RPS15 Expression between Tumour Cells of Human Colorectal ... analysis...Comparative Analysis of...

Comparative Analysis of RPS15 Expression between Tumour Cells of Human Colorectal System

Teh ZyYing

(44409)

Bachelor of Science with Honours (Resource Biotechnology)

2016

usat Khidmat 1lakJuma Akademik VNlVERSm MALAYSIA SARAWAJ(

Comparative Analysis of RPS15 Expression between Tumour CeUs of Human Colorectal System

Teh Zy Ying (44409)

A Thesis Submitted in Partial Fulfillment of the Requirement of

The Degree of Bachelor of Science with Honors (Resource Biotechnology)

Supervisor Assoc Prof Dr Edmund Sim Ui Hang

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2016

ACKNOWLEDGEMENT

First of all I would like to thank God for giving me an opportunity to complete this project

Thanks for accompanying me from beginning until the end ofthis project When facing any

problems you are the only one who accompany me to solve it until the end

I would like to acknowledge my research supervisor Associate Professional Dr

Edmund Sim Ui Hang who assisted me to finish my research project successfully

Throughout duration of doing my project he has been guiding and supervising me as well

as sharing his knowledge and advising me to accomplish my final year project I really like

to express my greatest appreciation to him for providing continuous mental and financial

support to accomplish this research Without his assistance throughout that time I feel that

I will be lost and wont be able to finish my project on time

My highest appreciation is also directed to all the postgraduate students of

Immunology Molecular Genetics Laboratory UNIMAS particularly MsKherlee Ng Ms

Shruti Talwar Ms Felicia Kavitha Thomas Ms Cassandra Chee Sheau Mei and Ms Yew

Keh Li Although they have packed schedules they still guide me by discussing the project

with me and sharing their valuable knowledge in order for me to finish up my final year

project I am also hereto thank my lab mates which are Selvamalar alp Mutsamy Ong Lian

Ween and Nur Atiqah binti Azman for their gracious help throughout my project

All in all I am grateful to my beloved family especially my parents Mr Teh Bun

Gak and Mrs Teoh Ai Chu in addition to my siblings Mr Teh Guan Wei and Mr Teh Guan

Hong with their love patient support and encouragement throughout the period while doing

the project Special thanks to everyone who have helped and contributed towards the

completion of my final year project

UNIVERSITI MALAYSIA SARAWAK

Grade _____

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DECLARATION OF ORIGINAL WORK

h IS f d flUMEThIs declaratIOn IS made on t e ay 0 2016

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Fakulti Sains dun TdI() l o~j SUl1b~r UNIVE~LA I bull bull RAW K

