|Year : 2018 | Volume
| Issue : 1 | Page : 220-225
Circulating microRNA-21 as a prognostic, biological marker in cholangiocarcinoma
Chen-Hai Liu1, Qiang Huang2, Zhi-Yuan Jin2, Fang Xie2, Cheng-Lin Zhu2, Zhen Liu2, Chao Wang2
1 Shandong University, Jinan 250012, Shandong, China
2 Department of General Surgery, Anhui Provincial Hospital, Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Hefei 230001, Anhui, China
|Date of Web Publication||8-Mar-2018|
Dr. Qiang Huang
Department of General Surgery, Anhui Provincial Hospital, Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Hefei 230001, Anhui
Source of Support: None, Conflict of Interest: None
Aims: The prognosis of cholangiocarcinoma (CCA) is generally poor because there is a lack of effective diagnostic tools including laboratory assessments and imageological examination. Therefore, a novel biological marker (biomarker) to effectively diagnose cancer is highly desirable in clinical. Previously, serum microRNAs as biomarkers of cancers have been reported. However, it was still unclear about the clinical significance of serum microRNA-21 (miR-21) expression levels for CCA.
Materials and Methods: The serum samples were separately collected from fifty patients of CCA, 15 patients of hepatolithiasis, and 15 healthy volunteers; quantitative real-time polymerase chain reaction was used for measuring miR-21 expression level in serum. The clinicopathological data were recorded before patients discharged.
Results: In the CCA serum, the expression level of miR-21 significantly upregulated (P < 0.05). With the tumor, node, and metastasis stage developed (Stage I vs. III and IV, P < 0.05), the serum miR-21 expression level increased, but there was no statistical difference between Stage I patients and hepatolithiasis patients or healthy control (P > 0.05 for both). Furthermore, the miR-21 level was significant difference between pre- and post-operative serum (P < 0.05) for the high miR-21 expression group. The serum miR-21 expression levels were defective in discriminating patients with CCA from healthy control subjects by receiver-operator curve analysis because the area under the curve (AUC) value was 0.871 which was not better than the conventional CCA markers—carbohydrate antigen 19-9 (AUC value = 0.96). However, in serum, high expression level miR-21 was significantly related to clinical stage, invasion depth, lymph vessel infiltration, metastasis status, differentiation status, whether to resection, and poor survival of CCA patients (P < 0.05 for all).
Conclusions: This study suggested that serum miR-21 was a promising biomarker for diagnosing the late stage CCA and would have potential to be a useful prognostic biomarker of CCA.
Keywords: Biological markers, cholangiocarcinoma, microRNA-21, serum
|How to cite this article:|
Liu CH, Huang Q, Jin ZY, Xie F, Zhu CL, Liu Z, Wang C. Circulating microRNA-21 as a prognostic, biological marker in cholangiocarcinoma. J Can Res Ther 2018;14:220-5
|How to cite this URL:|
Liu CH, Huang Q, Jin ZY, Xie F, Zhu CL, Liu Z, Wang C. Circulating microRNA-21 as a prognostic, biological marker in cholangiocarcinoma. J Can Res Ther [serial online] 2018 [cited 2020 May 27];14:220-5. Available from: http://www.cancerjournal.net/text.asp?2018/14/1/220/193125
| > Introduction|| |
Cholangiocarcinoma (CCA) arising from the cholangiocytes lining biliary trees has become the second most common primary hepatic malignancy. The incidence of CCA continues to increase worldwide, and the highest incidence area is in the Southeast Asia, especially in China, where liver fluke infection is endemic., Surgical resection including R1 resection offers the only chance of potentially curative treatment and long-term cure., Unfortunately, CCA is usually diagnosed in the late clinic stages, and the rates of survival resection are just only 23–50%, and the majority of patients with resection still develop recurrent or metastatic tumor., Hence, how to improve accuracy of CCA diagnosis at early clinical stages is critical for curative treatment.
