The 14th Korea Scholars’ Conference for Youth

siRNA directed silencing of TFAM decreased

the number of cancer colonies in gastric cancer
cells

2020 년 08 월

더 허드슨 스쿨 (The Hudson School, USA)

백선우 (Sunwoo Baek)

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국문초록

주제어: TFAM, 위암, Colony Forming Assay, 웨스턴 블랏, siRNA

미토콘드리아 전사 인자 A (TFAM)는 뉴클레오이드에서 발현되어 미토콘드리아 DNA

(mtDNA) 복제 및 전사를 조절하는 독특한 단백질입니다. TFAM 은 미토콘드리아 생물
발생의 조절에 중요한 역할을 하는 것으로 알려져 있기 때문에 자궁 내막 암, 전립선 암 및
방광암에서 인간 암에 대한 연구가 많이 되어 있습니다. 그러나 위암에서 TFAM 의 기능적
역할은 많이 연구되지 않았습니다. 이 연구에서 우리는 환자의 샘플을 두 그룹 (높은 TFAM
발현과 낮은 TFAM 발현)으로 나누어 환자의 생존율을 분석하기 위해 631 개의 위 환자의
유전자 발현 데이터를 분석했습니다. 높은 TFAM 발현 그룹은 낮은 TFAM 발현 그룹보다
낮은 생존율을 보였습니다. 또한 이 연구를 통해 위암에서 TFAM 유전자의 기능적 역할을
찾기 위해 MKN45 와 SNU-NCC-19 의 두 가지 위암 세포주를 사용했습니다. TFAM 을
표적으로하는 siRNA 는 두 세포주에서 TFAM 의 발현 수준을 성공적으로 억제 시켰습니다.
또한, 두 세포주에서 정착-독립적 성장 (anchorage-independent growth)를 확인하기 위해
콜로니 형성 분석을 수행하였습니다. TFAM 의 발현이 siRNA 로 인해 억제되었을 때 두 위암
세포주에서 암 콜로니의 수가 감소됨을 확인하였습니다. 결론적으로 이러한 결과는 높은
TFAM 유전자 발현이 암전이를 유도하고 환자 생존율을 감소시킬 수 있는 위암 세포의 악성
형질 전환에 중요한 요소임을 나타냅니다.

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Abstract

Key Words: TFAM, Gastric Cancer, Colony Forming Assay, Western Blot, siRNA

Mitochondrial Transcription Factor A (TFAM) is the unique protein that regulates mitochondrial
DNA (mtDNA) replication and transcription in the nucleoid. Since TFAM is known to play a key
role in the regulation of mitochondrial biogenesis, its involvement in human cancer has been
studied in endometrial, prostate, and bladder cancer. However, the functional role of TFAM in
gastric cancer has been poorly studied. In this study we analyzed 631 gastric patient’s gene
expression data to analyze the patient’s survival rate by splitting the patient’s samples into two
groups (high TFAM expression and low TFAM expression). The high TFAM expression group
showed lower survival rate than low TFAM expression group. To find the functional role of TFAM
in gastric cancer, two gastric cancer cell lines were used in this experiment: MKN45 and SNU-
NCC-19. siRNA targeting TFAM was designed to deplete the expression level of TFAM on both
cell lines. Then, the colony forming assay was performed to check the anchorage-independent
growth on both cell lines. When TFAM was depleted, the number of cancer colonies were
decreased on both cell lines. Overall these results indicate that high TFAM expression level is an
important factor for malignant transformation in gastric cancer cells, which may induce metastasis
and decrease patient survival rate.

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Contents

1. Introduction

2. Method and Materials

3. Result and Discussion

4. Conclusion

5. Reference List

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siRNA directed silencing of TFAM decreased the

number of cancer colonies in gastric cancer cells

더 허드슨 스쿨 (The Hudson School, USA)

백선우 (Sunwoo Baek)

1. Introduction
Gastric cancer is one of the top causes of human cancer deaths (1). Patients with metastasis

often showed decrease in survival rate in gastric cancer patients (2). As surgery is the only curative

treatment strategy and conventional chemotherapy has many limitations with low efficacy and various

side effects, molecular therapies are developed to overcome these limitations (3). Understanding the

molecular basis of gastric cancer in metastasis can be crucial to develop effective molecular therapies

to improve the survival rate of gastric cancer patients.

