MiR-141-3p Relative Expression Level from FFPE Samples as Biomarker of Prostate Adenocarcinoma Carcinogenesis in Yogyakarta.
Indonesia Sari Eka Pratiwi1.
Sri Nuryani Wahyuningrum2.
Rachma Greta Perdana Putri3.
Danarto4.
Didik Setyo Heriyanto5.
Nur Arfian6.
Sofia Mubarika Haryana7.
Indwiani Astuti8 Department of Biology and Pathobiology.
Faculty of Medicine.
Universitas Tanjungpura.
Indonesia Magelang Unit of Health Research and Development.
Ministry of Health.
Indonesia Department of Histology.
Faculty of Medicine.
Universitas Ahmad Dahlan.
Yogyakarta.
Indonesia Department of Urology.
Sardjito Hospital.
Yogyakarta.
Indonesia Department of Anatomy Pathology.
Faculty of Medicine.
Public Health and Nursing.
Universitas Gadjah Mada.
Yogyakarta.
Indonesia Department of Anatomy.
Faculty of Medicine.
Public Health and Nursing.
Universitas Gadjah Mada.
Yogyakarta.
Indonesia Department of Histology and Cell Biology.
Faculty of Medicine.
Public Health and Nursing.
Universitas Gadjah Mada.
Yogyakarta.
Indonesia Department of Pharmacology.
Faculty of Medicine.
Public Health and Nursing.
Universitas Gadjah Mada.
Yogyakarta.
Indonesia Correspondence:
Sari Eka Pratiwi.
Jl.
Prof.
Dr.
Hadari Nawawi.
Pontianak.
West Kalimantan.
Indonesia Zip Code: 78124 Email: sariekapratiwi@medical.
Received: September 7, 2021 Revised: October 1, 2021 Accepted: February 4, 2022 Published: April 28, 2022 DOI: 10.
33086/ijmlst.
Abstract Globally, prostate cancer (PCA) is the second leading cause of male cancer-associated mortality.
Micro-RNAs .
iRNA.
are small non-coding RNAs considered promising biomarkers for diagnosis, prognosis, and treatment options.
A miR-141 expression is frequently dysregulated and influences the development and progression of PCA.
This study aimed to identify miR-141 expression level as a marker to differentiate PCA from another prostate anomaly, especially in YogyakartaIndonesia.
Formalin-fixed paraffin-embedded (FFPE) tissues for each three groups: benign prostatic hyperplasia/BPH, high-grade prostatic intraepithelial neoplasia/HGPIN, and PCA .
=7/grou.
were stored in a commercial clinical laboratory in Yogyakarta.
The total RNA was extracted from FFPE sections using miRNeasy FFPE kit, followed by the quantification of miR-141-3p expression level by RT-PCR.
The result showed that miR141 relative expression level on PCA was higher than other groups and significantly different (P<0.
Kruskal Wallis The mean of the miR-141 relative expression level of BPH.
HGPIN, and PCA were 1.
04A0.
87, 6.
44A7.
8, and 06A8.
83, respectively.
The relative expression level of miR-141 can potentially be a prognostic biomarker in PCA and could differentiate aggressiveness in prostate anomaly, especially BPH.
HGPIN, and PCA.
Keywords BPH.
HGPIN, miR-141.
PCA.
Prostate Anomaly Markers.
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A2021 by author.
Sari Eka Pratiwi, et al.
RNAs .
RNA.
It silences the mRNAs by INTRODUCTION Prostate cancer (PCA) is the most common malignancy in men and the second leading cause of male cancer-associated miRNAs can lead to some diseases such as mortality around the world .
The early cancer, indicating their essential role in PCA stage is localized and can be treated carcinogenesis .
Ae.
The chemotherapy, radiation therapy, radical prostatectomy, cryotherapy, and others.
prognosis, and treatment options in several Unfortunately, following initial treatment, solid cancers, including PCA .
miRNAs around 23-40% of these patients will acquire tend to be oncomir or tumor suppressor miRNAs.
