Duality in Identifiable System Biology of DNA Injury and Contrasting DNA Repair
Lawrence M Agius*
Department of Pathology, University Of Malta Medical School, Malta Europe
Submission: November 01, 2018;Published: November 16, 2018
*Corresponding Address: Lawrence M Agius, Department of Pathology, Mater dei hospital, Tal-Qroqq, University of Malta Medical School, Malta, Europe
How to cite this article: Lawrence M Agius. Duality in Identifiable System Biology of DNA Injury and Contrasting DNA Repair. Canc Therapy & Oncol Int J. 2018; 12(4): 555846. DOI:10.19080/CTOIJ.2018.12.555846
Background: The aim of the current study was to assess the potential prognostic role of miRNAs as non-invasive biomarkers in different stages of prostate cancer (PC).
Methods: Quantitative RT-PCR analysis of plasma miRNAs from 50 PC patients [22 with localized (LPC) and 28 with metastatic disease (MPC)], 20 with benign prostatic hyperplasia (BPH) and 20 healthy normal controls (NC). The markers studied included total and free prostate-specific antigen (PSA), miR-375, miR-378, miRNA-141, and miR-18a.
Results: All markers were highly expressed in MPC and LPC group compared to BPH and NC (P<0.001), with a significant correlation with Gleason score and stage. miR-375 and miR-18a were significantly associated with clinically positive digital rectal examination (DRG), prostate volume, TNM staging of the tumor, lymph node and distant metastasis (P<0.05). The strong association of plasma miR-141, miR-375 and miR-18a with metastatic prostate cancer was observed. The sensitivity of miR-18a, miR-378, miR-375 and FPSA for the diagnosis of LPC was (95.5%, 86.4%, 81.8% and 4.5%; respectively, P<0.001) at 100% specificity. While for diagnosis of MPC, the sensitivity of miR-18a, miR-378, miR-375, miR-141 and FPSA were (60.7%, 53.6%, 78.6%, 53.6% and 96.4%; respectively) at 100% specificity.
Conclusion: circulating plasma miR-18a, miR-375, miR-141 and miR-378 could be used as prognostic markers for the differential diagnosis of localized and metastatic prostate cancer. The clinical utility of these novel biomarkers anticipates extra exploration.
Prostate cancer (PC) is considered the second most commonly diagnosed malignancy, and the fifth leading cause of cancer related deaths in males worldwide. It is associated with a variety of risk factors as age, family history, race, hormonal change and genetic abnormalities [1,2].An important prognostic indicator of prostate cancer is the histopathologic grading (Gleason score). Gleason-based grading allows classifying tumors according to their relative degree of differentiation by assigning a score from 1 (most differentiated) to 5 (least differentiated). However, it has been elucidated that tumors with similar histological patterns may provide different clinical outcomes, so Gleason score cannot exactly predict the aggressiveness of the disease although it is a powerful prognostic indicator .
prostate-specific antigen (PSA), which is the most widely used biomarker for diagnosis of PC, though it has a low specificity .also, using this marker for screening the population leads to over diagnosis of the patients with consequent overtreatment of indolent PC [5,6]. Therefore, identifying new biomarkers that could identify and differentiate different stages of PC (localized, advanced and metastatic) is highly needed.
MicroRNAs (miRNAs) are small endogenous single-stranded,
non-protein coding RNAs of about 15-22 nucleotides length.
They are important regulators of gene expression at the posttranscriptional
level because they degrade, or repress, target
mRNAs . They have an effect on different cellular processes
such as proliferation, apoptosis, differentiation, and regulation
of genes expression through modification of messenger RNA
Each individual miRNA may bind up to 200 gene targets, and
each gene also may have several binding sites for different miRNAs
. Consequently, dysregulation of the expression of miRNA may
contribute to the occurrence and progression of cancer. Recent
studies demonstrated that miRNAs even if derived from epithelial
tumors can be detected in blood, and some circulating miRNAs
derived from PC potentially correlate with the risk of disease
progression and aggressiveness [10-12]. These studies provided
evidence supporting the possible clinical use of circulating
miRNAs as non-invasive markers for monitoring disease
progression [11,13]. Therefore, the aim of the current study is
to assess the potential prognostic role of 4 different circulating
miRNAs namely: miR-375, miR-378, miR-141 and miR-18a in
patients with PC and their relation to different clinical stages.
