The association between circulating MicroRNA‐150 level and cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a rare tumor which requires a multimodality approach for its diagnosis. Carbohydrate antigen 19‐9 (CA19‐9) is currently the most commonly used tumor marker for CCA; nevertheless, it has certain limitations which need to be considered when using it as a tumor marker. MiRNA‐150 altered expression has been linked to the development and tumorigenesis of several cancers including CCA. This work aimed to study the serum level of CA19‐9 and miRNA‐150 expression in CCA patients and, also, to correlate their levels with tumor staging and different studied clinical and laboratory parameters. This work included 35 patients with CCA who were admitted to Hepatobiliary Unit, Alexandria Main University Hospital (Group I). Also, 35 age‐ and sex‐matched healthy subjects were included as a control group (Group II). All included subjects were submitted to measurement of serum CA19‐9 and MiRNA‐150 expression levels. Serum CA19‐9 levels showed an evident high median among CCA patients, while serum miRNA‐150 expression levels were evidently low among those patients. Moreover, combining miRNA‐150 with CA19‐9 made the accuracy of diagnosis of CCA much more reliable. Thus, miRNA‐150 can be considered as a non‐invasive, sensitive serum biomarker for the diagnosis of CCA especially when combined with CA 19‐9.

as an intrahepatic mass. 8 Imaging is a cornerstone in diagnosis, including ultrasonography, 9 computed tomography (CT), 10 and magnetic resonance cholangiopancreatography (MRCP) which is the current gold standard for non-invasive assessment of CCA, 11 as well as positron emission tomography (PET)-CT. 12 Endoscopic retrograde cholangiopancreatography (ERCP) also has diagnostic and therapeutic roles. 13 The most studied serum tumor marker for CCA is the carbohydrate antigen 19-9 (CA19-9). It is a sialylated Lewis blood group antigen targeted by the monoclonal antibody, which was described in 1979 as a tumor-associated antigen in colorectal cancer cell line. 14 CA19-9 has wide variation in sensitivity (50%-90%) and specificity (54%-98%) in the diagnosis of CCA. Also, serum CA-19-9 levels depend on the Lewis phenotype, where as many as 10% of the population have been found to be Lewis negative, resulting in undetectable CA19-9 levels; therefore, it has many limitations. 15 It has been reported that CA19-9 is elevated in colorectal cancers as well as nonmalignant conditions as pancreatitis, cholangitis, hepatolithiasis, and primary sclerosing cholangitis (PSC). 16,17 Thus, diagnosis of CCA still poses a challenge and is often underestimated due to the lack of a gold standard tumor marker of high specificity and sensitivity for early detection.
MiRNAs are short non-coding RNAs of 20-24 nucleotides that play an important role in virtually all biological pathways. 18 MiRNAs are present not only in tissues but also can be secreted from the cells and can gain access to different body fluids, and remain highly stable upon exposure to severe conditions that induce almost immediate degradation of free RNA, such as boiling, very low or high PH, and extended storage. 19 Therefore, circulating MiRNAs can be used as non-invasive, sensitive biomarkers for detecting different diseases. 20 In cancer, MiRNAs influence numerous cancer-related processes such as metabolism, migration, proliferation, apoptosis, cell cycle control, and differentiation. 18 Furthermore, considering that altered expression of some MiRNAs contributes to human carcinogenesis, MiRNAs have been reported to be useful as potential biomarkers for diagnosis, prognosis, and personalized therapy of different human cancers. 21 In CCA, dysregulation of miRNA expression has not been studied extensively as in many other cancers, perhaps due to the rarity of this pathological entity. The first report on miRNA expression in MiRNA-150 expression was up-regulated and linked to the development and tumorigenesis of breast cancer, 24 lung cancer, 25 and gastric cancer. 26 On the contrary, miRNA-150 overexpression inhibited the malignant behavior of pancreatic cancer. 27 Moreover, miRNA-150 has been down-regulated in esophageal squamous cell carcinoma 28 and colorectal cancer. 29 In IH-CCA, it has been reported that plasma levels of miRNA150 were higher than healthy subjects in a small sample of patients. 30 However, Wu et al 31 documented that serum miRNA-150 level was down-regulated in CCA patients. Thus, under different cellular microenvironments, miRNA-150 may carry out different functions. Therefore, the role of miRNA-150 in CCA has yet to be more elucidated.

