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Effect of RNA Interference Targeting STAT3 Gene Combined with Ultrasonic Irradiation and SonoVue Microbubbles on Proliferation and Apoptosis in Keratinocytes of Psoriatic Lesions

Background: Signal transducer and activator of transcription 3 (STAT3) was strongly expressed and activated in psoriatic keratinocytes (KCs) and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering RNA (siRNA) combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms. Methods: Psoriatic KCs were transfected under four experimental conditions: (1) STAT3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group); (2) STAT3 siRNA only carried by Lipofectamine 3000 (L group); (3) the negative control of siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (siRNA-NC); (4) not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration ([Ca2+]i). The expressions of cyclin D1 and Bcl-xL were detected both at the mRNA level by real-time reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance. Results: STAT3 siRNA inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1.28% in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the elevation of [Ca2+]i of KCs with the highest calcium fluorescence intensity mean of 1213.67 ± 60.51 in LUS group (P < 0.05 vs. L group, siRNA-NC, or Blank). STAT3 siRNA induced the downregulation of cyclin D1 and Bcl-xL expressions of KCs at mRNA and protein levels with the lowest expressions in LUS group with cyclin D1 expression of 51.81% ± 9.58% and 70.17% ± 4.22% at mRNA level and at protein level, respectively, and with Bcl-xL expression of 37.58% ± 4.92% and 64.06% ± 7.78% at mRNA level and at protein level, respectively (P < 0.05 vs. L group, siRNA-NC, or Blank). Conclusions: STAT3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca2+]i, and downregulating cyclin D1 and Bcl-xL expressions. Silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis.


IntroductIon
Psoriasis is an immune-mediated, chronic inflammatory skin disease with a complex etiology involving both genetic and environmental risk factors which affects approximately 1-3% of the population worldwide. [1] It had been reported that signal transducer and activator of transcription 3 (STAT3) was strongly expressed and activated in psoriatic keratinocytes (KCs) and participated in the hyperproliferation of KCs which was the main characteristic of psoriasis, and STAT3 could be recognized as a therapeutic target for psoriasis treatment. [2][3][4] Some studies discovered that small interfering RNA (siRNA) combined with ultrasonic irradiation and SonoVue microbubbles could trigger RNA interference (RNAi), enhance gene transfer efficiency, and be applied for the tissue-specific delivery. [5,6] Our previous studies had screened out the most effective sequence of siRNA targeted to the STAT3 gene of psoriatic KCs and also showed the optimal dose-and time-response of the siRNA combined with ultrasonic irradiation and SonoVue microbubbles on knocking down the STAT3 expression of psoriatic KCs. [7] In this study, the effects of STAT3 siRNA combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis of psoriatic KCs were investigated, and the relative mechanisms were discussed. This study aimed to provide a new way of exploring gene therapy in the treatment of psoriasis.

Patients and ethical approval
The study was approved by the Ethics Committee of Beijing Chaoyang Hospital, and the informed consent was obtained from all participants. Patients with active chronic plaque psoriasis were recruited for the study. They had not received any topical treatment for at least 4 weeks or any systemic treatment for at least 3 months before sampling. The biopsies were taken under local anesthesia from chronic psoriatic plaques. The biopsy specimens were used for isolating and culturing KCs.

Skin biopsy processing and keratinocytes culturing
Biopsies were rinsed with PBS containing antibiotics and incubated in 0.25% Dispase solution (Gibco, Thermo Fisher Scientific, USA) overnight at 4°C. On the following day, the epidermis was peeled from the dermis and incubated in the solution with 0.25% Trypsin-EDTA (Gibco) at 37°C for 10-15 min, and then the digestion was terminated with DMEM solution (Gibco) containing 10% fetal bovine serum (Gibco). The epidermis was blown and filtered, and then the KCs suspension was collected. The psoriatic KCs were seeded in culture flask filled with defined KC serum-free medium supplemented with growth supplement and bovine pituitary extract (Gibco). Cells were grown in the incubator at 37°C with 50 ml/L CO 2 and 95% relative humidity. At flaky confluence, KCs passage was made. The third generational KCs were applied for later experiments. Each experiment described below was performed three times using the same lot of KCs.

