O programa Statistical Package for the Social Science v. 20.0 (SPSS Inc., Illinois, EUA) foi utilizado para a análise estatística. As variáveis categóricas foram apresentadas em frequências relativas e absolutas, enquanto as variáveis contínuas foram apresentadas como mediana e intervalo interquartil.
A variável consumo alimentar frequente (categórica) foi analisada quanto a sua associação com cada um dos parâmetros de prognósticos histopatológicos do CPT (invasão, extensão, multifocalidade, metástases linfonodais e tamanho do tumor) por meio dos testes do qui-quadrado de Pearson e teste exato de Fisher.
Os valores séricos retinol e α-toceferol foram analisados de forma categorizada pela mediana em cada um dos 3 tempos de coleta, e ainda de forma continua por meio de testes não paramétricos, após teste de normalidade de Kolmogorov-
Smirnov. Em ambos os modelos, categórico e contínuo, foi investigado a associação com cada um dos fatores de prognóstico histopatológico do CPT separadamente.
O teste de Mann-Whitney foi utilizado para comparar os níveis séricos das vitaminas A e E com cada fator de prognóstico histopatológico do CPT de forma independente. E os testes não-paramétricos de Wilcoxon e Friedman foram utilizados para as comparações pareadas de duas ou mais amostras dos níveis séricos das vitaminas no pré (T0) e pós tireoidectomia (T1 e T2).
A regressão logística, nos modelos uni e multivarido, foi realizada a fim de se obter o valor relativo ao risco daquelas variáveis significativas para os testes de qui-quadrado. Os valores de p inferiores a 0,05 foram considerados estatisticamente significantes.
6.0 ARTIGOS CIENTÍFICOS
Os resultados obtidos nesta dissertação serão apresentados na forma de 3 artigos científicos, os quais são descritos nas páginas seguintes.
6.1 REVISÃO SISTEMÁTICA
Embora tenha sido objetivo desse estudo investigar as associações entre a modulação da expressão de miRNA’s induzidas pelas vitaminas A e E, só foram encontrados resultados suficientes para se conduzir um estudo de revisão sistemática para o manuscrito referente a vitamina A, visto que, o número de estudos que trabalharam com a vitamina E e seus derivados como agentes moduladores da expressão de miRNA’s não foi satisifatório para se atender o métodos de análise desta pesquisa.
A revisão sistemática “MODULATION OF ALL-TRANS RETINOIC ACID- INDUCED miRNA EXPRESSION IN NEOPLASTIC CELL LINES: A SYSTEMATIC REVIEW” foi submetida para publicação no periódico “Advances in Nutrition”, que possui fator de impacto 6,853, e Qualis A1 da CAPES para Nutrição. O comprovante de submissão encontra-se na página seguinte e as normas de publicação deste periódico no Anexo 3.
Modulation of All-trans Retinoic Acid-induced MiRNA Expression in Neoplastic Cell Lines: A 1
Systematic Review 2
3
Lara Clarisse de Lima Silva1,2, Thaísa Cristina Tavares de Melo², Diego Marques da Costa Santos2, Jéssica 4
Nayara Góes de Araújo2, Isabela Samária Fernandes Leite2, Camila Xavier Alves4, Julieta Genre², and 5
Vivian Nogueira Silbiger1,3 6
7
¹Department of Nutrition, Postgraduate Program in Nutrition, Federal University of Rio Grande do 8
Norte, Natal, Brazil; 9
²Laboratory of Bioanalysis and Molecular Biotechnology, Federal University of Rio Grande do Norte;
10
3Department of Clinical and Toxicological Analysis, Federal University of Rio Grande do Norte, Natal, 11
Brazil; 12
4Faculty of Health Sciences of Trairi, Federal University of Rio Grande do Norte, Santa Cruz, Brazil.
13 14
Address correspondence to VNS (email: [email protected]) 15
Professor, Departamento de Análises Clínicas e Toxicológicas, Universidade Federal do Rio Grande do
16
Norte, Natal, Rio Grande do Norte, Brazil. Av. General Gustavo Cordeiro de Faria S/N, Petrópolis, 59012-
17 570. Natal - RN, Brazil. 18 Telephone: +55 84 3342 9807; Fax: +55 84 3342 9833. 19 20
Word count: 6941 Figures: 1; Tables: 5
21
Running title: ATRA-induced miRNAs in neoplasia: a systematic review 22
Funding: This work had no financial support. 23
Conflict of interest: The authors declare that there is no conflict of interest regarding the publication of this 24
paper.
