Dermatologica Sinica

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 37  |  Issue : 3  |  Page : 139--146

Explore the action of MiRNA-21 on shikonin and epidermal growth factor in regulating the proliferation and Apoptosis of HaCaT Cell


Xiaohong Yang1, Fengling Xing2, Maocan Tao1, Lili Ma1, Wei Ding3, Hongbin Luo1, Yi Cao4,  
1 Department of Dermatology and STD, Zhejiang Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang, China
2 Department of Chinese Medicine Surgery, Zhejiang Chinese Medical University, Hangzhou, China
3 Department of Dermatology, Zhejiang Chinese Medical University, Third Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine, Hangzhou, China
4 Department of Dermatology and STD, Zhejiang Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang; Department of Chinese Medicine Surgery, Zhejiang Chinese Medical University, Hangzhou, China

Correspondence Address:
Dr. Yi Cao
Department of Dermatology and STD, Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Youdian Rd., 54th, Hangzhou, 310006
China

Abstract

Purpose: The aim of the study is to investigate the effect of MicroRNA-21 (miR-21) and its interaction with epidermal growth factor (EGF) and shikonin on the proliferation, and apoptosis of HaCaT cell line. Materials and Methods: HaCaT cells were cultured under different concentrations of EGF and shikonin, and to calculate their optimal effect dosages. The transfection was performed using Lipofectamine2000, and then gene expression of miR-21 was detected by quantitative real-time polymerase chain reaction (RT-PCR). MTT assay and flow cytometry were applied to test cell proliferation and apoptosis. Western blot and RT-PCR were used to detect the proliferation (proliferating cell nuclear antigen [PCNA], NF-κB/IKKβ) and apoptosis (caspase-3/caspase-9, bcl-2) signals of HaCaT cell. Results: MTT assay showed that miR-21 mimic and EGF promoted, whereas, shikonin and miR-21 inhibitor inhibited cell viability of HaCaT cell. MiR-21 was upregulated by miR-21 mimic and EGF, while downregulated by shikonin and miR-21 inhibitor. Besides, EGF and miR-21 mimic promoted proliferation-associated signals (PCNA, NF-κB/IKKβ) expression, which were suppressed by shikonin and miR-21 inhibitor. Yet, shikonin and miR-21 inhibitor induced apoptosis-related signals (caspase-3/caspase-9, bcl-2) expression while reversed by EGF and miR-21 mimic which were confirmed by the result of flow cytometry. Conclusions: MiR-21 promotes the process of EGF-induced cell growth of HaCaT. The antagonized effect of shikonin in EGF-induced proliferation and apoptosis might be mediated by suppressing the expression of miR-21.



How to cite this article:
Yang X, Xing F, Tao M, Ma L, Ding W, Luo H, Cao Y. Explore the action of MiRNA-21 on shikonin and epidermal growth factor in regulating the proliferation and Apoptosis of HaCaT Cell.Dermatol Sin 2019;37:139-146


How to cite this URL:
Yang X, Xing F, Tao M, Ma L, Ding W, Luo H, Cao Y. Explore the action of MiRNA-21 on shikonin and epidermal growth factor in regulating the proliferation and Apoptosis of HaCaT Cell. Dermatol Sin [serial online] 2019 [cited 2019 Dec 7 ];37:139-146
Available from: http://www.dermsinica.org/text.asp?2019/37/3/139/258939


Full Text



 Introduction



Psoriasis is a chronic dermatologic disorder, characterized by epidermal proliferation and inflammatory erythema scales, affects individuals of any age.

Although not life-threatening, a relatively high incidence of psoriasis in young adults has affected their psychosocial function and quality of life badly.[1] Moreover, its frequency increases with air pollution and social pressure. The study of the pathogenesis of psoriasis has become an important topic in the field of dermatology.[2] The hyperproliferation of keratinocyte and inflammation is considered as crucial factors in the pathogenesis of psoriasis, and expected to be controlled as potential therapeutic target. Exploratory in controlling psoriatic development is still ongoing.[3]

Recently, 1000 of human-derived microRNAs (miRNAs) have been detected and verified, and some have been widely studied for their pathogenesis role in the skin and malignant diseases.[4],[5] So far, miR-21, one of the first identified mammalian miRNAs, was frequently reported high expressed in solid tumors, and considered as an “onco-miR.” Besides, the present studies suggested that the elevated expression level of miR-21 on keratinocyte associated with psoriatic lesion.[6] It was demonstrated that miR-21 promoted HaCaT cell proliferation, and mediated vascularization and immune response in the process of psoriasis. We speculated that further study of miR-21 in the pathogenesis of psoriasis may provide opportunity for available therapeutic option for psoriasis.

