MiRNA expression changes in arsenic-induced skin cancer in vitro and in vivo.

193 Pages·2017·2.64 MB·English

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Electronic Theses and Dissertations
8-2017
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Laila Al-Eryani
University of Louisville
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Al-Eryani, Laila, "MiRNA expression changes in arsenic-induced skin cancer in vitro and in vivo." (2017).
Electronic Theses and Dissertations. Paper 2767.
https://doi.org/10.18297/etd/2767
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MIRNA EXPRESSION CHANGES IN ARSENIC-INDUCED
SKIN CANCER IN VITRO AND IN VIVO

By

Laila Al-Eryani

A Dissertation Submitted
To The Faculty of the School
Of Medicine of the University Of Louisville
In Partial Fulfillment of the Requirements
For The Degree Of

Doctor of Philosophy in Pharmacology and Toxicology

Department Of Pharmacology and Toxicology
University Of Louisville
Louisville, Kentucky

August 2017

MIRNA EXPRESSION CHANGES IN ARSENIC-INDUCED
SKIN CANCER IN VITRO AND IN VIVO

By

Laila Al-Eryani

Dissertation approved on
August 03, 2017
By the following Dissertation Committee:

J. Christopher States, Ph.D.

Carolyn M. Klinge Ph.D.

Shesh N. Rai, Ph.D.

Chendil Damodaran, Ph.D.

Theodore Kalbfleisch, Ph.D.
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ACKNOWLEDGMENTS
I would like to express my deepest gratitude to my mentor Dr. J.
Christopher States for his persistent help, guidance, motivation, patience and
very big and kind heart through my Ph.D. journey. I want also to thank my
committee members for their guidance, support and assistance starting form Dr.
Damodaran and his lab members and Dr. Tyagi and Dr. Pal for all their technical
help. I would also like to thank Dr. Shesh Rai and Dr. Pan for running the
statistical analyses. I want also to thank Dr. Kalbfleisch for his help with the NGS
data and Dr. Klinge for her advice and helping with troubleshooting. I also
sincerely thank the DNA Core manager at the University of Louisville, Sabine
Waigel, for all the tremendous help with hybridization microarray analyses and
other analyses and being always welcoming and welling to help. Special thank
you goes to Vennila Arumugam for all the technical support. My gratitude also
goes to members of the Price Institute of Surgery at the University of Louisville,
in particular Dr. Galandiuk, M. Robert Eichenberger, Dr. Chien, Dr. Sarojini for
the access to instruments and enormous technical help for the LCM and skin
lesions study. I would like to thank Dr. Malone for her tremendous help for
diagnosing the skin lesions. I sincerely thank Dr. Jala for his technical help with
the cell cycle assays and Josiah Hardesty for his technical help with western blot
troubleshooting.
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I want also to thank Vanessa States for running the skin lesions preliminary
miRNA expression data and Gregory States for designing the program used to
facilitate measuring the cells migration. I wish also to thank all the summer
students who contributed to the project, Sam Jenkins, who helped tremendously
with the LCM, and Jana Peremarti. I also sincerely thank the Fulbright program
for giving me the chance to pursue my graduate studies. Without their initial
support during my master, I would have been able to pursue my Ph.D. Many
thanks to the Integrated Program in Biomedical Sciences (IPIBS) and the
department of Pharmacology and Toxicology for supporting me throughout my
Ph.D.
Last but not least I am very thankful to my amazing family, mom, dad and
lamia along with my friends Banrida, Sabrin, Safa, Heather, Linda and Holly for
all their continuous support which without I would never be where I am now.
This work was supported in part by NIH grants R21ES023627,
R01ES011314, T35ES014559, R25CA134283, and a Competitive Enhancement
Grant from the University of Louisville Office of the Executive Vice President for
Research and Innovation.

Finally, I dedicate this dissertation to the memory of a beautiful soul and a
very special person that left us too soon, my bother, Luai Al-Eryani. I will always
love you, remember you and cherished you and every moment we spent
together, you are deeply missed!