Laboratory assessments are very important for detecting the early carcinoma. However, now, there are not high sensitive and specific serum markers for CCA detection. Currently, the serum marker carbohydrate antigen 19-9 (CA19-9) concentration determination as a common measurement method is routinely applied in most laboratories for CCA detection in spite of its sensitivity (89%) and specificity (86%) is not very satisfactory. In addition, the sensitivity of CA19-9 is just only 58% in the early stage (I) of CCA., Hence, identification of new tumor markers in the serum would be beneficial in the clinical management of CCA.
It is well-known that microRNAs (miRNAs) are noncoding RNAs with 18–25 nucleotides in length, which are recently demonstrated to have a major role in regulating virtually all cellular processes  and gene expression., Recently, numerous groups have reported that several aberrantly-expressed serum and tissue miRNAs could be the diagnostic or prognostic indicators in multiple cancer types.,, Especially, the aberrant miRNAs expression is associated with the genesis and homeostasis of CCA. However, tissue miRNAs as a CCA diagnostic biomarker are needed to extract from human CCA cell. It is invasive to body and not conductive to popularity in clinical. Fortunately, previous reports have suggested that serum miRNAs which are easily available also could be as biomarkers to diagnose several cancers with high sensitivity and specificity. Although the origin and physiologic function of cell-free miRNAs in the serum remain to be fully elucidated, expression characteristic of miRNAs in different cancers should be identified to explore these molecules as potential diagnostic and prognostic biomarkers.
In our early report and other preliminary studies, tumor tissue microRNA-21 (miR-21) level of CCA patients have been proven to upregulate assessing by in situ hybridization and real-time polymerase chain reaction (PCR) in the paraffin section, and it is associated with genesis and homeostasis of CCA.,, Furthermore, serum miR-21 as biomarkers has been detected in various carcinoma, but the diagnostic and prognostic values of serum miR-21 were different., Hence, determining the value of serum miR-21 for CCA was very important.
In this study, we systematically investigated the clinical significance of serum miR-21 in CCA. First, we evaluated the diagnostic value of serum miR-21 for CCA patients. Second, we analyzed the clinical significance of serum miR-21 which has potential to be a prognostic biomarker of CCA patients.
| > Materials and Methods|| |
Patients and specimens
Collection and studies of all the samples were approved by the Research Ethics Committee. All patients willingly donate their blood samples for research. In this study, a total of fifty patients with CCA from 2011 to 2014 were enrolled. Thirty-two patients received curative surgery and 18 received palliative resection. CCA patients enrolled did not receive radiotherapy or chemotherapy before operation. All tumors were clinically and histologically diagnosed as CCA. Inclusion criteria for all cases were (i) unambiguous histology and absence of mixed tumor types and (ii) absence of any treatment before surgery; the clinicopathological features of patients are shown in [Table 1]. At the same time, 15 patients of hepatolithiasis and 15 healthy volunteers (HVs) were enrolled as controls of this study. A total of eighty serum samples were obtained from fifty patients with CCA and thirty controls.
|Table 1: Association between serum miR-21 expression and clinicopathological variables of cholangiocarcinoma patients (American Joint Committee on Cancer Staging Manual 2010)|
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RNA extraction and quantitative real-time polymerase chain reaction
For miRNA quantification, total miRNA was extracted from the samples using miRNeasy RNA Isolation Kit (Qiagen, Inc.). TaqMan miRNA quantitative real-time PCR (qRT-PCR) (Thermo, CA, USA) was used to detect and quantify miRNA expression. 7500 software v. 2.0.1 (Thermo, CA, USA) was used for analyzing the data. Each sample was respectively detected three times, and then, the total PCR products were compared with internal reference (RNU6B).
