Role of microRNA-33a in regulating the expression of PD-1 in lung adenocarcinoma
Boldrini et al. Cancer Cell Int
Role of microRNA-33a in regulating the expression of PD-1 in lung adenocarcinoma
Laura Boldrini 0
Mirella Giordano 0
Cristina Niccoli 0
Franca Melfi 0
Marco Lucchi 0
Alfredo Mussi 0
Gabriella Fontanini 0
0 Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa , Via Roma 57, 56126 Pisa , Italy
Background: MiRNAs are vital in functioning as either oncogenes or tumor suppressors in the cell cycle. Target transcripts for immune checkpoint molecules such as PD-1/PD-L1 and (programmed cell death-1/its ligand and cytotoxic T-lymphocyte antigen 4) have proven to be beneficial against several solid tumors, including lung adenocarcinoma. Methods: Simultaneous quantification of the expression level of miR-33a and PD-1, PD-L1 and CTLA4 mRNAs with NanoString technology was performed in 88 lung adenocarcinoma specimens. A cohort of 323 lung adenocarcinoma patients from the cancer genome atlas (TCGA) database was further analyzed, in order to test our hypothesis. Potential interference of PD-1, PD-L1 and CTLA4 gene expression by miR-33a was predicted using the microRNA target prediction program RNA22. Results: High miR-33a expression was significantly associated with younger (p = 0.005), female (p = 0.04), patients with low grade (p < 0.0001), early stage (p = 0.03) tumors, and better survival. The hypothesis of the involvement of miR-33a in PD-1/PD-L1/CTLA4 mechanisms was corroborated by the finding of putative miR-33a binding sites in all three genes using the RNA22 method. We found an inverse correlation between miR-33a and PD-1 levels (p = 0.01), as well as for PD-L1 (p = 0.01) and CTLA4 (p = 0.03) expression, and a significant better prognosis for patients with high miR-33a/low PD-1. TCGA database analysis confirmed that miR-33a high levels were associated with low PD-1 expression and with longer survival on a larger population. Conclusions: Our study emphasizes the notion of a potential value of miR-33a as a favorable prognostic marker through PD-1 regulation.
miR-33a; PD-1; Lung adenocarcinoma
MicroRNAs (miRNAs) are small non-coding RNA
molecules that function as indispensable regulators of an
increasing number of cellular processes [
are vital in regulating cell proliferation and apoptosis,
in addition to functioning as either oncogenes or tumor
suppressors in the cell cycle [
]. In a recent study [
miR–33a was identified as having potential
tumor–suppressive activity. Overexpression of miR–33a was
demonstrated to inhibit the growth of lung cancer cells, but the
exact role of individual miRNAs strictly depends on their
expression pattern and their targeted genes. Lung cancer
is the most common cancer worldwide and despite recent
progress with molecularly targeted agents, its prognosis
is usually poor and new strategies need to be developed.
Cancer immunotherapy is emerging as a very promising
therapeutic strategy and recently various clinical trials
exploring the use of anti-programmed cell death protein
1 (PD-1, also known as CD279), PD-L1 (programmed
cell death-1 ligand, also known as CD274) and CTLA4
(cytotoxic T-lymphocyte antigen 4) inhibitors have
successfully shown antitumor activities in lung cancer [
For most cancer biomarkers, determination of the
levels of such immune function markers in formalin-fixed,
paraffin-embedded (FFPE) samples has been generally
performed by conventional immunohistochemistry using
various antibodies with various levels of validation.
Different thresholds have been used to define PD-1/PDL1
positivity, but cut-off points as the proportion of
membrane-positive tumor cells were subjective in terms of
estimated visual levels, such that reproducibility has not
been formally assessed in the clinical setting.
Moreover, there are limited data on the prognostic
significance of PD-1/PD-L1, with some showing poor
], better prognosis [
] or no prognostic role [
Another approach to regulating the immune response in
the tumor microenvironment is by modulating the level
of miRNAs. A recent review [
] emphasized the role of
miRNAs as modulators of immune checkpoint molecules
and consequently as potential cancer therapeutic targets.
Here, we investigated the potential role of miR-33a and
demonstrated its potential value as prognostic marker by
regulating PD-1/PD-L1 and CTLA4 expression in lung
Materials and methods
Eighty-eight lung adenocarcinoma patients were
retrospectively selected among those operated at the Unit of
Thoracic Surgery of the A.O.U.P. Histological diagnoses
were independently formulated according to the World
Health Organization classification [
pathological characteristics were collected whenever
available for all the patients.