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III

lusat Khidrnal l akI rna r l~utmjk UNlVERSm MALAYSIA SARAWAK

Table of Contents

Content Page

Acknowledgement I

Table of Contents IV- V

List of Figures VII - IX

Declaration II - III

List of Abbreviations VI

List of Tables VII

Abstract 1

10 Introduction 2-4

20 Literature Review 5

21 Gene expression 5

22 Cancer 6

221 Colorectal carcinoma 6-8

222 Significance of Colorectal Carcinoma 9-10

23 Ribosomal Protein 10 -11

231 RPS15 11 -l2

232 Ribosomal protein associated with disease 12

24 Cell line 13

241 SW480 13 -14

242 HCT116 14 - 15

25 Housekeeping genes 15

251 GAPDH 16

26 Reverse Transcriptase Polymerase Chain Reaction 16 - 17

IV

30 Materials and Methods 18

31 Cell Lines and Cell Cultures 18

32 Primer Designing 18 -20

33 RNA Extraction 21

34 RNA Quantification 22

35 RNA Quality Check with Agarose Gel Electrophoresis 22 -23

36 Reverse Transcription 24

37 Polymerase Chain Reaction 24-25

38 Agarose Gel Electrophoresis 25

39 Sequencing 26

310 Data analysis 26

40 Results 27



43 Polymerase Chain reaction 28

431 Optimization of Gradient PCR 28-29

44 Sequencing 30 - 31

45 Expression Profiling 32

46 Data Analysis 33 -34

50 Discussion 35 -39

60 Conclusion 40

70 References 41-45

80 Appendices 46-53

v

I

I

AGE

BLAST

cDNA

CRC

DBA

dH20

EtBr

GAPDH

M-MLVRT

mRNA

PBS

PCR

RNA

RPs

RPS15

rRNA

RT-PCR

tRNA

IJg

~

JlM

WHO

List of Abbreviations

Agarose Gel Electrophoresis

Basic Local Alignment Search Tool

Complementary Deoxyribonucleic Acid

Colorectal Carcinoma

Diamond-Blackfan Anaemia

Distilled Water

Ethidium Bromide

G 1 yceraldeh yde-3 -phosphate Dehydrogenase

Moloney Murine Leukemia Virus Reverse Transcriptase

Messenger RNA

Phosphate Buffered Saline

Polymerase Chain Reaction

Ribonucleic Acid

Ribosomal Proteins

Ribosomal Protein SmaIl 15

Ribosomal Ribonucleic Acid

Reverse Transcriptase Polymerase Reaction

Transfer Ribonucleic Acid

Microgram

Microliter

Micromolar

World Health Organization

VI

I

List of Tables

Table Page

31 List of Materials 18

32 List of Apparatus 18

33 Sources of cell lines 18

34 Infonnation on the primers selected 20

35 The Components ofPCR Mix 25

36 Thennal Cycle Condition 25

41 Quantification of total RNA extracted from UV-spectrometer 27

42 Optimized PCR thennal cycling condition 32

43 Infonnation of band intensity volumes 33

44 Nonnalized values of both cell lines 34

45 Statistical analysis of expression of RPS15 in both cell lines 34

VII

List of Figures

21 Location of Colorectal Carcinoma 7

22 Tumour Differentiation of CRC 8

23 Location ofRPS15 in Chromosome 19 12

24 Morphology ofSW480 14

25 Morphology ofHCT116 15

41 RNA quality check of cell lines 28

42 PCR amplified of GAPDH housekeeping gene to test the primer for 29 optimum annealing temperature

43 PCR amplified of RPS15 gene of interest to test the primer for optimum 29 annealing temperature

44 Authentication ofRPS15 sequences using forward primer sequencing by 30 blastn

45 Authentication ofRPS15 sequences using reverse primer sequencing by 30 blastn

46 Authentication of GAPDH sequences using forward primer sequencing by 31 blastn

47 Authentication of GAPDH sequences using reverse primer sequencing by 31 blastn

48 PCR amplification ofRPS15 and GAPDH in SW480 and HCT116 32

49 Average band intensity of RPS15 and GAPDHin SW480 and HCT116 33

A Features ofboth forward and reverse primers of RPS15 46

B The reverse primer of RPS15 span on exon-exon junction 46

C Oligocalc result on RPS15 forward primer 47

D Oligocalc result on RPS15 reverse primer 47

VIII

E OligoAnalyzer result on RPS15 forward primer 48

F OligoAnalyzer result on RPS15 reverse primer 48

G Features of both forward and reverse primers of GAPDH 49

H Oligocalc result on GAPDH forward primer 49

I Oligocalc result on GAPDH reverse primer 50

J OligoAnalyzer result on GAPDH forward primer 50

K OligoAnalyzer result on GADPH reverse primer 51

L Information oft-distribution tables 53

IX

1


Teh Zy Ying (44409)

Resource Biotechnology Department ofMolecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Ribosomal proteins (RPs) are components of ribosomes which shown different expression patterns in cancer cells The changes of RP expression pattern are associated with the development and progression of tumour cells There is lack of existing data comparing the expression levels of RPSI5 in cancer cell lines The main aim of our research is to detect and compare the expression patterns of RPSI5 in two cancer colon cell lines Gene expression of RPSI 5 was studied in CRC cell lines (SW480 and HCTlI6) with GADPH as a reference gene Optimization of reverse transcriptase polymerase chain reaction (RT -PCR) was utilized and amplicon produced was 115bp in length before identifying the expression patterns ofRPSI5 RT-PCR was used to study expr ion levels of RPSI5 and then run through agarose gel electrophoresis (AGE) to gain the result Nonnalization of the band intensity of RPSI5 was done against the reference gene Forward sequencing of RPSI5 showed 96 similarity while reverse sequencing indicated 100 match RPSI5 was significantly expressed in both cell lines Band intensity ofRPSI5 in HCTl16 is higher than that in SW480 Null hypothesis was rejected and significant differences were shown between SW480 and HCTl16 cell lines by using independent 2-tailed students t-test (p-value = 0015571 lt 005) According to results of sequencing RPSI5 the DNA differences may suggest DNA polymorphism of RPSI 5 DNA polymorphism may lead to differential expre ion ofRPSI5 Nonnal colon cell lines and biological replicates should be added into future expression studies of this gene

