MicroRNA-103a-3p Promotes Cell Proliferation and Invasion in Non-Small-Cell Lung Cancer Cells through Akt Pathway by Targeting PTEN

Abstract

Background: Increasing evidence has suggested that microRNA- (miR-) 103a-3p is crucial for cancer progression. However, the specific mechanism of miR-103a-3p in non-small-cell lung cancer (NSCLC) remains unclear until now. So, it is particularly urgent to clarify the mechanism between them.

Methods: qRT-PCR and western blot were used to measure the expression of miR-103a-3p, PTEN, Akt, and p-Akt. Cell biology experiment was applied to detect the biological function of miR-103a-3p in NSCLC cell lines. Moreover, bioinformatics analysis, luciferase reporter assay, and functional complementation analysis were carried out to investigate the target gene.

Results: miR-103a-3p was highly expressed in primary NSCLC samples and cell lines. miR-103a-3p mimics promoted the proliferation and invasion of NSCLC cells; miR-103a-3p inhibitor had the opposite effect. A double luciferase reporter gene experiment revealed that miR-103a-3p directly targets the PTEN mRNA 3'UTR region. siPTEN inhibited the proliferation and invasion of NSCLC cells. Further mechanistic studies showed that both overexpression of miR-103a-3p and PTEN knockdown reduced the expression of the p-Akt protein. Overexpression of PTEN partially reversed the cancer-promoting effect of miR-103a-3p.

Conclusion: miR-103a-3p promotes the progression of NSCLC via Akt signaling by targeting PTEN, highlighting the role of miR-103a-3p/PTEN/Akt signaling and suggesting miR-103a-3p as a novel therapeutic target for NSCLC.

Figure 1

miR-103a-3p promotes NSCLC cell proliferation and invasion. (a) TCGA database analysis of miR-103a-3p expression in NSCLC samples and normal lung samples. (b) Relative expression of miR-103a-3p in NSCLC cell lines (A549, NCI-H157, NCI-H1299, and NCI-H1975) and BEAS-2B were detected by qRT-PCR. A549 and NCI-H1299 cells were transfected with miR-103a-3p mimics or inhibitor for 48 h before the following detections. (c) Relative expression of miR-103a-3p was measured by qRT-PCR. (d) Effect of miR-103a-3p on cell proliferation was examined by MTT assay. (e) Effect of miR-103a-3p on colony-forming ability was detected by the colony formation assay. (f) Effect of miR-103a-3p on invasive capability was assessed by Transwell invasion assay (^∗∗∗p < 0.001, ^∗∗p < 0.01, and ^∗p < 0.05).

Figure 2

PTEN is a target gene of miR-103a-3p. (a) Sequence diagram of miR-103a-3p with its binding site in PTEN 3′-UTR. (b) Luciferase reporter assay detection for HEK293T transfected with miR-103a-3p mimics or PTEN-WT or PTEN-MUT. Effect of miR-103a-3p on PTEN mRNA (c) and protein (d) expressions was detected by qRT-PCR and western blot (^∗∗p < 0.01, ^∗p < 0.05).

Figure 3

siPTEN promotes the proliferation, invasion, and Akt signaling in NSCLC cells. Relative expression of PTEN mRNA (a) and protein (b) were detected by qRT-PCR and western blot after transfected with PTEN siRNA or NC siRNA for 48 h. (c) MTT assay on effect of PTEN knockdown. (d) Transwell invasion assay on effect of PTEN knockdown. Relative expression of p-Akt/Akt protein was detected by western blot after PTEN knockdown (e) or transfected with miR-103a-3p mimics/inhibitor (f) (^∗∗p < 0.01, ^∗p < 0.05).

Figure 4

Overexpression of PTEN partly reverses the cancer-promoting effect of miR-103a-3p. A549 and NCI-H1299 cells were cotransfected with miR-103a-3p mimics with or without PTEN expression plasmid for 48 h. Relative expression of PTEN protein (a) and p-Akt/Akt protein (b) was detected by western blot. (c) MTT assay. (d) Transwell invasion assay (^∗∗p < 0.01, ^∗p < 0.05).