MicroRNA-16 inhibits cell proliferation and migration by targeting heat shock protein 70 in heat-denatured dermal fibroblasts

Article information

Korean J Intern Med. 2019;34(3):634-642

Publication date (electronic) : 2018 January 5

doi : https://doi.org/10.3904/kjim.2016.315

Chun Zhang ¹, Jinhua Dai ², Youfen Fan ¹, Xianghui He ¹, Renxiong Wei ³

¹Department of Burn Ningbo No.2 Hospital, Ningbo, China

²Department of Clinical Laboratory, Ningbo No.2 Hospital, Ningbo, China

³Department of Clinical Laboratory, Ningbo Municipal Hospital of Traditional Chinese Medicine, Ningbo, China

Correspondence to Renxiong Wei, B.D. Department of Clinical Laboratory, Ningbo Municipal Hospital of Traditional Chinese Medicine, No. 819, Liyuan North Rd, Ningbo 315010, China Tel: +86-574-87089086 Fax: +86-574-87089086 E-mail: weirenxiong1001@126.com

Received 2016 September 28; Revised 2017 February 8; Accepted 2017 April 17.

This article has been retracted. See "Retraction notice to “MicroRNA-16 inhibits cell proliferation and migration by targeting heat shock protein 70 in heat-denatured dermal fibroblasts”" in Volume 35 on page 1532.

Abstract

Background/Aims

This study aimed to investigate the precise mechanism and function of miR-16 in heat-denatured primary human dermal fibroblasts.

Methods

Primary human dermal fibroblasts were separated from normal human skin samples. Under heat stress, the levels of miR-16 and heat shock protein 70 (HSP70) were detected in primary human dermal fibroblasts by quantitative real-time polymerase chain reaction (qRT-PCR). Next, heat-denatured cells were transfected with synthetic scrambled negative control (NC) RNA (NC group), miR-16 mimics, miR-16 inhibitor or miR-16 inhibitor accompanied by small interfering RNA targeting HSP70, then the mRNA level of HSP70 was detected by qRT-PCR, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and AlamarBlue assay, cell migration was examined by Transwell assay and cell apoptosis was assessed by transferase dUTP (deoxyuridine triphosphate) nick end labeling (TUNEL) assay. In addition, cell apoptosis-related proteins, Bax and Bcl-2, were detected by Western blotting.

Results

Heat stress significantly reduced miR-16 level and increased the mRNA level of HSP70 compared with untreated cells (p < 0.05). Overexpressed miR-16 reduced the mRNA level of HSP70, suppressed cell proliferation (p < 0.05 or p < 0.01), migration (p < 0.05), and promoted cell apoptosis (p < 0.001) compared with the NC group. Down-regulated miR-16 exerted an opposite effect on primary human dermal fibroblasts with heat-denaturation. Furthermore, effects of miR16 down-regulation on cell proliferation and migration were reversed by HSP70 silence.

Conclusions

MiR-16 might have an inhibitory effect on cell proliferation and migration in heat-denatured human dermal fibroblasts, and HSP70 might be associated with the cell proliferation and migration as a target gene of miR-16.

Keywords: MIRN16 microRNA; Wound healing; HSP70; Cell proliferation; Cell movement

INTRODUCTION

Healing in wounded skin is a dynamic physiological process, which involves in various cell types and some multiplicate stages, such as inflammation, re-epithelialization, hemostasis, and remodeling [1,2]. Skin burns usually cause denatured dermis that is closely related to pathologically morphological changes, cell metabolism disorders and functional impairment [3]. Furthermore, the denatured dermis is considered to participate in skin morphological and functional recovery during wound healing [3]. Therefore, it is necessary to investigate and search for the effective method for preservation of denatured dermis in the process of wound healing.

Heat shock proteins (HSPs), a highly conserved proteins family, are associated with heat-related pathology, and can be induced by various cellular stresses [4,5]. A previous study has shown that HSPs play key roles in cell cycle control, cell apoptosis and cytoskeletal rearrangement [6]. HSP70 is considered as a major stress-inducible HSP and remarkably up-regulated by heat stress in B lymphocytes [7]. Schmitt et al. [8] have demonstrated that HSP70 has anti-apoptotic and pro-proliferative effects under a variety of lethal stimuli, including heat stimuli. However, the precise mechanism of HSP70 in wound healing induced by heat stress is still unclear.

