METHODS
In this study, we enrolled plasma samples of 65 CRC patients (50 stages III/IV and 15 stages I/II) and
plasma samples of 31 individuals in the control group of similar ages. Thereafter, we performed plasma
separation and total RNA extraction; then, RNAs were reversely transcribed to complementary DNA.
And then, we analyzed using a quantitative real?time polymerase chain reaction technique for lncRNA
expression analysis.
RESULTS
Our results showed that the expression levels of ZNRF3IT1 (p=0.011), CCAT1 (p=0.007), CRNDE
(p=0.002), and XLOC_000303 (p=0.001) were significantly upregulated in the CRC when it was compared
to with the control group. ZNRF3IT1, CCAT1, CRNDE, and XLOC_000303 lncRNAs were observed
to have similar discriminating power. The calculated area under the curve of receiver operating
characteristic was 0.66, 0.67, 0.70, and 0.70, respectively.
CONCLUSION
Our results revealed a high discriminatory power of ZNRF3-IT1, CCAT1, CRNDE, and XLOC_000303
lncRNA in distinguishing CRC patients from healthy individuals. We also found that increased
XLOC_000303 expression is a protective factor against metastasis formation. This study demonstrated
that ZNRF3-IT1, CCAT1, CRNDE, and XLOC_000303 circulating lncRNAs may be used as a potential
non-invasive biomarker in CRC. We think that the increased expression of XLOC_000303 may be a
protective factor against metastasis formation.
Keywords: Circulating lncRNA; colorectal cancer; plasma; real-time polymerase chain reaction
Understanding the molecular basis of colorectal
carcinogenesis is important for both the prognosis and
treatment of CRC. It is suggested that individualization
of the treatment according to the pathological and
molecular characteristics of the tumors and the better
evaluation of the disease stage may have effective results
in the progress of the disease. Many studies have
been carried out in recent years to elucidate the molecular
mechanisms of genetic factors that are effective
in the development of CRC. Data, from these studies,
have shown that circulating ncRNAs such as miRNA
and lncRNA play a role in the progression of tumorigenesis,
invasion, and metastasis in carcinomas.[
Long non-coding RNAs represent a heterogeneous
group of RNAs ≥200 bp in size. LncRNAs effect to the
regulation of numerous processes such as the cell cycle,
apoptosis, histone modifications, chromosome
imprinting, and cell differentiation.[
The present study evaluated the possibilities of using
circulating candidate lncRNAs as a non-invasive
biomarker in CRC and also discussed, which is the
power of these circulating lncRNAs to discriminate between
patient and control groups.
Study Population
In this study, 96 participants were enrolled and divided
into two groups. The first group included
plasma samples of 65 patients who had been diagnosed
with colon cancer or rectum cancer in the
Department of Medical Oncology between February
2007 and April 2021. The second group included 31
age- and sex-matched healthy individuals who did
not have a history of malignant disease in the study as
a control group. In addition, the case group was evaluated
in two subgroups as early-stage (15 patients)
and advanced stage (50 patients).
Plasma Preparation and Total RNA Extraction
Peripheral blood samples were collected from each participant
in the study and were placed in the EDTA-anticoagulant
tube. Blood samples were centrifuged within
2 h after collection at 3000 rpm for 15 min at +4ºC,
followed by 3000 rpm for 10 min at +4 ºC, and plasma
was separated. The supernatant plasma was recovered
and stored at -80ºC until analysis. We extracted total
RNA from 800 µl plasma by Trizol reagent according to
the protocol of the manufacturer"s instructions (A.B.T.
Blood/Tissue RNA Purification Kit for Leukemia, Atlas
Biotechnology Laboratory Materials Industry and
Trade Ltd. Şti, Ankara, Turkey) and stored at -80°C until
analysis. The purity and concentration of RNA were
assessed using the Qubit device (Qubit 3 Fluorometer,
Invitrogen, by Thermo Fisher Scientific, Malaysia).
cDNA Synthesis
Total RNA was reversely transcribed and cDNAs were
synthesized using a reverse transcription kit (RevertAid
H Minus First Strand cDNA Synthesis Kit;
Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania)
according to the manufacturer's instructions. RNA
was added to reverse transcription master mix (4 µl) to
a final volume of 20 µL. Then, incubation period (including
5 min at 65ºC), cDNA was synthesized with the
following program of 5 min at 25ºC, 60 min at 42ºC, and 5 min at 70ºC. cDNA Synthesis reactions were
completed in a Thermocycler (GeneAmp PCR System
9700, Applied Biosystems, Singapore).