Key words ribosomal proteins expression patterns RPSI5 CRC

ABSTRAK

Protein ribosom (RP) adalah komponen ribosom yang menunjukkan corak ekspresi yang berbeza dalam selshysel kanser Perubahan corak ekspresi RP berkaitan dengan pembangunan dan perkembangan sel-sel tumor Terdapal kukurangan data sedia ada yang membandingkan tahap ekspresi RPSI5 dalam talian sel kanser Tujuan utama kajian ini adalah untuk mengesan dan membandingkan corak ekspresi RPSI5 dalam dua talian sel kanser usus besar Ekspresi gen RPSI5 telah dikaji dalam talian sel CRC (SW480 dan HCTJ I 6) dengan GAPDH sebagai gen rujukan Pengoptimuman Reverse transcriptase polymerase chain (RT-PCR) telah digunakan dan amplicon dihasilkan adalah 115bp panjang sebelum mengenal pasti corak ekspresi RPSI5 RT-PCR lelah digunakan untuk mengkaji tahap ekspresi RPSI5 dan kemudian dijalani melalui elektroforesis gel agarose (A GE) untuk mendapatkan keputusan Normalisasi intensiti band RPSI5 telah dilakukan terhadap gen rujukan Sequencing hadapan menunjukkan 96 persamaam manakala sequencing belakang menyatakan 100 persamaan RPSI5 telah didapati diekspres ketara dalam kedua-dua sel kanser Intensiti band RPSI5 dalam HCTJJ 6 adalah lebih tinggi daripada SW480 Hipotesis null telah dUolak dan perbezaan yang signifikan Lelah ditunjukkan antara sel SW480 dengan sel HCTJ 16 dengan menggunakan ujian-t bebas 2-ekor ((p-value = 0015571 lt 005) Menurut keputusan sequencing RPSI5 perbezaan DNA mungkin mencadangkan polymorphism DNA RPSI5 Polymorphism DNA boleh membawa kepada perbezaan corak ekspresi RPSJ5 Sel kolon normal dan ulangan biologi perlu dUambah ke dalam kalijian ekspresi masa depan Ice atas gen in

Kata Kunci protein ribosom corak ekspresi RPSI5 CRC

I

I

10 Introduction

Cancer is a disease that arises from abnormal cells that grow uncontrollably due to damage

DNA Cancers develop from the combined effects of genetic mutation and environmental

sources Colorectal cancer could cause a lump in either colon or rectum which is digestive

system in the body According to National Cancer Society Malaysia (2016) colorectal

cancer is the second most causes of cancer in Malaysia Each year around 2900 Malaysians

are suffering from this cancer and most of them are 50 years old and above (National Cancer

Society Malaysia 2016) The risk factors that increase the possibility to develop colorectal

cancer are based on family history genetics sex age lifestyle and race

Ribosome has two subunits RNA and protein Ribosomes generally function in

protein biogenesis Besides ribosome also contains extra-ribosomal proteins which is

independent of protein synthesis Recently ribosomes protein genes are widely studied in

disease or cancer research A substantial number of researchers had proven that alternation

of RP genes expression triggers genetic disease such as Tuner syndrome BardetBiedl

syndrome Diamond-Blackfan anaemia syndrome Camurati-Engelmann disease and

Noonan syndrome and once happens in tissue cells it leads to cell deaths particularly on

colon prostate breast cervix liver and nasopharyngeal (Lai amp Xu 2007) Any disturbances

in RPs either up-regulated or down-regulated in the cells can cause tumourigenesis or cancer

Besides the changes occuning in RP genes also affect the cellular pathway apoptosis and

metastasis of CRC

Lai and Xu (2007) mentioned that the changes of expression levels in ribosomal

proteins cause the development of CRe Previous investigation had showed that the RPs are

highly expressed in CRC such as RPSll and RPL7 (Shenoy et at 2012) Moreover RPs

such as S6 S8 S12 L5 and PO will influence CRC and adenomatous polyps by increasing

2

l I

the mRNAs levels (Lai amp Xu 2007) Thus the expression of RPs strongly affects the

development mechanism of CRC

RPS15 is found in 40S subunit of ribosome and situated in chromosome 19 The

biological processes of RPS15 include osteoblast differentiation rRNA processing

translation assembly and biogenesis in small ribosomal small unit as well as export from

the nucleus RPL37 RPS15 and RPS20 could attach to Mdm-2 and stimulate p53 that acts

as tumour suppressor (Daftuar et at 2013) Shenoy et at (2012) mentioned that RPS15 is

also involved in a disease named Diamond-Blackfan anemia (DBA)