25
ABSTRACT 26
Cancer comprises a genetic and epigenetic disease that requires the inactivation of tumor suppressor genes
27
along with proto-oncogene activation. All-trans retinoic acid (ATRA), a retinoic acid derivative, is involved 28
in the onset of differentiation and apoptosis in a wide variety of normal and cancer cells, functioning as an 29
anti-cancer agent for a variety of neoplasms. Several studies have shown that ectopic changes in the 30
expression of certain microRNAs (miRNAs) occur in response to ATRA, leading to phenotypic alterations 31
in neoplastic cell lines. Moreover, the modulation of miRNA patterns upon ATRA-treatment may constitute 32
an effective strategy for chemopreventive and anticancer therapy. Thus, the present systematic review was 33
performed to provide an overview of the modulation of ATRA-induced miRNA expression in different types 34
of neoplastic cells and identify the efficacy of intervention factors (i.e., concentration and time of treatment) 35
and how they influence the expression profile of miRNAs targeting oncogenesis. A systematic search of the 36
published literature was undertaken using the US National Library of Medicine`s MEDLINE/PubMed 37
bibliographic search engine. The search identified 23 experimental studies with human cell lines from four 38
different cancer types (neuroblastoma, myeloid and promyelocytic leukemias, breast and lung cancer) 39
treated with ATRA concentrations ranging between 5 pmol and 10 mmol for 3 to 7 days. The concentrations 40
and time of treatment of cancer cells with ATRA varied widely. The presence of ATRA in culture medium 41
containing cancer cells could modulate the expression of an additional 300 miRNAs, with the changes in 42
miRNA expression profiles potentially resulting in the promotion of apoptosis, growth control, invasion, 43
migration, metastasis, and in some cases, total tumor remission. ATRA may thus be broadly effective for 44
neoplasm treatment and prevention, although these studies may not accurately represent in vivo conditions. 45
Additional studies are required to elucidate the ATRA-induced miRNA modulations during neoplasm 46
treatment. 47
48
Keywords: Cancer, All-trans retinoic acid, MiRNA, Expression modulation 49
50 51 52
Introduction 53
Cancer comprises a genetic and epigenetic disease that requires the inactivation of tumor suppressor genes
54
and the activation of proto-oncogenes (1). Cancer constitutes a public health problem worldwide, with the 55
anticipated impact of this disease on the world population corresponding to over 17 million cases by the year 56
2020 (2). 57
Retinoic acid (RA)3, the major bioactive metabolite of retinol or vitamin A, serves as a potent regulator
58
of cell growth, differentiation, and matrix formation of various cell types during embryogenesis (3, 4). In
59
addition, all-trans retinoic acid (ATRA), a derivative of RA, is involved in the onset of differentiation and 60
apoptosis in a wide variety of normal and cancer cells (5) and has been shown to function as an anti-cancer 61
agent for a variety of neoplasms (6). Notably, several studies have shown that ectopic changes in the
62
expression of certain microRNAs (miRNAs) occur in response to ATRA, leading to phenotypic alterations
63
(7). Thus, a potential role for miRNAs with regard to the anti-tumor action of ATRA has been implied by 64
studies performed in various cellular contexts (8). Moreover, the modulation of miRNA patterns upon
65
ATRA-treatment (9) has been suggested as a potential strategy for chemopreventive and anticancer therapy 66
(6). 67
miRNAs comprise a class of endogenous, small non-coding RNAs that control gene expression by 68
binding to their target messenger RNAs for degradation and/or translational repression (5). These molecules
69
have been extensively associated with the pathology of cancer, as they are involved in the regulation of gene
70
expression and play important roles in regulating biological processes such as development and
71
differentiation, cell proliferation,
72
3 Abbreviations: AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ATRA, all-trans retinoic acid; miRNA, microRNA; NB, neuroblastoma; RA, retinoic acid
apoptosis, tumorigenesis, and maintenance of stem cell pluripotency (10, (11). Furthermore,
73
the deregulation of miRNA expression has been considered to constitute a key factor in the 74
pathogenesis of various types of cancer (12) as they may function as oncogenes or tumor 75
suppressors, with such dysregulated patterns representing promising cancer diagnostic and 76
prognostic markers as well (13). 77
The identification of genetic and epigenetic elements responsible for the transcriptional 78
