| Introduction Cholangiocarcinoma(CCA)is a kind of cancer from the biliary epithelium;it includes intrahepatic cholangiocarcinoma(ICC)arises from the liver and extrahepatic cholangiocarcinoma(ECC)from the extrahepatic bile ducts.ECC also includes hilar cholangiocarcinoma(HCC)and distal cholangiocarcinoma.ICC,which is the kind of primary liver cancer before,has separated to study lonely because of the different clinic pathology relatively to the hepatocellular carcinoma.The incidence rates of CCA have increased for these years.The highest prevalence rates of CCA have been reported in the south Asian including china,an area with a high prevalence of liver fluke infestations and hepatolithiasis.Although the CCA has been paid attention and taken more efforts,the outcome of CCA is not satisfactory and the 5-years survive rate doesnot reach to 30%.And the serious problem is the low rate of diagnosis for the early CCA.According to the reports,Resectability rates range between 19 and 74%.Recurrence rates are usually high around 60-65%.Survival rates depend on R0 resection and lymph node status.Following R0 resection,5-year survival rates are 23-42%versus 0%after R + resection.Five-year survival rates in patients with N1 status following surgical resection is 0-9%and up to 43%in NO disease.However,there are no large randomized controlled trials demonstrating a survival benefit of neoadjuvant or adjuvant chemotherapy.So we need to take two measures to improve the outcome of CCA,First,we need to improve the diagnostic rate of early CCA,Second,we need to explore the new and effective targets of chemotherapy.microRNAs is a kind of noncoding RNAs with 18-25 nucleotides in length,its function has been proved to play a major role in the regulation of gene expression,and regulates the target mRNA by binding to the sites of mRNAs.Recently,a lot of studies have shown that aberrant expression of microRNAs is related to the genesis and development of CCA.Many microRNAs are detected in CCA cell lines or human tissues.But using microRNAs as a diagnostic biomarker in CCA have many obstacle,for example the specimen?invasive and not conductive to popularity.Recently,several studies have shown that microRNAs could be detected in human serum of cancer and could be the biomarkers to diagnose with high sensitivity and specificity in many cancers.There is an advantage which further highlighted that the potential of circulating microRNAs is a non-invasive diagnostic and prognostic biomarkers in cancerous diseases.In our early experiment and others preliminary studies,we found that expression of microRNA-21 was up-regulation in the paraffin section of CCA patients,further reaserch found the expression of microRNA-21 is associated with pathological stage,Now circulating microRNA-21 has been detected to be a biomarker in various carcinoma,but the value of circulating microRNA-21 was different.So we should make sure the value of serum microRNA-21 for CCA.In this study,we investigated the role of serum microRNA-21 in CCA using two parts.In the first part.We analysed the feasibility of detecting of serum microRNA-21 for CCA patients.In the second part,we evaluated the clinical significance of microRNA-21 as a biomarkers of diagnosis and prognosis in CCA patients.Aim1.To confirm the abnormal expression of microRNA-21 in the tissue and serun of CCA2.To confirm the serum microRNA-21 to be a biomarker for CCA by analysing the correlation between serum microRNA-21 and clinic pathologyMaterials and methodsPatients and specimensCollection and studies were approved by the Research Ethics Committee of AnHui Province Hospital,All patients signed the written paper to agree to donating their samples for research.A total of 50 patients with CCA in the Anhui Province Hospital from Jan 2011 to Dec 2014 were enrolled in this study.18 patients received palliative resection and 32 patients received curative resection.32 tissue samples and 79 serum samples were obtained.All patients didn't accept the radiotherapy or chemotherapy before operation.All tumors were histologically diagnosed as biliary duct carcinoma after operation.RNA extraction and quantitative real-time polymerase chain reaction(qRT-PCR)For miRNA quantification,total miRNA was extracted from the samples using miRNeasy RNA isolation Kit according to the manufacturer's instructions(Qiagen,Inc.).TaqMan miRNA quantitative real-time PCR(qRT-PCR)(Thermo,CA,USA)were 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(U6).Statistical analysesSPSS 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.Kaplan-Meier and Cox's proportional harzard model were used to analyzing the survival of CCA,Two-sided test was employed to determine the P values,and P<0.05 was considered to be statistical significance.ResultsPart one Relative expression of microRNA-21 in the tissue and serum of