According to Lai and Xu (2007) the alternation of ribosomal proteins such as RPS15

could lead to colorectal cancer however the information is very little The identification of

gene expression levels of many RPs are found to have close relationships with various

cancers However the comparison of the gene expression of RPS15 in tumour cell lines in

colon have not been studied to date and thus the corresponding data is not available yet Thus

the expression level ofRPS15 in CRC will be determined in this study This research could

bridge the knowledge boundaries on the relationship between RPS15 and CRC as well as

spike the interest of the potential ofRPS15 as biomarkers in CRC Hypothesis of this project

is whether the expression of RPS15 is significant different between SW480 and HCTl16

cell lines Hence the RPS15 gene of SW 480 and HCT 116 were chosen for research by first

utilizing with the TRIzol methods to extract RNA quantification RNA reverse transcriptase

polymerase chain reaction (RT -PCR) and followed by agarose gel electrophoresis and data

analysis

The purpose of this research is to compare the expression levels of RPS15 between

two different cell lines colon adenocarcinoma (SW480) and colorectal carcinoma (HCT 116)

3

The objectives of this study

1 To detect the expression of RPS15 in two distinct tumour cell lines of human

colorectal system

2 To compare the differential expression patterns ofRPS15 gene between two different

tumour cell lines ofhuman colorectal system

4

usat Khidmat MakJumat Akademik UNlVERSTTI MALAYSIA SARAWAK

20 Literature Review

21 Gene Expression

Gene expression is a process in which functional gene products are fonned by specific genes

and it also occurs throughout an organisms life (Morris 2008) Morris (2008) stated the

gene products are divided into two parts protein and non-protein coding genes for instance

ribosomal RNA (rRNA) gene or transfer RNA (tRNA) genes In general the gene is firstly

encoded with infonnation and later converted into messenger RNA (mRNA) and then

transfonned into a protein (Morris 2008) According to Morris (2008) the first phase of the

expression of the genes is transcription by progression of RNA polymerase such as RNA

polymerase I II and III to create mRNA Few factors could be used to control on the timing

of transcription which involves DNA accessibility regulation from other genes and the

signals send to genes from other cells by way of honnones (Robinson 2010) After the

mRNAs are produced the mRNAs would undergo the process oftranslation to fonn amino

acid (Robinson 2010) This is one of the critical step for gene expression There are two

ways to control gene expression in translation which include the machinery that carries out

translation for instance by increasing or decreasing the effectiveness of translation that

occurs during the interaction of initiator proteins with ribosomes and the mRNA which

carries a message that controls when and how it is translated (Robinson 2010) Besides

Robinson (201 0) indicated that the structure and function of products of translation the

amino acid chains could be affected by gene expression Majority ofRP are involved in gene

regulation and expression in their organisms include RPS15 (Bhavsar et al 2010)

5

1

22 Cancer

Cancer is the diseases that associated with out-of-controlled growth and abnormal cells in

the body as the spread of abnormal cells are getting serious it would cause fatal (American

Cancer Society 2011) Most of the cancer cells grow divide and re-divide to create new

abnonnal cells instead of dying some of them are passed through other parts via lymph

vessels or blood circulation There are different types of cancers are be diagnosed include

breast lung colon skin and prostate cancers According to Sudhakar (2009) Greek

physician Hippocrates was the one who established the word of cancer He used the term

karkinos to depict carcinoma tumours In the early the human bone cancer was discovered

in mummies in ancient manuscripts and ancient Egypt as well as the bone cancer was named

as osteosarcoma (Sudhakar 2009)

Moreover cancer can be treated but could not be cure Although cancer is be treated

cancer will be returned after a period time Cancer is hardly to be cured due to it involves

different mechanisms Different treatments are used to treat different cancers for instance a

person who attacks by kidney cancer could not use prostate treatment to treat it Thus the

search for the ultimate cure for cancer goes on and on while therapies for example

chemotherapy radiation therapy and surgery are being continuously developed

221 Colorectal carcinoma

CRC is colon cancer or rectum cancer that develops in the large intestine (large bowel) in

the colon or rectum as shown in Figure 21 Colon can be divided into four parts which are

sigmoid colon transverse colon descending colon and ascending colon Ascending and

transverse colon are referred as proximal colon whereas descending and sigmoid are

belonged as distal colon Tumour cells could be classified into benign that could not cause 6

1

direct danger while the other one is malignant that would cause death (Ferreira et al 2013)

