Plasma miR-22-3p, miR-642b-3p and miR-885-5p as diagnostic biomarkers for pancreatic cancer

Abstract

Background

Diagnosis of pancreatic cancer (PC) by using sensitive and specific biomarkers is considered necessary. MiRNAs are master regulators of gene expression and several biological processes, and they are dysregulated in various cancers, where they play a vital role in either cancer progression or suppression. So, this study was designed to investigate the role of plasma miR-22-3p, miR-642b-3p and miR-885-5p expression as possible diagnostic markers in PC patients as compared to serum CA19-9. In addition, the correlation of those miRNAs and CA19-9 with clinical characteristics of PC patients was analyzed.

Methods

The expression levels of selected miRNAs and serum CA19-9 concentration were determined for 35 patients with PDAC and 15 healthy controls by quantitative real-time RT-PCR and electro-chemiluminescence immune assay, respectively. The sensitivities of miRNAs as biomarkers of PC were evaluated and compared with CA19-9 using a receiver operating characteristic analysis.

Results

The levels of three miRNAs (miR-22-3p, miR-642b-3p and miR-885-5p) and CA19-9 were significantly higher in PC patients, even those with early-stage disease (IB and IIB), than in healthy control. Both miRNAs and CA19-9 were associated with tumor stage. The high sensitivities of the three selected miRNAs and CA19-9 were observed.

Conclusion

The measurement of miR-22-3p, miR-642b-3p and miR-885-5p may prove to have clinical utility in diagnosis of PC. Those miRNAs are ideal early biomarkers for PC diagnosis. So, they can effectively be used with serum CA19-9 for PC screening in early tumor stage.

Introduction

Pancreatic cancer (PC) represents the 10th most frequent diagnosed malignancy in the world and recognized as 4th leading cause of deaths due to cancer with a 5-year survival rate of only 6 % (Rachagania et al. 2015). The most common type of PC is pancreatic ductal adenocarcinoma (PDAC) that arises from the exocrine pancreas and represents about 95 % of pancreatic cancers and threats about 1.5 % of men and women life (Becker et al. 2014; Chitkara et al. 2015). The majority of patients present late with locally advanced or distant metastatic disease, making surgical resection impossible. Also, the lack of early diagnostic and prognostic markers for PC makes pancreatic carcinoma a lethal disease with high mortality and poor prognosis. So, novel trustful biomarkers are urgently needed to detect this disease at an early stage (Rachagania et al. 2015). Among these biomarkers, microRNAs (miRNAs) become an attractive focus of research.

MiRNAs are a family of short, noncoding endogenous single RNA molecules (19–24 nucleotides) that regulate the gene expression at posttranscriptional level through binding to the 3′-untranslated regions (UTRs) of specific mRNA causing inhibition of translation or mRNA degradation depending on the degree of complementarily between miRNA and its target mRNA (Negrini et al. 2007; Abue et al. 2015).

MiRNAs are involved in different biological processes and pathways, for example cell proliferation, metabolism, differentiation, apoptosis, and tissue morphogenesis. On the other hand, miRNAs were found to be having altered expression in a variety of diseases including cancer (Yu et al. 2011). Where they play a vital role in either cancer progression or suppression (Zhang et al. 2007). The aberrant expression of miRNAs may function either as oncogenic miRNAs (oncomiRs) or tumor suppressor miRNAs (TSmiRs), and they appear to play necessary roles in the initiation, progression and metastasis in PDAC (Hawa et al. 2016).

MiRNAs have high stability in tissues and fluids, small size, abundance, tissues specificity (Yu et al. 2011), can be measured in very low quantities of material (Gayral et al. 2014), are located in fragile chromosomal regions that affected in various human cancers (Zhang et al. 2011), and moreover, their expression profiling signature can distinguish cancer from benign tissues (Nelson and Weiss 2008), so that miRNAs can be used as biomarkers to detect and monitor the cancer (Yu et al. 2011).