SPSS statistical software (version 19.0) (International Business Machines Corporation, Armonk, NY, USA) was used for statistical analysis. Mann–Whitney U-test, Fisher's exact test, or Chi-square test was used for assessing the difference between the groups, and Steel–Dwass test was used for multiple comparisons. To compare the serum miR-21 expression before and 7 days after surgery, we applied Wilcoxon test. To identify the diagnostic role of the miR-21 expression level, we designed the receiver-operator curve (ROC) to analyze. The area under the curve (AUC) values were used for identifying the probability of CCA patients. Youden index was applied to determine the optimal cutoff thresholds of diagnosis. Then, the predictive values of positive and negative (PPVs and NPVs), specificity value, and sensitivity value were assessed based on the optimal cutoff value. Two-sided test was employed to determine the P values, and P < 0.05 was considered to be statistical significance.
| > Results|| |
Relative expression of serum microRNA-21 in cholangiocarcinoma patients
qRT-PCR was used for investigating the concentration level of serum miR-21 in fifty patients with CCA, 15 patients with hepatolithiasis, and 15 HVs. We found that the expression level of serum miR-21 in CCA patients was obviously higher than in hepatolithiasis patients or healthy controls (n = 15) (P< 0.05) while there was no statistical difference between hepatolithiasis patients and healthy controls. Furthermore, we analyzed the clinical stage of the same fifty CCA patients to determine the correspondence relationship between expression level of serum miR-21 and the clinical stage of CCA patients. In the [Figure 1], it showed that the serum miR-21 expression level were increased as the tumor, node, and metastasis (TNM) stage development, and the expression level of serum miR-21 in TNM I stage patients was statistically significantly lower than III or IV patients (P< 0.05), TNM II stage patients was also statistically significantly lower than III or IV patients. However, there was no statistical difference between Stage I patients and Stage II patients. In addition, we compared Stage I patients with healthy control or hepatolithiasis patients, and there was no statistical difference between them (P > 0.05) [Figure 1].
|Figure 1: Expression of microRNA-21 among cholangiocarcinoma patients, hepatolithiasis, and healthy volunteers. One-way ANOVA was used to analyze the significant differences. *P < 0.05|
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The potentiality of serum microRNA-21 as a cholangiocarcinoma diagnostic biomarker
Based on the above results, ROC curves were designed to evaluate the potentiality of serum miR-21 as a noninvasive CCA diagnostic biomarker. According to the ROC analysis, it suggested that serum miR-21 level was defective in distinguishing CCA from healthy controls as its AUC value was 0.871. Hence, it did not seem to be better than the conventional CCA markers—CA19-9 (AUC value = 0.96) [Figure 2]a. The sensitivity values, specificity values, PPVs, and NPVs were 66.7%, 99.33%, 96.97%, and 46.7%, respectively, with the 1.99 as cutoff value of serum miR-21. By contrast, assigned 21.6 U/mL as cutoff value of CA19-9, the sensitivity, specificity, PPV, and NPV were 91.7%, 99.33%, 89.8%, and 77.8%, respectively [Figure 2]. Based on these results, we proposed that the miR-21 in serum did not show the better sensitivity and specificity than CA19-9.
|Figure 2: Serum microRNA-21 as a potential diagnostic biomarker for cholangiocarcinoma patients. (a) Serum microRNA-21 yielded an area under the curve value of 0.871, with 70.8% sensitivity and 86.7% specificity in distinguishing cholangiocarcinoma from normal control subjects. (b) Comparison of serum microRNA-21 levels from all cholangiocarcinoma patients (n = 32). Two-tailed Student's t-test was used to analyze the significant differences. *P < 0.05|
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Alteration of serum microRNA-21 expression level in pre- and post-operative cholangiocarcinoma patients
Subsequently, we analyzed the serum of pre- and post-operative in 32 CCA patients. As shown in [Figure 2]b, the expression level of miR-21 obviously plummeted after curative resection (P< 0.001) [Figure 2]b. Meanwhile, we found that the change of serum miR-21 between high expression group and low expression group was different and according to the expression of miR-21 was ≥2. Obviously, in the high expression group, miR-21 significantly plummeted, but in the low expression group, it remained steady. Hence, based on the data of these two groups, no statistically significant difference showed in the low expression group before or after surgery (P = 0.068). On the contrary, there was significant difference in the high expression group (P = 0.001). Generally speaking, these data implied that serum miR-21 maybe not a particularly effective biomarker of CAA, especially for the early stage of CCA.