However the benign tumour can change and tum into a dangerous tumour As for colorectal

cancer the benign tumours are adenomatous polyps (adenomas) and hyperplastic polyps and

inflammatory polyps but malignant tumour is actually colorectal carcinoma

ictndlng colon ()~ eending colon

Colon cancer

Cucum

ittmoid collin Rectum Rclum cancer

Figure 21 Location ofcolorectal carcinoma Adapted from Colon and rectal cancer by CancerTreatmentsorg 2015 Retrieved from httpllwwwcancertreatmentsorgcolon-and-rectal-cancer Copyright 2015 by CancerTreatmentsorg Reprinted with permission

In general CRC is known as malignant tumour conversely the benign tumours

appeared either in colon or rectum are named as colorectal adenomatous polyps (Unal amp

Ozturk 2015) According to Fleming et al (2012) most ofCRC commonly are identified as

adenocarcinoma which is derived from epithelial cells of colorectal mucosa however there

are other types of CRC including adenosquamous neuroendocrine spindle cell squamous

cell and undifferentiated carcinomas World Health Organization (WHO) reported that

adenocarcinoma could be subdivided into nine groups that are signet ring cell mucinous

micropapillary medullary cribriform comedo-type serrated undifferentiated spindle cell

and adenosquamous (Fleming et al 2012)

Figure 22 illustrates that the adenocarcinoma are classified into 4 categorises

undifferentiated carcinoma poorly differentiated adenocarcinoma moderately differentiated

7

adenocarcinoma and well differentiated adenocarcinoma (Fleming et al 2012) The grades

of tumours show how the cells are likely to grow or spread for well- and moderately

differentiated adenocarcinoma are low grades but poorly differentiated in addition

undifferentiated carcinomas are high grades (Hamilton 2014) High grades of tumours are

defined as tumours that would invade spread and grow faster surrounding the body

(Hamilton 2014) Colorectal cancer is also categorised into five stages which are stage 0 I

II III and IV These stages show how far the cancer spread in muscles lymph nodes and

colon layers TNM staging is commonly used for tumour node and metastasis which it is

also replaced the Dukes classification used previously

Figure 22 Tumour differentiation of eRe (A) Well differentiated adenocarcinoma (B) Moderately differentiated adenocarcinoma (C) Poorly differentiated adenocarcinoma and (D) Undifferentiated carcinoma Adapted from Pathology and Genetics of Tumours of the Digestive System (p 109) by S R Hamilton amp L A Aaltonen 2000 Lyon France IARe Press Copyright 2000 by the International Agency for Research on Cancer Reprinted with permission

8

222 Significance of Colorectal Carcinoma

In previous investigation incidence rates ofcolorectal cancer increased from 1975 thru midshy

1980 from 2008 to 2010 the incident rates started to decrease (Alteri et aI 2014)

According to Siegel et al (2014) over 136 830 human beings were diagnosed and around

50310 died in United Stated because of colorectal cancer in 2014 This cancer is the second

leading cause of death in US Besides around 60 cases showed that colorectal cancer

occurred among 65 years and older and 70 of them died of it There is a higher mortality

and incident rate of this cancer in men than women by approximately 30-40 Each year

approximately 2900 Malaysians are affected by colorectal cancer which is the second most

widespread cancer (National Cancer Society Malaysia 2015) Alteri et al (2014) have

indicated that tumour cells arise in proximal colon of women and elder however men and

younger patients are commonly affected in distal colon Majority individuals died of it

because the symptoms of colon cancer seldom appear in the early stage Consequently the

individuals only realise this cancer in late stage

Moreover some scientists found that disturbance of RPs in CRC generated

tumourigenesis or dysregulated in tumours (Gou et al 2010) Gou et al (2010) indicated

that RPs are co-related with tumours by affecting progression differentiation or metastasis

ofCRC There are some evidences showing that different expression patterns ofRPs could

cause CRC Increase in gene expression of S3 S6 S8 S12 L5 will result in CRC

development (Lai amp Xu 2007) The overexpression of RPs is mostly like to increase the

speed ofgrowth oftumours rather than cause tumourigenesis (Lai amp Xu 2007) Nevertheless

some RP genes such as L5 L6 L15 L29 L31 L39 are down-regulated in metastatic CRC

this indicates that the relationship between RPs and CRC are significant (Lai amp Xu 2007)

9

Alteration of tumour suppressor (pS3) or oncogene (pro-oncogene product - MYC) might

regulate cell proliferation and induce tumourigenesis in CRC (Shenoy et al 2012)