MiR-22-3p, miR-642b-3p and miR-885-5p are among those miRNAs which reveal differential expression in pancreatic cancer. MiR-22-3p is located at a fragile cancer-relevant genomic region in chromosome 17 (17p13.3) (Gurha et al. 2012), close to p53. MiRNA-22-3p might induce complex changes and extensive cooperation with p53 (Calin et al. 2004). MiR-22-3p plays essential roles in various types of cancer, such as breast cancer, colon cancer, lung cancer and gastric cancer (Xiong et al. 2010; Yamakuchi et al. 2011; Ling et al. 2012; Wang et al. 2013). MiR-642 is located on chromosome 19 and has two members miR-642a and miR-642b. The sequences of miR-642 members are certainly similar and only diverge by one base in their 3′end. Actually, miR-642b is positioned on the opposite strand to miR-642a. MiR-642b-3p is found to be over expressed in several types of diseases as adipogenesis (Zaragosi et al. 2011), ovarian cancer (Kumar et al. 2011) and human dermal papilla cells (Bae et al. 2014). MiR-885-5p is positioned in the 3p25.3 genomic region, and it has been shown to be a possible serum marker for liver pathologies including, liver cirrhosis, chronic hepatitis C virus and hepatocellular carcinoma (Tian et al. 2012).

So, the aim of this study was to investigate the role of plasma miR-22-3p, miR-642b-3p and miR-885-5p expression as possible diagnostic tumor markers in PC patients as compared to serum CA19-9. In addition, the correlation of miR-22-3p, miR-642b-3P, miR-885-5p and CA19-9 with clinical characteristics was analyzed.

Subject and methods

The present study was carried out on 50 participants. Fifteen healthy participants with no family history of pancreatic cancer, (age range, 25–58 years) and 35 patients with PC (age range, 35–75 years) were selected from those admitted to Experimental and Clinical Surgery Department, Medical Research Institute Hospital, Alexandria University, from 2014 to 2015.

Patients who had prior pancreatic cancer surgery, or other concomitant cancers, or a history of HIV infection, were excluded from the study. All patients were subjected to full medical history taking and routine laboratory investigation including: complete blood picture and liver function tests.

Patients were diagnosed by abdominal computerized tomography (CT) scan (pancreatic-protocol) for disease staging (n = 33 early clinical stage and n = 2 advanced clinical stage). All patients had X-ray and/or CT chest. Cytological diagnosis was performed to all patients with CT-guided fine-needle aspiration (All of them had PDAC).

Patients with early clinical stage (IB and IIB) were referred for surgical decision, while those with late stage were referred for endoscopic drainage. Whipple operation was performed for 5 patients, distal pancreatectomy with splenectomy for 3 patients and bypass surgeries in the form of hepaticojejunostomy anastomosis without resection of the primary tumor for 12 patients. Endoscopic drainage with stent insertion was performed for 15 patients (8 patients unfit for surgery, 5 patients starting neoadjuvant chemotherapy protocol, and 2 patients with liver metastasis). Seven patients had postoperative complications (delayed gastric emptying, pancreatic anastomotic leaks and wound infection) and treated conservatively.

This study was approved by the local ethical committee of the Medical Research Institute, Alexandria University. Also assigned informed consent was obtained from all the participants in the present study.

Blood specimen collection and processing

Five milliliters blood samples was drawn from all participants. 2 mL blood was collected into tube with sodium citrate to separate plasma. The remaining 3 mL blood was pipetted into tube without anticoagulants and then left for 15 min at 37 °C before centrifuge to separate serum. Harvested plasma and serum were stored at −80 °C until used for determination of plasma miRNAs expression levels by real-time RT-PCR and serum CA19-9 concentration by electro-chemiluminescence immune assay (ECLIA).

Quantification of miR-22-3p, miR-642b-3p and miR-885-5p expression by real-time RT-PCR (Ganepola et al. 2014)

miRNA extraction

The miRNeasy serum/plasma Kit (Qiagen Inc., Valencia, CA, USA-cat. no. 217184) is designed for purification of cell-free total RNA, primarily miRNA and other small RNA. To confirm the presence of miRNA in each sample, the total RNA was measured as ng/μL by using “Thermo Scientific NanoDrop instrument” (total RNA range 2.10–14.50 ng/μL).