As a tumor suppressor, miRNA frequently spread to the bone and other targets the oncogenic mRNA .
miR-141 organs, resulting in death .
is one member of the miR-200 family .
ir- Prostate Today, miRNA with a range of treatments, including Although PCA is ordinary, there are no .
that plays a role in proliferation, diagnostic or prognostic biomarkers that can epithelial-mesenchymal transition (EMT), be used to determine its aggressiveness.
The migration, invasion, and drug resistance prostate-specific antigen (PSA) test, utilized .
in the clinic as a standard assay, is not miR-141 specific for PCA.
Infections, inflammation, expression is frequently dysregulated and hyperplasia, and other things can affect PSA influences the tumor's development and levels to vary.
These limitations of PSA level (MicroRNA.
in many types of cancer also overtreatment .
Recent research has modulates cellular motility and regulates demonstrated the capability of nucleic acids miR-141 is commonly known to as a disease marker.
MicroRNA .
iRNA) is have a dual role in tumor development, as a a type of RNA that has been demonstrated to tumor suppressor gene or an oncomir be a disease marker .
Ae.
depends on the type and severity of cancer.
Dysregulation miR-141 Micro-RNAs .
iRNA.
are small This phenomenon provides more benefit of non-coding RNAs and one of the regulatory miR-141 utilization for therapeutic targeting RNAs arranged by 18-25 nucleotides.
agents, diagnostic or prognostic biomarkers miRNAs .
transcriptional regulation of gene expression degrading or inhibiting the mRNA translation by targeting almost 30% of all messenger miR-141-3p .
icroRNA-141-3.
directly inhibits mRNA ZEB1 .
inc finger EIna.
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box binding homeobox .
and ZEB2 .
inc neoplasia (HGPIN) and benign prostatic finger E-box binding homeobox .
, a gene hyperplasia (BPH), based on formalin-fixed that plays an essential role in the EMT paraffin-embedded (FFPE) tissue sample of process of invasive tumors .
Conversely, patients from Yogyakarta.
Indonesia.
one study in pancreatic, colorectal, and breast cancer cell lines showed that ZEB1 triggers a MATERIALS AND METHODS miRNA-mediated feed-forward loop by Sample collection directly inhibiting miR-141 transcription.
The study was approved by the Medical which stabilizes EMT and promotes invasion and Health Ethics Committee.
Faculty of of cancer cells .
Medicine.
MiR-141 also repressed STAT4 (Signal Universitas Gadjah Mada.
Yogyakarta, with approval number of Transducer and Activator of Transcription .
KE/FK/0888/EC/2018.
and targeting transcriptional co-activator collected from Formalin-Fixed Paraffin- with PDZ-binding motif that regulates cancer Embedded (FFPE) blocks stored in a invasion and proliferation in gastric cancer commercial clinical laboratory in Yogyakarta .
Meanwhile, as an oncomir, miR-141 from 2017 until 2018.
As many as seven was found up-regulates in many prostate FFPE blocks from each BPH.
HGPIN, and cancer studies compared to healthy controls PCA group were sliced into five sections with .
a 10-AAm thickness each and prepared for bone-metastatic Samples prostate cancer, the miR-141 expression level RNA extraction.
was elevated and positively correlated with Total the bone lesion .
Transcriptase-PCR (RT-PCR) RNA Reverse Total RNA extracted from FFPE sections a deeper understanding of tumorigenesis and with miRNeasy FFPE Kit (Qiagen.
Germany.
Cat No.
After FFPE blocks were prognostic biomarker in the development of sliced, the sections were placed in safe lock prostate cancer.
Studies of miR-141 in tubes .
5 mL), and 160 AAL Xylene was prostate cancer and another prostate anomaly added to each tube for the deparaffinization were rarely done in Indonesia, especially in The RNAs were eluted using 30 AAL Special Region of Yogyakarta, in the form of RNase-free water.
cDNA synthesis was FFPE sample.
Therefore, in this study, we performed with miRCURY LNA RT Kit aimed to identified miR-141 expression level (Qiagen.
Germany.
Cat.
No 339.
For the as a marker to differentiate prostate cancer synthesis of cDNA from miRNA, the RNA (PCA) to high-grade prostatic intraepithelial template was diluted by adding nuclease-free Ina.
