中国肿瘤临床
中國腫瘤臨床
중국종류림상
CHINESE JOURNAL OF CLINICAL ONCOLOGY
2014年
9期
555-559
,共5页
陈凯%常东方%段绍坤%李渝凉
陳凱%常東方%段紹坤%李渝涼
진개%상동방%단소곤%리투량
微小RNA%鳞状细胞癌%荧光定量PCR
微小RNA%鱗狀細胞癌%熒光定量PCR
미소RNA%린상세포암%형광정량PCR
microRNA%squamous cell carcinoma%fluorescence-based quantitative PCR
目的:探讨miR-31对皮肤鳞状细胞癌(鳞癌)生长的影响及作用机制。方法:在鳞癌细胞中转染miR-31的2'-羟基甲基化的反义寡核苷酸(antisense oligonueleotide,ASO),运用平板克隆形成和体外成瘤实验检测miR-31对鳞癌生长的影响。运用Western blot印迹及GFP报告基因实验验证miR-31的靶基因。在鳞癌细胞中转染靶基因的siRNA,检测其对细胞生长的影响。最后,运用实时定量PCR以及免疫组织化学检测miR-31和靶基因在鳞癌组织中的水平。结果:转染miR-31反义寡核苷酸(miR-31 ASO)可以抑制克隆数目及体内裸鼠肿瘤体积(P<0.05)。LATS2(large tumor suppressor homolog 2)是miR-31的直接靶基因。抑制LATS2表达后,鳞癌细胞克隆数目增加(P<0.05)。miR-31在鳞癌中高表达,与LATS2的表达呈负相关。免疫组织化学结果也显示LATS2在鳞癌组织中低表达。结论:miR-31通过负调控LATS2抑制鳞癌的生长。因此,miR-31具有作为鳞癌分子治疗靶标的潜能。
目的:探討miR-31對皮膚鱗狀細胞癌(鱗癌)生長的影響及作用機製。方法:在鱗癌細胞中轉染miR-31的2'-羥基甲基化的反義寡覈苷痠(antisense oligonueleotide,ASO),運用平闆剋隆形成和體外成瘤實驗檢測miR-31對鱗癌生長的影響。運用Western blot印跡及GFP報告基因實驗驗證miR-31的靶基因。在鱗癌細胞中轉染靶基因的siRNA,檢測其對細胞生長的影響。最後,運用實時定量PCR以及免疫組織化學檢測miR-31和靶基因在鱗癌組織中的水平。結果:轉染miR-31反義寡覈苷痠(miR-31 ASO)可以抑製剋隆數目及體內裸鼠腫瘤體積(P<0.05)。LATS2(large tumor suppressor homolog 2)是miR-31的直接靶基因。抑製LATS2錶達後,鱗癌細胞剋隆數目增加(P<0.05)。miR-31在鱗癌中高錶達,與LATS2的錶達呈負相關。免疫組織化學結果也顯示LATS2在鱗癌組織中低錶達。結論:miR-31通過負調控LATS2抑製鱗癌的生長。因此,miR-31具有作為鱗癌分子治療靶標的潛能。
목적:탐토miR-31대피부린상세포암(린암)생장적영향급작용궤제。방법:재린암세포중전염miR-31적2'-간기갑기화적반의과핵감산(antisense oligonueleotide,ASO),운용평판극륭형성화체외성류실험검측miR-31대린암생장적영향。운용Western blot인적급GFP보고기인실험험증miR-31적파기인。재린암세포중전염파기인적siRNA,검측기대세포생장적영향。최후,운용실시정량PCR이급면역조직화학검측miR-31화파기인재린암조직중적수평。결과:전염miR-31반의과핵감산(miR-31 ASO)가이억제극륭수목급체내라서종류체적(P<0.05)。LATS2(large tumor suppressor homolog 2)시miR-31적직접파기인。억제LATS2표체후,린암세포극륭수목증가(P<0.05)。miR-31재린암중고표체,여LATS2적표체정부상관。면역조직화학결과야현시LATS2재린암조직중저표체。결론:miR-31통과부조공LATS2억제린암적생장。인차,miR-31구유작위린암분자치료파표적잠능。
Objective:To investigate the roles and regulation mechanism of miR-31 in human cutaneous squamous cell carcino-ma (cSCC) growth. Methods:cSCC cells were transfected with the antisense oligonucleotide (ASO) of miR-31, and the cSCC growth was tested by colony formation and in vivo tumor formation assays. The target gene of miR-31 was validated by Western blot and green fluorescent protein (GFP) reporter assay. The cells were then transfected with the siRNA of the target gene, and the effect of the target gene on cell growth was preformed by colony formation assay. Finally, real-time PCR and immunohistochemistry were used for analy-sis of the expression of miR-31 and its target gene. Results:miR-31 ASO resulted in a low number of cell colonies and small tumor vol-ume (P<0.05). Western blot showed that the cells with miR-31 ASO had a higher protein level of large tumor suppressor homolog 2 (LATS2) than the control. The 3' UTR of LATS2 had a binding site with miR-31, and miR-31 ASO increased the GFP intensity con-trolled by LATS2 3' UTR, whereas no effect was observed on the mutant LATS2 3' UTR. Western blot showed that LATS2 siRNA inhib-ited the expression of LATS2 protein by about 80%. Knocking down of LATS2 increased the colony number by about 70%or 1.3-fold in cSCC cells. Real-time PCR showed that miR-31 was overexpressed in most cSCC tissues, compared with normal tissues. An inverse relationship existed between miR-31 and LATS2 expression levels. Immunohistochemistry validated that LATS2 was downregulated in cSCC tissues. Conclusion:miR-31, which functions as an oncogene, promotes cSCC growth by suppressing LATS2 expression. Our da-ta suggest that miR-31 is a potential miRNA-based therapeutic target for cSCC growth.