miR-155在肺腺癌A549细胞侵袭和转移中的作用

Role of miR-155 in invasion and metastasis of lung adenocarcinoma A549 cells

目的探讨miR-155在肺腺癌侵袭和转移中的作用。方法采用实时荧光定量PCR和原位杂交检测miR-155在肺腺癌和癌旁组织以及转移淋巴结中的表达，采用划痕实验和Transwell迁移实验检测miR-155对A549细胞的迁移和侵袭能力的影响；利用生物信息学软件预测miR-155的靶基因，并采用双荧光素酶检测靶基因；采用Westernblot和实时荧光定量PCR检测miR-155调控靶基因鼠抗人第10号染色体同源缺失性磷酸酶张力蛋白（PTEN）的表达水平。结果实时荧光定量PCR检测显示，肺腺癌组织、癌旁正常组织和转移淋巴结中的miR-155表达水平分别为9．6±3．1、4．1±0．5和7．8±2．2。原位杂交检测显示，肺腺癌组织、癌旁正常组织和转移淋巴结中miR-155阳性表达率分别为（75．4±20．2）％、（23．2±15．3）％和（60．4±25．1）％。划痕实验检测显示，miR-155模拟物组、miR-155模拟物对照组、miR-155抑制剂组和miR-155抑制剂对照组24h伤口愈合率分别为（43．2±2．2）％、（21．3±4．2）％、（24．3±5．3）％和（35．2±5．1）％，而48h伤口愈合率分别为（75．2±4．5）％、（52．6±5．2）％、（39．4±4．2）％和（51．5±4．3）％。双荧光素酶报告基因检测显示，miR-155模拟物组与含有PTEN3’UTR野生型和突变型质粒共转染的荧光素酶值分别为4．7±0．5和7．3±0．7，而miR-155模拟物对照组与含有PTEN3’UTR野生型和突变型质粒共转染的荧光素酶值分别为7．8±0．9和7．5±0．8。实时荧光定量PCR检测显示，miR-155模拟物组、miR-155模拟物对照组、miR-155抑制剂组和miR-155抑制剂对照组的PTENmRNA相对表达水平分别为0．5±0．3、1．0±0．1、2．2±0．2和1．2±0．1。Westernblot检测显示，miR-155模拟物组、miR-155模拟物对照组、miR-155抑制剂组和miR-155抑制剂对照组的PTEN蛋白相对表达水平分别为0．4±0．1、1．0±0．3、2．8±0．2和1．4±0．1。miR-155模拟物组与miR-155模拟物对照组、miR-155抑制剂组与miR-155抑制剂对照组的PTENmRNA和蛋白表达差异均有统计学意义（均P〈0．05）。结论miR-155可能是通过下调靶基因PTEN的表达而促进肺腺癌的侵袭和转移。

Objective To investigate the role and mechanism of miR-155 in invasion and metastasis of lung adenocareinoma A549 cells. Methods Real-time PCR and fluorescence in situ hybridization were used to detect the miR-155 expression in patients＇ lung adenocarcinoma and adjacent tissue and lymph nodes. Scratch test and Transwell migration assay were used to assess the effect of miR-155 on the A549 cell migration and invasion capability. Bioinformatics software was used to predict the target genes of miR-155, and using luciferase to assay the target gene. Western blot and real-time PCR were performed to confirm the role of miR-155 expression in the regulation of target gene PTEN. Results The real-time quantitative PCR showed that the miR-155 expression levels in adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were 4.1±0.5, 9.6 ± 3.1 and 7.8± 2.2, respectively. The in situ hybridization showed that the expression rates of miR-155 in the adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were （23.2± 15.3） %, （75.4±20.2） % and （60.4±25.1） %, respectively. The Scratch assay showed that the wound healing rates in the miR-155 mimics group, miR-155 mimics NC group, miR-155 inhibitor group and miR-155 inhibitor NC group at 24 h were （43.2±2.2）%, （21.3±4.2）%, （24.3±5.3）%, and （35.2± 5.1）%, and that at 48 h were （75.2±4.5）%, （52.6±5.2）%, （39.4±4.2）%, and （51.5±4.3）%, respectively. Dual lueiferase reporter gene assay showed that the value of the luciferase in the miR-155 mimics group co-transfeeted with PTEN 3＇UTR-containing wild-type and mutant plasmids were 4.7±0.5 and 7.3±0.7, and the miR-155 mimics luciferase values of the control group co-transfected with PTEN 3＇UTR- containing wild-type and mutant plasmids were 7.8±0.9 and 7.5±0.8, respectively. The real-time quantitative fluorescence PCR showed that the relative expression of PTEN protein in the miR-155 mimics group, miR- 155 mimics control group, miR-155 mimics inhibitor group, and miR-155 inhibitor control group were 0.5± 0.3, 1.0± 0. 1, 2.2± 0.2 and 1.2± 0. 1, respectively. The Western blot assay detected that the relative expression of PTEN protein levels in the miR-155 mimics group, miR-155 mimics control group, miR-155 inhibitor group and miR-155 inhibitor control group were 0.4±0.1, 1.0±0.3, 2.8±0.2 and 1.4±0.1, respectively. The differences in PTEN mRNA and protein expressions of the four groups were statistically significant （P〈0.05 for all）. Conclusions miR-155 may promote the invasion and metastasis of lung adenocarcinoma through reducing the target PTEN gene expression.