The reverse transcriptase (RT) reaction

TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used for synthesis cDNA from total RNA. The reverse transcriptase reactions contained 5 μL of RNAs, 0.15 μL of 100 mM dNTPs, 1 μL 50 U/μL of MultiScribe™ Reverse Transcriptase, 1.5 μL of 10× Reverse Transcription Buffer, 0.19 μL of 20 U/μL RNase inhibitor, 4.16 μL of nuclease-free water and 3 μL of stem-loop RT primer. The reaction samples were incubated in PCR thermocycler (Applied Biosystems) for 30 min at 16 °C, 30 min at 42 °C, 5 min at 85 °C and then held at 4 °C. After the RT run, store the cDNA at 15–25 °C.

Real-time PCR

The quantification of each miRNA expression (miR-22-3p, miR-642b-3p and miR-885-5p) was performed using TaqMan MicroRNA Assay kit (Applied Biosystems, Foster City, CA, USA). 1.33 μL of cDNA was amplified with 10 μL of TaqMan Universal PCR Master Mix (2X), 1 μL of gene-specific primers/probe, and 7.67 μL of nuclease-free water in a final volume of 20 μL. The reaction mixtures were incubated at 50 °C for 2 min then 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing/extension at 60 °C for 60 s. Quantitative RT-PCR was run on the real-time PCR instruments (Applied Biosystems one step, Foster City, CA, USA). The TaqMan miRNA assays used were: hsa-miR 22-3p (ID#000398), hsa-miR-642b-3p (ID#462949_mat), hsa-miR-885-5p (ID#002296) and hsa-miR-3196 “housekeeping gene” (ID#241941_mat) (Ganepola et al. 2014).

Primer sequence of miR-22-3p:

• 5′-AAGCUGCCAGUUGAACUGU-3′

• 3′-GGAUCAUUGACCAGGUCAC-5′

Primer sequence of miR-642b-3P:

• 5′-AGACACAUUUGGAGAGGGACCC-3′

• 3′-GAGUGUGCCCAAGAGAAAGUUU-5′

Primer sequence of miR-885-5P:

• 5′-UCCAUUACACUACCCUGCCUCU-3′

• 3′-CUUGCCGUGUCGUUUCAUUCUC-5′

Primer sequence of miR-3196:

• 5′-CGGGGCGGCAGGGGCCUC-3′

• 3′-UAAAAUAAUGAAAAUUCU-5′

The relative quantification (RQ) of each miRNAs expression was calculated using the comparative cycle threshold Ct (2^−ΔΔCt) method with miR-3196 as the endogenous control to normalize the quantitative PCR results (Fig. 1).

$$\mathrm{\Delta }\text{Ct}={\text{Ct}}_{\text{the studied gene}}-{\text{Ct}}_{\text{the corresponding one of housekeeping gene}}$$

$$\mathrm{\Delta }\mathrm{\Delta }\text{Ct}=\mathrm{\Delta }\text{Ct}-\mathrm{\Delta }{\text{Ct}}_{\text{calibrator}}$$

$$RQ=2^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$$

$$\mathrm{\Delta }{\text{Ct}}_{\text{calibrator}}=\text{the mean}{\text{Ct}}_{\text{the control cases of the studiedgene}}-\text{the mean}{\text{Ct}}_{\text{the control cases of the house keeping gene}.}$$

Fig. 1.

Amplification curve for a housekeeping miR-3196, b miR-22, c miR-642b-3p and d miR-885-5p

Determination of CA19-9 by ECLIA

The assay is indicated for the serial measurement of CA 19-9 to aid in the management of patients diagnosed with pancreatic cancer. The analyzer automatically calculates the concentration of each sample in U/ml where the reference range is up to 33 U/mL.

Statistical analyses

Data were fed to the computer and analyzed using IBM SPSS software package version 20. P values <0.05 were statistically significant. Chi-square test was used for categorical variables, to compare between different groups. For normally quantitative variables, Student’s t test was used to compare between two studied groups. For abnormally quantitative variables, Mann–Whitney and Kruskal–Wallis tests were used to compare between two and more than two studied groups, respectively. Spearman coefficient was used to correlate between two abnormally quantitative variables. Receiver operating characteristic curve (ROC) is generated by plotting sensitivity (TP) on Y-axis versus 1-specificity (FP) on X-axis at different cutoff values. The area under the ROC curve denotes the diagnostic performance of the test. Area more than 50 % gives acceptable performance, and area about 100 % is the best performance for the test.