Selected small interfering RNA and transfection condition
The preliminary studies of the project showed that the siRNA targeting to STAT3 (target sequence: C C G T G G A A C C A T A C A C A A A , s e n s e : 5'-CCGUGGAACCAUACACAAAdTdT-3' and antisense: 3'-dTdTGGCACCUUGGUAUGUGUUU-5') achieved the highest knockdown effect among three designed siRNAs and was selected for the following experiments. The negative control of siRNA (siRNA-NC), with random sequences, did not target any known mammalian gene. The siRNAs were chemically synthesized by Guangzhou Ribobio (Ribobio, China). The preliminary studies of the project also showed that 100 nmol/L siRNA carried by Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, USA) combined with ultrasonic irradiation at the parameters of 0.5 W/cm 2 and 30 s and 2.5 μg/μl SonoVue microbubbles (Bracco Suisse SA, Switzerland) could significantly downregulate the protein expression of the STAT3 gene in KCs at 72 h after transfection, and thus the same transfection conditions were used in this research. The ultrasonic transfer apparatus was from Institute of Ultrasound Imaging of Chongqing Medical University.

Evaluation of cell proliferation (Cell Counting Kit-8 assay)
Growth curves were plotted with Cell Counting Kit-8 (Dojindo, Japan) according to the manufacturer's instructions. KCs were seeded into sterilized 96-well plastic plates made of polystyrene and transfected with 100 nmol/L STAT3 siRNA (LUS group) or siRNA-NC carried by Lipofectamine 3000 associated with SonoVue microbubbles and then irradiated by the probe of ultrasonic transfer apparatus through ultrasound couplant (Yalian, China) from the bottom of the plates. At the same time, one group of cells was transfected with 100 nmol/L STAT3 siRNA only carried by Lipofectamine 3000 (L group), and one group of cells was not treated (Blank). KCs were incubated for 24, 48, 72, or 96 h. Ten microliters Cell Counting Kit-8 (CCK-8) reagent was added to the cells, and KCs were incubated for 2 h. Absorbance at 450 nm was measured using the Rayto RT-6000 enzyme microplate reader (Rayto, China). Results were derived from the mean of triplicate wells. Cell proliferation was expressed as a percentage ratio of exposed cells to blank control cells.

Annexin V-FITC apoptosis analysis
KCs were seeded into sterilized plastic dish and transfected under four experimental conditions: (1) LUS group, (2) L group, (3) siRNA-NC, and (4) Blank. KCs were incubated for 72 h. Attached cells were trypsinized, pooled with the detached population, and subsequently treated according to the manufacturer's instructions of FITC Annexin V apoptosis detection kit (BD Biosciences). The cells were analyzed by flow cytometer using the CellQuest analysis program (BD Biosciences). There were four populations of cells that were, respectively, distributed in four quadrants. Upper-left, upper-right, lower-left, and lower-right quadrants indicated, respectively, the percentages of cells damaged, in late apoptosis, viable, and in early apoptosis.

Real-time reverse transcription-polymerase chain reaction and Western blotting assays for cyclin D1 and Bcl-xL expressions
KCs were seeded into sterilized plastic dish and transfected under four experimental conditions: (1) LUS group, (2) L group, (3) siRNA-NC, and (4) Blank. KCs were incubated for 72 h. The incubated cells were harvested and the expressions of cyclin D1 and Bcl-xL were detected at mRNA level by real-time reverse transcription-polymerase chain reaction (RT-PCR) and at protein level by Western blotting.