Quantitative Real-Time Polymerase Chain Reaction
(PCR) for lncRNA Expression Analysis
Quantitative RT-PCR (qRT-PCR) was performed using
CFX96 real-time PCR systems (BIO-RAD, C1000
Touch Thermal Cycler, Bio-Rad Laboratories, Inc, California,
USA). PCR amplification mix contained 10 µl
SYBR Green Master Mix (Thermo Scientific? Maxima
SYBR Green/ROX qPCR Master Mix (×2), Thermo
Fisher Scientific Inc, California, USA), 2 µl of cDNA
product, 2 ?l of primer, and 6 ?l of RNase-free water.
Thus, a final volume was completed to 20 ºL. Β-actin
was used as a reference gene. The real-time PCR amplification
mix was incubated at 95°C for 10 min for Taq
activation, followed by 95 ºC for 15 s for denaturation
and 60ºC for 40 s annealing and extension during 40
cycles. The changes in the plasma expression levels of
candidate lncRNAs were investigated using a qRT?PCR
technique by lncRNA?specific oligonucleotide primers
(Table
Statistical Analysis
All data were statistically analyzed and graphically
represented using the Statistical Package for the Social
Sciences (SPSS) 21.0 software (SPSS Inc., Chicago, IL).
The conformity of quantitative variables to normal distribution
was evaluated with the Shapiro-Wilk test. The
comparison of the two groups was made with the t-test for normally distributed variables, and with the Mann-
Whitney U-test for non-distributed variables. The
relationship between qualitative variables was evaluated
with Chi-square analysis, and the relationship between
quantitative variables was evaluated with Spearman
correlation analysis. The discriminating power of
IncRNA expressions, which are suggested to be used in
distinguishing the patient and control groups, the areas
under the curve (AUC), sensitivity, and specificity values
were evaluated by receiver operating characteristic
(ROC) analysis. Survival analysis evaluations and life
functions graphs of the diagnosis period were obtained
by the Kaplan-Meier method. Cox regression analysis
was used to assess the metastasis risk of candidate circulating
lncRNAs. P<0.05 was considered significant.
This work received ethics committee approval (Ethics
committee no: 2019-349). A signed and written informed
consent form was obtained from the individuals
in the patient and control groups.
Evaluation of Plasma lncRNAs Expression Levels
in CRC Patients, Compared to the Control Group
In our results, ZNRF3-IT1, CCAT1, CRNDE, and
XLOC_000303 circulating lncRNAs (Fig.
CRC: Colorectal cancer; lncRNAs: long non-coding RNAs.
Evaluation of Expression Level in Candidate Circulating
lncRNAs between Stages in CRC Patients
The findings showed that only the XLOC_000303
lncRNA was significant between the stages (p=0.001). In addition, according to Cox regression analysis, it
was determined that the XLOC_000303 lncRNA was
a protective factor against the metastasis formation
(p=0.045, HR=0.90, 95%CI=0.812-0.997) (Table
Prognostic Value of Expression Level of Candidate
lncRNAs (ROC and AUC Analyzes)
To use candidate circulating lncRNAs as a biomarker,
the AUC of ROC of the test was calculated. The patient
group and control group were compared. The AUC of
ROC of ZNRF3-IT1 (95% confidence interval [CI]:
0.531-0.790; p=0.011; AUC: 0.660), of CCAT1 (95%
CI: 0.534-0.810; p=0.007, AUC: 0.672), of CRNDE
(95% CI: 0.586-0.814; p=0.002, AUC: 0.700), and of XLOC_000303 (95% CI: 0.579-0.833; p=0.001, AUC:
0.706) was detected (Fig.
CRC: Colorectal cancer.
Survive Analyzes of Candidate lncRNAs
The median value for the overall survival analysis of the
cases could not be calculated. Therefore, overall survival
analysis could not be evaluated. The mean survival time
after diagnosis of metastasis was 69.95±14.528 months
(95% CI: 41.480-98.429) and a median of 39.00±3.968
months (95% CI: 31.223-46.777) (Fig. 3). The mean
progression-free survival time was 86.864±17.564
months (95% CI: 52.439-121.289), and the median was
39.00±5.544 months (95% CI: 28.134-49.866) (Fig.