Results

Demographic data and clinical characteristics

In PC patient, the mean level of total bilirubin, direct bilirubin, SGOT and SGPT was significantly increased as compared to control group (P < 0.001, Table 1).

Table 1.

Demographic data and laboratory parameters in control and PC patient groups

	Control group (n = 15)	PC patients group (n = 35)	P
Sex
Male	4 (26.7 %)	14 (40.0 %)	0.368
Female	11 (73.3 %)	21 (60.0 %)
Age (years)	40.93 ± 9.26	57.43 ± 10.82	<0.001*
Total bilirubin	0.81 ± 0.09	9.11 ± 6.01	<0.001*
Direct bilirubin	0.20 ± 0.03	5.69 ± 4.03	<0.001*
SGOT	18.67 ± 3.74	98.87 ± 53.67	<0.001*
SGPT	21.33 ± 4.24	100.29 ± 64.27	<0.001*
WBC	7.39 ± 1.83	8.37 ± 2.50	0.181

P value for Mann–Whitney, Kruskal–Wallis or Student’s t test for comparing PC patients with the control group

n = Number of cases in each group

* Statistically significant at P ≤ 0.05

Table 2 demonstrates the CT findings for PC patients: All patients had mass size >2 cm, only one patient had tail mass (2.9 %), 2 patients had body mass (5.7 %), 4 patients had neck mass (11.4 %), and 28 patients had head mass (80 %). Only 24 patients (68.6 %) had common bile duct (CBD) dilatation. Out of all patients, only 2 patients (5.7 %) had liver metastasis, 10 patients (28.6 %) with regional lymph nodes, while no patients had ascites. Finally, there were 33 patients at an early stage {23 (65.7 %) with stage IB and 10 (28.6 %) with stage IIB} and 2 patients (5.7 %) with stage IV.

Table 2.

Association between miR-22-3p, miR-642b-3p, miR-855-5p expression (copies), CA19-9 concentration (U/mL) and clinical criteria of PC patients

	n (%)	miR-22-3p	miR-642b-3p	miR-885-5p	CA19-9
Gender
Male	14 (40)	1.65	6.45	4.90	507.0
Female	21 (60)	2.20	4.60	2.80	342.0
MW P value		0.71	0.51	0.49	0.51
Mass size(>2 cm)	35 (100)
Mass site
Head	28 (80)	2.09	4.70	2.80	429.60
Neck	4 (11.4)	1.5	1.10	2.30	11.30
Body	2 (5.7)	2.23	9.60	5.33	775.90
Tail	1 (2.9)	1.79	10.0	7.95	97.0
KW P value		0.89	0.04*	0.58	0.10
CBD dilatation
Positive	24 (68.6)	2.20	5.20	3.20	571.50
Negative	11 (31.4)	1.82	4.60	4.80	141.30
MW P value		0.28	0.85	0.49	0.13
Liver metastasis
Positive	2 (5.7)	2.62	5.35	6.55	371.10
Negative	33 (94.3)	1.98	5.20	3.60	443.0
MW P value		0.92	0.36	0.67	0.72
Lymph nodes
Positive	10 (28.6)	1.76	5.20	3.28	466.0
Negative	25 (71.4)	2.20	5.20	3.60	443.0
MW P value		0.67	0.66	0.80	0.61
Ascites
Positive	0
Negative	35 (100)
Stage of PC
IB	23 (65.7)	2.20	5.20	3.60	443.0
IIB	10 (28.6)	1.76	5.20	3.28	466.0
IV	2 (5.7)	2.62	8.35	6.55	371.10
KW P value		<0.001*	<0.001*	<0.001*	<0.001*

The data were given as median

CBD Common bile duct, n number of PC patient, ^KW P value for Kruskal–Wallis test, ^MW P value for Mann–Whitney test

* Statistically significant at P ≤ 0.05

Molecular and biochemical results

The mean values of miR-22-3p, miR-642b-3p and miR-885-5p expression as well as CA19-9 concentration were significantly higher in PC patients than the corresponding values in the control group (P < 0.001) (Table 3). Also, there was significant increase in those miRNAs expression and CA19-9 concentration in patients with stages IB, IIB and IV as compared to the control group (Fig. 2a–d).

Table 3.

