关键词: 非小细胞肺癌细胞, 淋巴结侵袭, 磷酸酶和张力蛋白同源基因10, 微RNA-29a/b/c

Abstract:

Objective To investigate the regulation and the underlying molecular mechanism of microRNA(miRNA/miR)-29 family on the cancer cell proliferation and invasion of human lymph-node invasive non-small cell lung cancer (NSCLC). Methods TCGA data base was used to analyze phosphatase and tension homology deleted from chromosome 10 (PTEN) and the mRNA expression between the non-lymph node invasive and lymph node invasive tissue in NSCLC patients. The PTEN protein expression in non-lymph node invasive NSCLC A549 cells and lymph node invasive NSCLC H1299 cells was detected by Western blotting. Three different online software were used to predict the miRNAs targeting PTEN mRNA. Real-time quantitative reverse transcriptase mediated (qRT-PCR) was used to detect miR-29 family expression in A549 cells and H1299 cells. The binding sites between PTEN mRNA 3’ untranslated region(UTR) and miR-29 were predicted by using database and confirmed by luciferase report assays. miR-a/b/c mimics or inhibitors were transfected to A549 or H1299 cells, and the PTEN protein expression was detected by Western blotting. miR-29 family knock-down cells were established by CRISPR cas9 technology. Cell counting kit-8(CCK-8) assay was used to detect the difference of proliferation between negative control group (NC group) and miR-29 family knock-down group, and transwell invasion chamber test was used to detect the difference in invasion. p-Akt,Akt, phosphorylation focal adhesion kinase（p-FAK）, FAK and survivn expression was detected by Western blotting. Survivin mRNA expression was measured by qRT-PCR. CCK-8 method was used to detect the cell proliferation and cell invasion was detected by transwell method. Results The PTEN mRNA expression in the non-lymph node invasive NSCLC patients was higher than that in lymph node invasive NSCLC patients by analyzing the TCGA database. The PTEN protein expression was higher in A549 cells than that in H1299 cells, while miR-29 family expression was lower in A549 cells than that in H1299 cells. The luciferase report assays confirmed that PTEN mRNA 3’UTR was the target of miR-29 family. The PTEN protein expression was decreased after the A549 cells transfected miR a/b/c mimics for 36 h, and PTEN protein expression was increased after H1299 cells transfected miR a/b/c inhibitors for 36 h. The miR-29 family were knocked down in H1299 cells by CRISPR cas9 technology and the proliferation and invasion were significantly decreased compared with NC group. Compared with NC group, the phosphorylation level of Akt, surviving mRNA and protein expression, and the phosphorylation level of FAK were all decreased after miR-29 family was knocked down. Conclusions The miR-29 family promoted the proliferation and invasion of lymph node invasive NSCLC cells by decreasing PTEN expression and abnormally activating p-Akt and p-FAK signaling pathway.

Key words: Non-small cell lung cancer, Lymph node invasion, Phosphatase and tension homology deleted from chromosome 10, MicroRNA-29a/b/c