MicroRNA-148a (miR-148a) has been reported to be deregulated in different tumor types, whereas the biological function of miR-148a in renal cell carcinoma (RCC) largely remains unexplored. In the present study we investigated the clinical significance, biological effects, and the underlying molecular mechanisms of miR-148 in RCC. Here, we showed that miR-148a was significantly downregulated in RCC tissues and cell lines. Low expression of miR-148a in RCC tissues was associated with large tumor size, advanced TNM stage, and lymph node metastasis. Functional assays revealed that overexpression of miR-148a significantly inhibited RCC cell proliferation, colony formation, migration and invasion in vitro and suppressed RCC xenograft tumor growth in vivo. In addition, using quantitative RT-PCR (qRT-PCR), western blot analysis and luciferase reporter assays, AKT2 was confirmed to be a direct target of miR-148a. AKT2 expression was upregulated, and was negatively correlated with miR-148a expression in RCC tissues (r=-0.641, P<0.001). Silencing of AKT2 phenotypically copied miR-148a-induced phenotypes, whereas re-expression of AKT2 reversed the suppressive effects of miR-148a in RCC cells. Further mechanistic investigations showed that miR-148a exerted its antitumor activity via inhibition of the AKT pathway in vitro and in vivo. Taken together, these findings suggest that miR-148a functions as tumor suppressor in RCC by targeting AKT2.
To identify the molecular effects of Tobacco bushy top virus (TBTV) evolution on the degeneration of tobacco bushy top disease, three TBTV isolates with mild virulence were compared with wild-type TBTV to assess the translation of p35, which relies on a BYDV-like translation element (BTE) in a cap-independent manner. The in vitro expression of p35 in the mild isolates was only 20% to 40% of the expression observed in wt TBTV. Based on translation data from chimeric TBTV RNA, low-level p35 expression in the three mild isolates was associated with two regions: the 5′ terminal 500 nt region (RI) and the 3′ internal region (RV), which included the BTE. For the RV region, low level p35 expression was mainly associated with structural alterations of the BTE instead of specific sequence mutations within the BTE based on SHAPE structural probing and mutation analysis. Additionally, structural alteration of the TBTV BTE resulted from mutations outside of the BTE, implying structural complexity of the local region surrounding the BTE. This study is the first report on the structural alteration of the 3′ cap-independent translation element among different isolates of a given RNA virus, which is associated with variations in viral virulence.
BackgroundDuring the past decade, tobacco bushy top disease, which is mainly caused by a combination of Tobacco bushy top virus (TBTV) and Tobacco vein-distorting virus (TVDV), underwent a sudden appearance, extreme virulence and degeneration of the epidemic in the Yunnan province of China. In addition to integrative control of its aphid vector, it is of interest to examine diversity and evolution among different TBTV isolates.Methods5’ and 3’ RACE, combined with one step full-length RT-PCR, were used to clone the full-length genome of three new isolates of TBTV that exhibited mild pathogenicity in Chinese fields. Nucleotide and amino acid sequences for the TBTV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates.ResultsThe genomes of three isolates, termed TBTV-JC, TBTV-MD-I and TBTV-MD-II, were 4152 nt in length and included one distinctive difference from previously reported TBTV isolates: the first nucleotide of the genome was a guanylate instead of an adenylate. Diversity and phylogenetic analyses among these three new TBTV isolates and five other available isolates suggest that ORFs and 3’UTRs of TBTV may have evolved separately. Moreover, the RdRp-coding region was the most variable. Recombination analysis detected a total of 29 recombination events in the 8 TBTV isolates, in which 24 events are highly likely and 5 events have low-level likelihood based on their correlation with the phylogenetic trees. The three new TBTV isolates have individual recombination patterns with subtle divergences in parents and locations.ConclusionsThe genome sizes of TBTV isolates were constant while different ORF-coding regions and 3’UTRs may have evolved separately. The RdRp-coding region was the most variable. Frequent recombination occurred among TBTV isolates. Three new TBTV isolates have individual recombination patterns and may have different progenitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0340-2) contains supplementary material, which is available to authorized users.
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