23 Ribosomal Protein

Ribosomes consist of ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) which are

also vital for protein synthesis in cellular organelles (Lai amp Xu 2007) The human ribosomal

proteins would undergo a fundamental process which the genetic information is translated

to create a new protein at ribosomes (Vladtmirov et al 1996) As mentioned by Bhavsar et

al (2010) protein is produced by the progression of transcription and translation which is

carried out by ribosomes tRNA and several protein factors Furthermore majority of

ribosomal proteins in prokaryotic and eukaryotic are involved in regulation of gene

expression at different levels (Bhavsar et al 2010)

Lai and Xu (2007) declared that RP genes are responsible for stabilizing the specific

rRNA structure at correct position during mature ribosomal protein and also promoting the

correct folding of rRNA during ribosomes assembly The disturbance of RP genes might

cause genetic disease disorder or cancer (Lai amp Xu 2007) In addition the structure of

ribosome in prokaryotes and eukaryotes are different however this research is emphasised

in eukaryotes for prokaryote it has only 70 ribosome while in eukaryotic it has a small 40S

and a 60S subunit that is 80 ribosome (Bhavsar et al 2010) Here Bhavsar et al (2010)

stated sma1l40S subunit of eukaryotes contains one 18S molecule of rRNA and around 33

proteins while 60S subunit contains 3 rRNA that are SS 28S and S8S and around SO protein

Annachea et al (2010) declared the ribosomes of eukaryotic contain additional RNA which

is named as expansion segments (ES) including r-protein extensions and many additional r-

proteins therefore the structure is becoming more complex and larger besides the additional

10

r-proteins might affect the complexity of translation regulation by increasing them in the

eukaryotic cells According to Doudna and Rath (2002) the function of small subunit of

protein is to ensure that the base pairings between codons and aminoacylated tRNAs are

accurate by binding and decoding mRNA Moreover RPs also play role independent of

ribosome biosynthesis known as extra ribosomal protein and their functions are DNA

replication transcription DNA repair RNA processing cell growth proliferation apoptosis

and development regulation and cellular transformation (Lai amp Xu 2007)

231 Ribosomal Protein Small 15 RPS15

Figure 23 showed the location of RPS15 of Homo sapiens (human) at chromosome 19 and

it is located at 19p133 (GeneCards 2015) RPS15 is the ribosomal protein S15 that is

encoded in the 40S subunit and belongs to S 19 protein family which is located in cytoplasm

(GeneCards 2015) Some researchers tried to block RPS15 synthesis by using siRNAs and

the RPS15 mRNA was down-regulated effectively by siRNA Besides RPS15 was

distinguished as pre-ribosomal nuclear export 40S particles in mammalian cells (Rouquette

et al 2005) Rouquette et al (2005) also stated RPS15 could be used for nuclear

transportation of 40S subunit Walter et al (1996) suggested that highly transcribed RPS15

might result in a substantial number of human tumours including colon cancer esophageal

cancer and neuroblastomas RPS15 gene encodes 145 amino acids while highly expressed

RPS15 leads to insulinomas disease (Walter et al 1996) RPS15 is highly osteogenic in the

tissues cells which is shown in bone of human (Roforth et al 2015)

11

() -t N ()() v v v () () () () -t -t -t -t rr rr rr rr

- I bull __

Figure 23 Location of RPSJ5 in chromosome 19 Adapted from RPSJ5 GeneCards by GeneCards 2015 Retrieved from httpwwwgenecardsorgcgi-binicarddispplgene=RPSI5 Copyright 2015 by GeneCards Reprinted with pennission

232 Ribosomal protein associated with disease

Ribosomal proteins may also be involved in disease condition as the abnonnal expression

levels or expression ofmutated genes occurs (Bhavsar et al 2010) One of the most common

diseases caused by RP genes is Diamond-Blackfan anaemia (DBA) which is a congenital

erythroid aplasia whereby the mutated ribosomal protein S 19 cause a patient suffering from

this disease to have low number of red blood cells and failure in bone marrow (Bhavsar et

al 2010) Consequently Bhavsar et ale (2010) mentioned this disease happens due to

different types of expression in RPs such as S17 S15 S24 S7 L5 and L11 Several of

diseases like Turner syndrome Noonans syndrome and 5q syndrome could occur when

RPs are expressed in different levels (Bhavsar et al 2010) Daftuar et ale (2013) suggested

that numerous RPs such as RPL37 RPS15 and RPS20 could activate a RP-Mdm2-p53

signalling pathway thereby induce cell cycle arrest senescence or apoptosis by attaching

to MDM2 and stimulating tumour suppressor p53 Furthennore Naora and Naora (1999)

indicated that while the RPS3a is expressed it would lead to cell death and transfonnation

