Genetic and epigenetic silencing of mircoRNA-506-3p enhances COTL1 oncogene expression to foster non-small lung cancer progression

Abstract

Although previous studies suggested that microRNA-506-3p (miR-506-3p) was frequently downregulated, and functioned as a tumor suppressor in several cancers, the biological role and intrinsic regulatory mechanisms of miR-506-3p in non-small cell lung cancer (NSCLC) remain elusive. The present study found miR-506-3p expression was downregulated in advanced NSCLC tissues and cell lines. The expression of miR-506-3p in NSCLC was inversely correlated with larger tumor size, advanced TNM stage and lymph node metastasis. In addition, we also found patients with lower expression of miR-506-3p had a poor prognosis than those patients with higher expression of miR-506-3p. Function studies demonstrated that aberrant miR-506-3p expression modulates tumor cell growth, cell mobility, cell migration and invasion in vitro and in vivo. Mechanistic investigations manifested that coactosin-like protein 1 (COTL1) was a direct downstream target of miR-506-3p. Knockdown of COTL1 mimicked the tumor-suppressive effects of miR-506-3p overexpression in A549 cells, whereas COTL1 overexpression enhanced the tumorigenic function in HCC827 cells. Importantly, we also found GATA3 transcriptionally actives miR-506-3p expression, and the long non-coding RNA urothelial carcinoma-associated 1 (UCA1) exerts oncogenic function in NSCLC by competitively 'sponging' miRNA-506. Together, our combined results elucidated genetic and epigenetic silencing of miR-506-3p enhances COTL1 oncogene expression to foster NSCLC progression.

Keywords: COTL1; LncRNA; UCA1; miR-506-3p; non-small cell lung cancer.

Figure 1. Downregulated expression of miR-506-3p predicts poor prognosis in NSCLC patients

(A) Expression of miR-506-3p in 52 matched pairs of primary NSCLC tissues and their corresponding adjacent samples. The expression level of miR-506-3p was detected using qPCR and normalized against an endogenous control (U6) mRNA. (B) Patients with a lower expression of miR-506-3p had a poor prognosis than the patients with high expression of miR-506-3p.

Figure 2. Abnormal expression of miR-506-3p alters the growth of NSCLC cells

(A) Alarmar Blue assay showed that overexpression of miR-506-3p inhibits cell growth of A549 cells in 72 h, whereas inhibition of miR-506-3p in HCC827 cells promotes cell proliferation in 72 h. (B) Colony formation assay showed that colony ability of A549 cells was inhibited after treatment of miR-506-3p mimics in 6 days, while silencing of miR-506-3p in HCC827 cells promoted cell colony formation in 6 days. (C) FACS assay showed that overexpression of miR-506-3p promotes cell apoptosis of A549 cells in 48 h, whereas inhibition of miR-506-3p in HCC827 cells inhibits cell apotosis in 48 h. (D) Wound healing assay showed that cell mobility ability was inhibited when transfected by miR-506-3p mimics in A549 cells in 48 h, whereas silencing of miR-506-3p in HCC827 cells promotes cell mobility in 48 h. (E–F) Transwell assay showed that overexpression of miR-506-3p inhibits cell migration and invasion ability of A549 cells in 48 h, while inhibition of miR-506-3p in HCC827 cells promotes cell migration and invasion in 48 h. (G–H) The mean volume and weight of the xenograft tumors in miR-506-3p-A549 group was smaller than those of miR-NC-A549 group, whereas silencing of miR-506-3p in HCC827 cells promotes tumor weight than those of siRNA-miR-NC-HCC827 xenografted tumors. Asterisk (*) indicated P < 0.05.

Figure 3. miR-506-3p directly targets to COTL1 3′UTR

(A) Targetscan software indicated that COTL1 was a potential target of miR-506-3p. (B) Luciferase reporter assay showed that COTL1 was a direct downstream target of miR-506-3p in A549 and HCC827 cells. (C–D) Overexpression of miR-506-3p inhibits the mRNA and protein expression of COTL1 in A549 cells, whereas silencing of miR-506-3p in HCC827 cells promotes COTL1 mRNA and protein levels. The oncogene N-Ras, a direct gene of miR-506-3p, has been acted as a positive control. (E) Western blot analysis demonstrated that COTL1-no 3′UTR abrogates that miR-506-3p mediated induction of COTL1, but COTL1-wt 3′UTR was found to decrease COTL1 protein expression in A549 cells. (F) Alarmar Blue assay showed that COTL1-no 3′UTR promoted the proliferation of miR-506-3p transfected A549 cells; however, COTL1-wt 3′UTR cells had no significant impact in the miR-506-3p transfected A549 cells. (G–H) Transwell assays showed that COTL1-no 3′UTR in A549 cells increased migration and invasion in miR-506-3p transfected cells; however, COTL1-wt 3′UTR cells had no significant impact in the miR-506 transfected cells. Asterisk (*) indicated P < 0.05.