Plasma miR-22-3p, miR-642b-3p, miR-855-5p expression (copies) and CA19-9 concentration (U/mL) in control and PC patient groups

	Control group	Pancreatic cancer group	P
miR-22-3p	0.89 ± 0.29	2.27 ± 1.18	<0.001*
miR-642b-3p	0.38 ± 0.42	6.41 ± 5.10	<0.001*
miR-885-5p	1.0 ± 0.08	5.18 ± 4.44	<0.001*
CA19-9	25.67 ± 5.95	751.37 ± 1018.38	<0.001*

P value for Mann–Whitney test for comparing PC patients with the control group

* Statistically significant at P ≤ 0.05

Fig. 2.

a MiR-22-3p, b MiR-642b-3p, c MiR-885-5p, d CA19-9 (U/mL) in control and PC patients groups with different stages

There was no association between miRNA-22-3p, miR-642b-3p, miR-885-5p, CA19-9 and the abdominal CT finding (CBD dilatation, liver metastasis and peripancreatic Lymph nodes metastasis) in PC group except a significant association between miR-642-3p and pancreatic mass site (P = 0.042). For miRNAs and CA19-9, an association with tumor stage was found, as relative median expression of miRNAs increased with stage (P ≤ 0.001), while the relative median of CA19-9 decreased with stage (P ≤ 0.001) (Table 2).

There was no correlation between miR-22-3p, miR-642b-3p, miR-885-5p expression, CA19-9 concentration and clinical parameters of PC patients except that there was significant positive correlation between CA19-9 and SGOT, SGPT and WBCs (P = 0.005, 0.003 and 0.011, respectively) (Fig. 3). In addition, there was no significant correlation between each of miRNAs expression and CA19-9 concentration.

Fig. 3.

Correlation between the CA19-9 concentration and a SGOT, b WBCs and c SGPT in patients group

The ROC curve analysis was used to compare the diagnostic values of miR-22-3p, miR-642b-3p, miR-885-5p and CA 19-9 depending on AUC. The higher AUC corresponds to a better diagnostic test. MiR-22-3p showed a significant AUC (94.3 %, P < 0.001) with sensitivity (97.14 %) and specificity (93.33 %) at cutoff 0.96. Both miR-642b and miR-885-5p showed a significant AUC (100 %, P < 0.001) with sensitivity (100 %) and specificity (100 %) at cutoff 0.99 and 1.24, respectively. CA 19-9 showed a significant AUC (92.4 %, P < 0.001) with sensitivity (91.43 %) and specificity (100 %) at cutoff 34 (Table 4; Fig. 4).

Table 4.

Agreement (sensitivity, specificity and accuracy) for miR-22-3p, miR-642b-3p, miR-855-5p expression level and CA19-9 concentration

		Control	Cases	AUC	P	Sensitivity	Specificity	Accuracy
MiR-22-3p
≤0.96		14	1	0.943*	<0.001*	97.14	93.33	96.0
>0.96		1	34
MiR-642b-3p
≤0.99		15	0	1.000*	<0.001*	100.0	100.0	100.0
>0.99		0	35
MiR-885-5p
≤1.24		15	0	1.000*	<0.001*	100.0	100.0	100.0
>1.24		0	35
CA19-9
≤34		15	3	0.924*	<0.001*	91.43	100.0	94.0
>34		0	32

Asterisks indicate higher area under the curve corresponds to a better diagnostic test

Fig. 4.

ROC curve for miR-22-3p, miR-642b-3p, miR-855-5p expression level and CA19-9 concentration to diagnose PC patients group

Discussion

Diagnosis of pancreatic cancer patients by using sensitive and specific biomarkers is considered necessary (Ko 2015). Although carbohydrate antigen 19-9 (CA 19-9) is the most common predictable diagnostic maker for PC patients, its levels often increase in the absence of PC (such as chronic pancreatitis or benign biliary obstruction) and it has poor sensitivity in the PC diagnosis (Winter et al. 2013).

MiRNAs have been well-known as master regulators of gene expression and many biological processes. MiRNAs are dysregulated in various cancers and can contribute to regulation of tumorigenesis and development by playing tumor suppressive and/or oncogenic roles (Price and Chen 2014). Definite miRNAs are involved in fundamental molecular processes associated with pancreatic cancer initiation and progression including cell cycle, DNA repair, proliferation, differentiation, apoptosis, metastasis and sensitivity to therapy through a mechanism including aberrant regulation of proto-oncogenes and tumor suppressor genes expression (Halkova et al. 2015).

