Structured Reports for the Pathological Society Awards: Career
Development Fellowships (CDF), Path Soc / J Shanks Fellowship, PhD
Studentships & Small Grant Awards.
Reports from recipients of Career Development Fellowships (CDF), Path Soc / J
Shanks Fellowship, PhD studentships & Small Grants should be set out using the
following subheadings and should consist of:
Annual Reports: 1 A4 page of text
Final reports: 2 A4 pages of text
Title: ‘The role of long non-coding RNAs in the cellular response to DNA
damage’.
(Small grant award)
Name & Address: Dr Francois Kwong, MRC Cancer Cell Unit, Hills Road,
Cambridge CB2 0XZ
Background:
The DNA in each cell of the human body is under constant attack by endogenous
and environmental agents, with the cellular responses collectively known as DNAdamage response (DDR). An improved understanding of the control of DDR proteins
is necessary to understand diseases with defects in DDR, such as cancer. Lately,
there has been a rash of publications on long non coding RNAs (lncRNAs), arbitrarily
defined as non coding transcripts over 200 nucleotides long. They can bind and alter
the activity of transcription factors, act as scaffolds for chromatin-modifying
complexes and mediate long range chromatin interactions. Very little is known of the
role of lncRNAs in DDR.
The role of microRNAs in DNA damage response is better understood. MicroRNAs
are relatively more stable than expected, suggesting that there are additional
regulatory mechanisms to regulate their activity in DNA damage response. LncRNAs
are possible candidates to inactivate or sequester microRNAs involved in DNA
damage response, in a specific manner. We therefore speculate that a lncRNA could
be a “micro RNA sponge” for miR-34 following DNA damage.
Original Aims (copied from original application):
1. Demonstrate the induction of TUG1 in response to DNA damage
2. The time course in expression of miR- 34 and TUG1 will be performed.
3. Demonstrate the sub cellular localization of TUG1 (nucleus v/s cytoplasm)
following DNA damage.
4. Demonstrate physical interaction between TUG1 and miR-34 family.
Results:
I spent much time initially on proving the induction of multiple lnc RNAs following
DNA damage. The lncRNA response in DNA damage was dependent on the type of
DNA damaging agents I used, ie ionising radiation or chemotherapeutic drugs such as
doxorubicin and on the type of cell lines.
TUG 1
Linc RNA p21
Panda
Gas 5
HCT 116
+
No change
+
+
Cal 51
No change
+
No change
No change
Hek 293
No change
No change
+
Table: LincRNA induction in different cell lines.
Previous studies suggested that the lncRNAs Tug1, lincRNA p21 and Panda were
induced following DNA damage, an effect which was p53 dependent. I have now
identified the cell lines in which particular lncRNAs are consistently induced
following DNA damage (see above table). Gas 5 is is expressed in cells deprived of
nutrients, but is not known to be induced in DDR. There is an 8 fold induction of
Gas5 following ionising radiation, an effect which is p53 dependent.
However, the induction of these lncRNAs have a similar kinetics to mir 34 a
induction in DDR. Mir 34 a level does not decrease with increasing lncRNA levels.
Therefore, the hypothesis that lncRNAs act as a sponge for mir 34a was rejected.
In addition to DNA damage, I have become interested in the role of lncRNAs in the
pathogenesis of lung adenocarcinoma. I have acquired ethical approval to obtain
fresh frozen lung tissue from 6 patients who have undergone a lung adenocarcinoma
resection at Papworth hospital. Total RNA has been extracted from the tissue and
next generation sequencing performed to determine the differential expression of long
non coding RNAs between normal and cancer tissue. The RNA-Seq have identified
12 potential lncRNAs which may be differentially expressed and I am now validating
these results with qPCR.
Figure: Gel electrophoresis of total RNA extracted from fresh frozen lung tissue from
patients with lung adenocarcinoma
Following confirmation of the differential expression of lncRNAs, I plan to identify
their functions.
Conclusions:
In DNA damage response, mir34a level does not appear to be reduced by an
increase in the lncRNAs tug1, panda, linc RNA p21 and gas 5.
How Closely Have the Original Aims been Met:
Original aim 1 and 2 were met, but not the following aims as the hypothesis was
rejected.
Subsequent effort was directed in identifying lncRNAs involved in lung
carcinogenesis.
Outputs from the study in which the Pathological Society has been
acknowledged:
None up to now. But, it is hoped that a publication will result from ongoing work.