Amsterdam et ale (2004) found that quite a lot of RP genes are involved in cancer genes by

fonning tumours in variety of tissues and also act as haploinsufficient tumour repressors in

fish Mutation of ribosomal proteins RPS15 could cause colorectal carcinogenesis (Lai amp

11 2007)

12

24 CeU line

Cell line is described as the cells growth after the subculturing of primary culture by which

it is used extensively for investigation research and analysis (Skelin et al 2010) Cell line

is commonly intended as the models ofexperiment ofneoplastic disease which is originated

from human tumours (Ross et at 2000) Langdon (2004) mentioned George Gey discovered

HeLa as the first human cancer continuous cell line in 1951 In general cancer cell line is

likely to use for the investigation ofgenetic epigenetic and cellular pathway (Ferreira et at

2013) According to Ferreira et al (2013) there are some benefits of using cell line in the

experiment which includes they are easy to manage and manipulate low heterogeneity

abundant cell lines are available low degree of dissimilarity with the initial tumour

instantaneous accessibility infinite auto-replicative supply convenient substitution and high

reproducibility of results As the Musashi-l (MSI 1) of human colon cell line is knocked

down it will reduce the invasion migration proliferation of colorectal cancer (Smith et al

2015)

241 SW480

SW480 human colon adenocarcinoma cell line is the tumour in the colon derived from

intestinal gland cells (Figure 24) The prefix adeno defines that it is from the glandular

tissues The tumour has a tendency to begin from inner layer of colon The properties of this

cell line are adherent and epithelial It is from a 50 years old Caucasian male Compared to

others it is neither fast nor slow growing cell lines (Ahmed et al 2013) This cell line is

isolated from Dukes B stage tumour which has grown through muscle layer of bowel at

descending colon (Flatmark et at 2004) Tumour grade of this cell line SW480 is grade III

to IV that is moderately differentiated (Romano et al 2009 Niu et al 2012)

13

usat Khidmat 1lakJuma Akademik VNlVERSm MALAYSIA SARAWAJ(

Comparative Analysis of RPS15 Expression between Tumour CeUs of Human Colorectal System

Teh Zy Ying (44409)

A Thesis Submitted in Partial Fulfillment of the Requirement of

The Degree of Bachelor of Science with Honors (Resource Biotechnology)