regulation of miRNAs implicated in cancer might lead to a better understanding of the 79
pathways governing oncogenes. Thus, their detailed examination will contribute to a better 80
comprehension of the molecular basis of human diseases and might allow the characterization 81
of novel cancer biomarkers and therapeutic targets (13,14). Accordingly, the objective of the
82
present systematic review was to provide an overview of the modulation of ATRA-induced 83
miRNA expression in different types of neoplastic cells. 84
85
Methods 86
This review was written in accordance with The PRISMA Statement for Reporting 87
Systematic Reviews and Meta-Analyses of Studies That Evaluate Health Care Interventions: 88
Explanation and Elaboration (15). 89
90
Search strategy 91
A systematic search of the published literature form January 1, 2007 to March 12, 2018 was
92
undertaken by using US National Library of Medicine`s MEDLINE/PubMed
93
(www.ncbi.nlm.nih.gov/pubmed) bibliographic search engine. Multiple PubMed searches
94
were conducted by using the combination of keywords “(miRNA OR microRNA) AND
95
(“retinoic acid” OR “vitamin A” OR retinol) AND (cancer OR neoplasm OR tumor).” The
96
manuscripts were compiled into a single database and duplicates were removed. An initial
screening of manuscripts was performed by assessing the title and abstract. After reading the 98
selected manuscripts, citations on the reference section of each text were analyzed for 99
additional sources of new manuscripts. 100
101
Study selection 102
For this review, we selected studies written in the English language that evaluated changes in 103
the expression profile of miRNAs in neoplastic human cell lines after treatment with ATRA. 104
The exclusion criteria were as follows: studies carried out over 10 years ago, other systematic 105
reviews on the subject, bioinformatics analyses, short communications, and supporting 106 information. 107 108 Data extraction 109
Data extraction was conducted by one reviewer (LCLS) and verified by the other authors.
110
Extracted data included the type of cancer, cell type analyzed, conditions of ATRA treatment
111
(dose and time), miRNAs evaluated, and significant findings. For those studies that
112
determined ATRA-induced miRNA expression at multiple time points, the values of the last
113
day were considered. The studies were described according to the type of cancer and cell line 114 employed. 115 116 RESULTS 117
From searches using 18 combinations of key terms, we identified 854 manuscripts. A total of
118
142 records were identified through database searching. After screening the abstracts, 92
119
records were excluded, and 50 full-text manuscripts were accessed and assessed for
120
eligibility. Reasons for manuscript exclusion included studies describing ATRA-mediated 121
effects not related to miRNA expression or mentioning genes that exhibited modifications 122
induced by ATRA without describing the miRNA involved, systematic reviews and 123
manuscripts of bioinformatics analysis, short communications or supporting information, and 124
studies that were unclear regarding the description of ATRA treatment (Figure 1). 125
The characteristics and findings of the total of 23 studies included in this systematic
126
review are summarized in Tables 1 to 5 according to type of cancer. In particular, four
127
different types of cancer were addressed: neuroblastoma (NB) (n = 11), leukemia (n = 8), 128
breast carcinoma (n = 2), and lung cancer (n = 2). All studies were based on human 129
neoplastic cell lines treated with ATRA, followed by evaluation of changes in miRNA 130
expression patterns. One study (22) used human and animal cell lines; however, only results 131
related to human cells were included in this review. The results, grouped according to the 132
type of cancer, are presented below. 133
134
Neuroblastoma (NB)
135
NB, which originates from the aberrant development of primordial neural crest cells, 136
constitutes the most common extracranial solid tumors in childhood and the most common 137
tumors in infants(16). Several lines of evidence support a role for miRNAs in NB 138
pathogenesis as well as the usefulness of miRNA profiles for NB diagnostics, classification, 139
and prognosis (16). Furthermore, the involvement of miRNAs in ATRA-induced 140
differentiation of NB cells was recently reported (16). In particular, cell lines derived from
141
NB, such as SK-N-BE, SH-SY5Y, and LAN-5, can be induced to undergo neural cell
142
differentiation by treatment with ATRA and thus are often used as a model system to study
143
biochemical pathways involved in the process of differentiation (17).