In this study, seven lncRNAs (ARHGAP5-AS1, LOC152578, SNHG16, ZNRF3-IT1, CCAT1, CRNDE, and XLOC_000303) were selected to investigate their expression profiles in the plasma of CRC patients. Expression analysis results showed that the case group had significantly higher plasma expression levels of ZNRF3-IT1 (p=0.011), of CCAT1 (p=0.007), of CRNDE (p=0.002), and XLOC_000303 (p=0.001). By further analysis, it was revealed that there is a high discriminatory power of these genes (ZNRF3-IT1 [AUC=0.66; p=0.012], CCAT1 [AUC=0.67; p=0.007], CRNDE [AUC=0.70; p=0.002], and XLOC_000303 [AUC=0.706; p=0.001]) in plasma. Moreover, we found that there may be potential for use as clinical biomarkers in plasma.
ZNRF3-IT1 is lncRNA that belongs to a class of
sense intronic ncRNA. It has been reported to cause
cellular proliferation in various cancer types such as
CRC, breast cancer, and hepatocellular carcinoma.[
In our study, we were determined that the ZNRF3-
IT1 lncRNA was upregulated an average of 0.22-fold
in the CRC patient group when compared to the control
group. We found that the expression profile of
this circulating lncRNA showed a statistically significant
difference in the patient group, while it was compared
with the controls (p=0.011), but no difference
was found according to the stages. This suggests that
ZNRF3-IT1 may alter the tumor suppressor function
of target gene ZNRF3 of it. Our findings showed that
lncRNA ZNRF3-IT1 has a strong distinguishing feature
between the patient and control group, and also it
can be used as a biomarker.
CCAT1 has been identified as an oncogene in
CRC. It was found that CCAT1 expression was highly
upregulated in the tumor sample, regional node, distant
liver metastasis, and plasma samples in CRC.[
In this study, we determined CCAT1 lncRNA was
upregulated by an average of 0.006-fold in the CRC
patient group, and there was a significant difference
between the patient and the control (p=0.007), but no
significant difference was found between the stages. A
strong correlation was found between the expression
level of CCAT1 and tumor size (p=0.021). Our results
support the informations in direction that CCAT1 increases
proliferation and invasion in CRC. It has been
observed that there is a strong discrimination feature
between the patient group and control group. These
data suggest that CCAT1 lncRNA can be used as a
biomarker in CRC patients.
CRNDE lncRNA is localized next to the IRX5 gene.
[
In this study, it was found that CRNDE was upregulated
by an average of 0.008-fold in the CRC patient
group, with a statistically significant difference
in the patient group (p=0.002), but there was no significant
difference between the stages. A strong association
was found between the expression level of
CRNDE, tumor size (p=0.010), tumor differentiation
(p=0.023), and primary tumor location (p=0.009).
Our findings are concordant with the literature and
support the information that CRNDE is effective in
processes such as proliferation, migration, invasion,
and suppression of apoptosis. However, when the relationship
between CRNDE and neoadjuvant therapy
was examined in our study, no statistically significant
difference was found. This finding contradicts
the knowledge that CRNDE has a role in oxaliplatin based chemotherapy resistance.[
XLOC_000303 is a newly identified intergenic
lncRNA. It has been suggested that it is highly expressed
in the plasma of CRC patients and can be
used as a new and rapid diagnostic biomarker.[
Limitations of the Study
Our study has some limitations. First, the study population
was relatively small. Working with a larger population
in the future may improve the diagnostic and
prognostic accuracy of the lncRNAs. Second, median
values could not be calculated, because the follow-up
period was short and the number of deaths were not
sufficient in the overall survival analysis of the patients.
In addition to the expression profile, additional studies
on polymorphic and mutant variants may contribute to
explain the molecular mechanism.
Acknowledgements: This work accepted as a doctoral thesis in the field of medical genetics.
Peer-review: Externally peer-reviewed.
Conflict of Interest: All authors declared no conflict of interest.
Ethics Committee Approval: The study was approved by the Eskisehir Osmangazi University Non-interventional Clinical Research Ethics Committee (No: 2019-349, Date: 10/12/2019).
Financial Support: This study was supported by Eskişehir Osmangazi University Scientific Research Projects Coordination Unit with the project code 202011036.
Authorship contributions: Concept - S. Arslan, O.Ç., M.D., A.E.S., S. Artan; Design - S. Arslan, O.Ç., M.D., A.E.S., S. Artan; Supervision - O.Ç.; Funding - None; Materials - D.B., D.A.; Data collection and/or processing - S. Arslan, D.B., E.E.G., S.K., D.A., H.Ö., B.D.A.; Data analysis and/or interpretation - S. Arslan, A.E.S., E.E.G., S.K., D.A., H.Ö., M.D., B.D.A., S. Artan, O.Ç.; Literature search - S. Arslan, O.Ç.; Writing - S. Arslan, D.B., E.E.G., S.K., B.D.A., O.Ç.; Critical review - O.Ç.
