Role of microRNA-30c targeting ADAM19 in colorectal cancer.

MicroRNAs (miRNAs) are deregulated in a number of cancers including colorectal cancer. MiR-30c belongs to miR-30 family, and is involved in a variety of malignant diseases. In this study, we detected the expression of miR-30c in colon cancer cell lines and clinical colon cancer specimens. MiR-30c was shown to be dramatically down-regulated both in cell lines and cancer tissues. Additionally, miR-30c could inhibit cancer cell growth, migration and invasion in vitro. Consistently, stable over-expression of miR-30c inhibited the growth and lung metastasis of colon cancer cell xenografts in vivo. Furthermore, bioinformatics algorithm and luciferase reporter assay indicated ADAM19 as a direct target of miR-30c. Of interest, further experiments demonstrated that inhibition of ADAM19 by miR-30c partially mediated the anti-tumor effect of miR-30c. Overall, our study provides the new insight that miR-30c inhibited colon cancer cells via targeting ADAM19. Thus, miR-30c might serve as a promising therapeutic strategy for colon cancer treatment

MiR-30c inhibited cell migration and invasion ability of colon cancer cells.

<p>(A-B) Effects of miR-30c on migration were analyzed by Transwell migration assay. A, representative photos; B, quantitative analysis. (C-D) Effects of miR-30c on invasion were analyzed by Transwell invasion assay. C, representative photos; D, quantitative analysis. (E-F) F, Wound healing assay was adopted to evaluate the effect of miR-30c on migration. The artificial gap was through the central axis when cells reached a density of 80%. Photos of cells were taken at 0 and 24 h. E, representative photos; F, quantitative analysis. *<i>p</i><0.05; ** <i>p</i><0.01.</p

Primers for plasmid construction, qRT-PCR, and oligonucleotide.

<p>Primers for plasmid construction, qRT-PCR, and oligonucleotide.</p

MiR-30c inhibited tumor growth in vivo.

<p>(A) The stable overexpression of miR-30c in HCT116 cell clones was determined with qRT-PCR. (B) 5×10⁶ HCT116 cells which were stably transfected with miR-30C or empty vector (mock) were subcutaneously injected into nude mice (n = 3). Mice were sacrificed 28 days after injection. (C) Tumors were harvested and representative tumors were shown. (D) Tumors were weighted and the miR-30C overexpression group had a lower weight compared with the mock group. (E) MiR-30c overexpression resulted in inhibition of growth rate. (F) The expression of miR-30c was detected with qRT-PCR in tumors. (G-H) Hematoxylin and eosin staining of sections of lung and statistical results of metastasis nodes. ** <i>p</i><0.01.</p

Characteristics and miR-30c expression in colon cancer patients.

<p>* The correlation between miR-30c and CEA and CA199 is determined by Spearman’s correlation.</p><p>Characteristics and miR-30c expression in colon cancer patients.</p

MiR-30c inhibited cell proliferation in vitro.

<p>(A) Relative expression levels of miR-30c in five colon cancer cell lines were detected with the quantitative real-time PCR (qRT-PCR). Columns, average of three independent experiments; bars, S.E. (B) HCT116 or Lovo cells were transiently transfected with miR-30c mimic or NC mimic and inhibitor or NC inhibitor respectively. The expression of miR-30c was determined by qRT-PCR after 24h. (C-D) Effects of miR-30c on the proliferation were examined by MTT assay. Points are the average of three independent experiments; bars represent S.E.; *<i>p</i><0.05; ** <i>p</i><0.01. (E) Effects of miR-30c on the proliferation of HCT116 cells were examined by colony formation assay. (F) The number of clones was quantitatively analyzed. *<i>p</i><0.05; **<i>p</i><0.01.</p

ADAM19 was a direct target of miR-30c.

<p>(A) The predicted targeting site with miR-30c of ADAM19 3’-UTR; MiR-30c targeting sequences of ADAM19 3’-UTR are evolutionarily conserved through eight species (Hsa: human; Ptr: chimpanzee; Mml: rhesus; Oga: bushbabay; Tbe: treeshrew; Mmu: mouse; Rno: rat; and Ocu: rabbit.). The targeting sites are highlighted in red. (B-C) qRT-PCR and western blot analysis was applied to detect mRNA and protein expression of ADAM19 in HCT116 cells after transfection of miR-30c mimic or NC mimic. (D) ADAM19 mRNA and miR-30c expression level was determined in five colon cancer cell lines by qRT-PCR. (E) ADAM19 mRNA and miR-30c levels were inversely correlated in colon cancer tissues as determined by qRT-PCR. β-actin and U6 were used as the endogenous controls, respectively. (F) HEK293T cells were co-transfected with wild-type or mutant reporters and the miR-30c mimic or negative control (NC mimic). After 48h, Luciferase/Renilla activity was measured. (G) ADAM19 was upregulated in colon cancer tissues compared with normal tissues, as indicated by Western blotting.</p

MiR-30c was down-regulated in colon cancer tissues.

<p>MiR-30c expression was examined by qRT-PCR in 60 pairs of human colon cancer tissues. 54 out of 60 tumor samples showed decreased expression of miR-30c compared with the adjacent normal mucosa tissues. MiR-30c expression was normalized to that of U6 in each sample.</p

Polyploidy Index and Its Implications for the Evolution of Polyploids

Polyploidy has contributed to the divergence and domestication of plants; however, estimation of the relative roles that different types of polyploidy have played during evolution has been difficult. Unbalanced and balanced gene removal was previously related to allopolyploidies and autopolyploidies, respectively. Here, to infer the types of polyploidies and evaluate their evolutionary effects, we devised a statistic, the Polyploidy-index or P-index, to characterize the degree of divergence between subgenomes of a polyploidy, to find whether there has been a balanced or unbalanced gene removal from the homoeologous regions. Based on a P-index threshold of 0.3 that distinguishes between known or previously inferred allo- or autopolyploidies, we found that 87.5% of 24 angiosperm paleo-polyploidies were likely produced by allopolyploidizations, responsible for establishment of major tribes such as Poaceae and Fabaceae, and large groups such as monocots and eudicots. These findings suggest that >99.7% of plant genomes likely derived directly from allopolyploidies, with autopolyploidies responsible for the establishment of only a few small genera, including Glycine, Malus, and Populus, each containing tens of species. Overall, these findings show that polyploids with high divergence between subgenomes (presumably allopolyploids) established the major plant groups, possibly through secondary contact between previously isolated populations and hybrid vigor associated with their re-joining