax software (Partec, Munich, Germany). The fraction of the cell population in different quadrants was analyzed using quad-rant statistics. The lower left quadrant contained

intact cells, the lower right quadrant contained

Fig. 1.

fi broblasts were transfected with miR-21 mimic, scramble control, miR-21 inhibitor, and nega-tive control, respectively. At 24, 48, and 72 hours later, relative expression levels of miR-21 were analyzed by quantitative real-time polymerase chain reaction. Results showed that miR-21 levels had changed greatly in cells compared with related controls (*p < 0.0001), whereas they were not signi fi cantly di ff erent between time points (p = 0.43).

6OLUME .UMBER s MI2 2EGULATES +ELOID &IBROBLASTS

565e

early apoptotic cells, and the upper right quad-rant contained necrotic or postapoptotic cells.Statistical Analysis

The data are expressed as mean ± SD. The t test was conducted to compare the difference between the expression levels of PTEN in the 23 keloid samples and normal skin tissues. Transient transfection results and proliferation of keloid cells were compared by analysis of repeated measure-ments for four groups. The effect of DNA synthesis was measured by using the chi-square test. Western blot analysis was performed with ImageJ software (National Institutes of Health, Bethesda, Md.), and the experimental result was handled by analysis of variance. All statistics were calculated using the STATISTICA program (StatSoft, Inc., Tulsa, Okla.). A value of p < 0.05 was considered signi?cant.

RESULTS

miR-21 Inhibitor and Mimic Regulation of miRNA Expression

To evaluate the biological impact of cutting down miR-21 levels in human keloid ?broblasts, we transfected the keloid cells with miR-21 mimic, scramble control, miR-21 inhibitor, and negative control, respectively. To determine the ef?ciency of transfection, the amount of miR-21 was detected by quantitative real-time polymerase chain reaction at 24, 48, and 72 hours after transfection. We found that the levels of miR-21 were strikingly changed after transfection at 24, 48, and 72 hours compared with relative control (Fig. 1). Compared with nega-tive controls, expression of miR-21 was signi?cantly enhanced in cells transfected with miR-21 mimics (p < 0.0001), whereas miR-21 expression was obvi-ously decreased in cells transfected with

miR-21

Fig. 2. Transient transfection of miR-21 inhibitor inhibits DNA synthesis and cell proliferation in keloid cells, opposite the e ff ect of miR-21 mimic. Cells were treated with 5-ethynyl-2′-deoxyuridine (EdU ) and nuclei were stained with Hoechst after 24 hours’ trans-fection. Photographs are shown at 100× magni fi cation by imaging with fl uorescence microscopy. Scale bar = 200 μ

m.

566e

Plastic and Reconstructive Surgery s /CTOBER

inhibitor (p < 0.0001). There is no statistical signi?-cance between control groups or between different time points (p = 0.43).

miR-21 Modulates Keloid Fibroblast DNA Synthesis and Proliferation

DNA synthesis was measured by using a nucleotide analogue of thymidine, 5-ethynyl- 2′-deoxyuridine, which is incorporated into DNA during S phase. The incorporation rate can re?ect DNA synthesis speed (i.e., the cell vitality). In the inhibitor group, the incorporating rate was 28.89 percent (Fig. 2), whereas in the cells transfected with miR-21 mimics, the cell banding rate was 54.05 percent (Fig. 3). The groups of negative and scram-ble control were 43.2 percent and 42.71 percent,

respectively. All of these results indicated that low expression of miR-21 markedly inhibited keloid cell DNA synthesis (p = 0.035), and overexpression of miR-21 visibly increased keloid ?broblast DNA synthesis (p = 0.005). From the MTT assay (Fig. 4), proliferation of keloid cells after small interfering RNA transfection was signi?cantly reduced com-pared with control and that overexpression of miR-21 led to elevated proliferation. It is indicated that the aberrant expression of miR-21 in keloid ?bro-blasts may contribute to the overmultiplication.Down-Regulation of miR-21 Inhibits Apoptosis of Keloid Fibroblasts

Furthermore, we performed ?ow cytometry

to analyze the effect of miR-21 on cell apoptosis

Fig. 4. Proliferation of keloid cells after transfection was signi fi cantly reduced in the miR-21 inhibitor group compared with the control group and increased in the mimic group. *p < 0.05 (signi fi cant di ff

erence).