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ABSTRACT
MIRNA EXPRESSION CHANGES IN ARSENIC-INDUCED SKIN CANCER IN
VITRO AND IN VIVO
Laila Al-Eryani
August 03, 2017
Arsenic is a naturally prevalent metalloid. Chronic arsenic ingestion
through drinking water causes skin cancer. Arsenic-induced cancer mechanisms
are not well defined. Epigenetic changes, including microRNA expression
changes, might be playing a role. This dissertation investigates the impact of
miRNA expression changes in arsenic-induced skin cancer. MiRNA expression
was measure and compared using 3 different techniques, RTq-PCR,
hybridization arrays and RNA-sequencing. MiRNAs differential expression in skin
lesions was phenotype- and stage-related. Immortalized human keratinocytes
(HaCaT) were transformed by chronic low arsenite exposure serving as a model
for arsenic-induced skin carcinogenesis. Early changes in miRNAs and target
mRNAs contribute to arsenic-induced carcinogenesis. Throughout the time
course of arsenic exposure, dysregulation of cells’ growth and cancer-related
pathways were identified. Comparisons between the miRNA profiles in lesions
and cells predict some miRNAs may serve as biomarkers and/or therapeutic
targets for arsenic-induced tumors. This dissertation provides strong evidences
of epigenetic changes related to carcinogenesis in arsenic-induced skin cancer.
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TABLE OF CONTENTS
Acknowledgements.…………………………………………………...………….. iii
Abstract………………………...……………………………………………….… v
List of Tables……………..…………………………………………………….… viii
List of Figures…………………………………………………………….……… ix
Chapter 1: General Introduction……………………………………………… 1
Chapter 2: MiRNA Expression Profiles of Premalignant and Malignant

Arsenic-Induced Skin Lesions
Introduction…………………………………………………….…..
13
.

Materials and Methods…………………………………………… 19

Results…………………………………………………….……….. 21

Discussion.…………………………………………………….…... 33
Chapter 3: Differentially Expressed MiRNAs and MRNAs at Early Stages
of Transformation of HaCaT Cells Chronically Exposed to
Low Arsenite

Introduction…………………………………………………….….. 40

Materials and Methods…………………………………………… 43

Results…………………………………………………….……….. 47

Discussion………….……………………………………………… 61
Chapter 4: Cell Cycle Pathway Dysregulation in Human Keratinocytes at

Early Stages of Chronic Exposure to Low Arsenite

Introduction…………………………………………………….….. 68
Materials and Methods…………………………………………… 70

Results…………………………………………………….……….. 73
Discussion………….……………………………………………… 78
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Chapter 5: A Longitudinal Study on Small RNA And MRNA Expression

Profiles in The Chronic Exposure HaCaT Model
Introduction…………………………………………………….….. 80
Materials and Methods…………………………………………… 83
Results…………………………………………………….……….. 87
Discussion………….……………………………………………… 109
Chapter 6: MiRNA Expression Profiles Comparisons Across Three

Different Techniques
Introduction…………………………………………………….….. 112
Materials and Methods…………………………………………… 114
Results…………………………………………………….……….. 116
Discussion………….……………………………………………… 126
Chapter 7: Overall Discussion, Conclusions and Future Directions .……. 129
References……………………………………...…………………………….….. 135
Appendix: List of Abbreviations……………………………………………… 171
Curriculum Vitae……………………………………………...………………….. 174

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LIST OF TABLES
Table 2.1. Demographics of study population 22
Table 2.2. Differentially expressed miRNAs in Arsenic-induced 27
Skin Lesions and Their Expression
Table 3.1. Differentially expressed small RNAs at both 3 and 7 52
weeks of exposure to arsenite
Table 3.2. Top 10 pathways of the differentially expressed 38 54
mRNAs at both 3 and 7 weeks that are predicted
targets of miRNAs differentially expressed at both 3
and 7 weeks
Table 4.1. Pathways populated by differentially expressed mRNAs 75
7 weeks
Table 5.1. MiRNAs differentially expressed at more than one time 88
point
Table 5.2. Pathway analysis of differentially expressed genes at 7, 90
19 and 28 weeks
Table 5.3. Pathway analysis of differentially expressed mRNAs 104
targets of differentially expressed miRNAs
Table 6.1. Differentially expressed miRNAs at 3 weeks of 117
chronically arsenic- exposed/unexposed HaCaT cells
using RT-qPCR array cards
Table 6.2. Differentially expressed miRNAs at 3 weeks in exposed 119
cells vs. unexposed cells to those of unexposed cell vs.
skin lesions
Table 6.3. Differentially expressed miRNAs at 3 weeks of 121
chronically arsenic-exposed HaCaT cells in both
hybridization microarrays and RT-qPCR array cards
Table 6.4. Differentially expressed miRNAs at 7 weeks of 123
chronically arsenic-exposed HaCaT cells in both
hybridization microarrays and Next-Generation
Sequencing (NGS)
Table 6.5. MiRNAs differentially expressed in chronically arsenic- 125
exposed HaCaT cells and arsenic-induced skin lesions
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