Micro-RNAs (miRNAs), a kind of small noncoding RNAs (approximately 22 nucleotides in length), can inhibit the expressions of target genes through binding to the 3’-untranslated region (3’UTR) of the target mRNAs at the post-transcriptional level [9,10]. The regulatory role of miRNAs has been proved in wound healing, skin morphogenesis and cancer [11]. Sand et al. [12] have reported that multiple miRNAs such as miR-105, miR-125b, miR-15, miR-16, miR-29, and miR-192 are differentially expressed during wound healing. Notably, a recent study has shown that miR-16 remarkably reduces the expression of HSP70 in Parkinson disease [13]. Currently, several studies have demonstrated the anti-tumor effect of miR-16 by regulating cell cycle and cell apoptosis [14-16]. In addition, miR-16 is also reported to be involved in the granulation phase during wound healing [12]. However, few studies have investigated the mechanism and the function of miR-16 in the wound-healing process.

In the present study, we detected the levels of miR-16 and HSP70 in primary human dermal fibroblasts under heat stress. Next, we up- or down-regulated the miR-16 level in heat-denatured primary human dermal fibroblasts, and further explored the effect of miR-16 on HSP70 expression, cell proliferation, migration, and apoptosis in heat-denatured primary human dermal fibroblasts.

METHODS

Cell culture and transfection

This study was approved by the Ethics Committee (IACUC-14-047) of our University and normal human skin samples were obtained from patients who performed plastic surgery. Written informed consents were obtained from patients before the surgery. Then, primary human dermal fibroblasts were separated from these normal human skin samples. Briefly, skin samples were cut into small pieces (0.1 × 0.1 cm) and digested using trypsin-EDTA (Sigma, St. Louis, MO, USA). Then, the digested pieces were incubated with Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Sigma) and penicillin/streptomycin (1%; Gibco) with 5% CO₂ at 37℃. Subsequently, the separated human dermal fibroblasts were cultured at 52℃ with a water bath for 30 seconds and cells treated with a bath at 37℃ were served as the control group.

After heat-denaturation for 24 hours, primary human dermal fibroblasts were cultured in 6-well plates. On the following day, the cells were transfected with synthetic scrambled negative control (NC) RNA (NC group), miR-16 mimics, miR-16 inhibitor or small interfering RNA targeting HSP70 (si-HSP70, Ambion, Austin, TX, USA) in equal amounts using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The cells were collected after transfection for 24 hours for the following experiments.

Quantitative real-time polymerase chain reaction

Trizol reagent (Invitrogen) was used to extract total RNA, and mirVana miRNA Isolation Kit (Ambion) was used to collect miRNAs. Then, the expressions of HSP70 mRNA and miR-16 were detected with One-Step RT-PCT kit (TaKaRa Bio Inc., Tokyo, Japan). The comparative threshold (Ct) cycle method (2^–ΔΔCt) was used for relative quantification normalized with β-actin for HSP70 and U6 SnRNA for miR-16.

Cell proliferation

Under heat stress, human dermal fibroblasts were incubated in 96-well plates. On the following day, cells were transfected with scrambled NC RNA, miR-16 mimics or miR-16 inhibitor, respectively. After transfection for 12, 24, or 48 hours, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and AlamarBlue assay. For MTT assay, 10 μL of MTT (5 mg/mL; Sigma) was added into each well for 4 hours at 37℃. Next, 100 μL of dimethyl sulfoxide (Sigma) was used to dissolve the formazan crystals. A microplate reader (Molecular Devices, Silicon Valley, CA, USA) was used to measure the absorbance at 490 nm. For AlamarBlue assay, the medium was replaced by DMEM containing 10% AlamarBlue (Invitrogen) and the mixture was incubated for 3 hours at 37℃. The absorbance was measured at 570 nm (600 nm background subtraction) with a microplate reader (Molecular Devices).

Cell migration assay

Cell migration was evaluated using Transwell assay. The transfected cells were suspended with DMEM containing 0.5% FBS, and then added into the upper chamber. The lower chamber was filled with 500 μL DMEM containing 10% FBS. After cultured for 16 hours, the cells on the upper side of the membrane were wiped carefully with cotton swab. Then, the cells on the lower side of the membrane were fixed by 4% paraformaldehyde for 30 minutes and were stained with crystalline violet for 15 minutes. The cells that moved to the lower membrane were counted in four randomly selected visions (× 200) under an inverted microscope (Olympus, Tokyo, Japan).