Total RNA were isolated from a representative area
selected and marked on the surface of 10 micron
sections of formalin-fixed, paraffin-embedded (FFPE)
tissues using the miRNeasy FFPE Kit (Qiagen Inc., Hilden,
Germany) according to the manufacturer’s instructions.
The quality and concentration of RNA was assessed using
a NanoDrop spectrophotometer (Thermo-Scientific,
NanoString nCounter® assay, data normalization
Expression of the selected miR-33a, PD-1, PD-L1 and
CTLA4 genes among a miRGE panel was measured using
the NanoString nCounter technology (NanoString
Technologies, Seattle, WA). The nCounter measures total
counts of mRNAs/miRNA by a multiplex hybridization
assay followed by scanning and digital readouts of
fluorescent probes [
]. Raw NanoString counts for each
gene were subjected to a technical normalization,
considering the counts obtained for positive control probe
sets, followed by a biological normalization, using three
reference genes included in the CodeSet, according to
NanoString Technologies’ guidelines. The nCounter
custom code set used in this study included three genes
(PD-1, PD-L1 and CTLA4) with five housekeeping genes
for reference [clathrin heavy chain 1 (CLTC),
beta-glucuronidase (GUSB), tubulin beta (TUBB), hypoxanthine
phosphoribosyltransferase (HPRT1), phosphoglycerate
kinase 1 (PGK1)]. MiR-33a expression was tested
simultaneously with other selected miRNAs and the
normalization was performed using a scaling factor based on
miRNAs with the lower variability coefficients.
The Cancer Genome Atlas (TCGA) database. From
the TCGA data portal (http://tcga.cancer.gov/; accessed
October 2017), we extracted PD-1 and miR-33a
expression together with the corresponding
clinical–pathological characteristics and survival data for 323
adenocarcinoma patients (LUAD).
Potential regulation of PD-1, PD-L1 and CTLA4 gene
expression by miR-33a was predicted using the
microRNA target prediction program RNA22, a pattern-based
approach for the discovery of microRNA binding sites
and their corresponding microRNA/mRNA complexes
Once the RNA hybridization data had been correctly
prepared, the data were subjected to 2-way
hierarchical clustering analysis (HCA) using nSolver 2.5
Analysis Software. Differential expression was determined by
applying the non-parametric Wilcoxon test in order to
determine the association between miR-33a expression
and the clinical–pathological parameters. Survival
analyses were performed using the Kaplan–Meier method
with log-rank test and the Cox proportional hazard
model. Statistical analyses were performed using JMP10
software (SAS, Milan, Italy), and a two-tailed p value
< 0.05 was considered significant.
This study included 88 patients with lung
adenocarcinoma, 56 males and 32 females, with an age at
diagnosis ranging from 30 to 81 years (mean 58.9 years, median
54.5). The predominant histologic patterns were
characterized as follows: lepidic (29/88, 33%), solid (26/88,
29.5%), acinar (22/88, 25%), and papillar (11/88, 12.5%)
variants. According to the degree of differentiation,
three tumors were G1, whereas 58 and 27 were G2 and
G3, respectively. The adenocarcinomas were all invasive.
Their stages were classified as IA (17), IB (23), IIA (13),
IIB (9), IIIA (23), IIIB (1), and IV (2). The survival data,
with disease-free interval (DFI) and overall survival (OS),
were available for all the patients and was last updated
in March 2015. Concerning the smoking habits, there
were 17 non-smokers, 16 former smokers, and 23 current
smokers; for 33 patients, smoking history was unknown.
miR‑33a expression and clinical–pathological
miR-33a expression was compared with the patients’
clinical–pathological characteristics. High miR-33a
expression was significantly associated with younger
(p = 0.005), female (p = 0.04) patients with low grade
(p < 0.0001), early stage (p = 0.03) tumors (Table 1).
miR‑33a and survival analysis
A Kaplan–Meier survival analysis using DFI (range
0–148, median 21 months) and OS (range 3–148, median
31.5 months) as endpoints and miR-33a expression as a
dichotomous variable, distinguishing tumors with low
from tumors with high expression, showed a significant
better prognosis in patients with high miR-33a levels
(Wilcoxon test, p = 0.02 and p = 0.008, for DFI and OS,
respectively) compared to those with low levels (Fig. 1).