Supervisor Assoc Prof Dr Edmund Sim Ui Hang

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2016

ACKNOWLEDGEMENT
























Grade _____











II



















III


Table of Contents

Content Page

Acknowledgement I





List of Tables VII

Abstract 1

10 Introduction 2-4



22 Cancer 6




231 RPS15 11 -l2


24 Cell line 13

241 SW480 13 -14

242 HCT116 14 - 15


251 GAPDH 16


IV










39 Sequencing 26


40 Results 27









60 Conclusion 40

70 References 41-45

80 Appendices 46-53

v

I

I

AGE

BLAST

cDNA

CRC

DBA

dH20

EtBr

GAPDH

M-MLVRT

mRNA

PBS

PCR

RNA

RPs

RPS15

rRNA

RT-PCR

tRNA

IJg

~

JlM

WHO







Distilled Water

Ethidium Bromide



Messenger RNA



Ribonucleic Acid

Ribosomal Proteins





Microgram

Microliter

Micromolar


VI

I

List of Tables

Table Page












VII

List of Figures



















VIII









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13

ACKNOWLEDGEMENT
























Grade _____











II



















III


Table of Contents

Content Page

Acknowledgement I





List of Tables VII

Abstract 1

10 Introduction 2-4



22 Cancer 6




231 RPS15 11 -l2


24 Cell line 13

241 SW480 13 -14

242 HCT116 14 - 15


251 GAPDH 16


IV










39 Sequencing 26


40 Results 27









60 Conclusion 40

70 References 41-45

80 Appendices 46-53

v

I

I

AGE

BLAST

cDNA

CRC

DBA

dH20

EtBr

GAPDH

M-MLVRT

mRNA

PBS

PCR

RNA

RPs

RPS15

rRNA

RT-PCR

tRNA

IJg

~

JlM

WHO







Distilled Water

Ethidium Bromide



Messenger RNA



Ribonucleic Acid

Ribosomal Proteins





Microgram

Microliter

Micromolar


VI

I

List of Tables

Table Page












VII

List of Figures



















VIII









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13


Grade _____











II



















III


Table of Contents

Content Page

Acknowledgement I





List of Tables VII

Abstract 1

10 Introduction 2-4



22 Cancer 6




231 RPS15 11 -l2


24 Cell line 13

241 SW480 13 -14

242 HCT116 14 - 15


251 GAPDH 16


IV










39 Sequencing 26


40 Results 27









60 Conclusion 40

70 References 41-45

80 Appendices 46-53

v

I

I

AGE

BLAST

cDNA

CRC

DBA

dH20

EtBr

GAPDH

M-MLVRT

mRNA

PBS

PCR

RNA

RPs

RPS15

rRNA

RT-PCR

tRNA

IJg

~

JlM

WHO







Distilled Water

Ethidium Bromide



Messenger RNA



Ribonucleic Acid

Ribosomal Proteins





Microgram

Microliter

Micromolar


VI

I

List of Tables

Table Page












VII

List of Figures



















VIII









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13



















III


Table of Contents

Content Page

Acknowledgement I





List of Tables VII

Abstract 1

10 Introduction 2-4



22 Cancer 6




231 RPS15 11 -l2


24 Cell line 13

241 SW480 13 -14

242 HCT116 14 - 15


251 GAPDH 16


IV










39 Sequencing 26


40 Results 27









60 Conclusion 40

70 References 41-45

80 Appendices 46-53

v

I

I

AGE

BLAST

cDNA

CRC

DBA

dH20

EtBr

GAPDH

M-MLVRT

mRNA

PBS

PCR

RNA

RPs

RPS15

rRNA

RT-PCR

tRNA

IJg

~

JlM

WHO







Distilled Water

Ethidium Bromide



Messenger RNA



Ribonucleic Acid

Ribosomal Proteins





Microgram

Microliter

Micromolar


VI

I

List of Tables

Table Page












VII

List of Figures



















VIII









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13


Table of Contents

Content Page

Acknowledgement I





List of Tables VII

Abstract 1

10 Introduction 2-4



22 Cancer 6




231 RPS15 11 -l2


24 Cell line 13

241 SW480 13 -14

242 HCT116 14 - 15


251 GAPDH 16


IV










39 Sequencing 26


40 Results 27









60 Conclusion 40

70 References 41-45

80 Appendices 46-53

v

I

I

AGE

BLAST

cDNA

CRC

DBA

dH20

EtBr

GAPDH

M-MLVRT

mRNA

PBS

PCR

RNA

RPs

RPS15

rRNA

RT-PCR

tRNA

IJg

~

JlM

WHO







Distilled Water

Ethidium Bromide



Messenger RNA



Ribonucleic Acid

Ribosomal Proteins





Microgram

Microliter

Micromolar


VI

I

List of Tables

Table Page












VII

List of Figures



















VIII









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13










39 Sequencing 26


40 Results 27









60 Conclusion 40

70 References 41-45

80 Appendices 46-53

v

I

I

AGE

BLAST

cDNA

CRC

DBA

dH20

EtBr

GAPDH

M-MLVRT

mRNA

PBS

PCR

RNA

RPs

RPS15

rRNA

RT-PCR

tRNA

IJg

~

JlM

WHO







Distilled Water

Ethidium Bromide



Messenger RNA



Ribonucleic Acid

Ribosomal Proteins





Microgram

Microliter

Micromolar


VI

I

List of Tables

Table Page












VII

List of Figures



















VIII









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13

AGE

BLAST

cDNA

CRC

DBA

dH20

EtBr

GAPDH

M-MLVRT

mRNA

PBS

PCR

RNA

RPs

RPS15

rRNA

RT-PCR

tRNA

IJg

~

JlM

WHO







Distilled Water

Ethidium Bromide



Messenger RNA



Ribonucleic Acid

Ribosomal Proteins





Microgram

Microliter

Micromolar


VI

I

List of Tables

Table Page












VII

List of Figures



















VIII









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13

List of Tables

Table Page












VII

List of Figures



















VIII









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13

List of Figures



















VIII









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13









IX

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13

1


Teh Zy Ying (44409)



ABSTRACT



ABSTRAK



I

I

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13

10 Introduction



















metastasis of CRC





2

l I





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13





















analysis



3



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13



colorectal system



4



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13



21 Gene Expression




















5

1

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13

22 Cancer























1






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13






Colon cancer

Cucum














7











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13











8
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13
























9
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13
























10





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13





















11


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13


- I bull __



















11 2007)

12

24 CeU line













2015)

241 SW480









13

24 CeU line













2015)

241 SW480









13

RPS15 Expression between Tumour Cells of Human Colorectal ... analysis...Comparative Analysis of...

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Transcript of RPS15 Expression between Tumour Cells of Human Colorectal ... analysis...Comparative Analysis of...