In China, oral Chinese herbal medicine (CHM) penetrated into clinical practice for 1000 of years, especially in difficult miscellaneous or chronic diseases, such as psoriasis, known as “white crust.” In traditional Chinese medicine (TCM), profound experts' experiences in the treatment of psoriasis has been summarized and developed.[7] At present, data analysis on the application of CHM in the treatment of psoriasis gradually constructed, in which, lithospermum is one of the most preferred herbs with functions of cooling blood-heat and detoxification. Our previous clinical practice found that, as monarch drug in Qing Re Liang Xue Decoction, lithospermum significantly reduced the Psoriasis Area and Severity Index (PASI) scores of patients with blood-heat syndrome, and downregulated their serum level of miR-21. Shikonin, the main active ingredient of lithospermum, has been detected to own various pharmacological properties including anti-inflammatory, antitumor/bacterial/fungal, immunoregulation.[8] Although the efficacy of shikonin treatment for psoriasis is well-demonstrated, the potential mechanism remains largely unknown.

Therefore, the present studies highlight potential efficiency of CHM to regulate the proliferation, apoptosis of HaCaT cells through the molecular pathway. Recently, the NF-κB/IKKβ pathway has been identified to be associated with the proliferation, immune response of psoriasis, in which, PCNA also curial to its process. Besides, classic apoptosis signal pathway (caspase-3/-9, bcl-2) also has been checked frequently. In thisin vitro study, human immortalized keratinocyte (HaCaT cell line) was applied as a model for epidermal keratinocytes of psoriasis. We explored the effect of miR-21 and its interaction with shikonin and epidermal growth factor (EGF) in regulating the proliferation and apoptosis of HaCaT cell.

 Materials and Methods



Reagents

EGF (PeproTech, USA), shikonin (Sigma, USA), miR-21 inhibitor (miR20000076-1-5) and miR-21 mimic (miR10000790-1-5) (Ruibo, Guangzhou, China), and Lipofectamine2000 (Life Technologies, USA). Primary antibodies: polyclonal rabbit antibodies to human caspase-3, monoclonal antibodies to human Bcl-2, β-actin (Santa Cruz, USA), rabbit antibodies to human proliferating nuclear antigen (PCNA), NF-κB, and IKKβ (Abcam, USA); secondary antibodies conjugated with horseradish peroxidase: Mouse anti-rabbit immunoglobulin (Promega, USA) and goat anti-mouse (Santa Cruz, USA).

Cell culture and grouping

HaCaT cells were purchased from the Chinese Academy of Sciences Kunming Cell Bank, and cultured with Dulbecco's Modified Eagle's Medium, 10% fetal bovine serum (Life Technologies, UK). Cells in the logarithmic phase of growth were chosen for further experiments. The miR-21 mimics and inhibitors were transfected based on the manufacturer's instructions for Lipofectamine 2000, then HaCaT cells were divided into eight groups: vehicle group, EGF group, shikonin group, EGF + shikonin group, miR-21 inhibitor group, EGF + miR-21 inhibitor group, and miR-21 mimic group, shikonin + miR-21 mimic group.

MTT assay

Cell proliferation ability was assessed by MTT assay (Sigma, USA). HaCaT cell cultured under different concentration of EGF (0, 5, 10, and 25 μmol/L), and shikonin (0, 1, 5, and 10 ng/ml) separately for 24 h and 48 h, their dose-effect curves were generated. Then, HaCaT cells of eight groups were seeded in flasks and treated with corresponding agents for 48 h. MTT was added to each well and incubated at 37°C for 24 h. The optical density (OD) was measured by an enzyme-linked immunosorbent assay detector (Bio Tek, China) at 490 nm, all experiments were performed in triplicates.

Real-time polymerase chain reaction

A total RNA was isolated with TRIzol (Invitrogen Bio, USA) according to the manufacturer instructions, and 2 μg of total RNA was reverse transcribed into complementary DNA using PrimeScript™ RT Reagent Kit (ABI Bio, USA) by PCR instrument (Applied Biosystems, USA). Then, the qRT-PCR was performed following the Amplification kit (TaKaRa Bio, Japan) protocol on the ABI-7900HT system (USA). Thermocycling conditions were as follows: 1 min at 94°C followed by 40 cycles (95°C for 10 s, 58°C for 10 s, 72°C for 10 s). Gene expression of miR-21 is expressed relative to U6, and the others were compared with endogenous glyceraldehyde-3-phosphate dehydrogenase. The 2–ΔΔCt was calculated to compare the difference in threshold cycle (Ct) between samples and control.

