摘要
目的探讨微小RNA-330-3p(miR-330-3p)对口腔鳞癌细胞增殖和凋亡的调控作用。筛选并验证相关靶基因。方法实时荧光定量聚合酶链反应(PCR)检测口腔鳞癌细胞系、口腔鳞癌组织与癌旁正常上皮组织中miR-330-3p的表达情况。应用miR-330-3p抑制物(miR-330-3p inhibitor)和miR-330-3p模拟物(miR-330-3p mimics),分别转染口腔鳞癌细胞系(HN13)细胞,实时荧光定量PCR检测miR-330-3p的转染效率;然后应用细胞增殖、细胞克隆形成以及流式细胞术等实验技术,分别检测miR-330-3p上调和下调对口腔鳞癌细胞增殖及凋亡的影响。通过生物信息网站miRanda和TargetScan与本课题组前期基因芯片数据比对预测了9个可能的靶基因,并进一步用相关实验验证miR-330-3p对靶基因的负向调控作用。结果与癌旁正常上皮组织相比,口腔鳞癌组织中miR-330-3p的表达显著上调,平均表达水平为癌旁正常组织的1.75倍(n=29,t=5.282,P=0.0045);miR-330-3p抑制物和miR-330-3p模拟物,能分别显著降低或升高HN13细胞中miR-330-3p的表达量;转染miR-330-3p抑制物组HN13细胞增殖能力明显降低、凋亡率升高;而miR-330-3p模拟物组细胞的增殖能力明显升高、凋亡率降低。miR-330-3p能够结合PDCD4的3’UTR,负向调控PDCD4的表达。结论 miR-330-3p能够与PDCD4的3’UTR结合抑制PDCD4的转录后翻译作用,从而促进了口腔鳞癌细胞的生长;其抑制物能发挥有效的抗癌作用,研究结果为口腔鳞癌的靶向基因治疗提供候选分子。
Objective To investigate the regulatory function of microRNA-330-3p（miR-330-3p） on the cell growth and apoptosis in oral squamous cell carcinoma（OSCC）, and to screen and verify the revelant target genes. Methods The expressions of miR-330-3p were measured in OSCC tissues, adjacent normal tissues and OSCC cell lines by quantitative real-time PCR. Then miR-330-3p inhibitor and mimics were transfected into OSCC cell line HN13 cells, and their transfection efficiency was detected by quantitative real-time PCR. And then, cell proliferation assay, cell cloning experiments and flow cytometry were conducted to determine the effects of miR-330-3p on the proliferation and apoptosis of OSCC cells. We found 9 putative targets through miRanda and TargetScan that matching the data of related gene chip. Then quantitative real-time PCR, Western blot test and Dual-Luciferase Reporter assay were used to verify the putative targets of miR-330-3p. Results compared with adjacent normal tissues, the expression of miR-330-3p was upregulated in OSCC tissues and cell lines.The inhibitor and mimics of miR-330-3p could significantly downregulate or upregulate the expression of miR-330-3p in transfected HN13 cells. miR-330-3p-inhibitor-transfected HN13 cells had lower proliferation rate and higher apoptosis rate. And HN13 cells showed an opposite change of proliferation rate and apoptosis when transfected with miR-330-3p mimics. MiR-330-3p can negatively regulate the translation of PDCD4. Conclusions miR-330-3p is a tumor promoter in OSCC by repressing the post-transcriptional translation of PDCD4 through binding with 3 ＇UTR, and its inhibitor has the anticancer potential. So our study provides a new candidate molecule to gene-targeting treatment in OSCCs.
出处
《中华口腔医学研究杂志（电子版）》
CAS
2015年第1期10-14,共5页
Chinese Journal of Stomatological Research(Electronic Edition)
基金
国家自然科学基金重大研究计划(培育)项目(91229103)
教育部高等学校博士学科点专项科研基金(20110073110078)
