Notably, coexpression of mir-200c with Cre significantly reduced the number of tumors formed (Fig. L-741626 These mutations impair the GTPase activity of KRAS and enable constitutive activation of downstream pathways impartial of exogenous regulatory signals. The abnormal activation of downstream effectors in KRAS pathways, such as RAFCextracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/AKT, had been found to contribute to KRAS-driven tumorigenesis, which is usually characterized by cellular transformation, resistance to apoptosis, and metastasis (1,C6). Moreover, downstream transcription factors of KRAS pathways, such as FOS, JUN, nuclear factor B (NF-B), and Fra1, are required for cancer cell survival, proliferation, migration, and invasion (7,C10). Although the molecular mechanisms dictating how the aberrant activation of KRAS pathways affects transformed phenotypes and tumorigenesis have been well studied, the role of SPRY4 noncoding genes in mediating KRAS function is still largely unknown (11). MicroRNAs (miRNAs) are endogenous 18- to 25-nucleotide noncoding small RNAs that regulate gene expression in a sequence-specific manner via the degradation of target mRNAs or inhibition of protein translation (12,C14). MicroRNA 200 (mir-200) is usually a well-characterized, highly conserved miRNA family, consisting of five members that are located in two miRNA gene clusters (mir-200b/a/429 and mir-200c/141) on different chromosomes. Each cluster is usually transcribed into a single primary miRNA transcript (pri-miRNA) and processed by the Drosha/DGCR8 complex into individual precursor transcripts (pre-miRNA), which are further sliced by Dicer into mature miRNAs. The five mature miRNAs L-741626 of the family contain highly comparable seed sequences, which leads them to share a wide range of biological functions, such as regulation of development (15,C17), cellular senescence (18), apoptosis (19), tumor metastasis (20,C27), angiogenesis (28), and immunosuppression of lymphocytes (29). These biological functions L-741626 of mir-200 were disclosed by the discovery of its target genes, such as those coding for ZEB1/2 (21, 22, 24,C26), SEC23 (30), CXCL1/IL-8 (28), and PD-L1 (29), in different cellular contexts. Similar to other miRNAs involved in tumorigenesis (31), the expression levels of mir-200 family members were deregulated in cancer cells by different mechanisms, implying their crucial roles in normal physiological processes. For example, repressive epigenetic markers were present in the promoter regions of mir-200 gene clusters in cancers (32,C34). In addition, mir-200 was suppressed by ZEB1/2 in mesenchymal cancer cells (21, 22, 26, 35). These results, taken together, indicate that mir-200 functions as a tumor suppressor in multiple cancer types. Restoring the expression of mir-200 was sufficient to rescue the transformed phenotypes (20, 24, 25), implicating a novel strategy for cancer therapy by targeting mir-200. The present study aimed to identify novel miRNA factors regulating KRAS functions by using array-based miRNA profiling in cells expressing oncogenic KRAS. The expression of the mir-200 family was revealed potently suppressed by KRAS activation, and mir-200 represents a novel suppressor of KRAS oncogenic functions. MATERIALS AND METHODS Plasmids. KRASG12D/pBabe vector was used for enforced overexpression (Addgene plasmid 58902). The shKRAS/pLKO construct was generated by inserting a short hairpin RNA (shRNA) with sequences targeting (GACGAATATGATCCAACAATA) into pLKO.1 vector (Addgene). The luciferase reporter plasmid made up of the mir-200b/a/429 promoter region was kindly provided by Gregory J. Goodall. mir-200c.Cre was generated by replacing the luciferase gene in Luc.Cre vacant vector (Addgene plasmid 20905) with a cDNA fragment encoding primary mir-200c from the mir-200c/pLV expression vector (a gift from Qihong Huang). Wild-type and mutant gene 3 UTR into psiCHECK2 vector (Promega) (italic letters represent the artificially mutated binding site of mir-200 in the BCL2 3 UTR). Cell culture. IMR90 cells were cultured in Eagle’s minimum essential medium (ATCC) supplemented with 10% fetal bovine serum (FBS) (GIBCO), and 1% penicillin-streptomycin (GIBCO). MCF10A cells were cultured in Dulbecco’s altered Eagle’s mediumCF-12 (DMEMCF-12) (GIBCO) with 5% horse serum (GIBCO), 10 g/ml epidermal growth factor (Sigma), 10 mg/ml insulin (Sigma), 0.1 mg/ml cholera toxin (Sigma), 2 mg/ml hydrocortisone (Sigma). The 293T, PT67,.