Fig. 3. The black histogram represents the ratio of the percentage of 5-ethynyl-2′-deoxyuridine (EdU ) incorporation rate and is expressed as the ratio of 5-ethynyl-2′-deoxyuridine–positive cells versus total cells. *p = 0.035 (signi fi cant di ff erence); ?p = 0.005 (signi fi cant di ff

erence).

6OLUME .UMBER s MI2 2EGULATES +ELOID &IBROBLASTS

567e

by conducting annexin V and propidium iodide double-staining. As shown in Figure 5, the annexin V–positive early apoptotic cells were increased in cells transfected with miR-21 inhib-itor (14.72 percent) compared with the nega-tive control group (Table 1). The percentage of early-phase apoptosis was decreased after over-expression of miR-21. These results indicate that down-regulation of miR-21 induces apopto-sis. Immuno?uorescent assay and Western blot were used to determine the miR-21–induced apoptosis in keloid ?broblast cells. As shown in Figures 6 and 7, the expression of active caspase 3 was much stronger in cells transfected with miR-21 inhibitor than in the other groups.

Low Expression of PTEN in Keloid Tissue Compared with Normal Skin

PTEN mRNA expression of 23 keloid and normal skin samples was detected by real-time polymerase chain reaction analysis. As shown in Figures 8 and 9, PTEN mRNA levels were signi?cantly higher in the normal skin samples (p < 0.0001). PTEN protein was greatly decreased in keloid compared with nor-mal skin samples. This result con?rmed the lower expression level of PTEN in keloid samples.miR-21 Suppresses PTEN and Negatively

Regulates the AKT Pathway in Keloid Fibroblasts

We estimated whether the regulation of miR-

21 could affect PTEN protein expression in keloid

Fig. 5. Keloid cells were transfected with miR-21 inhibitor, mimics, and their controls. Cells were collected for the next procedure after transfection. Flow cytometric analysis was used to assess cell apoptosis. (Above , left ) Cells transfected with miR-21 inhibitor. (Above , right ) Cells transfected with negative control. (Below , left ) Cells transfected with scramble control. (Below , right ) Cells transfected with miR-21 mimics. PI , propidium iodide; FITC , fl uorescein isothiocyanate.

568e

Plastic and Reconstructive Surgery s /CTOBER

cells. Total cellular protein was isolated from cells transfected with miR-21 inhibitor, miR-21 mimics, and negative and scramble control, respectively. The miR-21 inhibitor signi?cantly increased PTEN protein expression (Fig. 10). AKT was a considerable downstream target of PTEN; thus, the expression of phosphorylated AKT and AKT was measured at the same time (Fig. 11). The down-regulation of miR-21 resulted in a signi?-cant increase of PTEN protein, and up-regulation of miR-21 induced the opposite result. Meanwhile, the diversity of phosphorylated AKT protein was

inversely related to the change of PTEN protein. The expression of phosphorylated AKT was con-comitantly decreased by miR-21 inhibitor.Mimics miR-21 Transfection in Normal Skin Fibroblasts Increases Proliferation

We wondered whether raising the miR-21 level of normal ?broblasts could result in changes of the cell phenotype. Therefore, experiments to increase miR-21 expression by transfecting miR-21 mimics were conducted. Our results showed that overexpression of miR-21 caused an

increased

Fig. 7. Immunocytochemical staining of active caspase 3 in keloid fi broblast cells after transfection with miR-21 inhibitor. Caspase 3 was visualized by counterstaining with an anti–fl

ized with 4′,6-diamidino-2-phenylindole (fl uorescence microscopy; original magni fi Scale bar = 50 μm.

Fig. 6. Western blot analysis showed endogenous levels of full-length caspase 3 (35 kDa) in each group and cleaved caspase 3 (17 kDa) in miR-21 inhibitor transfected keloid cells.

