The novel miR-9500 regulates the proliferation and migration of human lung cancer cells by targeting Akt1

MicroRNAs have crucial roles in lung cancer cell development. They regulate cell growth, proliferation and migration by mediating the expression of tumor suppressor genes and oncogenes. We identified and characterized the novel miR-9500 in human lung cancer cells. The miR-9500 forms a stem-loop structure and is conserved in other mammals. The expression levels of miR-9500 were reduced in lung cancer cells and lung cancer tissues compared with normal tissues, as verified by TaqMan miRNA assays. It was confirmed that the putative target gene, Akt1, was directly suppressed by miR-9500, as demonstrated by a luciferase reporter assay. The miR-9500 significantly repressed the protein expression levels of Akt1, as demonstrated via western blot, but did not affect the corresponding mRNA levels. Akt1 has an important role in lung carcinogenesis, and depletion of Akt1 has been shown to have antiproliferative and anti-migratory effects in previous studies. In the current study, the overexpression of miR-9500 inhibited cell proliferation and the expression of cell cycle-related proteins. Likewise, the overexpression of miR-9500 impeded cell migration in human lung cancer cells. In an in vivo assay, miR-9500 significantly suppressed Fluc expression compared with NC and ASO-miR-9500, suggesting that cell proliferation was inhibited in nude mice. Likewise, miR-9500 repressed tumorigenesis and metastasis by targeting Akt1. These data indicate that miR-9500 might be applicable for lung cancer therapy.MicroRNAs (miRNAs) are small, non-coding RNAs, 18–25 nucleotides (nt) in length that regulate gene expression by binding to the 3′-untranslated region (UTR) of their target genes,^{1, 2} and these RNAs are processed from introns, exons or intergenic regions.³ First, miRNAs are transcribed by RNA polymerase II into primary miRNA (pri-miRNA) molecules that contain several thousand nucleotides. The pri-miRNAs are then sequentially processed by a microprocessor, such as Drosha RNase III endonuclease and DiGeorge syndrome region gene 8 protein (DGCR8), to form ∼70 nt-stem-loop intermediates known as miRNA precursors (pre-miRNAs).^{4, 5} The pre-miRNAs are then exported from the nucleus into the cytoplasm via Exportin-5 (EXP5), with its cofactor Ran-GTP; in the cytoplasm, these pre-miRNAs are processed into 18–25 nt mature miRNA duplexes by the RNase III endonuclease Dicer.^{6, 7} The mature miRNA duplexes, along with the Argonaute proteins, are integrated as single-stranded RNAs into an RNA-induced silencing complex, which induces either the cleavage or the translational inhibition of the targeted mRNAs.^{8, 9, 10} miRNAs have been implicated in a variety of biological processes associated with cancer development, including cell proliferation and invasion,¹¹ and miRNA expression is deregulated in many forms of cancer.¹²Cancer is a major public health problem worldwide. Lung cancer represents one of the most predominant types of cancer, with high mortality rates in both men and women. Epithelial lung cancer can be categorized into one of two types: small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). NSCLC accounts for ∼80% of lung cancer cases, and these cases can be further categorized as adenocarcinoma (40%), squamous cell carcinoma (30–35%), and large cell carcinoma (5–15%). NSCLC has a 5-year survival rate of only 16%.^{13, 14, 15} Current studies have shown that miRNAs are deregulated in various cancers, including NSCLC, and may act as oncogenes or tumor suppressor genes.¹⁶ For example, the Let-7 family,¹⁷ miR-15a/16,¹⁸ miR-17-92,¹⁹ miR-107 and miR-185,²⁰ are deregulated in lung cancer.Some studies have reported that phosphatidylinositol 3-kinase (PI3K) signaling is activated in human cancers^{21, 22} and has an important role in the progression of NSCLC. The PI3K pathway modulates several cellular mechanisms, such as cell survival, proliferation, migration and motility, and thereby significantly affects the growth of tumors.^{23, 24} The primary regulator of the PI3K pathway is Akt, a protein kinase B that mediates cell survival, cell death,²⁵ cell growth, cell migration and angiogenesis.^{26, 27, 28} The silencing of the Akt1 gene has been shown to inhibit the proliferation of gastric cancer cells both in vitro and in vivo.²⁹ Other studies have shown that aberrant AKT activation has a critical role in tumorigenesis.³⁰In this study, we identified small RNAs in lung cancer cells. To analyze a novel miRNA signature, we examined the structure and sequence of the small RNAs, analyzed the expression patterns of the novel miRNAs in lung cancer tissues and assessed the miRNA target genes. Our data revealed that miR-9500 regulates certain human lung cancer cell functions, including cell growth, proliferation, and migration.