RPL34基因敲低对人皮肤鳞状细胞癌SCL-1细胞的影响

Effect of ribosomal protein L34 gene knockdown on a human cutaneous squamous cell carcinoma cell line SCL-1

目的研究RPL34基因对皮肤鳞状细胞癌(鳞癌)细胞增殖、凋亡的影响。方法收集2016年1月至2017年1月内蒙古医科大学附属医院皮肤性病科14例皮肤鳞癌和16例正常皮肤组织石蜡标本,通过免疫组化分析组织中RPL34的表达。构建RPL34基因干扰慢病毒,感染皮肤鳞癌SCL-1细胞(shRNA组),通过RT-PCR与Western印迹验证目的基因敲减。感染后72 h,通过流式细胞仪检测敲低RPL34基因后SCL-1细胞周期及凋亡情况,MTT法检测细胞增殖活力。两组间比较采用t检验或秩和检验。结果免疫组化显示,皮肤鳞癌组织细胞质RPL34表达评分(2.143±1.956)高于正常对照组织(0.500±0.516,z=3.53,P< 0.05)。RT-PCR显示,shRNA组SCL-1细胞RPL34mRNA相对表达(0.149±0.016)低于对照组(1±0.018,t=36.95,P< 0.05);Western印迹显示,shRNA组SCL-1细胞RPL34蛋白相对水平明显低于对照组。shRNA组S期细胞比例高于对照组(t=13.76,P< 0.05),G1期细胞比例低于对照组(t=36.62,P< 0.05);shRNA组细胞凋亡率(9.42%±0.16%)高于对照组(4.58%±0.41%,t=19.02,P< 0.05)。MTT结果显示,继续培养120 h,shRNA组细胞活力(0.815±0.005)低于对照组(1.886±0.005,t=265.91,P< 0.05)。结论 RPL34基因在皮肤鳞癌组织中过表达,敲低RPL34基因可干扰SCL-1细胞周期,降低增殖活力,促进凋亡。

Objective To evaluate the effect of ribosomal protein L.34(RPL34)gene knockdown on the proliferation and apoptosis of human cutaneous squamous cell carcinoma(cSCC)cells.Methods From January 2016 to January 2017,14 paraffin-embedded skin samples of cSCC and 16 paraffin-embedded normal skin tissue samples were collected from Department of Dermatology and Venereology,the Affiliated Hospital of Inner Mongolia Medical University,and RPL34 expression in the skin tissues was analyzed by immunohistochemical study.A lentivirus vector containing short hairpin RNA targeting RPL34 gene was constructed and used to transfeet a human eSCC cell line SCL-1(shRNA group),SCL-1 cells transfected with an empty lentivirus vector served as control group,and the knockdown efficiency was verified by real-time quantitative PCR(RT-PCR)and Westerm blot analysis.At 72 hours after the transfection,flow cytometry was performed to analyze the cell cyele and detect apoptosis of SCL-1 cels,and methyl thiazolyl tetrazolium(MTT)assay to evaluate the cellular proliferative activity of SCL-1 cells.Comparisons between 2 groups were performed by using 1 test or rank sum tesl.Results Immunohistochemical study showed that the cytoplasmie expression score of RPL34 was significantly higher in the cSCC tissues(2.143±1.956)than in the normal control tissues(0.500±0.516,z=3.53.P<0.05).RT-PCR showed that the relative mRNA expression of RPL34 in the SCL-I cells was significantly lower in the shRNA group(0.149±0.016)than in the control group(1±0.018,t=36.95,P<0.05);Westerm blot analysis revealed that the relative protein expression of RPL34 in the SCL-1 cells was significantly lower in the shRNA group than in the control group.Compared with the control group,the shRNA group showed a significantly increased proportion of S-phase cells(1=13.76,P<0.05),but a significantly decreased proportion of G I-phase cells(1=36.62.P<0.05);the apoptosis rate was significantly higher in the shRNA group(9.42%±0.16%)than in the control group(4.58%±0.41%,1=19.02,P<0.05).MTT assay showed that the cell viability was significantly decreased in the shRNA group(0.815±0.005)than in the control group(1.886±0.005,t=265.91,P<0.05)after additional 120-hour culture.Conclusion The RPL34 gene was overexpressed in the cSCC tissues,and knockdown of the RPL34 gene in SCL-1 cells could interfere with cell cycle,decrease their proliferative activity,and promote their apoptosis.