Figure 1. TFAM is a nucleus encoding gene and functions in mitochondrial transcription and

replication

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TFAM is a nucleus encoding gene that mainly functions in mitochondria (4). When TFAM

protein enters mitochondria, it regulates mitochondrial transcription and replication(4). Through

transcription, 2 rRNAs, 13 mRNAs, and 9 tRNAs are transcribed to maintain the biogenesis of

mitochondria (5). TFAM also supports mitochondrial DNA replication and protects the DNA from

various toxic substances produced in mitochondria (6,7). Since TFAM is a mitochondrial protein that

supports ATP synthesis and mitochondrial biogenesis, I hypothesized that this protein may have an

important function in cancer cells.

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2. Method and Materials(영문: Times New Roman, 굵게, 14 포인트)

1. Overall patient survival analysis

631 gastric patients’ gene expression data were downloaded from GEO, EGA, and TCGA. The

database is analyzed by PostgreSQL server, which integrates gene expression and clinical data.

The prognostic value of a particular gene were analyzed by splitting the patient samples into two

groups by the quantile expression of the proposed biomarker

2. Gastric cancer cell culture

MKN45 and SNU-NCC-19 were maintained in RPMI-1640 medium (Gibco) supplemented with 10%

fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin and streptomycin (Gibco) in a 5%

CO2 atmosphere at 37 °C.

3. siRNA transfection

Negative control scrambled siRNA med GC (12935–300) and TFAM siRNA were purchased from

Thermo Fisher Scientific.

4. Western blot

Antibody targeting TFAM and actin (cell signaling) was used to detect protein expression level using

western blot

5. Soft agar colony forming assay

Anchorage-independent growth was analyzed with soft agar colony forming assay using agar.

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3. Result and discussion

Figure 2. Effect of TFAM expression level on patient survival rate with 631 gastric patient’s

data.

To determine if the expression levels of TFAM can be predictive for gastric cancer patient

survival, a set of 631 gastric patient samples were analyzed using the public patient database enabling

the validation of gastric cancer survival biomarker candidates (8). This database provides gene

expression data from microarray analysis with overall survival information to analyze the prognostic

value of a particular gene (9). The patient samples were split into two groups according to various

quantile expressions of TFAM ’ gene expression data was divided into two groups, patients of high

TFAM expression indicated in red line and those of low TFAM expression indicated in black line. As

a result, high TFAM expression group showed lower survival rate than that of low TFAM expression

group. According to the data, High expression level of TFAM may support gastric cancer progression.

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Figure 3. Western blot was performed to analyze the protein expression level of TFAM and actin

Since the patient survival analysis provides the evidence that high level of TFAM expression

may be associated with aggressive tumor behaviors, we performed in vitro assay to find the functional

role of TFAM in gastric cancer cell lines. Here, two gastric cancer cell lines were used which are

MKN45 and SNU-NCC-19. siRNA targeting TFAM was transfected on both cell lines with negative

control (transfected with control siRNA). By using the Western Blot, TFAM expression level was

successfully depleted by siRNA transfection. The TFAM expression level was normalized with actin

and TFAM expression level showed lower protein level. Since the transfection was successfully

optimized, colony forming assay was performed to find the novel effect of TFAM on gastric cancer

cell lines.

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Figure 4. Effect of TFAM depletion by siRNA on number of colonies formation in MKN45 cells

The colony formation assay is an in vitro cell survival assay based on the ability of a single

cell to proliferate into a colony(10). At least 50 cells are required to become a colony. The assay tests

every cells in the population for its capability for unlimited division(11). Although colony forming

assay was initially described for studying the effects of radiation on cells and have played an essential

role in radiobiology, this method is now widely used to examine the effect of agents or gene with

potential application in the clinic. Therefore, colony forming assay was performed in this experiment

to analyze the effect of the role of TFAM on gastric cancer cell lines. When the MKN45 cell lines

were transfected with siRNA targeting TFAM, the number of colonies were significantly decreased.

Therefore, this result shows that TFAM expression level may support the gastric cancer cells to

survive and proliferate to produce colonies.