| Aim of work
The aim of the current work was to study the serum expression level of MiRNA-150-5P in CCA patients and, also, to correlate its level with tumor staging and different clinical and laboratory findings of the studied patients.

| SUBJEC TS AND ME THODS
The present study included 35 patients with CCA (randomly selected both intra-and extrahepatic CCA), who were admitted to Hepatobiliary Unit, Alexandria Main University Hospital (Group I).
Also, 35 age-and sex-matched healthy subjects were included as a control group (Group II) to obtain normal range of biochemical assays.
Diagnosis of CCA depended on CT and/or MRCP, ERCP with brush cytology (whenever needed). Also, the studied CCA patients had positive CA19-9. Patients with other malignancies, systemic diseases (as hepatic, renal, cardiac, or respiratory diseases), sepsis, and collagenic diseases (as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis) were excluded. Also, all our studied CCA patients (Group I) and normal control subjects (Group II) had negative HCV Abs and HBs Ag.
All included patients were submitted to the following: • Detailed history taking with emphasis on age, gender, yellow skin and sclera, itching, abdominal pain, anorexia and nausea, vomiting, weight loss, and change in the color of urine and stool.
• Clinical examination including both general and local abdominal examination (hepatomegaly, abdominal mass, and palpable gallbladder).

| Method
Total serum RNA was isolated using miRNeasy Mini Kit (Q IAGEN) Cat No./ID: 217004, according to the manufacturer's protocol.
Briefly, 5 μL of 5 nmol/L Syn-cel-miR-39 (miScriptmiRNA Mimic) was added to each sample as a spike-in control, and then, total

| Statistical analysis
Quantitative data of the present work were analyzed, using F-test

| Demographic data
In the present study, age showed a median of 54 years and 57 years in Group I and Group II, respectively, with no statistical significant difference between both groups. As regards sex, males predominated females in both studied groups. Table 1 showed comparison between both studied groups according  Table 2 showed negative correlation between serum CA19-9 and miRNA-150-5p expression levels, where P = .001.

| D ISCUSS I ON
CCA is a tumor with high mortality rate and is consistently asymptomatic in the early stages. 32 Its diagnosis and staging require a multimodality approach involving laboratory, radiologic, endoscopic, and pathologic analyses. Therefore, the identification of new diagnostic and prognostic biomarkers for CCA with proper sensitivity and specificity is urgently needed. 33 Studies have revealed that circulating miRNAs may be potential di-   Based on this hypothesis, miRNA-150 is an exocrine agent released by peritumoral non-cancerous cells and may act as an important negative feedback regulating agent. 42 Yet, the exact mechanism for this difference between tissue and plasma miRNA profiles is not clear and needs to be further studied.
In the present study, negative correlation between serum  reported that combining miRNA-150 with CA19-9 made the sensitivity and specificity for the diagnosis of CCA 80% and 100%, respectively.

F I G U R E 2 ROC analysis to study the ability of serum
MicroRNA-150-5p expression for the diagnosis of CCA F I G U R E 3 ROC analysis to study the ability of combination of both serum CA19-9 and MiRNA-150-5p expression for the diagnosis of CCA

ACK N OWLED G M ENTS
The authors thank all the contributors of this work.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest in this work.

AUTH O R S CO NTR I B UTI O N S
Dr Perihan Salem formed the idea of the research, assessed the included patients, and wrote the paper. Dr Rasha Ghazala performed the laboratory work and revised the paper. Dr Ahmed El Gendi involved in collection of the cases and revised the paper. Dr Doaa Emara involved in imaging and revised the paper. Dr Nesma Ahmed involved in collection of the cases, performed the statistical analysis, and revised the paper.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
The study was approved by the Research Ethics Committee of the Faculty of Medicine, University of Alexandria, and was conducted Good Clinical Practice guidelines. Also, an informed consent was obtained from all subjects included in the study.

CO N S E NT FO R PU B LI C ATI O N
All the contributing authors agreed for the publication in this journal.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data related to this work are available at the corresponding author.