Problem was the eighth-most common cancer globally with 456,000

Problem to be addressed:

I am an
undergraduate student studying Microbiology Honours ,which attributes to my
ardent interest in studies related to cancer. I would like to address the
problem of chemoresistance of oesophageal cancer cells which causes treatment
of this cancer difficult and makes it one of the most fatal malignancies in the
world. (50)

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Background to the problem:

Oesophageal
cancer(OC) is the cancer growing from the food pipe that runs through throat
and stomach. As of 2012, oesophageal cancer was
the eighth-most common cancer globally with 456,000 new cases during the
year.  Rates vary widely among countries, with about half of all cases
occurring in China. It is around three times more common in men than in
women. It is the fourth most common cause of death due to cancer in India.
Approximately, 47,000 new cases are reported each year and the reported deaths
reach up to 42,000 each year in India. Outcomes are related to the extent
of the disease and other medical conditions, but generally tend to be fairly poor, as diagnosis is often late.
Chemoresistance restricts therapeutic outcome of oesophageal cancer. It
has been reported that a Long noncoding RNA
(lncRNA) gene, PCAT-1, showed higher
expression in OC tissues. Overexpression of PCAT-1 increased the proliferation
rate and growth of OC cells. Inhibition of PCAT-1 decreased proliferation and
growth of OC cells, and increased cisplatin chemosensitivity. PCAT-1–mediated cell proliferation is
dependent on cMyc overexpression. Prevention of c-Myc prtein can help in
inhibiting cell proliferation due to PCAT-1.
Downregulation of PCAT-1 gene and gene inhibition of c-myc gene may prove to be
helpful in control of oesophageal cancer and thus become a part of the
therapeutic strategy required to treat oesophageal cancer.(225)

Experiment to address the
problem:

Oesophageal cancer
cell lines may be purchased and maintained in standard media. The expression of
lncRNA PCAT-1 can be measured by Real-Time PCR. The cell lines showing high expression
of PCAT-1 is  selected. A cancer specific
Cell Penetrating Peptide(CPP) is chosen(for example BR2) to specifically
transport siRNA to the oesophageal cancer cell lines. Anti-c-myc ribozyme gene is
designed and cloned. A retrovirus vector that expresses the ribozyme is  used to transfect the previously made stable cells.
The stable transfected cells are selected with the help of Dual Luciferase
reporter assay. Cell proliferation assay must be carried out then with the help
of trypan blue staining. The stained cell are counted under microscopy at
24h,48h and 72h after transfection. Total RNA is extracted and then whole
genome gene expression of the sample is  measured( OneArray Plus chips may be used).Western
Blotting technique can be used to analyse the protein formed with the help of a
chemiluminescent agent or protein band densitometry .The tumor suppressor role
of the c-myc specific ribozyme and siRNA can be evaluated in vivo using mouse
oesophageal cancer cell xenografts. Cancer specific CPP is used to transfer
siRNA into the cell and then the cell is  transfected using the expression vector
plasmid, containg anti-c-myc ribozyme gene, that is constructed .Tumor volumes
must be measured everyday and compared to a standardized data. The difference
in data among the cell lines used for the experiment can be calculated using
one way analysis of variance. If experiment is carried out using 2 cell
lines,Student’s t test may also be used.(259)

Expected outcomes and alternative strategy

It is expected that
targeting of lncRNA PCAT-1 using siRNA with the help of a cancer specific CPP will
lead to association of siRNA with RNA-induced silencing complex which will bind
to PCAT-1 and cause its degradation. The c-myc specific ribozyme formed in the
transfected cell will inhibit c-myc gene by cleaving and degrading the gene.
Both the actions may inhibit oesophageal cancer cell proliferation and allow
the uptake of chemotherapeutic agents by the cells. If the experiment fails
then other methods like drug delivery by nanoparticles, gene silencing by RNA
interference may be used to fight OC cancer.(99)