Correlation between serum microRNA-21 expression level and clinicopathological features in cholangiocarcinoma patients
In [Table 1], serum miR-21 was significantly associated with clinical stage development (P = 0.003), invasion depth increasing (P = 0.002), lymph vessel infiltration (P = 0.007), metastasis status occurrence (P = 0.001), differentiation status changed (P = 0.003), and curative resection (P = 0.01). On the other hand, the miR-21 expression level was not associated with other clinical features, such as age and gender (P > 0.05).
Relationship between serum microRNA-21 expression level and survival or prognosis in cholangiocarcinoma patients
Finally, Kaplan–Meier analysis and log-rank test were used for investigating the prognostic value of serum miR-21 expression in CCA and analyzing the correlation between the serum miR-21 expression level and clinical prognosis. The statistically significant difference in overall survival and progression-free survival between the high serum miR-21 expression group and low serum miR-21 expression group is shown in [Figure 3]a and [Figure 3]b. Overall survival and progression-free survival for each patient are showed in [Table 2]. Compared to the patients in low serum miR-21 expression group, the patients in high serum miR-21 expression group obviously have shorter overall and progression-free survival time.
|Figure 3: Association of serum microRNA-21 expression with progression-free and overall survival. (a and b) Kaplan–Meier graphs representing the probabilities of progression-free and overall survival in cholangiocarcinoma patients according to expression level of microRNA-21. Two-tailed Student's t-test was used to analyze the significant differences|
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|Table 2: Association of serum miR-21 expression with progression-free and overall survival|
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| > Discussion|| |
CCA accounts for <2% of all human malignancies, but it is the second most common primary liver tumor. It is more commonly seen in Asia with incidences as high as 113 per 100,000 men and 50 per 100,000 women. CCA was diagnosed mainly by the imaging analysis in the late clinical stage which the tumor was unresectable. Serum biomarkers were not always the first choice to diagnose tumors due to lack of sensitivity, especially for the early CCA. Aberrant miRNAs expression patterns have been found in various cancers, and alteration of certain miRNAs expression correlate highly with progression, diagnosis, and prognosis of specific human malignant diseases. Mitchell et al. reported that serum miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. It revealed that the circulating miRNAs have potential for as a valuable and noninvasive diagnostic biomarker of cancers. The poor prognosis associated with CCA mainly due to lack of minimally invasive and early detection method. Therefore, identifying a circulating miRNA in serum as biomarker for CCA seems to be very urgent and important.
In this study, we first evaluated the important role of serum miR-21 in diagnosing CCA. We found that the serum miR-21 expression level in CCA patients was to some extent higher than healthy controls, and it closely correlated with the TNM stage of CCA patients. However, the correlation was just between Stage I + II patients and Stage III + IV patients. There was no statistical difference between Stage I patients and Stage II patients. More interestingly, there was no statistical difference between Stage I patients and healthy control. This result would prompt us serum miR-21 was not an effective biomarker for early CCA. Then, we identified the important role of serum miR-21 in diagnosing the CCA by AUC and comparing the AUC curves of serum miR-21 with conventional CAA marker CA19-9. We found the AUC value, sensitivity, specificity, and NPV of serum miR-21 were all lower than CA19-9. At last, we investigated the change of serum miR-21 expression between pre- and post-operative patients, and it showed significant difference in the high miR-21 expression group, but no difference in the low miR-21 expression group. We speculate the reasons for this difference might be two aspects. (1) Previous reports have proved that miR-21 expression will also increase with inflammation or inflammatory diseases., Hence, the inflammatory response of surgery would change the miR-21 expression. (2) Some patients in low miR-21 expression group are in the early clinical stage of CCA. Moreover, to the best of our knowledge, in the early clinical stage of CCA, serum miR-21 was low expression level, so small change appeared in expression level. These results suggested that extraction and identification of individual serum miR-21 to diagnosing CCA are feasible although serum miR-21 might be not an effective diagnostic biomarker, especially for the early clinical stage of CCA because of its low sensitivity and specificity. This conclusion was different with Kishimoto et al.