This cohort study including 90 subjects; 50 PC patients [22
with localized prostate cancer (LPC) before radical prostatectomy
(RP) and 28 with metastatic prostate cancer (MPC)], 20 patients
with benign prostatic hyperplasia (BPH) and 20 healthy normal
controls (NC). All Patients were histologically and radiologically
proven prostate carcinoma who received no medical or surgical
therapeutic intervention before enrollment in the study. The
exclusion criteria were any severe co-morbidity or prostatitis.
The study protocol was approved by the ethical committee of
the National Cancer Institute (NCI), Cairo University, Egypt, in
accordance with the 2011 declaration of Helsinki. Informed
written consent was obtained from each participant before
enrolment. PC patients were admitted and treated at urology unit
of NCI during the period between June 2015 and January 2018.
BPH patients were recruited from Andrology unit at Kasr Al-Ainy
hospital, and the NC subjects were healthy volunteers.
All subjects underwent routine laboratory investigations and
imaging diagnosis. Staging of PC patients was done by using the
TNM staging system . Tumor aggressiveness was detected
by histological tumor grading system in the Gleason score with
≤ 7 considered low grade, and score >7 was considered highgrade.
Peripheral blood samples (7ml) were withdrawn from PC
patients before the start of any active treatment and divided into
two tubes: the first containing k2EDTA for plasma separation and
the second for serum collection. Fresh blood samples on k2EDTA
were immediately centrifuged at room temperature for 10 min at
3000 R.P.M. The plasma samples were then liquated and frozen at
˚C 80 until RNA extraction. Serum collecting tubes were left to clot
for 30 minutes and then centrifuged at 4000 R.P.M for 10 minutes.
Serum was then used for the determination of total prostatespecific
antigen (TPSA) and free prostate-specific antigen (F.PSA)
by chemiluminescence assays (Architect i1000SR Immunoassay
Analyzer, Abbott, and the U.S.A) according to the manufacturer’s
Total RNA including miRNA was separated from 200μL of
plasma using the miRNeasy Mini Kit [cat. no. 217004, Qiagen,
Germany] according to the manufacturer’s recommended
protocol.Concentration and purity of RNA samples were
measured using Nanodrop 1000 [Thermo Scientific NanoDropTM
spectrophotometer ND 1000 Wilmington USA]. Then, The RNA
was eluted in 40μL of RNase- free water and stored at -80°C until
reverse transcription (RT) reactions.
Reverse transcription of the total RNA (100 ng) was carried
out using mi Script II RT Kit [catalog no. 218161 Qiagen, Germany]
according to the manufacturer’s instructions.complementary
DNA (cDNA) was then synthesized in a thermal cycler [IGEM:
MIT/2005/Thermo cycler]. The cDNA was stored at -80°C until.
Quantitative Real-Time Polymerase Chain Reaction2.5μl of cDNA
was amplified using 10μl of TaqMan 2X Universal PCR Master
Mix II [Applied Biosystems; Thermo Fisher Scientific], 1μl of
gene-specific primers of the target miRNA and 6.5μl of nucleasefree
water in a final volume of 20μl. qPCR was run on the Step
One Real-Time PCR system [Applied Biosystems, Foster City, CA,
USA].The reaction mixtures were incubated at 95˚C for 10 min
to stimulate HotStarTaq DNA polymerase, followed by 40 cycles:
(denaturation for 15 s at 94 °C, annealing for 30 s at 55°C, finally
extension for 30 s at 70°C).
The expressions of selected miRNAs in the blood were
normalized to the expression of U6 small nuclear RNA (RNU6B).
The data obtained from the miRNA expression levels were
evaluated by the cycle threshold (Ct) method .ΔCt was
calculated by subtracting the Ct values of RNU6B from the Ct
values of the target miR-RNA. ΔΔCt was then calculated by
subtracting the average ΔCt of the healthy control samples from
the ΔCt of the patient’s samples (LPC, MPC, and BPH). The fold
change in the miRNA expression level was calculated (fold change
= 2-ΔΔCt) to determine the relative quantitative levels of target
All data were statistically analyzed using SPSS software
version 25 [IBM SPSS, Armonk, NY, USA]. Data were presented as
median and range. Comparisons between patients’ groups were
analyzed using Kruskal- Wallis test, and Mann- Whitney. MPC, and
BPH. Receiver operating characteristic (ROC) curves were used
to assess miRNAs as biomarkers, and the area under the curve
(AUC) was reported. P value < 0.05 (two-tailed) was considered
This is a retrospective cohort study conducted on 22 patients
with localized prostate cancer (LPC), twenty-eight patients with
metastatic prostate cancer (MPC) and 20 cases with benign
prostate hyperplasia (BPH), compared to 20 normal control group (NC).