Caspase-3 32KD Caspase-3 17KD

6OLUME .UMBER s MI2 2EGULATES +ELOID &IBROBLASTS

569e

ability of ?broblasts to synthesize DNA (Fig. 12). The percentage of cells incorporating 5-ethynyl-2′-deoxyuridine transfected with miR-21 mimics was 47.06 percent compared with the scramble control, which was 38.09 percent (p = 0.0006) (Fig. 13), which means that miR-21 may be a stim-ulus to abnormal growth.

DISCUSSION

To date, the cause of keloid formation is still vague, and it is necessary to further explore the pathogenesis of keloids. The main conclusion of this study is that miR-21 may serve as a novel etiologic factor for keloid by negatively regulating PTEN. MicroRNA-21, one of the most prominent miRNAs, is implicated in human malignancy. Its expression is obviously up-regulated in diverse

types of malignancies such as breast cancer, lung cancer, esophageal cancer, gastrointesti-nal cancer, and others.10,12,17 PTEN, identi?ed as a tumor suppressor, could induce cell apoptosis and control cell growth, invasion, migration, and angiogenesis through interference with several signaling pathways.23–25 It has been proven that miR-21 suppresses PTEN by direct binding to the 3′ untranslated region of PTEN.26 Conversely, the links between miR-21, PTEN, and keloids have not been described.

Recent reports indicate that PTEN can be reg-ulated by miR-21 in many tumor cells.23,24,27 This is consistent with our ?ndings. In this work, the PTEN regulation effect by miR-21 and its possible mecha-nism was studied in keloid tissue ?rst. Expression of PTEN in 23 keloid tissues and adjacent nor-mal skin tissues was investigated by

quantitative

Fig. 8. PTEN expression in keloid and normal skin samples, signi fi -cant di ff erences (p

< 0.0001).

Fig. 9. The protein level of PTEN in keloid and normal skin samples detected by Western blot assay (N1 to N4, normal skin samples; K1 to K4, keloid samples). Bands were quantitated by densitometric analysis. Fold change represents the protein level of keloid and samples to the fi rst normal skin sample and the resulting protein levels were then normalized to the β

-actin protein.

570e

Plastic and Reconstructive Surgery s /CTOBER

real-time polymerase chain reaction and Western blot assay. The results showed that PTEN protein was decreased in keloid samples compared with normal skin samples (Fig. 9). In a previous study, we found that the expression of miR-21 was much higher in keloid tissues than in adjacent normal skin.6 Considered together, we can conclude that the protein level of PTEN was negatively corre-lated to the level of miR-21 in keloid tissues.

Apoptosis, a form of programmed cell death that occurs through activation of cell-intrinsic suicide machinery,28 has been considered as the major form of cell death in various physiologic events.29,30 Caspases are part of a growing family of cysteine proteases that have been involved in many forms of apoptosis.31 Activation of caspase proteases was required for the induction of apop-tosis in different cell types.32,33 Caspase 3 is one of the key executioners of apoptosis. On activation, caspase 3 can cleave ?ve substrates, including other effectors’ caspases and fodrin, which forms a cytoskeletal network.34

Fig. 10. Western blot analysis of PTEN, phosphorylated AKT, AKT, and β-actin with loss or gain of function of miR-21. Total cellular protein was isolated from cells transfected with miR-21 inhibitor, miR-21 mimics, and negative and scramble control, respectively. The miR-21 inhibitor enhanced PTEN expression. β-actin was used as a loading

control.

Fig. 11. Data were normalized to β-actin and presented in a column chart (?p < 0.01, *p < 0.05).