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water until the concentration achieved 5 ng/ After the iiCq results were AAL, then placed 2 AAL into 0.
2 mL tube.
obtained, the next step was to analyze the Quantitative Real Time PCR For miR- target microRNA expression by the Livak 141-3p method with the formula was 2Ae.
The Quantification miR-141-3p type of quantification in this study used ggUAGaUGGUCU--GUCACAAu 5') in relative quantification by comparing the FFPE section used miRCURY LNA miRNA groups of HGPIN and PCA to BPH.
The PCR Assay Kit (Qiagen.
Germany.
Cat No.
Mann-Whitney test and Kruskal-Wallis test 339.
in BIORAD CFX96 real-time were used to analyze the differences of miR- The preparation step was started by 141 expression levels in the tumor tissue diluting 1 AAL cDNA template with 59 AAL nuclease-free water .
, then placed 4 AAL characteristics (P value significant < 0.
diluted cDNA template into a white qPCR strip tube.
RESULTS
The initial PCR condition was 95 C for Subject characteristics 10 minutes, followed by 40 cycles of denaturation step at 95 C for 10 seconds, with This study involved 21 subjects that consisted of 7 BPH patients, 7 HGPIN annealing and extension steps at 58 C for 1 patients, and 7 PCA patients.
Patients were This study used U6 for internal dominated by the elderly, with an average of control and normalized miRNA.
H2O for age was 68 years old.
Gleason score for PCA negative control, and all the samples were patients showed 2 people having medium- performed in duplicate.
grade cancer .
and 5 people having miR-141-3p expression was analyzed high-grade cancer .
core 8 and .
Detail for based on the value of iCq of miR-141-3p, subject characteristics is shown in Table 1.
which was normalized with iCq of U6.
Then.
Mean difference of miR-141-3p relative the expression was calculated using the Livak MiR-141 relative expression level of The microRNA amplification curve was three groups of subjects was significantly read as Cycle Quantification (C.
The Cq of different (P<0.
Kruskal Wallis tes.
The miR-141-3p value was then normalized with difference showed that the miR-141 level in the Cq of U6 value, and the value of iCq was PCA samples was higher than HGPIN and Then the iiCq quantification BPH samples.
The mean difference of miR- with the comparison was the average value of 141 in the three groups is described in Figure the BPH groupAos iCq (As a comparative Ina.
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Table 1.
Subject characteristics of samples Characteristics of Subject Age .
, mean A SD .
in Ae ma.
BPH
HGPIN
PCA
Prostate anomaly status .
, %) BPH
HGPIN
PCA
Gleason score for PCA samples .
Score 7 Score 8 Score 9 Grade of PCA samples .
Grade 3
Grade 4
Grade 5
Relative expression mean of miR-141-3p
BPH
HGPIN
PCA
71A11.
- .
71A11.
- .
29A12.
- .
14A10.
- .
04A0.
44A7.
06A8.
BPH: Benign prostatic hyperplasia.
HGPIN: High-grade prostatic intraepithelial neoplasia.
PCA: Prostate cancer P=0.
P=0.
Mean of miR-141 realtive expression level .
old-chang.
P=0.
7,06
6,44
1,04
BPH
Pca
HGPIN
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: 1Ae9 Figure 1.
Clustered coloumn of miR-141 mean relative expression in benign prostatic hyperplasia .
=7.
BPH), prostate cancer .
=7.
PCA), and high-grade prostatic intraepithelial neoplasia .
=7.
HGPIN).
*P=0.
023,three-way Kruskal-wallis test.
**P=0.
009 BPH versus Pca, **P=0.
048 BPH versus HGPIN.
P=565 Pca versus HGPIN.
Mann-whitney test Sari Eka Pratiwi, et al.
early PCA but not in the advanced stages DISCUSSION Regulation of miR-141 in prostate cancer development involves complex mechanisms The results of this study are in line with between the target gene and other interfering studies conducted by Cheng et al.
, .
which Considering that regulation of miR-141 was cancer type-dependent, the expression level of miR-141 could be different based on cancer severity.
