摘要
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） STA T3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles （LUS group）; （2） STA T3 siRNA only carried by Lipofectamine 3000 （L group）; （3） the negative control ofsiRNA 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 （[Ca^2＋）. 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）. ST,4 T3 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 [Ca^2＋] 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）. STA T3 siRNA induced the downregulation ofcyclin D 1 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： STA T3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca^2＋], and downregulating cyclin DI 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.
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） STA T3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles （LUS group）; （2） STA T3 siRNA only carried by Lipofectamine 3000 （L group）; （3） the negative control ofsiRNA 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 （[Ca^2＋）. 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）. ST,4 T3 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 [Ca^2＋] 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）. STA T3 siRNA induced the downregulation ofcyclin D 1 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： STA T3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca^2＋], and downregulating cyclin DI 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.
基金
This work was supported by a grant from National Natural Science Foundation of China （No. 81441126）.