R N A e x t r a c t i o n a n d r e a l -t i m e r e v e r s e transcription-polymerase chain reaction
The total RNA was extracted from KCs using TRIzol reagent (Ambion, Thermo Fisher Scientific, USA) according to the manufacturer's protocol. First-strand cDNA was synthesized using the TIANScript RT-PCR reagent kit (TIANGEN, China) according to the manufacturer's instructions. The specific primers for cyclin D1 (forward: 5'-TAGCAGCGAGCAGCAGAGTC-3', reverse: 5'-TTTCCACTTCGCAGCACAGG-3', 174bp) and for Bcl-xL (forward: 5'-AGCTT TGAACAGGATACTTTTGTGG-3', reverse: 5'-GGTGGGAGGGTAGAGTGGAT-3', 183 bp) were designed according to the BatchPrimer3 software (NCBI Reference Sequence: NM_053056.2 and NM_001191.2, respectively) and synthesized by Sangon Biotech (Sangon, China). The specific primer for β-Actin was provided by Sangon Biotech. Real-time PCR was prepared using the SYBR Premix Ex Taq kit (TAKARA, China) according to the manufacturer's protocol, and amplification was performed on an ABI Prism 7000 detection system (Applied Biosystems, USA) according to the conditions recommended by the manufacturer, with an initial denaturation step at 95°C (2 min), followed by 40 cycles of denaturation at 95°C (15 s) and annealing at 60°C (40 s). At the end of the amplification, a melting curve (disassociation curve) was run to ensure that only a single specific product was amplified. The cycle threshold values were determined. The modification of the 2 −ΔΔCt method was used to calculate changes of the relative expressions of cyclin D1 and Bcl-xL normalized against β-Actin.

Protein extraction and Western blotting
The total protein was extracted from the KCs using RIPA lysis buffer and 1:100 dilution of a protease inhibitor cocktail (Sigma, USA). A total of 30 μg of the protein lysate was resolved electrophoretically on SDS-PAGE and blotted onto PVDF membrane (Immobilon, USA). After being blocked for 1 h in blocking buffer (5% nonfat dried milk and 0.1% Tween-20 in Tris-buffered saline [TBS]) and separately incubated with an antihuman cyclin D1 rabbit monoclonal antibody (1:1000) (Cell Signaling, USA), an antihuman Bcl-xL rabbit monoclonal antibody (1:1000) (Cell Signaling, USA), and an antihuman β-Actin rabbit monoclonal antibody (1:1000) (Cell Signaling, USA) for 2 h at room temperature, the blots were washed three times with TBST (0.1% Tween in TBS) and incubated for 1.5 h with goat antirabbit IgG/HRP (1:3000) (ZSJB-BIO, China) at room temperature, followed by washing three times with TBST. The signals were visualized with the enhanced chemiluminescence method and detected by Tanon 4200 chemiluminescence imaging analysis system (Tanon, China). The band density was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized against the density of the respective housekeeping β-Actin.

Statistical analysis
The obtained data were statistically evaluated by two-way analysis of variance (ANOVA) to compare the difference among groups and presented as mean ± standard deviation (SD) from three independent experiments. A value of P < 0.05 was considered statistically significant. All analyses were carried out using SPSS 22.0 version statistical software (SPSS Inc., Chicago, IL, USA).

STAT3 small interfering RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles inhibited the growth of psoriatic keratinocytes
The results were obtained in CCK-8 assays, revealing that STAT3 siRNA inhibited the growth of KCs in a time-dependent manner when compared with siRNA-NC and Blank, showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72 h (P < 0.05 vs. L group, siRNA-NC, or Blank), the next in L group with proliferation ratio of 64.82% ± 7.17% at 72 h (P < 0.05 vs. siRNA-NC and Blank), and no significant difference between siRNA-NC and blank at 72 h (P = 0.54) [ Figure 1].