Therefore, this study investigates the role of plasma miR-22-3p, miR-642b-3p and miR-885-5p expression levels as possible diagnostic tumor markers in PC patients as compared to serum CA19-9.

The plasma miRNAs expression levels (miR-22-3p, miR-642b-3p and miR-885-5p) identified in this study were shown to be significantly up-regulated in PC patients as compared to the control group, and these results are in accordance with other studies (Ganepola et al. 2014; Li et al. 2013). The elevated level of circulating miRNAs (miR-22-3p, miR-885-5p and miR-642b-3p) in PC patients may be attributed to leak or release of tissue or organ-specific intracellular miRNAs into the circulation during processes accompanying cell death or apoptosis due to cell turnover, cellular destruction or pathological injury (Mitchell et al. 2008). In addition, the high level of miRNAs in the circulation could reflect a metabolic imbalance of these miRNAs in vivo (Gui et al. 2011).

In pancreatic cancer, the over-expression level of miRNA-22-3p induces inhibition of cell growth, metastasis and apoptosis through suppression its target genes; Sp1 transcription factor (SP1) and estrogen receptor 1 (ESR1) (Park et al. 2011). Furthermore, miRNA-22-3p is proposed to be a putative tumor suppressor by repressing the EVI1 oncogene expression (Patel et al. 2011). MiRNA-22-3p inhibits cell cycle progression by post-transcription repression of Myc-associated factor x (Max) and ErbB3 expression, mediates the effects of the tumor-suppressor p53 and suppresses interferon gene expression by blocking interferon regulatory factor-5 (Ling et al. 2012; Ting et al. 2010; Polioudakis et al. 2013).

MiR-642 has putative targets involved in several important pathways including, mitogen-activated protein kinase (MAPK), insulin-like growth factor (IGF), transforming growth factor-β (TGF-β), apoptosis, tumor-suppressor protein (p53) and other signaling pathways. MAPK signaling is the most affected pathway by miR-642, and its activation by phosphorylation can result in either cell proliferation or apoptosis. TGF-β is involved in cell proliferation, cell adhesion, cell migration and cell differentiation. MiR-642 may regulate TGF-β-induced apoptosis and cell cycle (Kumar et al. 2011; Kim et al. 2000).

MiR-885-5p is recognized to have a tumor suppressive function by activating cell cycle arrest and induce apoptosis (Afanasyeva et al. 2011). MiR-885-5p inhibits cancer cell proliferation through induction of p53-dependent cell cycle arrest and leads to down-regulation of cyclin-dependent kinase (CDK, proliferation-related genes) and mini-chromosome maintenance protein (MCM5). In addition, MiR-885-5p suppresses the expression of matrix metallopeptidase 9 and caspase genes such as CASP-3, which is an important mediator for apoptotic process (Afanasyeva et al. 2011; Yan et al. 2011; Guan et al. 2013). The overexpression of miR-885-5p in PC causes inhibition of tumor growth, angiogenesis and metastasis.

The data obtained from this study observed that the expression of three miRNAs (miR-22-3p, miR-642b-3p and miR-885-5p) was significantly higher in PC patients with different stages IB, IIB and IV than that of the control group. Moreover, the expression of miRNAs tends to increase with PC stages. Previous study revealed that miRNA released from tumors tissue into the circulation by selective process of tumor derived exosomes, these may occur at an early stage in tumorigenesis as a mechanism for function regulation and miRNAs have been recognized in the blood stream at different stages of the disease (Taylor and Gercel-Taylo 2008; Schwarzenbach et al. 2014). Additionally, the plasma miRNAs expression level may be reflecting the degree of organ inflammation (Gui et al. 2011) and alterations of the entire organism homeostasis (Vietsch et al. 2015). From that, the aberrant high expression of miRNAs in circulation leads to use miRNAs as diagnostic marker of cancer at an early stage.