All the primers were designed and synthesized by Shanghai Sangon Biological Engineering Technology and Services, China. The details of primers for the amplification of miR-21 as showed in [Table 1], and primers for Bcl-2, PCNA, NF-κB-p65, IKKβ, Caspse-3, and Caspse-9 were shown in [Table 2].{Table 1}{Table 2}

Western blot

HaCaT cells were collected after digested by RIPA (Pierce, China). According to the manufacture's instruction, the total protein concentration of each group was detected by BCA assay (Sigma, USA). Total protein (20 μg/well) mixed with 2X loading buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, then transferred to polyvinylidene difluoride membrane (Amersham, USA) with the wet transfer method (Bio-Rad, USA). After incubation at room temperature and washed with Tris Buffered Saline With Tween (TBST), then incubated with relevant primary antibodies and corresponding horseradish peroxidase conjugated secondary antibody. Finally, immunoreactive membrane visualized with DAB Kit (Pierce, China) and quantified with Image LabTM Software #1708265 (Bio-Rad, USA).

Flow cytometry

Annexin V/PI Apoptosis Detection Kit (Logji Bio, China) was applied according to the manufacturer's protocol. HaCaT cells of different groups were trypsin released and Annexin-V/PI stained at room temperature and keep in a dark place for 5 min. Apoptotic cells were measured by flow cytometer (BD, USA).

Statistical analysis

All variable data were analyzed using IBM SPSS Statistics 19.0 software (SPSS Inc., Chicago, IL, USA). The results of multiple experiments are presented as mean ± standard deviation. Measurement data were tested by one-way analysis of variance, P < 0.05 was considered as statistically significant.

 Results



In a dose-dependent way, the proliferation of HaCaT-cell inhibited by shikonin, while promoted by epidermal growth factor

It [Figure 1] showed that EGF promoted the proliferation of HaCaT cell in a concentration dependent manner (0–25 ng/ml) (P < 0.01), and the dosage of 10 ng/ml reached a significant stimulatory effect. On the contrary, shikonin inhibited the proliferation of HaCaT cell in a concentration-dependent way (0–10 μmol/L) (P < 0.01), and 5 μmol/L shikonin significantly inhibited cell viability.{Figure 1}

MiRNA-21 was upregulated by epidermal growth factor and miRNA-21 mimic, while downregulated by Shikonin and miRNA-21inhibitor

RT-PCR [Figure 2] indicated that gene expression of miR-21 on HaCaT cell was significantly increased by miR-21 mimic (P = 0.000), while decreased by miR-21 inhibitor (P = 0.000) and shikonin (P = 0.004). Shikonin partially reduced miR-21 mimic-induced hyper-expression (P = 0.000).{Figure 2}

Shikonin, miRNA-21 inhibitor suppressed proliferation of HaCaT cell, which were partially reversed by epidermal growth factor and miRNA-21 mimic

MTT assay [Figure 3] showed that the proliferation of HaCaT cell was suppressed by shikonin (P = 0.007), miR-21 inhibitor (P = 0.007), while promoted by EGF (P = 0.008) and miR-21 mimic (P = 0.007). In addition, EGF and miR-21 mimic induced cell growth effects were blocked by shikonin (P = 0.005, P = 0.003) significantly.{Figure 3}

Western blot method [Figure 4] indicated that the protein expression of PCNA on HaCaT cell was downregulated by shikonin and miR-21 inhibitor which could be upregulated by EGF and miR-21 mimic. In addition, the expression of PCNA mediated by EGF and miR-21 mimic was decreased by shikonin. In the NF-κB/IKKβ pathway signals, the two crucial proteins, NF-κB and IKKβ were overexpressed in EGF and miR-21 mimic groups, while expressed inversely in shikonin and miR-21 inhibitor groups.{Figure 4}

In RT-PCR test [Figure 4], gene expression of PCNA, NF-κB/IKKβ pathway signals showed the proliferation tendency of HaCaT cells. When compared with control group, shikonin and miR-21 inhibitor reduced mRNA expressions of PCNA, NF-κB, and IKKβ, while EGF and miR-21 mimic increased their expression. What is more, EGF confronted the function of shikonin and miR-21 inhibitor, and shikonin antagonized miR-21 mimic-induced effect.