144 145
SK-N-BE cell line
Das et al. (7) administered 5 pmol of ATRA in SK-N-BE NB cells by replacing the culture 147
medium every 24 h for 7 days to determine changes in the expression of 368 miRNAs after 148
ATRA treatment. Of these, they observed that seven were over expressed by at least 2-fold
149
and 17 exhibited a 2-fold or greater decrease in expression. This study showed that miR-152
150
increased 2.5-fold following ATRA treatment and that the ectopic up-regulation of this
151
miRNA negatively regulates cell invasiveness and anchorage-independent cell growth but
152
does not induce differentiation or inhibit cell growth. Moreover, miR-9, miR-125a, and miR-
153
125b were up-regulated in response to ATRA. Subsequently, Das et al. (18) repeated the
154
treatment applied in NB cell lines in his previous study (7) and observed a correlation
155
between changes in the expression profile of miRNAs and methylation following ATRA
156
treatment. In SK-N-BE cells, they identified 20 miRNAs as being up-regulated and 24 down-
157
regulated following ATRA treatment. Furthermore, miR-340 exhibited the most significant
158
up-regulation. 159
Using an ATRA concentration a thousand times greater (5 µmol/L) than that of Das et
160
al. (18), Chen and Stallings (19) evaluated the expression of 34 human miRNAs in SK-N-BE 161
cells treated with ATRA by replacing the culture medium every 24 h for 5 days. The results 162
revealed that the expression of 21 miRNAs examined was altered when compared to that in 163
untreated cells. Of these miRNAs, 17 were up-regulated in ATRA-treated cells and four were 164
down-regulated. In particular miR-184 showed the most significant alteration in expression, 165
being up-regulated by 9-fold following ATRA treatment. In comparison, 13 miRNAs 166
exhibited no significant changes in their expression profile. In SK-N-BE cells treated with 167
ATRA for 5 days, and then released from ATRA for 3 days, levels of miR-184 were 25-fold 168
higher than untreated cells, suggesting that the effects of ATRA on miRNA expression 169
continued even after ATRA treatment. 170
Foley et al. (17) also used 5 µmol/L ATRA; however, they followed the treatment for 171
an additional two days, changing the culture medium every 24 h for 7 days in total, and 172
analyzed the expression profile of 364 miRNAs. This study showed that ATRA treatment 173
induced changes in the expression profile of many miRNAs with highest significance 174
observed in 56, of which 30 were up-regulated and 26 down-regulated. miRNAs with the
175
most significant changes in expression (410-fold at day 7) included miR-132, miR-10a, and
176
miR-10b.
177
In turn, Laneve et al. (20) doubled the dose and reduced the time to 72 h and analyzed 178
the expression pattern of 70 miRNAs. The results showed that 14 miRNAs were up-
179
regulated, 33 did not change their expression, and 23 were undetectable. The miRNAs
180
that were up-regulated were miR-9, miR-124a, miR-125a, miR-125b, let-7a, let-7b, miR-
181
7, miR-22, miR-23a, miR-24, miR-26a, miR-30a-5p, miR-100, and miR-103. Expression of
182
the majority of these miRNAs was induced after 3 days upon ATRA treatment and
183
progressively accumulated at higher levels after terminal differentiation (10 days).
184
Moreover, they observed that the expression levels of miR-9, miR-125b, and miR-125a
185
increased 1.7, 2.2, and 2.6 folds, respectively, compared to that of control cells, and that
186
the increase in the levels of these miRNAs led to a strong decrease of NB cell proliferation
187
in vitro. In a similar manner, Ragusa et al. (21) analyzed the expression profile of miR-
188
152, miR-200b, and miR-338 using the same concentration of ATRA during 10 days of
189
treatment, observing up-regulation of all three miRNAs analyzed, with miR-200b
190
exhibiting the most significant increase in expression.
191 192
SH-SY5Y cell line
In order to test the effects of RA on the differentiation of NB and the development of
194
embryonic cells, Das et al. (18) also treated the SH-SY5Y cell line with 5 pmol ATRA during
195
7 days, showing that ATRA altered the expression of 13 miRNAs among which miR-340 was 196
significantly up-regulated. In addition, the ATRA treatment resulted in a decrease in cell 197
growth to that comparable to negative controls.
