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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.

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+]iof 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. :联合超声辐照和声诺维微泡靶向STAT3基因的RNA干扰对银屑病皮损角质形成细胞增殖和凋亡的影响摘要背景：信号传导子和转录活化子3（Signal transducer and activator of transcription 3, STAT3）在银屑病角质形成细胞（keratinocytes, KCs）中表达增加并活化，与银屑病严重程度相关。本研究目的是调查联合超声辐照和声诺维微泡的STAT3小分子干扰RNA（small interfering RNA, siRNA）对银屑病皮损KCs增殖和凋亡的影响及其机制。 方法：在以下4种实验条件下对银屑病KCs进行转染：①联合超声辐照和声诺维微泡的以Lipo3000为载体的STAT3 siRNA作为LUS组；②以Lipo3000为载体的STAT3 siRNA作为L组；③联合超声辐照和声诺维微泡的以Lipo3000为载体的阴性对照siRNA作为siRNA-NC组; ④不作处理的作为Blank组。CCK-8实验用于检测细胞增殖。细胞周期检测试剂盒联合流式细胞术分析细胞周期。FITC Annexin V凋亡检测试剂盒联合流式细胞术分析细胞凋亡。钙荧光探针联合流式细胞术分析胞内游离钙离子浓度([Ca2+]i)。应用实时RT-PCR 和Western blotting 分别在mRNA 和蛋白水平检测cyclin D1和Bcl-xL的表达。应用两因素方差分析对数据进行统计学分析。 结果：STAT3 siRNA以时间依赖方式抑制KCs增殖且尤以LUS组为著，72h时其增殖比率为45.38% ± 5.85% (与L 组、siRNA-NC组及Blank组相比，均P<0.05)。STAT3 siRNA 改变KCs细胞周期进程，LUS组G2/M期细胞百分比最高，为18.06% ± 0.36% (与L 组、siRNA-NC组及Blank组相比，均P<0.05)。STAT3 siRNA促进KCs晚期凋亡且尤以LUS组为著，其晚期凋亡百分比最高，为22.87% ± 1.28% (与L 组、siRNA-NC组及Blank组相比，均P<0.05)。STAT3 siRNA诱发KCs [Ca2+]i升高，LUS组钙离子荧光强度均值最高，为1213.67 ± 60.51 (与L 组、siRNA-NC组及Blank组相比，均P<0.05)。 STAT3 siRNA下调KCs cyclin D1和Bcl-xL的表达且尤以LUS组为著，其cyclin D1和Bcl-xL在mRNA 和蛋白水平表达比率分别为51.81% ± 9.58%、70.17% ± 4.22%和37.58% ± 4.92%、64.06% ± 7.78% (与L 组、siRNA-NC组及Blank组相比，均P<0.05)。 结论：STAT3 siRNA抑制银屑病KCs增殖并诱导其凋亡，可能部分通过改变细胞周期进程、升高[Ca2+]i、下调cyclin D1和Bcl-xL的表达。联合超声辐照和声诺维微泡的siRNA沉默银屑病皮损KCs靶基因STAT3，将有助于开创一种新的银屑病治疗方法。.

journal_name

Chin Med J (Engl)

journal_title

Chinese medical journal

authors

Ran LW,Wang H,Lan D,Jia HX,Yu SS

doi

10.4103/0366-6999.239297

subject

Has Abstract

pub_date

2018-09-05 00:00:00

pages

2097-2104

issue

17

eissn

0366-6999

issn

2542-5641

pii

ChinMedJ_2018_131_17_2097_239297

journal_volume

131

pub_type

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