Question:

Do miR132 loading MSCs-Derived Exosome Mimetics under hypoxia

involve in breast cancer EZH2 signaling and brain tropic metastasis?
Object:
Breast cancer most 80% are invasive and about 10–30% of patients
develop brain metastases during the course of their disease (). Metastatic
phenotypes of tumor cells have been the subjects of investigation that is
now understood tumour cells do not act alone,cancer cells interact with
their surrounding stroma whiched called”TME”(tumor microenviroment)
resulting Cross-talk consists of ECM, including fibrillar collagens,
mesenchymal stem cells (MSCs) and immune system cell may giving
theraputic effect.Uniquely,MSCs are recruited to the primary tumor site
from the bone marrow in response partly through their secretome
Recently study depict Polycomb histone methyltransferase enhancer of
zeste homolog 2 (EZH2) l
Hypothesis:
mesenchymal stem cell do possess some unique features ,ecruit to tumor
site ; anti-inflammatory and immunosuppressive properties) and mediates
distant metastasis . These abilities of MSCs track to tumor cell have
significant clinical potential ,may be employed for tracking or targeting
metastasis and tumors that are inaccessible for resection.Based on
previous research results,miR-132 was usually down-regulated in lung
cancer(),ovarian cancer() and breast cancer tissues compared with para-
tumor breast tissues.Further results revealed that miR- 132 was able to
repress EZH2 in breast cancer cells,which a Polycomb group
protein(PcG), as a critical oncogene specifically upregulated in hormone
receptor negative carcinomas and their metastasis when compared to
normal breast tissues . EZH2 expression is significantly associated with
poor clinical outcome .miR-132 is epigenetically downregulated EZH2
by targeting SOX4 H3K27me3 inhibits migration and invasion in lung
cancer cell .(Yang Li,et al ,2015)act as tumor suppressorsEZH2, a marker
of aggressive breast cancer that is involved in tumor progression
1.To determine the molecular mechanism of EZH2-silenced metastasis
suppressor miR132 in tumor metastasis.

2.To determine the role of EZH2/miR132 signaling under hypoxia in

tumor stroma MSCs interaction.
3.To determine the clinical significance of EZH2 inactivated metastasis
surpressor miRNA.
Approach:
Experimental Design
The first round of experimentation will study to observe the effects of
solely on hMSCs -3A6 cell,include bulding stable hypoxia
condition :24h/48h/72h in 1%O2,37℃ hypoxic incubator and control in
normoxia,then assay HIF-1α
EZH2 expression by western blot and q-PCR .(now in this step).
And knockdown EZH2 in 3A6 cell then assay miR132 expression,cell
proliferation and migration ,make sure that EZH2 is target for miR132
binding and surpress may also affect in MSCs proliferative ability.
Having defined miR132/EZH2 expression under nomoxia/hypoxia, will
now move on to experiment with MDA-MB-231, which an invasive and
poorly differentiated triple-negative breast cancer (TNBC) cell line.3A6
cell in co-culture with MDA-MB-231 by transwell under
nomoxia/hypoxia condition then collect the medium in upon chamber and
assay miR132/EZH2 expression by western blot or q-PCR .To investigate
the interaction while 3A6 contact with BCCs.
Having demonstrated the interaction in hMSCs and BCCs ,will
addresses ways i.Cell cultureor
Human mesenchymal/stromal cell lines (hMSCs) were used in this work,
the 3a6 hMSC ,Cultured in DMEM low-glucose nutrients + 10%
serum.
ii.Co-culture
MDA-MB-231 breast cancer cell co-culture with 3A6 hMSC in
transwell.
iii. Data Analysis
Discussion:
The signals may either result in increased growth and metastasis or lead
to inhibition of growth with increased cell death. It is quite confusing to
the definition of MSCs regarding it play a dual role in cancer progression