This study demonstrated that serum CA19-9 concentration was significantly higher in PC patients as well as in each stage of PC than the control group and these results concur with the findings of the previous studies (Ganepola et al. 2014; Li et al. 2013; Park et al. 2011). Since circulating CA19-9 is released from the cell surface of PC cells, the elevated level of CA19-9 in PC may be attributed to uncontrolled growth of aberrant epithelial cells and their continuous secretion of this antigen (Lin et al. 2014). In addition, any noxa (viral or toxic) causes stimulate tissue inflammatory damage and subsequently, reparative features with fibrotic tissue deposition and parenchymal regeneration can induce CA19-9 synthesis (Hekele and Muller 2006).

From the obtained data, the serum CA19-9 concentration tends to decrease with PC stages. CA19-9 is known to be sialylated Lewis (Le ^a) blood-group antigen. Serum level of CA19-9 varied according to the patient’s Lewis genotype and secretor status. In addition, the Lewis phenotype was determined by two independent genes: the Lewis gene or α1-4 fucosyltransferase (also known as FUT3) and the secretor gene or α1-2 fucosyltransferase (FUT2). Individuals who are Lewis blood type-negative (Le^a−b−) do not express the CA19-9. The lower level of CA19-9 even when tumor burden is high may be attributed to deficiency of fucosyltransferase (enzyme essential for CA19-9 biosynthesis) and Lewis gene (Vestergaard et al. 1999; Su et al. 2015).

The results demonstrated that there was no significant correlation between miRNAs (miR-22-3p, miR-642b-3p and miR-885-5p) and age, gender and some clinical parameters such as total bilirubin, direct bilirubin and tumor mass of PC patients. These results are in line with some previous studies by Gui et al. (2011) and Tian et al. (2012), who stated that there was no correlation between circulating miR-885-5p and traditional liver parameters (SGOT and SGPT). The lack of correlation may be the result of heterogeneity within the groups and/or to the small sample size (Garzon et al. 2008). Moreover, there was no correlation between miRNAs expression level and CA19-9 concentration and these was in accordance with previous study (Kojima et al. 2015).

There was a significant positive correlation between CA19-9 level and SGOT, SGPT and WBC’s count. These results agreed with Qureshi et al. (2006), Bertino et al. (2013), Amirkande et al. (2015). Serum CA19-9 up-regulation may be secondary to necroinflammatory processes, which in turn causes liver enzymes elevation in serum (Qureshi et al. 2006). On the other hand, there was no correlation between CA19-9 concentration and age, gender and some clinical parameters such as total bilirubin, direct bilirubin, and tumor mass of PC patients. The lack of correlation could be attributed to the diversity of histological types of PC included in this study (Wang et al. 2013).

ROC curve approaches have been extensively used in evaluating the diagnosis power of markers. The values of plasma miR-22-3p, miR-642b-3p miR-885-5p expression levels and serum CA19-9 concentration in diagnosis of PC were evaluated by ROC curve analysis. The use of the area under ROC curve was useful in the elucidation of the validity of a specific marker in the diagnosis of PC. For plasma miRNA-22-3p, AUC = 94.3 %, P < 0.001, for both miR-642b-3p and miR-885-5p, AUC = 100 %, P < 0.001, while for serum CA19-9, AUC = 92.4 %, P < 0.001. These results indicate the validity of using plasma miR-22-3p, miR-642b-3p miR-885-5p and serum CA19-9 as diagnostic markers for discriminating between PC patients and healthy controls. Furthermore, both plasma miR-642b-3p and miR-885-5p were superior to plasma miR-22-3p followed by serum CA19-9 for diagnosis of PC. These results are consistent with previous study which indicated that circulating tumor miRNAs may be aberrantly highly expressed and therefore might be used as diagnostic markers (Liu et al. 2012), but with difference in sensitivity and specificity (Ganepola et al. 2014) and that may be attributed to variation in sample size, PC stage and histological types.

In conclusion, since circulating plasma miR-22-3p, miR-642b-3p and miR-885-5p are significantly elevated in PC patients, their measurement may prove to have clinical utility in diagnosis of PC. Those selected plasma miRNAs are ideal early biomarkers for PC diagnosis. So, they can effectively be used with serum CA19-9 for PC screening in early tumor stage.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

All procedures performed in the study involving human participants were in accordance with the Local Ethical Committee of the Medical Research Institute, Alexandria University.

Informed consent

Written informed consent was obtained from all patients included in the study.