198
In turn, Meseguer et al. (16) showed that ATRA treatment of SH-SY5Y NB cells resulted 199
in profound changes in the expression pattern of miRNAs. In this study, NB cells were 200
treated with 1 µmol/L ATRA and changes in miRNAs expression were assessed at 0, 24, 48, 201
and 96 h, demonstrating that 42 miRNAs exhibited significantly changed expression levels 202
(26 up-regulated and 16 down-regulated). In particular, miR-10a and -10b showed the most
203
prominent expression changes. Furthermore, changes in the expression of these miRNAs, 204
induced by ATRA, contributed to the regulation of SH-SY5Y NB cell differentiation and in 205
the associated changes in migratory and invasive activities. 206
Foley et al. (17) utilized a 5-fold increased ATRA concentration and cultured SH-SY5Y
207
cells over the same duration. Similar to the observations for SK-N-BE cells that received the
208
same treatment, the miRNAs that showed greatest up-regulation were miR-132, miR-10a, and
209
miR-10b.
210
In comparison, Chen et al. (22) treated SH-SY5Y cells with 10 µmol/L ATRA for 2 days.
211
The results showed significant changes in the expression profile of 12 miRNAs, of which half
212
were up-regulated (miR-132, miR-16, miR-27a, miR-27b, miR-214, and miR-197) and half
213
down-regulated (miR-133a, miR-508-3p, miR-7, miR-1, miR-205, and miR-20b). Similarly, 214
Le et al. (23) also used the same concentration of ATRA during 5 days and analyzed the
215
expression profile of 175 miRNAs. This study revealed that the expression of 12 of these was
216
significantly modified during treatment, with miR-7, miR-124a, miR-125b, miR-199a, miR-
217
199a*, and miR-214 being up-regulated.
Using the same concentration as Chen et al. (22) and Le et al. (23), Evangelisti et al. 219
(24) measured miR-128 expression following treating the culture of cells with ATRA 220
every 48 h, with the treatment stopped six days after the first stimulation. They observed 221
that miR-128 expression was up-regulated approximately 3-fold when compared to that 222
in untreated cells. 223
Beverigde et al. (25) also treated SH-SY5Y cells with 10 µmol/L ATRA albeit for one
224
additional day than Evangelisti et al. (24). They identified 43 miRNAs with altered
225
expression after treatment, of which 32 were significantly down-regulated and 11 up-
226
regulated. Furthermore, six miRNAs that exhibited increased expression (miR-9, miR-124a,
227
miR-128a, miR-208, miR-210, and miR-423) were previously shown to comprise brain-
228
specific or brain-enriched miRNAs, and are considered to play an important role in brain
229
development, neuronal maturation, and neuronal differentiation. In addition, this study
230
revealed that treatment with ATRA induced the down-regulation of the entire miR-17 cluster.
231 232
LAN-5 cell line
233
Similar to the treatment performed in other NB cell lines (SK-N-BE and SH-SY5Y), Foley et
234
al. (17) treated LAN-5 cells with 5 µmol/L ATRA, changing the culture medium every 24 h 235
for 7 days. The results were similar among the three cell lines with the miRNAs that showed
236
significant up-regulation including miR-132, miR-10a, and miR-10b. These results support 237
the idea that miRNA regulation plays a key role in the differentiation of NB cells induced by 238
ATRA and in the phenotypic changes linked to the expression of genes associated with the 239 affected miRNAs. 240 241 Leukemia 242
In hematopoietic malignancies, a number of miRNAs have been reported to exhibit tumor- 243
suppressive or oncogenic roles in leukemogenesis (12). Accordingly, it has been suggested
244
that miRNAs might be important in the molecular pathogenesis of leukemia by interfering
245
with essential pathways for hematopoietic differentiation (26).
246 247
Acute promyelocytic leukemia (APL) 248
APL is characterized by chromosomal translocations involving the retinoic acid receptor-α
249
gene, which result in clonal expansion of hematopoietic precursors blocked at the
250
promyelocytic stage of differentiation (26). A discrete number of miRNAs up-regulated upon
251
ATRA treatment of APL cell lines have been already identified by different studies (26).
252
To evaluate changes in miRNA expression mediated by ATRA, Bräuer-Hartmann et al.
253
(9) cultured APL NB4 cell in the presence of 10−7 mol/L ATRA and analyzed the expression
254
of miR-181 family members (miR 181a-d) 24 h after treatment. In this study, a significant
255
down-regulation of all miR-181 family members was observed, among which miR-181a and
256
mi-181b showed similar expression levels, whereas miR-181c and miR-181d were
257
differentially expressed. They also observed that inhibition of the miR-181a/b-cluster by
258
ATRA treatment effectively represses cell proliferation and induces apoptosis in APL cells.