Proposal Title: Regulation of liver cancer cell's EMT status by

mortalin via micro-RNA

Background:
Previous studies showed mortalin expression in HCC was closly
associated with advanced tumor stage, metastasis and venous
infiltrations(1, 2).
The miR-200 family, contains microRNAs that are known to have
negative implication on epithelial-to-mesenchymal transition (EMT)
in cancer cells, are down-regulated in HCC. By in silico method, the
3'UTR of mortalin was found containing miR-200b/ miR-200c/ miR429 conserved targeting seqeuences.
Recent studies showed that mortalin interact with p53 and
sequester p53 to the cytoplasm to diminish its tumor suppressive
effect(3, 4). It's interesting that p53 regulate microRNA (e.g. miR-34
family) and repress EMT process. Thus, it would be interesting to
investigate whether mortalin play part of the EMT process via
controlling the p53 dependent miRNA expression profile(5, 6). Last
but not least, targeted therapy against mortalin may repress
malignant phenotype in HCC cells(7).
Hypothesis:
- Downregulation of miR-200 subfamily leaded to mortalin
overexpression in HCC.
- Overexpression of mortalin sequester p53 to cytoplasm which
leaded to miR-34 and miR-142-3p expression that are known to
repress the EMT markers.

Objective (1): To verify mortalin (HSPA9) as a direct target of miR-

200 family
To identify miRNAs that target mortalin, TargetScan 7.0 and
miRnada were used to predict miRNA targeting mortalin 3'UTR in
silico. As showed in Fig. 1, miR-200b/ -200c/ -429 targeting
conserved sequence were found in HSPA9 gene 3'-UTR.
To verify the results from in silico prediction, miR-200b/ -200c/
-429 (miR-200 subfamily) genes will be amplified MIHA cells'
genomic DNA and cloned into miRNA expression vector for stable
cell line establishment. Wildtype and mutated miR-200 subfamily
targeting site (100-106 nt) in HSPA9 3'UTR will be subcloned into the
dual-luciferase miRNA target expression vector. The endogenous
protein level of Mortalin will be checked using western blotting on
miR-subfamily expressing cell line. The stable cell line will then be
transfected by miRNA target expression vector, luciferase activities
will be compared between wildtype and mutated 3'UTR containing
vectors.
Objective (2): To examine the change in tumorigenic properties and
invasion ability
Since Stable liver cancer cell line expressing full length (FL),
Truncated-1 (T-1-180) mortalin will be created in the first hand. To
characterize the phenotypic effect caused by mortalin, MTT, anchorindependent colony formation, wound healing and transwell cell
migration assay would be performed using stable FL-mortalin or
parental cell line transfected with miR-200 subfamily gene . To
obtain a clearer comparsion, stable FL-mortalin or parental cell line
treated with siRNA against endogenous mortalin will be used for the
same sets of experiments.
Objective (3): To investigate the microRNAs responsible for mortalinp53 regulated EMT
To investigate the microRNAs regulated through mortalin-p53
axis, liver cancer cell (stable FL-mortalin, stable T-1-180, stable T180-300 and parental) treated with Mortalin siRNA will be subjected
to immunofluorscent, western blotting and RT-qPCR. Since p53 was
reported to bind mortalin via it's c-terminal tail and localized to
cytoplasm, immunofluorscence staining reveal whether p53 is
released upon mortalin depletion. Subsequently, RT-qPCR will be
employed to check for the expression of miR-142-3p (5) and miR-34
family to see if their expression are induced by p53 nuclear
translocation. Total protein extracted from each condition will also
be blotted against SNAIL1, SNAIL2, Vimentin, E-Cadherin and Ncadherin as the markers of epithelial or mesenchymal cell.

Objective (4) : Correlation between miR-200 subfamily/ miR-142-3p/

miR-34 family expression and the protein expression of mortalin/
p53 in clinical samples.
Total RNA were extracted from frozen tissue are used for RTqPCR to quantify the amount of miRNA investigated in this study,
the p53 and mortalin stained using IHC method, correlation will be
made between these two set of data and other clinicopathological
features.

Figure 1 - in silico prediction results for miRNA regulating mortalin

(HSPA9) using miRnada.

Reference
1. Yi X, Luk Jm Fau - Lee NP, Lee Np Fau - Peng J, Peng J Fau - Leng
X, Leng X Fau - Guan X-Y, Guan Xy Fau - Lau GK, et al. Association of
mortalin (HSPA9) with liver cancer metastasis and prediction for
early tumor recurrence.
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J-L, Ma Jl Fau - Huang M, Huang M Fau - Deng Y-R, et al.
Overexpression of Mortalin in hepatocellular carcinoma and its
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Mortalin-p53 interaction in cancer cells is stress dependent and
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feedback loop to regulate epithelial-mesenchymal transitions.
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