Our undergone metastasis compared to the serum finding shows that the miR-141 expression of healthy patients.
miR-141-3p levels of prostate cancer patients was Cancer metastases involve angiogenesis, significantly higher than HGPIN and BPH a formation of new blood vessels, which is Sample of prostate cancer tissue required for tumor expansion, and sufficient with upregulating miR-141 expression was tumor nutrition.
A previous study showed known as a prostate cancer patient in a that overexpression of miR-141 correlated condition of grades 3, 4, and 5.
In this term, with higher blood vessel formation in mouse we could report that miR-141 in the high models and inducing secretion of VEGFA stadium of prostate cancer, in this case, .
ascular endothelial growth factor-A), one grades 3-5, played a role as oncomir.
of the potent blood vessel growth factor Oncomirs or oncogenic miRNAs are miRNA .
Another mechanism that relates miR-141 which are associated with cancer.
Various miRNAs act as EMT repressors, in worsening tumor conditions is inducing particularly inhibiting the ZEB1 expression, oxidative stress response.
A prior study which involves miR-200s families .
iR- reported that miR-141 could modulate higher 200a, miR-200b, miR-200c, miR-141, and reactive oxygen species (ROS) in oxidative miR-.
The function of the miR-200s families is to promote MET and inhibit EMT microenvironment, by directly targeting the p38a gene that blocks proliferation and ways.
ZEB1 represses the gene transcription promotes apoptosis.
This condition implies of miR-200s by directly binding on the gene's disruption of cellular components, induces promoter region .
MiR-141-3p is cell proliferation, and stimulates tumor growth .
ZEB1.
mRNA ZEB1 and regulates the Androgen Receptor (AR) Fluctuation of miR-141 expression level mRNA expression by targeting the ARAos in any cancer was capable of distinguishing open reading frame (ORF), particularly at malignant or non-malignant tissue and used Ina.
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to differentiate cancer staging .
In a of the patients.
A large number of prostate prostate cancer, overexpression of miR-141 cancer patient subjects is required to validate is the most potent biomarker of metastatic diagnostic and prognostic relevance of miR- It can differentiate prostate cancer 141 to represent prostate cancer progression.
patients with local advanced from those with metastasis .
In line with our results, a CONCLUSIONS study reported that a higher level of miR-141 In conclusion, our research has shown was related to more aggressive and advanced that miR-141 level in a higher grade of PCA disease of prostate cancer .
igh Gleason plays a role as oncomir and can be a score, large tumor size, and lymph node prognostic biomarker.
The miR-141 level Upregulation of miR-141 also also could differentiate aggressiveness in associated with increased risk of biochemical BPH.
HGPIN, and PCA.
Other studies confirm that overexpression of serum miR-141 could be used to differentiate non-metastatic from AUTHOR CONTRIBUTIONS Sari Eka Pratiwi: conceptualization, .
Nuryani Sri Otherwise, a recent study revealed that higher Wahyuningrum: software, data curation, and expression of miR-141 enforces a strong writing-editing.
Rachma Greta Perdana Putri:
epithelial phenotype and minimizes partial Danarto: conseptualization:
loss of mesenchymal phenotype by directly Didik Setyo Heriyanto: supervision.
Nur binding to the ZEB1 gene, an EMT Arfian:
This Sofia Mubarika Haryana:
correlated with miR-141 down-regulation in supervision and validation.
Indwiani Astuti:
metastatic prostate cancer patients .
Our study, to our knowledge, is the first reviewing and editing.
study to look for the possible role of miR-141 on prostate cancer compared to high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia in Indonesian people using FFPE samples.
Our result suggests that elevating miR-141 expression shows a higher stadium of prostate cancer.
This study had Ina.
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: 1Ae9 This work was supported by a research grant from Faculty of Medicine.
Public Health, and Nursing 2018.
This work was conducted at the Laboratory of Anatomy.
Molecular
Biology Laboratory, integrated laboratory of Faculty of Medicine, limitations in representing the small number
ACKNOWLEDGEMENTS
Sari Eka Pratiwi, et al.
Public Health Nursing.
UGM.
Yogyakarta.
CONFLICT OF INTEREST
There are no conflicts of interest.
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