miR‑33a target prediction
To investigate the putative involvement of miR-33a in
the regulation of the most studied immune checkpoint
molecules (PD-1, PD-L1, and CTLA4), we used RNA22
software for the discovery of microRNA binding sites and
their corresponding microRNA/mRNA complexes. The
RNA22 method suggests that the number of microRNA
binding sites may be greater than hypothesized and
that microRNA regulation may be effected through
the 5′UTR and CDS of gene transcripts in animals, in
addition to 3′UTRs. Specifically, the hypothesis of the
involvement of miR-33a in PD-1/PD-L1/CTLA4
mechanisms was corroborated by the observation that the 3′
untranslated region (UTR) of the PD-1 mRNA carries a
putative miR-33a binding site at position 1789. Two
miR33a binding sites for CTLA4, at positions 96 and 253 of
the CDS region, and one in the 3′UTR of PD-L1 (position
1701) were also found (Fig. 2).
Correlation between miR‑33a and PD‑1 expression
We evaluated the abundance of PD-1, as well as of
PDL-1 and CTLA4, mRNA by NanoString technology.
The samples were divided into high and low expression
groups based on the median fold-change value (1.76-fold
change value ± 7.34 for PD-1; 2.79 ± 11.5 for PDL-1, and
30.23 ± 46.08 for CTLA4). Samples with low PD-1 mRNA
levels also demonstrated low protein expression, assessed
in FFPE tissue samples by immunohistochemistry using
the anti-PD-1 mouse monoclonal antibody NAT105
(Ventana Medical Systems, Inc. USA) (data not shown).
We then analyzed their relationship with the
expression level of miR-33a and found a statistically
significant inverse correlation between miR-33a and PD-1/
PD-L1 expression. MiR-33a levels were lower in patients
with higher PD-1 (Chi square test, p = 0.01), PD-L1
and CTLA4 expression (p = 0.01 and p = 0.03,
respectively) (Fig. 3). To evaluate possible relationships between
patients’ survival, miR-33a, and PD-1, we grouped lung
adenocarcinoma patients according to the co-expression
of both factors (miR-33a and PD-1). We then compared
the difference in survival between the two groups linked
to a negative association, high miR-33a/low PD-1 and
low miR-33a/high PD-1 expression. We found that the
first group of patients, with high miR-33a and low
PD1, showed a better prognosis, either for DFI or OS, than
the opposite group (p = 0.04 and p = 0.007, respectively)
(Fig. 4), implicating miR-33a as a good prognostic marker
as a consequence of PD-1 regulation.
TCGA data analysis
A cohort of 323 lung adenocarcinoma patients from
TCGA database was further analyzed, in order to
validate our findings on a larger population. The samples
were divided into high and low miR-33a expression
groups based on the median value, and statistical analysis
revealed that high miR-33a levels were significantly
associated with low PD-1 expression (t test; p = 0.03), as we
found by NanoString methodology. Moreover, survival
analysis confirmed the association between miR-33a high
expression and a better survival (p = 0.004 for DFI, and
p = 0.007 for OS) (data not shown).
Gaining insight into the molecular basis of lung
cancers is of critical clinical relevance in order to identify
subgroups of patients for a more accurate management.
Several studies have identified miRNA signatures with
diagnostic and prognostic relevance [
potentially target hundreds of different mRNAs, thus
regulating a wide variety of cellular processes .
miR33a has been shown to have potential tumor-suppressive
activity by downregulating the expression of beta-catenin
], but an improved deeper understanding of the role of
this miRNA and its alternative antitumor mechanism is
needed. In the current study, we showed that high
miR33a expression was associated with several favorable
clinical–pathological characteristics, such as young age,
female gender, low tumor grade, and early stage.
