Increasing evidences suggest that circular RNAs (circRNAs) exert crucial functions in regulating gene expression. In this study, we perform RNA-seq and identify 6,154 distinct circRNAs from human bladder cancer and normal bladder tissues. We find that hundreds of circRNAs are significantly dysregulated in human bladder cancer tissues. We further show that circHIPK3, also named bladder cancer-related circular RNA-2 (BCRC-2), is significantly down-regulated in bladder cancer tissues and cell lines, and negatively correlates with bladder cancer grade, invasion as well as lymph node metastasis, respectively. Over-expression of circHIPK3 effectively inhibits migration, invasion, and angiogenesis of bladder cancer cells and suppresses bladder cancer growth and metastasis Mechanistic studies reveal that circHIPK3 contains two critical binding sites for the microRNA miR-558 and can abundantly sponge miR-558 to suppress the expression of heparanase (HPSE). Taken together, our findings provide evidence that circRNAs act as "microRNA sponges", and suggest a new therapeutic target for the treatment of bladder cancer.

The prevailing view that the evolution of cells in a tumor is driven by Darwinian selection has never been rigorously tested. Because selection greatly affects the level of intratumor genetic diversity, it is important to assess whether intratumor evolution follows the Darwinian or the non-Darwinian mode of evolution. To provide the statistical power, many regions in a single tumor need to be sampled and analyzed much more extensively than has been attempted in previous intratumor studies. Here, from a hepatocellular carcinoma (HCC) tumor, we evaluated multiregional samples from the tumor, using either whole-exome sequencing (WES) (n = 23 samples) or genotyping (n = 286) under both the infinite-site and infinite-allele models of population genetics. In addition to the many single-nucleotide variations (SNVs) present in all samples, there were 35 “polymorphic” SNVs among samples. High genetic diversity was evident as the 23 WES samples defined 20 unique cell clones. With all 286 samples genotyped, clonal diversity agreed well with the non-Darwinian model with no evidence of positive Darwinian selection. Under the non-Darwinian model, MALL (the number of coding region mutations in the entire tumor) was estimated to be greater than 100 million in this tumor. DNA sequences reveal local diversities in small patches of cells and validate the estimation. In contrast, the genetic diversity under a Darwinian model would generally be orders of magnitude smaller. Because the level of genetic diversity will have implications on therapeutic resistance, non-Darwinian evolution should be heeded in cancer treatments even for microscopic tumors.

The ADP-ribosylation factors (Arfs) are six proteins within the larger Arf family and Ras superfamily that regulate membrane traffic. Arfs all share numerous biochemical activities and have very similar specific activities. The use of dominant mutants and brefeldin A has been important to the discovery of the cellular functions of Arfs but lack specificity between Arf isoforms. We developed small interference RNA constructs capable of specific depletion of each of the cytoplasmic human Arfs to examine the specificity of Arfs in live cells. No single Arf was required for any step of membrane traffic examined in HeLa cells. However, every combination of the double knockdowns of Arf1, Arf3, Arf4, and Arf5 yielded a distinct pattern of defects in secretory and endocytic traffic, demonstrating clear specificity for Arfs at multiple steps. These results suggest that the cooperation of two Arfs at the same site may be a general feature of Arf signaling and provide candidates at several cellular locations that when paired with data on the localization of the many different Arf guanine nucleotide exchange factors, Arf GTPase activating proteins, and effectors will aid in the description of the mechanisms of specificity in this highly conserved and primordial family of regulatory GTPases.