6OLUME .UMBER s MI2 2EGULATES +ELOID &IBROBLASTS

571e

AKT (v-akt murine thymoma viral oncogene/protein kinase B) is a serine/threonine kinase that is involved in mediating various biological responses.35 The actions of AKT in the cell are numerous and diverse, including metabolism, protein synthesis, apoptosis pathways, transcrip-tion factor regulation, and the cell cycle. AKT exerts its effects in the cell by phosphorylating a variety of downstream substrates.36 Recent stud-ies have demonstrated that miR-21 increases tumor cell proliferation, migration, and invasion through targeting PTEN ,37 a tumor suppressor

3-kinase by removing the 3′ untranslated region phosphate of phosphatidylinositol 3,4,5-trisphos-phate. It is well known that PTEN negatively regu-lates the phosphatidylinositol 3-kinase–protein kinase B pathway 38 and the mitogen-activated pro-tein kinase/extracellular signal regulated kinase 1/2 pathway.39

In various cancer cells (e.g., renal cancer, breast cancer, ovarian carcinomas), researchers have provided evidence for an inverse correla-tion between miR-21 levels and PTEN abun-dance. Furthermore, they have demonstrated that miR-21–sensitive PTEN regulates prolifera-tion and migration of cancer cells by means of activation of AKT.40–42 Thus, we evaluated the expression of phosphorylated AKT in cells trans-fected with miR-21 inhibitor, miR-21 mimics, and negative and scramble control, respectively, to further study the apoptosis pathway induced by PTEN in keloid ?broblasts. Data showed that miR-21 inhibitor increased expression of PTEN and inhibited phosphorylated AKT expression (Fig. 10), which suggested that miR-21 inhibi-tor transfection induced cell apoptosis by means of blocking the phosphatidylinositol 3-kinase/AKT pathway. It seems that miR-21 suppresses PTEN and negatively regulates the AKT pathway in keloid ?broblasts. To further demonstrate the effect of miR-21 on PTEN expression and cell proliferation and apoptosis, miR-21 mimics was transfected in normal skin ?broblasts. Results

Fig. 12. Normal skin fi broblasts were transfected with miR-21 mimics and scramble control, respectively. Twenty-four hours later, cells were treated with 5-ethynyl-2′-deoxyuridine (EdU )– and Hoechst-stained nuclei. Photographs are shown at 100× magni fi ca-tion. Scale bar = 200 μm.

Fig. 13. The black histogram represents the ratio of the per-centage of DNA synthesis and was expressed as the ratio of 5-ethynyl-2′-deoxyuridine–positive (Edu ) cells versus total cells. *p = 0.0006 (signi fi cant di ff erence).

572e

Plastic and Reconstructive Surgery s /CTOBER

showed that cell proliferation was increased when normal skin ?broblasts were transfected with miR-21 mimics (Fig. 12). These data also supported that miR-21 could negatively regulate PTEN expression and then affect cell apoptosis by means of the AKT pathway.

There must be many other factors and path-ways that can affect these processes; we can only show that the phosphatidylinositol 3-kinase/AKT pathway is one of them, but it is certainly not the one and only pathway. Further studies will be required to investigate precise molecular mechanisms on how miR-21 affects related gene expression and functions. Our data thus suggest that miR-21 in?uences cell proliferation and apoptosis, perhaps by modulating the expression of PTEN in keloid ?broblasts. These ?ndings pro-vide some hints toward effective applications of

miR-21 as a therapy target for keloids.

Zhibo Xiao, Ph.D.

Plastic and Aesthetic Department

Second Af?liated Hospital of Harbin Medical University

Harbin 150086, People’s Republic of China

xiaozhibodoctor@https://www.360docs.net/doc/a712221665.html,

A CKNOWLEDGMENTS The research was supported by the National Natu-ral Science Fund of China (no. 81271711 and no.

81301635) and the Natural Science Foundation of Heilongjiang Province of China (no. 2011-D-61). The authors thank the scienti?c research center of Second Af?liated Hospital of Harbin Medical University for donating their time, equipment, and personnel.

REFERENCES

1. Atiyeh BS. Nonsurgical management of hypertrophic scars:

Evidence based therapies, standard practices, and emerging methods. Aesthetic Plast Surg . 2007;31:468–492.

2. Juckett G, Hartman-Adams H. Management of keloids and

hypertrophic scars. Am Fam Physician 2009;80:253–260.

