The HSP70 family is the most highly conserved of the high molecular weight HSP's in both non-neuronal and neuronal systems. Recent studies have also shown that the overexpression of HSP70 is neuroprotective for stroke and the polyglutamine (PolyQ) disorders. Hence understanding the mechanisms that regulate HSP70 may allow new therapeutic strategies to be developed. MicroRNAs are a subset of small non-coding RNAs that have been shown to mediate an entirely new level of post-transcriptional gene regulation by inhibiting the translation of target mRNAs. MiRNAs bind complementary sequences within the 3 'UTR of specific mRNAs and mediate their translational repression. An aim of this study was to identify the miRNAs that regulate HSP70 and investigate their involvement in regulating HSP70 during a heat shock response. To achieve this I initially used the online algorithms designed to predict miRNA targets. The predictions made by the different algorithms showed considerable variation and we hence also carried out an empirical analysis. MiR-206 and miR-21 were found to down regulate luciferase expression when linked to the HSP70 3 'UTR. In order to study the role these two miRNAs play in controlling HSP70 expression further, a lentivirus expressing miR-206 and miR-21 was used. Transduction of HeLa cells with this virus and an HSP70 3'UTR luciferase reporter found the virus mediated a dose dependent decrease in translational activity. HeLa and C2C12 cells were used to study function and preliminary experiments showed that HSP70 mRNA expression was increased 1-3 hours after the induction of a heat shock (HS). HSP70 protein levels were also increased following a HS. Following heat shock miR-206 levels were found to fall significantly within the first hour after heat shock (in C2C12 cells) and then start to recover twenty-fours later. The fall in miR-206 levels correlated with increased HSP70 levels, though this trend did not reach statistical significance. Transduction with viruses expressing miR-206 and miR-21 were shown to significantly decrease HSP70 protein but not mRNA expression following a HS. Furthermore, miR-206 specific antagornirs mediated a decrease in endogenous miR-206 levels and a trend towards increased basal levels of HSP70 protein was also seen. These results suggest that miR-206 may be one of a number of miRNAs that help ensure conditions within a cell are permissive or non-permissive for the expression of heat shock proteins following a stress or consitutively.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:550325 |
| Date | January 2011 |
| Creators | Xu, Shenjun |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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