MicroRNAs (miRNAs) have essential roles in carcinogenesis and tumor progression. of miR-143 in NSCLC cells. (A) wild type Limk1 3′-untranslated regions (3′-UTR) (WT) or Mutated Limk1 3′-UTR (Mut) of Limk1 sequence; (B) HEK-293 cells were co-transfected with miR-143/miR-NC with WT/Mut … 2.5. miR-143 Cyclophosphamide monohydrate IC50 Suppresses NSCLC Development by Targeting Limk1 The involvement of Limk1 in miR-143-induced suppression of NSCLC cell growth and invasion was examined using the CCK-8 assay (Figure 5A), migration (Figure 5B), and invasion (Figure 5C). In each case, Limk1 overexpression significantly attenuated the suppressive effects of miR-143 in NSCLC cells. The effect of Limk1 overexpression was confirmed by qRT-PCR (Figure 5D). Figure 5 miR-143 suppresses NSCLC development by targeting Limk1. (A) NCI-H23 cells were transfected with miR-143 with/without Limk1 overexpression plasmid (pcDNA-Limk1), and CCK-8 assay was performed; (B) Migration and (C) invasion assays of NCI-H23 cells; and … 2.6. miR-143 Expression Is Inversely Correlated with Limk1 Expression in NSCLC Tissues Expression of Limk1 in 24 samples of NSCLC tissue, and the corresponding normal controls, was examined by qRT-PCR. Limk1 mRNA was markedly increased in NSCLC tissues (Figure 6A). Furthermore, the level of Limk1 mRNA expression was inversely correlated with that of miR-143 NSCLC tissues (Figure 6B). Figure 6 miR-143 is negatively correlated with Limk1 in NSCLC tissues. (A) The expression of Limk1 in 24 pairs of NSCLC tissues and the matched NC was measured by qRT-PCR; and (B) Limk1 mRNA level was inversely correlated with miR-143 level in Cyclophosphamide monohydrate IC50 NSCLC tissues (Spearmans … 3. Discussion miRNAs have been reported to play critical roles in carcinogenesis and tumor progression [20,21]. Aberrant miRNA expression has been implicated in almost all aspects of tumor biology, including proliferation, apoptosis, migration, and invasion, and they can act as either tumor suppressors or oncogenes. Here, we focused on miR-143, which acts as a tumor suppressor in human malignancies. Ng reported that miR-143 was significantly down-regulated in breast cancer, and that its overexpression suppressed proliferation and soft agar colony formation of breast cancer cells by down-regulating DNA methyltransferase 3A (DNMT3A) expression [15]. Zeng found that miR-143 regulated NSCLC cell apoptosis by inhibiting PKC [18], and Ma reported that miR-143 inhibited migration and invasion of NSCLC cells through the targeting of CD44v3 [19]. miR-143 and miR-145 are co-expressed miRNAs which form a bicistronic cluster in 5q33.1, and have been widely studied as potential tumor suppressors [23]. Viana reported that miR-143 and miR-145 might be involved in the pathogenesis of benign prostatic hyperplasia via regulating target genes and proteins [24]. Kojima found that in prostate cancer miR-143/145 cluster suppressed cell migration and invasion by targeting Golgi membrane protein 1 (GOLM1) [13]. Zhang showed that down-regulation of miR-143 and miR-145 might be associated with overexpression of DNA methyltransferase 3B (DNMT3B) and worse prognosis in endometrioid carcinomas [25]. Cho revealed that restoration of miR-145 suppressed cancer cell growth in lung adenocarcinoma patients Mouse monoclonal antibody to Calumenin. The product of this gene is a calcium-binding protein localized in the endoplasmic reticulum (ER)and it is involved in such ER functions as protein folding and sorting. This protein belongs to afamily of multiple EF-hand proteins (CERC) that include reticulocalbin, ERC-55, and Cab45 andthe product of this gene. Alternatively spliced transcript variants encoding different isoforms havebeen identified who contained epidermal growth factor receptor (EGFR) mutation [26]. These studies suggest that miR-143 and miR-145 may co-contribute to the progression of several cancers. In NSCLC, miR-143 and miR-145 have been found to suppress the progression of NSCLC by targeting different targets [18,19,27]. Thus, whether miR-143 and miR-145 may co-contribute to the progression of NSCLC needs further studies. In the present study, we showed that miR-143 was significantly down-regulated in NSCLC tissues and cell lines. Forced overexpression of miR-143 effectively suppressed NSCLC cell proliferation, enhanced apoptosis, and inhibited migration and invasion. Limk1 is a key regulator of the actin cytoskeleton, cell motility, and invasion, and is thought to be a therapeutic Cyclophosphamide monohydrate IC50 target for metastatic disease [28]. Limk1 is frequently overexpressed in many malignancies and functions as an important oncogene [9,29,30]. Downregulation of Limk1 suppressed migration of NSCLC cells and enhanced their sensitivity to chemotherapy drugs [29]. Increased Limk1 expression has been found in prostate tumor tissues, and is involved in regulating the invasiveness of prostate cancer cells [30]. Here, we demonstrate that Limk1 is a direct target of miR-143, which we confirm by luciferase activity and western blot. We found that the tumor suppressive effects of miR-143 on NSCLC cells were partially reversed by overexpression of Limk1. Finally, we have shown that Limk1 is significantly elevated in NSCLC tissues and its expression is inversely correlated with the level of miR-143 expression. Together, these data suggest that miR-143 inhibits NSCLC growth and metastasis.