The lncRNA LINC01194/miR-486-5p Axis Facilitates Malignancy in Non-Small Cell Lung Cancer via Regulating CDK4

Abstract

Background: This experimental design was based on lncRNA LINC01194 to explore the pathogenesis of NSCLC.

Methods: RT-qPCR was used to detect the expression of lncRNA LINC01194 and miR-486-5p in NSCLC tissues and cell lines. CCK-8, colony formation, and transwell assays were used to examine the effects of lncRNA LINC01194 and miR-486-5p on NSCLC cell proliferation and migration invasiveness. For target gene prediction and screening, luciferase reporter assays were used to verify downstream target genes for lncRNA LINC01194 and miR-486-5p. The protein expression of CDK4 was detected using Western blotting. The tumor changes in mice were detected by in vivo experiments in nude mice.

Results: LncRNA LINC01194 was highly expressed in NSCLC tissues and NSCLC lines (A549, H1299, H460 cells, H1975), and lncRNA LINC01194 significantly promoted cell proliferation and migration of NSCLC cells. MiR-486-5p was identified as a potential target for LINC01194, and miR-486-5p was expressed at a low level in NSCLC tissues and NSCLC lines (A549, H1299, H460 cells, H1975). CDK4 was identified as a potential target for miR-486-5p. LncRNA LINC01194 was able to inhibit miR-486-5p expression and upregulate the expression level of CDK4. Finally, the results of in vivo animal models confirmed that lncRNA LINC01194 promoted NSCLC progression by modulating the miR-486-5p/CDK4 axis.

Conclusion: LncRNA LINC01194 promoted the progression of NSCLC by modulating the miR-486-5p/CDK4 axis.

Keywords: CDK4; invasion; lncRNA LINC01194; miR-486-5p; non-small cell lung cancer; proliferation.

Figure 1

Biological role of lncRNA LINC01194 in NSCLC. (A) Relative expression of NSCLC in NSCLC tissues and adjacent normal tissues (n=26). (B) lncRNA LINC01194 mRNA expression level in NSCLC cell lines. (C) lncRNA LINC01194 mRNA levels under different treatment conditions. (D) CCK8 measured cell viability. (E) Colony formation measured cell proliferation. (F, G) Transwell measured the number of cell invasion and migration. *P<0.05, n=3.

Figure 2

LINC01194 regulated the expression of miR-486-5p in NSCLC cells. (A) Expression of miR146a-5p mRNA levels in NSCLC cell lines. (B) Putative target sequence of miR-486-5p on the 3ʹ-UTR of LINC01194. (C) miR-486-5p mRNA levels in A549 cells under different treatment conditions. (D) Detection of luciferase activity by luciferase reporter assay. (E) LINC01194 expression levels in samples by biotinylated miR-486-5p or negative control. (F) Correlation between LINC01194 and miR-486-5p levels was using detecting RNA pull down. (G) Pearson’s correlation analysis of LINC01194and miR-486-5p in NSCLC tissues (n=26) (r=-0.672, P<0.01).* P <0.05, n = 3.

Figure 3

LINC01194 exerted a biological effect on NSCLC cells via miR-486-5p. (A) CCK8 measured the viability of A549 cells. (B, C) colony formation assay cell proliferation. (D, E) Transwell measured the number of cell invasions. (F, G) Transwell measured the amount of cell migration. *P<0.05, n=3.

Figure 4

CDK4 was a direct miR-486-5p target. (A) Putative target sequence of miR-486-5p on the 3ʹ-UTR of CDK4. (B) Detection of luciferase activity by luciferase reporter assay. (C) RT-PCR and Western blot analysis were used to determine (C and D) CDK4 mRNA and protein levels. (C) RT-PCR assay for CDK4 mRNA levels. (D) Western blot analysis was used to determine CDK4 protein levels. (E) Western blot analysis to determine CDK4 protein levels. *P<0.05, n=3

Figure 5

CDK4 mediated the role of the LINC01194/miR-486-5p axis in NSCLC cells. (A) CCK8 measured cell viability. (B) Colony formation assay cell proliferation. (C, D) Transwell measured the number of cell invasion and migration. *P<0.05, n=3.

Figure 6

LINC01194 promoted tumor growth in vivo. (A) Tumor volume. (B) Image of tumor tissue. (C) Tumor weight. (D) Ki67 immunochemistry. (E) Western blot analysis to determine CDK4 protein levels. *P<0.05.