Figure 4. Aberrant expression of COTL1 governs NSCLC cell growth

(A–B) COTL1 mRNA and protein levels were upregulated in NSLCL tissues compared with their corresponding adjacent samples. (C) miR-506 was inversely related to COTL1 mRNA in NSLCL tissues. (D) Kaplan-Meier survival curves depicted that patients with lower miR-506-3p and higher COTL1 expression had a poor prognosis than that with the patients with high miR-506-3p and lower COTL1 expression. (E–F) The mRNA and protein level of COTL1 were upregulated in A549, HCC827 and SPC-A1 cells compared with that of BEAS-2B cells. (G–I) COTL1 knockdown inhibited cell proliferation, migration and invasion ability in A549 cells, whereas COTL1 overexpression in HCC827 cells promotes cell growth, migration and invasion. Asterisk (*) indicated P < 0.05.

Figure 5. GATA3 transcriptionally modulates miR-506-3p expression in NSCLC

(A) The putative GATA3 binding site in mature miR-506 promoter regions. (B) ChIP assay showed that GATA3 expression was increased in the promoter of mature miR-506 in A549 and HCC827 cells. (C) Luciferase activity of pGL3-miR-506-3p was increased in GATA3-transfected A549 cells and HCC827 cells. (D–F) Inhibition of miR-506-3p (miR-506i) attenuated the decreased cell proliferation, migration and invasion of GATA3 overexpression in A549 cells, whereas overexpression miR-506-3p inhibited the elevated cell growth, migration and invasion of GATA3 knockdown (GATA3i) in HCC827 cells. (G) The mRNA levels of GATA3 were decreased in NSCLC tissues compared with their adjacent samples. (H) Spearman correlation analysis revealed that GATA3 expression was positively correlated with miR-506-3p mRNA in NSCLC tissues. (I) Kaplan-Meier survival curves depicted that patients with lower miR-506-3p and lower GATA3 expression had a poor prognosis than that with the patients with higher miR-506-3p and higher GATA3 expression. Asterisk (*) indicates P < 0.05.

Figure 6. UCA1 acts as a molecular sponge for miR-506-3p

(A) The association between UCA1, miRNA-506-3p and Ago2 was ascertained by analyzing A549 cells lysates by RNA immunoprecipitation with an Ago2 antibody. miR-143 act as a negative control. (B) Putative miR-506-3p binding sequence of UCA1 RNA. Mutation was generated on the UCA1 RNA sequence in the complementary site for the seed region of miR-506-3p. (C) A549 cells were transfected with the vector, wild-type UCA1 (UCA1-Wt) or mutant UCA1 (UCA1-Mut) with a mutation of the miRNA binding sites. Luciferase activity was measured using a dual-luciferase reporter gene assay system after 48 h transfection. (D–F) Inhibition of miR-506-3p (miR-506i) attenuated the decreased proliferation, migration and invasion effect of UCA1 knockdown (UCA1-KD) in A549 cells, whereas overexpression miR-506-3p inhibited the elevated cell growth, migration and invasion of UCA1-transfected HCC827 cells. (G) UCA1 mRNA levels were upregulated in NSLCL tissues compared with their corresponding adjacent samples. (H) miR-506-3p was inversely correlated with UCA1 mRNA in NSLCL tissues. (I) Kaplan-Meier survival curves depicted that patients with lower miR-506-3p and higher UCA1 expression had a poor prognosis than that with the patients with high miR-506-3p and lower UCA1 expression. Asterisk (*) indicated P < 0.05.

Figure 7. A schematic diagram deciphering the mechanism underlying GATA3 and UCA1 driven expression of miR-506-3p enhances COTL1 oncogene expression to foster NSCLC progression