Western blotting

The collected cells were treated with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) for 30 minutes on ice. Totally, 25 μg protein which quantified by BCA protein assay kit (Beyotime Institute of Biotechnology) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membrane. After being blocked with 3% defatted milk for 1 hour, the membrane was exposed to anti-β-actin (1:1,000; sc47778), anti-HSP70 (1:500; sc-137239), anti-Bax (1:1,000; sc-20067) or anti-Bcl-2 (1:1,000; sc-509) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4℃ overnight. After washing with phosphate-buffered saline (PBS), the membrane was incubated with secondary antibody marked by horseradish peroxidase for 2 hours at room temperature. The protein was detected with Enhanced chemiluminescence (ECL, Applygen Technologies Inc., Beijing, China). The signals were captured and the intensity of the bands was quantified using Image Lab software (Bio-Rad).

TUNEL assay

Cell apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP (deoxyuridine triphosphate) nick end labeling (TUNEL) staining with an In Situ Cell Death Detection Kit (Roche, Indianapolis, IN, USA) according to the supplier’s instructions. Briefly, the transfected cells were fixed by 4% paraformaldehyde for 30 minutes, followed by incubation with TUNEL buffer for 1 hour at 37℃. After rinsing by PBS, the number of TUNEL positive apoptotic cells and the total number of cells in five different random high power fields were counted using a microscope (Olympus) at a magnification of 400. The percentage of apoptotic cells was calculated as the ratio of the number of TUNEL-positive cells to the total number of cells.

Statistical analysis

Data was analyzed by SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as the mean ± standard deviation (SD) or mean + SD and analyzed by student t test or one-way ANOVA. Significant statistical differences were defined as a value of p < 0.05.

RESULTS

Effect of heat stress on the expressions of miR-16 and HSP70 mRNA in primary human dermal fibroblasts

We detected the expressions of miR-16 and HSP70 mRNA in primary human dermal fibroblasts with heat-denaturation by quantitative real-time polymerase chain reaction. The results showed that after treatment with heat stress, the level of miR-16 was significantly reduced and reached the lowest level at 24 hours, while the reduced miR-16 level displayed a slow upturn at 48 hours (p < 0.05) (Fig. 1A). Conversely, the mRNA level of HSP70 was markedly increased (p < 0.05) and had an opposite tendency compared with the level of miR-16 (Fig. 1B). Thus, the following experiments were performed at 24 hours after heat-denaturation.

Figure 1.

Heat stress reduces miR-16 level and elevates the mRNA levels of heat shock protein 70 (HSP70) in primary human dermal fibroblasts. (A) miR-16 level. (B) HSP70 mRNA level. The levels of miR-16 and HSP70 mRNA in heat stress treated primary human dermal fibroblasts at different time points were assessed by quantitative real-time polymerase chain reaction. Values are presented as mean ± SD. Each experiment was repeated five times. ^a p < 0.05 compared with control.

Effect of miR-16 on the expression of HSP70 in primary human dermal fibroblasts with heat-denaturation

The results revealed that miR-16 level was significantly elevated in cells transfected with miR-16 mimics compared with the NC group (p < 0.05) (Fig. 2A), and was markedly lowered in cells transfected with miR-16 inhibitor compared with the NC group (p < 0.05) (Fig. 2A). Furthermore, we found that in comparison with the NC group, the mRNA level of HSP70 was remarkably down-regulated in cells transfected with miR-16 mimics (p < 0.05) (Fig. 2B) and obviously up-regulated in cells transfected with miR-16 inhibitor (p < 0.05) (Fig. 2B).

Figure 2.

Overexpressed miR-16 inhibits the expression of heat shock protein 70 (HSP70) in primary human dermal fibroblasts with heat-denaturation. (A) miR-16 level and (B) HSP70 mRNA level. The levels of miR-16 and HSP70 mRNA after transfected with scrambled negative control (NC) RNA, miR-16 mimics or miR-16 inhibitor in primary human dermal fibroblasts were assessed by quantitative real-time polymerase chain reaction. Values are presented as mean + SD. Each experiment was repeated five times. ^a p < 0.05 compared with the NC group.

Effect of miR-16 on cell proliferation and migration in primary human dermal fibroblasts with heat-denaturation

Results of MTT and AlamarBlue assays showed that in comparison with the NC group, cell viability was significantly reduced in cells transfected with miR-16 mimics at 24 hours and 48 hours (p < 0.05 or p < 0.01) (Fig. 3A-3C). Also, down-regulated miR-16 obviously increased cell viability compared with the NC group at 24 and 48 hours (p < 0.05 or p < 0.01) (Fig. 3A-3C). In addition, Transwell results showed that cells with miR-16 mimics had a weakened migration than the NC group (p < 0.05) (Fig. 3D and 3E), while migration rate was significantly increased in cells with miR-16 inhibitor compared with the NC group (p < 0.05) (Fig. 3D and 3E). The results implied that miR-16 inhibited cell proliferation and migration in primary human dermal fibroblasts with heat-denaturation.