We also explored whether the serum miR-21 could be a prognostic biomarker of CCA. The results revealed that serum miR-21 expression level was closely correlated with adverse clinicopathological factors including clinical stage, invasion depth, lymph vessel infiltration, metastasis status, and differentiation status. More importantly, the serum miR-21 expression level was correlated with CCA patients whether to undergo the curative resection. At the same time, it was also significantly correlated with progression-free and overall survival of CCA patients. Our finding proved that in CCA patients, high level of serum miR-21 indicated a poor prognosis. Hence, this study implied that serum miR-21 was a potential prognostic biomarker to help predict metastasis, clinical stages, and survival time of CCA patients.
The role of miR-21 is extremely different in different diseases. It has reported that upregulated miR-21 plays an important role in progression of tumor and serves as an oncogenic miRNA in solid cancers. Just because of the upregulated miR-21 in cancer, serum miR-21 has been detected as a novel biomarker of cancer. From the meta-analysis reports, we found that diagnostic and prognostic value of serum miR-21 was different in different cancers. Some believed that it was an effective diagnostic biomarker, and more people supported it to be a prognostic biomarker., The current study indicated that serum miR-21 was a powerful prognostic biomarker but not an effective diagnostic biomarker because of its low sensitivity and specificity. At the same time, our study did not include the serum miR-21 expression of the inflammatory biliary diseases which were the precancerous lesions of CCA, such as primary sclerosing cholangitis, hepatolithiasis, and choledochal cyst. This might make our results to be controversial if the serum miR-21 would be a diagnostic biomarker of CCA.
| > Conclusions|| |
This study illustrated that serum miR-21 expression level was obviously regulated in CCA patients with clinical stage development and distant metastasis occurrence, and it was closely correlated with the progression-free and overall survival in CCA patients. However, serum miR-21 was not enough sensitive to diagnose the early stages CCA. Hence, serum miR-21 was an effective prognostic but not a diagnostic tool in patients with CCA.
Financial support and sponsorship
This study is supported by National Natural Science Foundation (No. 81272397) and University Science Research Project of Anhui province (No. KJ2013Z143).
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Gatto M, Bragazzi MC, Semeraro R, Napoli C, Gentile R, Torrice A, et al.
Cholangiocarcinoma: Update and future perspectives. Dig Liver Dis 2010;42:253-60.
Lubana SS, Singh N, Seligman B, Tuli SS, Heimann DM. First reported case of primary intrahepatic cholangiocarcinoma with pure squamous cell histology: A case report. Am J Case Rep 2015;16:438-44.
Bertuccio P, Bosetti C, Levi F, Decarli A, Negri E, La Vecchia C. A comparison of trends in mortality from primary liver cancer and intrahepatic cholangiocarcinoma in Europe. Ann Oncol 2013;24:1667-74.
de Martel C, Plummer M, Franceschi S. Cholangiocarcinoma: Descriptive epidemiology and risk factors. Gastroenterol Clin Biol 2010;34:173-80.
Murakami Y, Uemura K, Sudo T, Hashimoto Y, Nakashima A, Kondo N, et al.
Prognostic factors after surgical resection for intrahepatic, hilar, and distal cholangiocarcinoma. Ann Surg Oncol 2011;18:651-8.
Schiffman SC, Reuter NP, McMasters KM, Scoggins CR, Martin RC. Overall survival peri-hilar cholangiocarcinoma: R1 resection with curative intent compared to primary endoscopic therapy. J Surg Oncol 2012;105:91-6.
Cho MS, Kim SH, Park SW, Lim JH, Choi GH, Park JS, et al.
Surgical outcomes and predicting factors of curative resection in patients with hilar cholangiocarcinoma: 10-year single-institution experience. J Gastrointest Surg 2012;16:1672-9.
Ercolani G, Vetrone G, Grazi GL, Aramaki O, Cescon M, Ravaioli M, et al.
Intrahepatic cholangiocarcinoma: Primary liver resection and aggressive multimodal treatment of recurrence significantly prolong survival. Ann Surg 2010;252:107-14.