3. Nurul Syazana MS, Halim AS, Gan SH, Shamsuddin S.

Antiproliferative effect of methanolic extraction of tualang honey on human keloid ?broblasts. BMC Complement Altern Med . 2011;11:82.

4. Vincent AS, Phan TT, Mukhopadhyay A, Lim HY, Halliwell B,

Wong KP. Human skin keloid ?broblasts display bioenerget-ics of cancer cells. J Invest Dermatol . 2008;128:702–709.

5. Al-Attar A, Mess S, Thomassen JM, Kauffman CL, Davison

SP. Keloid pathogenesis and treatment. Plast Reconstr Surg . 2006;117:286–300.

6. Liu Y, Yang D, Xiao Z, Zhang M. miRNA expression pro-?les in keloid tissue and corresponding normal skin tissue. Aesthetic Plast Surg . 2012;36:193–201.

7. Zhang Z, Li Z, Gao C, et al. miR-21 plays a pivotal role in

gastric cancer pathogenesis and progression. Lab Invest . 2008;88:1358–1366.

8. Folini M, Gandellini P, Longoni N, et al. miR-21: An oncomir

on strike in prostate cancer. Mol Cancer 2010;9:12.

9. Kimura S, Naganuma S, Susuki D, et al. Expression of microR-NAs in squamous cell carcinoma of human head and neck and the esophagus: miR-205 and miR-21 are speci?c markers for HNSCC and ESCC. Oncol Rep . 2010;23:1625–1633.

10. Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an anti-apoptotic factor in human glioblastoma cells. Cancer Res . 2005;65:6029–6033.

11. Selaru FM, Olaru AV, Kan T, et al. MicroRNA-21 is over-expressed in human cholangiocarcinoma and regulates programmed cell death 4 and tissue inhibitor of metallopro-teinase 3. Hepatology 2009;49:1595–1601.

12. Si ML, Zhu S,Wu H, Lu Z, Wu F, Mo YY. miR-21-mediated tumor growth. Oncogene 2007;26:2799–2803.

13. Gao W, Xu J, Liu L, Shen H, Zeng H, Shu Y. A systematic-analysis of predicted miR-21 targets identi?es a signature for lung cancer. Biomed Pharmacother . 2012;66:21–28.

14. Yamamichi N, Shimomura R, Inada K, et al. Locked nucleic acid

in situ hybridization analysis of miR-21 expression during colorec-tal cancer development. Clin Cancer Res . 2009;15:4009–4016.

15. Dillhoff M, Liu J, Frankel W, Croce C, Bloomston M. MicroRNA-21 is overexpressed in pancreatic cancer and a potential predictor of survival. J Gastrointest Surg . 2008;12:2171–2176.

16. Zhang A, Liu Y, Shen Y, Xu Y, Li X. miR-21 modulates cell apoptosis by targeting multiple genes in renal cell carci-noma. Urology 2011;78:474.e13–474.e19.

17. Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007;133:647–658.

18. Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y, Klinge CM. Estradiol downregulates miR-21 expres-sion and increases miR-21 target gene expression in MCF-7 breast cancer cells. Nucleic Acids Res . 2009;37:2584–2595.

19. Iliopoulos D, Jaeger SA, Hirsch HA, Bulyk ML, Struhl K. STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking in?ammation to cancer. Mol Cell 2010;39:493–506.

20. Schmittgen TD, Lee EJ, Jiang J, et al. Real-time PCR quan-ti?cation of precursor and mature microRNA. Methods 2008;44:31–38.

21. Livak KJ, Schmittgen TD. Analysis of relative gene expres-sion data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–408.

22. Wang F, Zhao XQ, Liu JN, et al. Antagonist of microRNA-21 improves balloon injury-induced rat iliac artery remodeling by regulating proliferation and apoptosis of adventitial ?bro-blasts and myo?broblasts. J Cell Biochem . 2012;113:2989–3001.

23. Stewart AL, Mhashilkar AM, Yang XH, et al. PI3K block-ade by Ad-PTEN inhibits invasion and induces apoptosis in radial growth phase and metastatic melanoma cells. Mol Med . 2002;8:451–461.

