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

Abstract

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 i...

Targeted siRNA Therapy for Psoriasis: Translating Preclinical Potential into Clinical Treatments

“…Silencing the STAT3 gene in psoriatic keratinocytes using siRNA, combined with ultrasound irradiation and microbubble technology, inhibits keratinocyte growth and induces apoptosis. 35 Transfection of keratinocytes, whether treated or untreated with avidin, with siRNA targeting Signal Transducer and Activator of Transcription 1 ( STAT1 ) and STAT3 , confirmed that avidin regulates psoriatic keratinocyte proliferation via the STAT signaling pathway. 36 Inhibiting Casein Kinase 2 ( CK2 ) suppresses both the STAT3 and Threonine Kinase ( AKT ) signaling pathways in human keratinocytes, which inhibits epidermal hyperplasia, abnormal differentiation, and the inflammatory response.…”

Ultrasonic irradiation and SonoVue microbubbles-mediated RNA interference targeting PRR11 inhibits breast cancer cells proliferation and metastasis, but promotes apoptosis

“…Antisense DNA or siRNA has promising preclinical results on suppressing the expression of a target gene, and different from unlike traditional medication, gene silencing through RNA interference (RNAi) using viral vectors can suppress targeted genes with a high selectivity and great potency [ 14 ]. However, the application of viral vectors has also stimulated great concern over its safety and efficiency due, as it could cause toxicity, immunogenicity, and tumorigenicity [ 18 ].…”

“…For transfection using microbubble and ultrasound, SonoVue was used as previously described [ 14 ]. Six fluorinated sulfur (SF 6 ) was purchased from Bracco (Milan, Italy) and used as the agent of SonoVue (average diameter: 2.5 μm).…”

Ultrasound microbubble-mediated RNA interference targeting WNT1 inducible signaling pathway protein 1(WISP1) suppresses the proliferation and metastasis of breast cancer cells

“…In terms of another transfection method using ultrasonic irradiation and SonoVue microbubbles (US), SonoVue (Bracco, Italy), the phospholipid-encapsulated six fluorinated sulfur (SF6) with an average diameter of 2.5 μm was used as previously described [ 23 ]. Briefly, 5 ml normal saline and 50 μl siWISP1 or siNC were added into the SonoVue microbubble before use.…”