Berardi R, Mocchegiani F, Pierantoni C, Federici A, Nicolini D, Morgese F, et al.
Resected biliary tract cancers: A novel clinical-pathological score correlates with global outcome. Dig Liver Dis 2013;45:70-4.
Juntermanns B, Radunz S, Heuer M, Hertel S, Reis H, Neuhaus JP, et al.
Tumor markers as a diagnostic key for hilar cholangiocarcinoma. Eur J Med Res 2010;15:357-61.
Mendell JT. MicroRNAs: Critical regulators of development, cellular physiology and malignancy. Cell Cycle 2005;4:1179-84.
Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004;116:281-97.
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001;294:853-8.
Silakit R, Loilome W, Yongvanit P, Chusorn P, Techasen A, Boonmars T, et al.
Circulating miR-192 in liver fluke-associated cholangiocarcinoma patients: A prospective prognostic indicator. J Hepatobiliary Pancreat Sci 2014;21:864-72.
Wang LG, Gu J. Serum microRNA-29a is a promising novel marker for early detection of colorectal liver metastasis. Cancer Epidemiol 2012;36:e61-7.
Ng EK, Chong WW, Jin H, Lam EK, Shin VY, Yu J, et al.
Differential expression of microRNAs in plasma of patients with colorectal cancer: A potential marker for colorectal cancer screening. Gut 2009;58:1375-81.
Pisarello MJ, Loarca L, Ivanics T, Morton L, LaRusso N. MicroRNAs in the cholangiopathies: Pathogenesis, diagnosis, and treatment. J Clin Med 2015;4:1688-712.
Kim YK. Extracellular microRNAs as biomarkers in human disease. Chonnam Med J 2015;51:51-7.
Huang Q, Liu L, Liu CH, You H, Shao F, Xie F, et al.
MicroRNA-21 regulates the invasion and metastasis in cholangiocarcinoma and may be a potential biomarker for cancer prognosis. Asian Pac J Cancer Prev 2013;14:829-34.
Chusorn P, Namwat N, Loilome W, Techasen A, Pairojkul C, Khuntikeo N, et al.
Overexpression of microRNA-21 regulating PDCD4 during tumorigenesis of liver fluke-associated cholangiocarcinoma contributes to tumor growth and metastasis. Tumour Biol 2013;34:1579-88.
Selaru FM, Olaru AV, Kan T, David S, Cheng Y, Mori Y, et al.
MicroRNA-21 is overexpressed in human cholangiocarcinoma and regulates programmed cell death 4 and tissue inhibitor of metalloproteinase 3. Hepatology 2009;49:1595-601.
Wu K, Li L, Li S. Circulating microRNA-21 as a biomarker for the detection of various carcinomas: An updated meta-analysis based on 36 studies. Tumour Biol 2015;36:1973-81.
Wang Y, Gao X, Wei F, Zhang X, Yu J, Zhao H, et al.
Diagnostic and prognostic value of circulating miR-21 for cancer: A systematic review and meta-analysis. Gene 2014;533:389-97.
Tyson GL, El-Serag HB. Risk factors for cholangiocarcinoma. Hepatology 2011;54:173-84.
Ohtsuka M, Ling H, Doki Y, Mori M, Calin GA. MicroRNA processing and human cancer. J Clin Med 2015;4:1651-67.
Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al.
Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105:10513-8.
Tomimaru Y, Eguchi H, Nagano H, Wada H, Kobayashi S, Marubashi S, et al.
Circulating microRNA-21 as a novel biomarker for hepatocellular carcinoma. J Hepatol 2012;56:167-75.
Paraskevi A, Theodoropoulos G, Papaconstantinou I, Mantzaris G, Nikiteas N, Gazouli M. Circulating MicroRNA in inflammatory bowel disease. J Crohns Colitis 2012;6:900-4.
Kishimoto T, Eguchi H, Nagano H, Kobayashi S, Akita H, Hama N, et al.
Plasma miR-21 is a novel diagnostic biomarker for biliary tract cancer. Cancer Sci 2013;104:1626-31.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]