Shikonin and miRNA-21 inhibitor-induced apoptosis of HaCaT cell, which were reduced by epidermal growth factor and miRNA-21 mimic

In Western blot and RT-PCR [Figure 5], apoptotic conditions under different treatments were proved by gene expression and protein production. When compared with control group, both EGF and miR-21 mimic regulated gene expression of caspase-3, caspase-9 (reduced), and bcl-2 (induced) on HaCaT cell, which countered to the effect of shikonin and miR-21 inhibitor. That is to say EGF and miR-21 mimic prevent the HaCaT cell apoptosis by expressing more anti-apoptotic gene (bcl-2) and less pro-apoptotic gene (caspase-3/-9), which followed by generating more corresponding proteins, such as caspase-3 and caspase-9. It revealed that shikonin and miR-21 inhibitor displayed an opposite biofunction in apoptotic gene and protein generation.{Figure 5}

In flow cytometry [Figure 6], apoptosis of HaCaT cell induced by shikonin (5.5%) and miR-21 inhibitor (3.2%) were partially reversed by EGF (1.7%, 1.5%). The anti-apoptosis effect of miR-21 mimic (0.8%) was reversed by shikonin (7.2%) significantly. Consequently, all dates from these methods showed consistency.{Figure 6}

 Discussion



Psoriasis is a long-lasting, chronic-recurrent erythema scaly skin disease, characterized by epidermal proliferation and inflammation. The incidence of psoriasis was relatively high among young adults, especially under the environmental pollution and social pressure. The high morbidity, long duration, easy to relapse, and the skin lesion of psoriasis makes it much more suffering, which caused a heavy burden on their physical and psychological health.[9] At present, the psoriatic therapies mainly include topical external drugs, phototherapy, systemic therapy, and biotherapy. Treatments mainly regulate keratinocyte proliferation, antiactivity of T lymphocyte, or against some cellular cytokines associated with the onset of psoriasis.[10] Gradually, adverse reactions and heavy economic burdens of long-term application of these drugs uncovered, thus limited their clinical application to a certain extent.[11]

There were profound understandings of pathogenesis and treatment for psoriasis in the ancient medical publications of TCM. According to the pathogenesis and syndrome differentiation of psoriasis, the progressive stage of psoriasis was badly in need of treatment, in which cooling blood heat was regarded as the basic therapeutic principle. Thus, herbs in properties of heat remove and blood cool were useful, like lithospermum.

In our clinical trail, 30 psoriasis patients (blood-heat syndrome) were treated with Qing Re Liang Xue Decoction, in which, lithospermum was the monarch drug. After treatment patients' PASI decreased and TCM syndrome scales alleviated significantly, and followed by decreased serum level of EGF and miR-21. Thus, the correlation analysis implied that serum level of EGF and miR-21 was related to psoriatic stage and TCM syndrome type, which could be regulated by cooling blood-heat prescription. Consequently, we assumed that herbs could regulate psoriasis-related miRNA expression to achieve efficacious and safe therapy.

Modern pharmacology studies found that shikonin was the main active component of lithospermum, which has multimedical functions. It was active in modulating anti-proliferation and pro-apoptosis of keratinocyte in psoriasis which has been showedin vitro studies.[12],[13] Others[14],[15] also found that shikonin influenced the formation and activation of Th17 cells, inhibited the expression of vascular endothelial growth factor, IL-6 and IL-23 on keratinocytes, which benefit for the achievement of psoriatic remission. The significant efficacy of lithospermum was well-known in psoriasis therapy, either used alone or added into formula.[8] Here, we unfold the proliferation, apoptosis of HaCaT cell intervened by shikonin, miR-21, and EGF. Moreover, we explored the function of shikonin in regulating gene expression of keratinocytes preliminarily.

It is known that miRNAs involved in the regulation of proliferation, differentiation, and morphological development of cell, and its deregulation occurred in the pathogenesis of psoriasis and contributes to the dysfunction of the cross talk between resident and infiltrating cells. MiR-21, a subtype of miRNAs, was regarded as oncogene and promotes tumor proliferation. There are some similarities in the pathology of tumor and psoriasis, in which hyperproliferation, neovascularization, proinflammatory cytokine, and immune cell infiltration involved. For this point, it has led to a new scientific approach to regard psoriasis not only as inflammatory disease, but also as benign epidermal hyperplasia or tumor.[16] Gradually, the promising role of miR-21 played in psoriasis has been uncovered.