24. Tamura M, Gu J, Matsumoto K, Aota S, Parsons R, Yamada KM. Inhibition of cell migration, spreading, and focal adhe-sions by tumor suppressor PTEN. Science 1998;280:1614–1617.

25. Castellino RC, Durden DL. Mechanisms of disease: The PI3K-Akt-PTEN signaling node. An intercept point for the control of angiogenesis in brain tumors. Nat Clin Pract Neurol . 2007;3:682–693.

26. Zhang JG, Wang JJ, Zhao F, Liu Q, Jiang K, Yang GH. MicroRNA-21 (miR-21) represses tumor suppressor PTEN and promotes growth and invasion in non-small cell lung cancer (NSCLC). Clin Chim Acta 2010;411:846–852.

6OLUME .UMBER s MI2 2EGULATES +ELOID &IBROBLASTS

573e

27. Friedland DR, Eernisse R,Erbe C, Gupta N, Ciof? JA. Cholesteatoma growth and proliferation: Posttranscrip-tional regulation by miR-21. Otol Neurotol . 2009;30: 998–1005.

28. Vinatier D, Dufour P, Subtil D. Apoptosis: A programmed cell death involved in ovarian and uterine physiology. Eur J Obstet Gynecol Reprod Biol . 1996;67:85–102.

29. Sarraf CE, Bowen ID. Proportions of mitotic and apoptotic cells in a range of untreated experimental tumours. Cell Tissue Kinet . 1988;21:45–49.

30. Carson DA, Ribeiro JM. Apoptosis and disease. Lancet 1993;341:1251–1254.

31. Nicholson DW. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ . 1999;6:1028–1042.

32. Islam S, Islam N, Kermode T, et al. Involvement of cas-pase-3 in epigallocatechin-3-gallate-mediated apoptosis of human chondrosarcoma cells. Biochem Biophys Res Commun . 2000;270:793–797.

33. Hayakawa S, Saeki K, Sazuka M, et al. Apoptosis induction by epigallocatechin gallate involves its binding to Fas. Biochem Biophys Res Commun . 2001;285:1102–1106.

34. Hsu HF, Houng JY,Kuo CF, Tsao N, Wu YC. A novel phen-ylpropanoid from Glossogyne tenuifolia , induced apopto-sis in A549 lung cancer cells. Food Chem Toxicol . 2008;46: 3785–3791.

35. Testa JR, Tsichlis PN. AKT signaling in normal and malig-nant cells. Oncogene 2005;24:7391–7393.

36. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2:489–501.

37. Vogt PK, Gymnopoulos M, Hart JR. PI 3-kinase and cancer: Changing accents. Curr Opin Genet Dev . 2009;19:12–17. 38. Weng LP , Smith WM, Brown JL, Eng C. PTEN inhibits insulin-stimulated MEK/MAPK activation and cell growth by blocking

IRS-1 phosphorylation and IRS-1/Grb-2/Sos complex forma-tion in a breast cancer model. Hum Mol Genet . 2001;10:605–616.

39. Park MJ, Kwak HJ, Lee HC, et al. Nerve growth factor induces endothelial cell invasion and cord formation by promoting matrix metalloproteinase-2 expression through the phos-phatidylinositol 3-kinase/Akt signaling pathway and AP-2 transcription factor. J Biol Chem . 2007;282:30485–30496.

40. Dey N, Das F, Ghosh-Choudhury N, et al. microRNA-21 gov-erns TORC1 activation in renal cancer cell proliferation and invasion. PLoS One 2012;7:e37366.

41. Han M, Liu M, Wang Y, et al. Antagonism of miR-21 reverses epithelial-mesenchymal transition and cancer stem cell phenotype through AKT/ERK1/2 inactivation by targeting PTEN. PLoS One 2012;7:e39520.

42. Polytarchou C, Iliopoulos D, Hatziapostolou M, et al. Akt2 regulates all Akt isoforms and promotes resistance to hypoxia through induction of miR-21 upon oxygen depriva-tion. Cancer Res . 2011;71:4720–4731.