STAT3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles altered cell cycle distribution of psoriatic keratinocytes
STAT3 siRNA induced an altered cell cycle distribution of KCs when compared with siRNA-NC and Blank, showing increases in G2/M-phase population up to 18.06% ± 0.36% (P < 0.05 vs. L group, siRNA-NC, or Blank) in LUS group and up to 13.28% ± 2.26% (P < 0.05 vs. siRNA-NC and Blank) in L group, respectively, no significant difference in G2/M-phase population between siRNA-NC and blank (P = 0.89), and no significant differences in G0/G1-phase and S-phase populations among the four groups (P = 0.94, P = 0.42, respectively) [ Figure 2]. STAT3 small interfering RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles mediated apoptosis of psoriatic keratinocytes STAT3 siRNA induced late apoptosis of KCs significantly when compared with siRNA-NC and blank, with late apoptosis percentage located in the upper-right quadrant of 22.87% ± 1.28% (P < 0.05 vs. L group, siRNA-NC, or Blank) in LUS group and with late apoptosis percentage of 13.96% ± 4.98% (P < 0.05 vs. siRNA-NC and blank) in L group, respectively, without significant difference in late apoptosis percentage between siRNA-NC and Blank (P = 0.99), and there were no significant differences in the percentages of cells in the damaged and in early apoptosis among the four groups (P = 0.43 and P = 0.06, respectively) [ Figure 3]. There were 2 × 10 4 cells analyzed in every group.

STAT3 small interfering RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles induced the elevation of [Ca 2+ ] i of psoriatic keratinocytes
STAT3 siRNA induced the elevation of [Ca 2+ ] i of KCs significantly when compared with siRNA-NC and blank, with calcium fluorescence intensity mean of 1213.67 ± 60.51 (P < 0.05 vs. L group, siRNA-NC, or Blank) which indicated [Ca 2+ ] i in LUS group and the calcium fluorescence intensity mean of 1028.20 ± 111.40 (P < 0.05 vs. siRNA-NC and Blank) in L group, respectively, and without significant difference between siRNA-NC and blank (P = 0.92) [ Figure 4].

STAT3 small interfering RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles induced the downregulation of cyclin D1 expression of psoriatic keratinocytes
STAT3 siRNA induced the downregulation of cyclin D1 expression of KCs significantly when compared with siRNA-NC and Blank, with 51.81% ± 9.58% cyclin D1 expression at mRNA level (P < 0.05 vs. L group, siRNA-NC, or blank) and 70.17% ± 4.22% cyclin D1 expression at protein level (P < 0.05 vs. L group, siRNA-NC, or Blank) in LUS group, with 67.01% ± 10.06% cyclin D1 expression at mRNA level (P < 0.05 vs. siRNA-NC and Blank) and 80.87% ± 3.45% cyclin D1 expression at protein level (P < 0.05 vs. siRNA-NC and Blank) in L group, respectively, and without significant difference in cyclin D1 expression at mRNA or protein level between siRNA-NC and blank (P = 0.52, P = 0.61, respectively) [ Figure 5].

STAT3 small interfering RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles induced the downregulation of Bcl-xL expression of psoriatic keratinocytes
STAT3 siRNA induced the downregulation of Bcl-xL expression of KCs significantly when compared with siRNA-NC and Blank, with 37.58% ± 4.92% Bcl-xL expression at mRNA level (P < 0.05 vs. L group, siRNA-NC, or blank) and 64.06% ± 7.78% Bcl-xL expression at protein level (P < 0.05 vs. L group, siRNA-NC, or Blank) in LUS group, with 51.49% ± 13.90% Bcl-xL expression at mRNA level (P < 0.05 vs. siRNA-NC and Blank) and 77.02% ± 6.26% Bcl-xL expression at protein level (P < 0.05 vs. siRNA-NC and blank) in L group, respectively, and without significant difference in Bcl-xL expression at mRNA or protein level between siRNA-NC and blank (P = 0.83 and P = 0.20, respectively) [ Figure 5].