Figure 3.

Overexpressed miR-16 inhibits cell proliferation and migration in primary human dermal fibroblasts with heat-denaturation. Cells were respectively transfected with scrambled negative control (NC) RNA, miR-16 mimics or miR-16 inhibitor. (A) Cell viability upon heat stress by MTT assay, (B) cell viability upon heat stress by AlamarBlue assay, (C) true picture of cell viability at 24 hours after heat stress, (D) true picture of cell migration at 24 hours after heat stress, and (E) cell migration upon heat stress by Transwell assay (×400) at 24 hours after heat stress. (A, B) Values are presented as mean ± SD. (E) Values are presented as mean ± SD. Each experiment was repeated five times. ^a p < 0.05 and ^b p < 0.01 compared with the NC group.

Effect of miR-16 on cell apoptosis in primary human dermal fibroblasts with heat-denaturation

Western blotting results showed that up-regulated miR-16 obviously reduced the expression level of anti-apoptotic protein Bcl-2, while increased the expression of pro-apoptotic protein Bax compared with the NC group (p < 0.05) (Fig. 4A-4D). Conversely, down-regulated miR-16 had an increased expression of Bcl-2 and a decreased expression of Bax when compared to the NC group (p < 0.05) (Fig. 4A-4D). Moreover, the results of TUNEL assay in Fig. 4E demonstrated that cell apoptosis was remarkably enhanced by miR-16 up-regulation (p < 0.001) but repressed by miR-16 down-regulation (p < 0.01) when compared to the NC group.

Figure 4.

Overexpressed miR-16 promoted cell apoptosis in primary human dermal fibroblasts with heat-denaturation. Cells were respectively transfected with scrambled negative control (NC) RNA, miR-16 mimics or miR-16 inhibitor. (A, C) the protein level of Bax by Western blotting, (B, D) the protein level of Bcl-2 by Western blotting, and (E) cell apoptosis by transferase dUTP (deoxyuridine triphosphate) nick end labeling (TUNEL) assay. (C, D) Values are presented as the median of five independent experiments. Error bars indicate minimum and maximum. (E) Values are presented as mean ± SD. Each experiment was repeated five times. ^a p < 0.05, ^b p < 0.01, and ^c p < 0.001 compared with the NC group.

Effect of HSP70 silence on cell proliferation and migration in primary human dermal fibroblasts with heat-denaturation

To validate whether miR-16 functions through modulation of HSP70 expression, cells were respectively transfected with scrambled NC RNA, miR-16 inhibitor or miR-16 inhibitor accompanied by si-HSP70. The cell proliferation and migration were both assessed. In Fig. 5A and 5C, the increase of cell viability induced by miR-16 down-regulation was obviously reversed by HSP70 silence, resulting in significant decrease of cell viability at 24 and 48 hours after heat stress when compared to the NC group (p < 0.05 or p < 0.001). Similarly, the elevated cell migration by miR-16 down-regulation was markedly reversed by HSP70 silence, resulting in obvious decrease of cell migration at 24 hours after heat stress when compared to the NC group (p < 0.001) (Fig. 5B and 5D). Thus, we hypothesized miR-16 functioned via modulation of HSP70 expression.

Figure 5.

Effects of miR-16 down-regulation on cell proliferation and migration were reversed by heat shock protein 70 (HSP70) silence in primary human dermal fibroblasts with heat-denaturation. Cells were respectively transfected with scrambled negative control (NC) RNA, miR-16 inhibitor or miR-16 inhibitor accompanied by small interfering RNA targeting HSP70 (siHSP70). (A) Cell viability by AlamarBlue assay, (B) cell migration by Transwell assay, (C) true picture of cell viability at 24 hours after heat stress, and (D) true picture of cell migration at 24 hours after heat stress (×400). Values are presented as mean ± SD. Each experiment was repeated five times. ^a p < 0.05, ^b p < 0.01, and ^c p < 0.001 compared with the NC group.

DISCUSSION

The proliferation and migration of fibroblast played an important role in the process of wound healing induced by burn [17]. The present study showed that heat stress significantly reduced miR-16 level and increased the mRNA level of HSP70 in primary human dermal fibroblasts. Overexpressed miR-16 could reduce the mRNA levels of HSP70, suppress cell proliferation and migration, and promote cell apoptosis in primary human dermal fibroblasts with heat-denaturation, while down-regulated miR-16 had opposite effects on primary human dermal fibroblasts with heat-denaturation. Moreover, increases of cell viability and migration, induced by miR-16 inhibition, were reversed by HSP70 silence in primary human fibroblasts with heat-denaturation.