In the psoriatic miRNAome investigation, it has found that nearly 100 of miRNAs were highly expressed in the skin lesion and raised 2–42-fold, miR-21 was specify involved in vascularization of psoriatic skin lesion.[17] In the skin proto keratinocytes and HaCaT cells, miR-21 promoted cell proliferation and migration by inhibiting bone morphogenetic protein-dependent anti-oncogenes: phosphatase tensin homolog, programmed cell death 4, inhibitor of metalloproteinase 3, and tropomyosin 1.[18] In the therapy of psoriasis by ultraviolet B (UVB), the alleviated topical tissues showed decreased the expression of miR-21 level.[19] Moreover, miR-21 also mediated immune response during psoriasis process, especially, for T-cell activation. Immunohistochemical showed that both epidermis and dermis of psoriasis were widely infiltrated with T lymphocytes, and accompanied higher miR-21 than normal person, and elevated miR-21 could activate cells CD4+ T, CD8+ T cells.[20],[21],[22] Further studies also found that upraising of miR-21 causes activation-infiltration of T cells and suppresses T-cell apoptosis in psoriasis skin, it concluded that excessive expression of miR-21 plays an important role in T cells skin inflammation derived psoriasis patients.

Besides, it is known that the high level of EGF both in serum and skin lesion of psoriasis means poor prognosis.[23],[24] In this experiment, EGF promoted HaCaT cell proliferation in a dose-dependent way, and the optimal effect dosage was 10 ng/ml. What is more, we confirmed that EGF can increase the expression of cell PCNA which is recognized as the surest marker to evaluate cell proliferation state. Researchers[25],[26] found that PCNA highly expressed in the psoriatic lesions, especially strong positive in the whole layer, which can reflect the proliferation status of the keratinocyte. We also found that EGF could upregulate inflammation signal pathway IKKβ/NF-κB which could mediate cell proliferation. NF-κB was the key factor in modulating the expression of inflammatory gene. As essential molecular and main kinase, IKKβ plays a crucial role in NF-κB-dependent gene translation and NF-κB classical activation pathway, which mainly responsible for inflammatory response, proliferation, and survival of keratinocyte.[27]

Interestingly, we found that miR-21 inhibitors could significantly inhibit EGF-induced proliferation of HaCaT cells, and promote gene expressions of PCNA, KKβ, and NF-κB. While miR-21 mimic has a similar effect to the EGF. These results revealed a potential bio-function of miR-21 in the pathogenesis of EGF-mediated psoriasis.

One more, there was a dose-dependent inhibiting effect of shikonin on HaCaT cell proliferation, and 5 μmol/L showed optimal dosage effect. In subsequent tests, they showed that shikonin could downregulate gene expression of miR-21, and modulate cell proliferation which could be reversed partially by miR-21 mimic. Besides, shikonin also antagonized EGF-induced effect, which probably mediated by reducing the expression of miR-21.

The influences of shikonin, EGF, miR-21 inhibitor, and miR-21 mimic on the apoptosis of HaCaT cells had been investigated. We found that shikonin, miR-21 inhibitor could promote HaCaT cell apoptosis at early stage, which may through regulating gene expression of bcl-2 (downregulating) and caspase-3/-9 (upregulating); while this effect could be partially reversed by EGF and miR-21 mimic. What is more, protein production of caspase-3/-9 also showed an upward trend stimulated by shikonin and miR-21 inhibitor, while exhibited a downward trend induced by EGF and miR-21 mimic.

There were three types of cell apoptosis signal pathway: death receptor pathway, mitochondrial pathway, and endoplasmic reticulum pathway. They were mutual independent and coregulate cell apoptosis. Caspase-9 acted as the key protease in the process of mitochondrial apoptosis pathway, at the top of the caspases “waterfall” activation, followed by caspase-3 activation which could pyrolyze the DNA, induce apoptosis of profilin and skelemin, cell death occurred, finally.[28] In addition, expression of Bcl-2 family, the crucial protein of mitochondrial pathway would change when the intracellular death signal expressed to inhibit cell apoptosis. These important proteins of apoptosis pathway were exactly what we checked. Our results suggest that shikonin-induced apoptosis of HaCaT cell were associated with both death receptor pathway and mitochondrial pathway, while one of the crucial factors of shikonin-induced apoptosis of keratinocytes may relate to suppression of miR-21.

Consequently, we uncovered the special role of miR-21 on the regulation of HaCaT cell growth of psoriasis partially. Moreover, we also confirmed the effect of shikonin on HaCaT cell and its antagonistic effect to EGF-induced cell growth, which may through regulating miR-21 expression. It provided some experimental basis for the development and application of shikonin in psoriasis therapy.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (Grant No. 81502724; Grant No. 81774313). The funding sources had no involvement in the study design, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit the article for publication. The authors were responsible for all content and editorial decisions.

Financial support and sponsorship

Nil.

Conflicts of interest

Xiaohong Yang, Fengling Xing and Hongbin Luo are faculties and researches in this experiment. Lili Ma and Maocan Tao guided this experiment. Yi Cao interpreted the data and reviewed this manuscript. All of them have nothing to disclose.

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