cholangiocarcinoma patients1-Expression of microRNA-21 in the tissue of CCAqRT-PCR was used for investigating the concentration level of microRNA-21 in the 32 tissue samples of CCA patients.We found the expression of microRNA-21 in tumor tissues was significantly higher than control tissue(1.72±0.67 vs 0.86±0.29,P<0.05),this result shows that the abnormal expression of microRNA-21 was the important factor of occurrence of CCA.2.Expression of microRNA-21 in the serum of CCAWe detectd the relative expression of serum microRNA-21 in 50 CCA patients and 15 healthy people and found the serum microRNA-21 of CCA patients could be detected.The expression of serum microRNA-21 in CCA patients was significantly higher than healthy controls(n=15)(3.11±2.02 vs 1.12±0.55,P<0.05).This result took the basis for serum microRNA-21 as biomarker of CCA.3.The correlation of microRNA-21 expression in the tissue and serum of CCAWe compared the expression of microRNA-21 in tissue with in serum,and found that they showed consistent,there was correlative between them(R=0.839,P=0.000)Part two The clinical significance of microRNA-21 for CCA patients1.Clinical significance of microRNA-21 as a diagnostic biomarker for CCA patients1)Correlation between serum microRNA-21 and the clinic pathology of CCAwe analyzed the clinic stage of 50 CCA patients according to the AJCC Cancer Staging Manual(2010)and make sure the stage ? 7 cases,stage ?16 cases,stage ?12 cases and stage ?5cases.The expression of microRNA-21 was 1.56 ±0.51?2.48± 1.15.3.74±2.08 and 3,96±2.57 respectively.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.From Our data we could find the expression levels of serum microRNA-21 were increased as the TNM stage increased,the expression of serum microRNA-21 in TNM? stage patients was statistically significantly lower than in ? or IV patients(P<0.05),but there was no statistical difference between stage I patients and healthy controls(1.56 ± 0.51 vs 1.12± 0.55,P=0.581).2)Comparison between serum microRNA-21 and CA19-9 in CCA patientsBased 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 2a].The sensitivity values,specificity values,PPVs,and NPVs were 66.7%,99.33%,96.97%,and 46.7%,respectively,with the 2 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 CA19-9.3)Alterations of serum microRNA-21 expression levels in pre-and postoperative cholangiocarcinoma patientsSubsequently,we analyzed the serum of pre-and postoperative in 32 CCA patients who underwent curative resection.We found that serum levels of microRNA-21 obviously plummeted after curative resection(2.44± 1.1 vs 1.65±0.63,P<0.001).Meanwhile,we found that the change of serum microRNA-21 was different between high microRNA-21 expression group and low microRNA-21 expression group which according to the levels of microRNA-21(the value of 2-? ?CT)was equal or greater than 2.The high microRNA-21 expression group was plummeted obviously than low levels of microRNA-21.Moreover a little samples was greater than preoperative resection.So we analyzed the data of these two groups and found no statistically significant difference in the low microRNA-21 expression group before or after surgery(1.33±0.36 vs1.24±0.33,P=0.068),But there was significant difference in the high microRNA-21 expression group(3.12±0.72 vs 1.94±0.62,P=0.001).Taken together,these data suggested serum microRNA-21 was not a effective biomarker for the recurrence of CCA,sepcially for the early stage of CCA.2.Clinical significance of microRNA-21 as a prognostic biomarker for CCA patients1)Correlation between serum microRNA-21 expression level and clinicopathological features in cholangiocarcinoma patientsNext,correlation between serum microRNA-21 expression and clinicopathological characteristics of CCA patients was been explored.We found serum microRNA-21 was significantly associated with clinical stage development(P=0.003),invasion depth(P=0.002),lymph vessel infiltration(P=0.007),metastasis status(P=0.000),differentiation status(P=0.003),and whether to undergo curetive resection(P=0.01).On the other hand,the microRNA-21 expression level was not associated with other clinical features,such as age,gender(all at P>0.05).2)Relationship between serum microRNA-21 expression level and survival or prognosis in cholangiocarcinoma patientsFinally,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(36.23±7.6 vs 19.84±7.64;31.23±7.06 vs 17.47±8.12,P<0.05 respectively).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.Conclusion1.microRNA-21 expressed highly in the tissue and serum of CCA,it showed microRNA-21 effect the occurrence of CCA and might be a good biomarker for CCA.2.serum miR-21 was not a effective diagnostic biomarker for the early detection of CCA,but could be a potential and useful prognostic biomarker for CCA... |
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