Survival analysis also confirmed the good prognostic value
of miR-33a. Our analysis next focused on the putative
role of miR-33a in regulating the expression of immune
markers in order to account for the antitumor
function of miR-33a. The PD-1 receptor is a member of the
immunoglobulin CD28 family, playing a crucial role in
immune escape during tumor progression [
]. PD-L1 is
expressed in different cancer types, including lung
cancer, and its interaction with PD-1 plays an important role
in the blockade of the “cancer immunity cycle” [
of the most promising approaches in cancer, including
lung adenocarcinoma, is antibody blockade of the PD-1/
PDL-1 pathway [
]. This approach raises several
questions, in particular whether PD-1/PD-L1 expression
status is important in order to select patients eligible for
these treatments, which technique is best suited in
evaluating their levels, if these tumor immunity factors may be
regulated by miRNAs, and how to define a threshold for
positive PD-1/PD-L1 staining of tissue samples,
considering that certain patients respond to treatment targeting
PD-L1/PD-1, despite low or absent immunoreactivity for
these biomarkers. Previously published studies suggest
that tumor PD-L1 protein expression may be evaluated
on human cancers using immunohistochemistry (IHC)
in FFPE samples [
]; however, several factors may
impact the evaluation of PD-L1 positivity by
conventional IHC, such as the specificity and reproducibility
of the commercially available antibodies as well as the
subjectivity of the staining interpretation. Velcheti et al.
 reported a novel method of in situ measurement of
PD-L1 mRNA NanoString nCounter technology,
suggesting its utility for the accurate measurement of PD-1/
PDL-1 levels in predicting the response of lung
adenocarcinoma patients to targeted immunotherapy. NanoString
employs unamplified nucleic acids and no cDNAs or
enzymatic reaction step and is, therefore, less sensitive
to tissue fixation effects; in our experience, this platform
was extremely suitable to operate at very low levels of
expression. Our findings provide a proof-of-principle
that transcriptome-based analysis is a highly sensitive
and effective means for PD-1/PD-L1 testing.
Moreover, further analysis on a cohort of 323 lung
adenocarcinoma patients from the TCGA database confirmed our
findings on a larger population and also using a different
transcriptome-based technology, such as Illumina HiSeq
The finding that targeting the PD-1/PD-L1
mechanism is of crucial importance in lung adenocarcinoma
has led to increasing search for biomarkers predictive
of response. Even if Nivolumab, an antibody targeting
PD-1, has recently received Food and Drug
Administration (FDA) approval for NSCLC therapy, regardless
of the PDL1 expression status, a better prediction of
which patients are more likely to respond to this
cancer therapy may improve the treatment costs. Moreover,
the effects of PD-1/PD-L1 signaling in the outcome of
several neoplasms, including lung cancer, are not
completely understood due to contradictory results [
An alternative strategy in regulating immune response
to tumors could be the expression of miRNAs that
target immune checkpoint mRNAs. Using miRNA target
prediction tools, we demonstrated a direct interaction
of miR-33a and the 3′UTR of PD-1 and PD-L1 mRNAs,
with consequent downregulation of the expression of
both genes. In most circumstances, miRNAs bind to the
target mRNA at the 3′ UTR region  and few of them
were also reported to modulate genes through binding
with the CDS region [
]. The target genes of
miR33a were surveyed globally by examining 3′UTR and
CDS. Three putative binding sites of miR-33a-5p were
found in the CDS region of the CTLA4 gene with similar
Moreover, we identified a subset of lung cancer patients
with high miR-33a levels and low PD-1 expression that
had a favorable outcome, suggesting a better prognostic
value of miR-33a via PD-1 regulation. Taken together, our
findings identify a novel mechanism of tumor immune
evasion regulated by miR-33a via PD-1/PD-L1, with
potential application in clinical practice.
Our data present novel insights into PD-1/PD-L1
signaling, first, regarding a novel reliable method for their
evaluation, alternative to immunohistochemical testing,
second, concerning miRNAs as modulators of immune
checkpoint mechanisms, and finally regarding the
potential value of miR-33a as a favorable prognostic marker via
PD-1 regulation. MiRNA analysis combined with tumor
expression of immune-biomarkers will improve our
ability to select the best candidates to receive immune-based
therapies, with important clinical benefits.
LB, MG and GF conceived and designed the experiments. MG performed the
experiments. LB wrote the paper. GF diagnosed lung cancer. CN performed
FFPE sections. FM, ML, and AM performed lung surgery and follow-up. All
authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The data supporting the conclusions of this paper are included within the
Consent of publication
All authors are responsible for the submission of this article and accept the
conditions of submission.
Ethics approval and consent to participate
Our study was conducted in accordance with the ethical standards of our
institutional research committee and with the 1964 Helsinki declaration; all
the patients gave their informed consent to the molecular analyses.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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