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Figure 5. Effect of TFAM depletion by siRNA on number of colonies formation in SNU-NCC-19

cell line

To further test the effect of TFAM on gastric cancer, other cell line SNU-NCC-19 was used

to confirm the result of the colony formation ability in figure 4. In this experiment, when siRNA

targeting TFAM was transfected, the number of colonies were also decreased in SNU-NCC-19 cells.

These results indicates that TFAM support the gastric cancer cells to survive in hash conditions, where

the cells cannot attached to the surface. Since TFAM depletion efficiently decreased the number of

colonies, targeting TFAM may provide a novel method to treat gastric cancer.

Depletion of TFAM results in mitochondrial dysfunction through the loss of mtDNA and the

mitochondrial membrane potential. Therefore, depletion of TFAM expression by siRNA may mediate

the mitochondrial dysfunction to inhibit the ATP synthesis, which is required for cancer growth. Also,

when TFAM is depleted, mtDNA was lost in many different types of cancer cells. Since mtDNA

encodes oxidative phosphorylation complex genes, TFAM depletion may block the ATP synthesis by

deactivating the oxidative phosphorylation process.

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4. Conclusion

The patient expression data suggests that expression level of TFAM can be used as a

biomarker for patient survival. Through this study we found that the high expression level of TFAM is

correlated with the low survival rate in gastric cancer. When TFAM expression level was

downregulated through siRNA transfection, number of colonies were decreased in two different cell

lines: MKN45 and SNU-NCC-19. In conclusion, TFAM gene expression is an important factor for

malignant transformation in cancer cells, which may induce metastasis and decrease patient survival

rate. However, future studies are needed to confirm this result and to define the detailed molecular

mechanism on TFAM dependent malignant transformation in gastric cancer.

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5. Reference List

1. Sitarz R, Skierucha M, Mielko J, Offerhaus GJA, Maciejewski R, Polkowski WP. Gastric

cancer: epidemiology, prevention, classification, and treatment. Cancer Manag Res. 2018 Feb

7;10:239–248.

2. Gomi D, Fukushima T, Kobayashi T, Sekiguchi N, Sakamoto A, Mamiya K, et al. Gastric

cancer initially presenting as bone metastasis: Two case reports and a literature review. Oncol

Lett. 2018 Nov;16(5):5863–5867.

3. Orditura M, Galizia G, Sforza V, Gambardella V, Fabozzi A, Laterza MM, et al. Treatment of

gastric cancer. World J Gastroenterol. 2014 Feb 21;20(7):1635–1649.

4. Kang D, Kim SH, Hamasaki N. Mitochondrial transcription factor A (TFAM): roles in

maintenance of mtDNA and cellular functions. Mitochondrion. 2007 Apr;7(1-2):39–44.

5. Gunnarsdóttir ED, Li M, Bauchet M, Finstermeier K, Stoneking M. High-throughput

sequencing of complete human mtDNA genomes from the Philippines. Genome Res. 2011

Jan;21(1):1–11.

6. Pohjoismäki JLO, Wanrooij S, Hyvärinen AK, Goffart S, Holt IJ, Spelbrink JN, et al.

Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the

mode of mitochondrial DNA replication in cultured human cells. Nucleic Acids Res. 2006 Oct

24;34(20):5815–5828.

7. Araujo LF, Siena ADD, Plaça JR, Brotto DB, Barros II, Muys BR, et al. Mitochondrial

transcription factor A (TFAM) shapes metabolic and invasion gene signatures in melanoma. Sci

Rep. 2018 Sep 21;8(1):14190.

8. Goel MK, Khanna P, Kishore J. Understanding survival analysis: Kaplan-Meier estimate. Int J

Ayurveda Res. 2010 Oct;1(4):274–278.

9. Dudley WN, Wickham R, Coombs N. An Introduction to Survival Statistics: Kaplan-Meier

Analysis. J Adv Pract Oncol. 2016 Feb;7(1):91–100.

10. Borowicz S, Van Scoyk M, Avasarala S, Karuppusamy Rathinam MK, Tauler J, Bikkavilli RK,

et al. The soft agar colony formation assay. J Vis Exp. 2014 Oct 27;(92):e51998.

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11. Franken NAP, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in

vitro. Nat Protoc. 2006;1(5):2315–2319.

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