The mean age of the patients was 58.8±12.48. Of all 50 cancer
patients, 9 (18%) cases were stage I, eight (16%) were stage II,
five (10%) cases were stage III, and 28 (56%) patients were stage
IV. Positive digital rectal examination (DRE) was felt in 29 (32.2%)
cases. Gleason score was 6 in 14 (28%) of patients, 8 in 17 patients
(34%), and 9 in 12 (24%) patients, while only 2 (4%) patients
had Gleason score 10. Prostate volume was more than 50g in 37
(74%) patients. 15 (30%) patients had lymph node metastasis
while 28 (56%) patients had distant metastasis (Table 1).
There was a statistically significant difference among all
patients’ groups regarding TPSA plasma expression (P<0.001). However, regarding FPSA; there was no statistically significant
difference between BPH groups and LPC (P=0.91), while there
was a significant difference among NC, LPC and MPC groups
(P<0.001). Also, regarding F/TPSA; there was no statistically
significant difference between NC and BPH groups (P=0.56), while
there was a significant difference among NC, LPC and MPC groups
There was no statistically significant difference between NC
and BPH groups regarding plasma expression of miR-141, miR-
375 and miR-18a (P = 0.083, 0.661 and 0.684; respectively).
Moreover, there were significant differences among LPC, MPC and
non-malignant groups (NC and BPH) regarding plasma expression
of miR-141, miR-375 and miR-18a (P<0.001, for all), however
there was no significant difference in the expression level of miR-
141 between LPC and non-malignant groups (NC and BPH). There
was a statistically significant difference among all tested groups;
NC, BPH, LPC, and MPC ((P<0.001) regarding miR-378 expression
(Table 2)(Figure 1).
*Data are expressed as median and range
#Data having the same letters in the same variable are statistically similar
ROC curve analysis for the tested miRNAs and FPSA in
localized prostate cancer and non-malignant cases.
As relative median miRNAs expression was differentially
expressed in the peripheral blood of PC patients, BPH patients
and control subjects. The potential of peripheral blood oncogenic
ROC curve analysis was performed for FPSA, miR-375, miR-378,
and miR-18a to differentiate patients with LPC and those with
BPH and NC. MiR-375 showed AUC, 0.919; 95% CI, 0.828-1.000
(P<0.0001), miR-378 showed AUC, 0.939; 95% CI, 0.857-1.000
(P<0.0001), and FPSA showed the lowest AUC, 0.704; 95% CI,
0.577-0.831 (P<0.008). MiR-18a showed the highest AUC, 0.996;
95% CI, 0.987-1.000 (P<0.0001). The sensitivity of miR-18a, miR-
378, miR-375 and FPSA were (95.5%, 86.4%, 81.8% and 4.5%;
respectively) at 100% specificity (Figure 2A).
By adding miR-378 to miR-18a for detecting patients with
LPC, the sensitivity (95.5%) didn’t increase at a specificity 100
%, AUC was 0.997 and 95%CI was 0.988-1.0; <0.0001 (Table 3)
(Figure 2B).ROC curve analysis for the tested miRNAs in localized
prostate cancer and metastatic cases.ROC curve analysis was
performed for FPSA, miR-375, miR-141, miR-378 and miR-18a
to differentiate patients with LPC and those with MPC. It showed
that FPSA had the highest AUC, 0.996; 95% CI, 0.986-1.000
(<0.0001). miR-375 showed AUC, 0.911; 95% CI, 0.817-1.000
(<0.0001). MiR-141 showed AUC, 0.925; 95% CI, 0.854-0.996
(<0.0001). MiR-18a showed AUC, 0.966; 95% CI, 0.922-1.000
(<0.0001). While miR-378 showed the lowest AUC, 0.828; 95%
CI, 0.704-0.952 (P=0.002). The sensitivity of miR-18a, miR-378,
miR-375, miR-141 and FPSA were (60.7%, 53.6%, 78.6%, 53.6%
and 96.4%; respectively) at 100% specificity (Figure 3A).
