Abstract
Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.
Highlights
Cell Activities Affected by miR-328 To investigate the role of a microRNA in morphogenesis, it is essential to maintain its function for a period of time
We have generated a construct that can express the pre-microRNA of miR328 and green fluorescence protein (GFP), with an antibiotic selection marker (Figure 1A, all primers used in this study are listed as Supplementary Table S1)
Capillary formation affected by miR-328 targeting CD44 To have a better insight of cell behaviour while CD44 expression was repressed by miR-328, Matrigel cultures of the mixture containing both the miR-328- and the GFP-transfected cells were subjected to confocal microscopic examination
Summary
Once ignored completely or overlooked as cellular detritus, small RNA fragments (microRNA or miRNA) that were discovered over a decade ago recently took many by surprise because of their widespread expression and functions [1,2,3]. miRNAs are single-stranded RNA of 18-24 nucleotides, which are generated by sequential processing of long RNA transcripts by two key RNase III proteins, Drosha and Dicer [4,5,6,7,8]. miRNA functions as a guide molecule in post-transcriptional gene silencing by partially pairing with the 39-untranslated region (UTR) of target mRNAs, leading to translational repression [9]. miRNAs have key roles in diverse regulatory pathways, including control of development [10,11,12,13], cell proliferation [14,15,16], differentiation [17,18,19,20,21], apoptosis [22,23], cell cycle progression [2,24,25], immuno-response [21,26,27], protein secretion [28,29], viral infection [30,31,32,33,34,35], tumorigenesis [36,37,38,39,40] and many other physiological or pathological processes[21,41,42], Recent studies indicated that microRNAs are important for tissue morphogenesis [43,44,45,46]. A431 cells transfected with miR-328 (pooled cell line) or a control vector expressing GFP alone were cultured on tissue culture plates to confluence. It showed that addition of hyaluronan promoted aggregation of the GFP-transfected cells, expression of miR-328 was able to reduce hyaluronan’s effect on cell aggregation (Figure S5A).
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