MicroRNA 144 inhibits cell migration and invasion and regulates inflammatory cytokine secretion through targeting toll like receptor 2 in non-small cell lung cancer

Abstract

Introduction: MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules involved in modulation of cancer progression. Here, we investigated the possible role of miR-144 in non-small cell lung cancer (NSCLC) development.

Material and methods: The expression of miR-144 and TLR2 in NSCLC tissue and cell lines was determined by quantitative real-time PCR (qPCR). The TargetScan database was used to predict potential target genes of miR-144. Luciferase assay was used to verify the interaction between TLR2 and miR-144. TLR2 protein expression was measured by western blot. The secretion of interleukin (IL)-1β, IL-6 and IL-8 in A549 cells was detected by an ELISA kit. Cell migration and invasion were evaluated by wound healing assay and transwell assay, respectively.

Results: Our results showed that miR-144 was downregulated in NSCLC tissue and cell lines when compared with the normal tissues and cell line (p < 0.05). The protein level of TLR2 in NSCLC tissue and cell lines was significantly higher than that in normal lung tissues. Dual luciferase reporter gene assay showed that miR-144 could bind to the 3'UTR of TLR2 specifically. Up-regulation of miR-144 significantly decreased the expression of TLR2. Up-regulation of miR-144 or down-regulation of TLR2 could decrease cell migration, invasion and secretion of IL-1β, IL-6 and IL-8 in A549 cells. Moreover, overexpression of TLR2 rescued the inhibitory effects of miR-144 on migration, invasion and inflammatory factor secretion of A549 cells.

Conclusions: miR-144 could inhibit the migration, invasion and secretion of IL-1β, IL-6 and IL-8 through downregulation of TLR2 expression in A549 cells.

Keywords: MiR-144; TLR2; inflammation; non-small cell lung cancer.

Figure 1

Expression of miR-144 and TLR2 in non-small cell lung cancer (NSCLC) tissues and cell lines. A, B – Expression levels of miR-144 and TLR2 in 28 pairs of NSCLC tissues and their corresponding normal lung tissues were measured by qPCR. U6 snRNA and β-actin were used as internal controls, respectively. C, D – Expression levels of miR-144 and TLR2 in normal lung epithelial cell line (BEAS-2B) and 5 NSCLC cell lines (H460: large cell cancer cell line, H520: squamous cancer cell lines; A549: adenocarcinoma cell line; H157, SK-MES1) were measured by qPCR. ***P < 0.001. (vs. normal or BEAS-2B groups)

Figure 2

Overexpression of miR-144 inhibits cell proliferation, migration and invasion of A549 cells. A – A549 cells were infected with miR-144 mimic or miR-control, and the expression of miR-144 was detected by qPCR. B – Cell migration was detected by wound healing assay. C – Cell invasion was detected by transwell invasion assay. ***P < 0.001 (vs. miR-control group)

Figure 3

TLR2 is a downstream target of miR-144. A – Putative binding sequences of miR-144 in the TLR2 3ʹUTR. Mutation was generated in the TLR2 3ʹUTR by mutating 7 nt that is recognized by miR-144. Either wild-type (WT) or mutant (Mut) TLR2 3ʹUTR was subcloned into the dual-luciferase reporter vector. B – A549 cells were cotransfected with miR-144 mimic and luciferase reporter containing the TLR2 3′-UTR (WT) or a mutant (Mut). Luciferase activities were measured 48 h after transfection. **P < 0.001

Figure 4

miR-144 regulates expression of TLR2 and inflammatory cytokine secretion of A549 cells. A549 cells were infected with miR-144 mimic or miR-control. A, B – mRNA level and protein level of TLR2 were detected by qPCR (A) and western blotting (B), respectively. C – Inflammatory cytokine secretion was detected by ELISA after transfection with miR-144 mimic for 48 h. ***P < 0.001

Figure 5

Down-regulation of TLR2 inhibits cell proliferation, migration, invasion and inflammatory cytokine release of A549 cells. A549 cells were transfected with TLR2 shRNAs or scramble control vector. After transfection for 48 h. A – Expression of TLR2 was detected by western blot. B – Cell migration was detected by wound healing assay. C – Cell invasion was detected by transwell invasion assay. D – Inflammatory cytokine secretion was detected by ELISA. **P < 0.01, ***p < 0.001, vs. scramble group

Figure 6

Inhibitory effects of miR-144 on proliferation, migration, invasion and inflammatory cytokine release of A549 cells were reversed by TLR2 overexpression. A549 cells were co-transfected with miR-144 mimic and TLR2 lacking 3ʹUTR or control vector. After transfection for 48 h. A – Expression of miR-144 was detected by qPCR. B – Expression of TLR2 was detected by western blot. C – Cell migration was detected by wound healing assay. D – Cell invasion was detected by transwell invasion assay. E – Inflammatory cytokine secretion was detected by ELISA. *P < 0.05, **p < 0.01, **p < 0.001