By combining FPSA and miR-378 for differentiating patients
with MPC, it didn’t affect the sensitivity (96.4%) at a specificity
100%, AUC is 0.982 and 95%CI is 0.946-1.0; P<0.0001 (Table
4) (Figure 3B).Correlations between miRNAs, PSA and clinicpathological
features of the patients.There was significant
correlation between the tested miRNA (miR-18a, miR-378, miR-
375 and miR-141) with PSA, FPSA, Gleason score and pathological
stages of the tumor (P<0.01). while there was significant inverse
correlation with F/TPSA (P<0.05, (Table 5).
miR-375, miR-18a, TPSA, FPSA, and F/TPSA were significantly
associated with PR volume (P=0.005, 0.006, 0.001, 0.001 and
0.025; respectively), DRE (P=0.018, 0.013, 0.003, 0.006 and 0.007;
respectively) and nodal metastasis (P=0.016, 0.012, 0.001, 0.001
and 0.002; respectively). Also, miR-141 associated significantly
with nodal metastasis (P=0.005). miR-378 and miR-141 didn’t
associate significantly with age, PR volume and DRE (P>0.05)
miRNAs play an important promising role in the prediction
and progression of prostate cancer . In the current study, we
investigated the role of miR-375, miR-378, miR-141 and miR-
18a in the diagnosis of LPC and MPC compared to BPH and NC
subjects.Our results demonstrated that the expression levels
miR-375, miR-378 and miR-18a follow an increasing trend with
disease progression. These data agreed with Nguyen et al. 
who demonstrated an elevated expression of circulating miR-
375, miR-378 and miR-141 in patients with metastatic castration
resistant prostate cancer compared to those with low-risk LPC.
However, our data regarding the expression level of miR-141 is
controversial with other several studies, we found that miR-141 is
only significantly expressed in MPC, while there was no significant
difference between LPC and benign groups (BPH and NC). Our
data were consistent with that reported by Agaoglu et al. who
reported that plasma miR-141 could only distinguishes localized
from metastatic prostate cancer patients , as miR-141 is a
member of the miR-200 family, which has an essential function in
epithelial-to-mesenchymal transition [20,21].
However, it was in contrary to Mitchell et al. who demonstrated
elevated expression of circulating miR-375 and miR-141 in
patients with prostate cancer in comparison to healthy control
. Similarly, other previous studies reported increased
expression of miR-375 and miR-141 in prostate cancer patients
with advanced disease compared to patients with earlier stages
of prostate cancer or healthy tissues [8,10,22, 23]. miRNA-375
potentially down-regulates Sec23A in prostate cancer cell lines
resulting in enhanced proliferation, indicating that miR-375 may
have a role in promoting cell growth .
Regarding miRNA18a, we found increased expression
of miRNA18a in LPC and MPC patients compared to normal
control. These data were consistent with that reported by He
et al. . Hence, MiR-18a target a serine/threonine-protein
kinase 4 that acts as a tumor suppressor in prostate cancer .
The upregulation of miR-18a binds directly to the 30UTR of the
STK4 mRNA, and by downregulating STK4 at the protein level,
it thus suppresses apoptosis and promotes tumor survival .
We also found a significant association of miR-375 and miR-
18a with clinically positive digital rectal examination (DRG), prostate volume, TNM staging of the tumor, distant and lymph
node metastasis which confirm their possible uses as prognostic
markers for PC patients.Our data showed increased serum FPSA
level in metastatic groups and thus provide an easy and simple
way for detection of patients with metastasis. Therefore, it can be
used in the follow up of the disease progression, not only in early
detection of PC in patients with grey zone PSA level as approved
by FDA (5-9).
This was confirmed by assessing the diagnostic power of the
tested miRNAs together with FPSA. we found that FPSA could
differentiate accurately patients with MPC with 96.4% sensitivity
and 100% specificity, followed by miR-375 which achieved 78.6%
sensitivity and 100% specificity. Whereas for the diagnosis of
patients with LPC, our results showed that miR-18a could detect
LPC patients with 95% sensitivity and 100% specificity. As PC is a
heterogeneous and complex disease, thus it is potentially needed
to search for a panel of markers, including miRNAs that could help
in diagnosing of such patients.
In conclusion, our results confirmed the great potential of
circulating miR-18a, miR-375, miR-141 and miR-378 as prognostic
biomarkers for the differential diagnosis of metastatic prostate
cancer from localized indolent prostate cancer, which may
produce clinical different aspects of prostate cancer management,
and thus deserve further investigation in a large scale study to
further confirm its usefulness in the identification of occult micrometastasis
which is too small to be detected with conventional
imaging techniques. additional studies of these miRNAs’ roles in
pathogenesis of dissemination of metastases will still be needed
to developed targets to future curative therapy of PC.