Earlier studies on miR-16 mainly focused on its roles in cancer. Bonci et al. [18] had reported that miR-16 inhibits cell survival, proliferation and invasion by targeting BCL2, CCND1, and WNT3A in prostate cancer. Calin et al. [19] showed that miR-16 was involved in the development of chronic lymphocytic leukemia by regulating several cancer genes, including BCL2, ETS1, MCL1, and JUN. Xia et al. [20] also reported that overexpressed miR-16 inhibited the development of multidrug resistance in gastric cancer cells, which was associated with its target gene BCL2. In addition, several studies demonstrated that miR-16 played an potential role in myelodysplastic syndrome [21] and fetal growth [22]. A previous study had demonstrated that miRNAs are associated with the different phases during wound healing through regulating cell differentiation, angiogenesis, migration, and re-epithelialization [23]. Notably, abundant expression of miR-16 was observed in skin morphogenesis [24]. Meanwhile, a recent study has indicated that miR-16 plays a key role in the granulation phase during wound healing [12]. It had been reported that fibroblasts could be activated by heat denaturation, then the activated fibroblasts induced the production of collagen fibers and the expressions of several growth factors [25]. In the present study, we demonstrated that miR-16 was down-regulated in primary human dermal fibroblasts under heat stress. Furthermore, up-regulation of miR-16 inhibited cell proliferation and migration, as well as accelerated cell apoptosis by regulating the expressions of pro-apoptotic Bax and anti-apoptotic Bcl-2, whereas inhibition of miR-16 exerted the opposite effects. These functions of miR-16 in primary human dermal fibroblasts under heat stress were consistent with its role in various cancers [15,26-28]. These data suggested that miR-16 might play a regulatory role in human dermal fibroblasts during wound healing.

It was well-known that the function of miRNAs was exerted by regulating their target genes. BCL2, MCL1, CCND1, and WNT3A were identified as the targets genes of miR-16 in human cancers [14]. A recent study revealed that up-regulated miR-16 could reduce the expression of HSP70 in Parkinson disease and HSP70 might be a target gene of miR-16 [13]. Additionally, a previous study had demonstrated that the transcriptional up-regulation of HSPs could be induced by hypoxia, hyperoxia, heat, or surgical stress in a number of cell lines [7]. Liang et al. [29] had shown that HSP70 is highly expressed in cancer cells and had an anti-apoptotic effect. In the keloid fibroblasts, HSP70 was also proved to be overexpressed [30]. Moreover, approximately 5-fold increase in HSP70 at mRNA level was demonstrated in fibroblasts upon heat shock [31]. Furthermore, in an earlier research, HSP70 was proved to promote cell proliferation and migration in renal cell carcinoma cells [32]. Thus, we hypothesized the regulation of miR-16 might be associated with HSP70 expression. Consistently, this study illustrated that up-regulated miR-16 could inhibit HSP70 expression, whereas down-regulated miR-16 could increase the mRNA level of HSP70 in human dermal fibroblasts with heat-denaturation. Besides, our results also indicated that HSP70 was up-regulated in human dermal fibroblasts under heat stress, and which was negatively correlated with miR-16 level. In addition, we interestingly found that HSP70 silence could reversed the effect of miR-16 down-regulation on cell proliferation and migration in human dermal fibroblasts with heat-denaturation. All these results indicated that HSP70 might be involved in the regulatory role of miR-16 in the proliferation and migration of human dermal fibroblasts under heat stress.

In conclusion, miR-16 might have an inhibitory effect on cell proliferation and migration in human dermal fibroblasts under heat stress, and HSP70 might be associated with the cell proliferation and migration as a target gene of miR-16.

KEY MESSAGE

1. Heat stress reduces miR-16 level in primary human dermal fibroblasts.

2. Heat stress increases heat shock protein 70 (HSP70) level in primary human dermal fibroblasts.

3. HSP70 may be a target gene of miR-16.

4. miR-16 suppresses cell proliferation and migration but promotes cell apoptosis.

5. Effect of miR-16 inhibition is reversed by HSP70 silence.

Notes

No potential conflict of interest relevant to this article was reported.

Acknowledgements

This study was funded in full by the Key Subjects of Ningbo No.2 Hospital (No.2016-55).

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