dIscussIon
Psoriatic skin lesions are typically characterized by the redness, thickness, and scaling induced by inflammation and epidermal hyperproliferation with disordered KCs differentiation, and these symptoms can severely impair patient quality of life. [1,8] It is generally accepted that various cytokines including TNF-α, IFN-γ, IL-6, IL-12, IL-17, IL-20, IL-22, and IL-23 result in the excessive proliferation and aberrant differentiation of KCs which produce various kinds of immune-related cytokines such as IL-1α, IL-1β, and IL-8, leading to an amplified immune response. [9][10][11] The cytokines play roles through signaling pathways. It has been demonstrated that Janus kinase (JAK)/STAT signaling pathway is altered in psoriatic skin, specifically the expression of STAT3 is correlated with the severity of psoriasis, STAT3 participates in the hyperproliferation of KCs and inflammatory infiltration, and STAT3 is a possible important link between KCs and immunocytes which is crucial to the development of psoriasis. [2,4,10,[12][13][14] Thus, blockading the JAK-STAT signaling pathway, especially STAT3, could be a potential strategy for the treatment of psoriasis so that the immunocytes/cytokines/KCs network cannot be formed. [2,10,12,15] STAT3 is a potential therapeutic target for psoriasis.  Over the last decade, targeted gene suppression by antisense DNA and siRNA has shown promising preclinical results. [16] Gene silencing through RNAi could inhibit all classes of gene targets with high selectivity and potency, unlike traditional drugs. [17] However, the use of viral vectors is a major concern because this vector system is associated with numerous safety concerns including toxicity, immunogenicity, and tumorigenicity. [5] The present studies show that ultrasound-mediated microbubble destruction may have the potential to become a new approach for targeted gene transfection. [5] Our previous studies had shown STAT3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down and downregulate the protein expression of STAT3 gene in psoriatic KCs. [7] Subsequently, we applied this kind of experimental method to investigate the effects about STAT3 gene silencing on the proliferation and apoptosis of psoriatic KCs and the relative mechanisms. The same study was not found in previous reports.
Previous studies have shown that STAT3 is a key transcriptional factor involved in the regulation of cell proliferation, and the inhibitions of STAT3 phosphorylation or expression can result in decreased proliferation of normal human KCs or HaCaT cells. [12,18] In our study, STAT3 siRNA inhibited the growth of psoriatic KCs in a time-dependent manner. The siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles showed higher proliferation inhibition compared with that only carried by Lipofectamine 3000.
Several studies have suggested that STAT3 promotes the cell cycle, and the inhibitions of STAT3 phosphorylation or phosphorylated STAT3 translocation may be related with accumulation of cells in the G1 or G2/M phase in normal human KCs or HaCaT cells. [15,19] In the present study, Previous studies suggest that the ability of psoriatic KCs to resist apoptosis is a key mechanism in psoriatic pathogenesis, STAT3 activation is involved in modulating cell apoptosis, and the inhibition of STAT3 signaling or dephosphorylation of STAT3 induces apoptosis of HaCaT KCs or normal KCs. [4,14,20] In our study, STAT3 siRNA   Potential target genes of STAT3 are cell survival genes including Bcl-xL and Bcl-2 and cell cycle regulators such as cyclin D1, and the downregulation of STAT3 has been shown to downregulate cyclin D1 and Bcl-xL of HaCaT KCs. [14,23] The present study demonstrated that STAT3 siRNA induced the significant downregulations of cyclin D1 and Bcl-xL expressions in psoriatic KCs at both mRNA and protein levels. The siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles showed lower cyclin D1 and Bcl-xL expressions compared with that only carried by Lipofectamine 3000. The data suggest that the apoptosis mediated by silencing STAT3 of psoriatic KCs may be induced partly through downregulating the Bcl-xL expression as an antiapoptotic protein and the suppressed G2/M-phase switching mediated by silencing STAT3 of psoriatic KCs may be induced partly through downregulating the cyclin D1 expression as a gene regulating cell cycle progression, although the mechanisms about apoptosis and cell cycle are complicated and further investigations are necessary to elucidate them.
In conclusion, the results suggest that STAT3 siRNA inhibited the growth and induced the apoptosis of psoriatic KCs likely partly through altering the cell cycle distribution, elevating [Ca 2+ ] i , and downregulating cyclin D1 and Bcl-xL expressions. The siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles showed more significant effects than that carried by Lipofectamine 3000 alone. We conclude that silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis. The experiment needs to be performed in vivo to further support the conclusion, and imiquimod-induced psoriasis mice model may be applied for the relative study.

Financial support and sponsorship
This work was supported by a grant from National Natural Science Foundation of China (No. 81441126).