| ?Background and Objective? Liver cirrhosis is defined as the histological development of regenerative nodules surrounded by fibrous connective tissue in response to chronic liver injury,causing portal hypertension and end stage liver disease.It is the 14 th leading cause of death in the whole world,and has an increasing tendency in developed country.Recently,the new concept of the treatment of liver cirrhosis is to stabilize the progress of the disease through prevention and early intervention,to delay the progress of liver cirrhosis and avoid liver transplantation.Therefore,it is necessary for early diagnosis of liver cirrhosis.Liver biopsy,the gold standard for fibrosis assessment,still has some limitations in clinic,including severe complications,observer variability and sampling variation.Therefore,developing a reliable and convenient non-invasive method for the diagnosis and prognosis evaluation of liver cirrhosis will be a promising advance in the clinical application.In clinic,the united serum biomarkers have been used to improve the accuracy of the diagnosis of liver cirrhosis,such as APRI(AST/PLT ratio index),FIB-4(fibrosis index based on the 4 factors),AAR(AST/ALT ratio),which are used widely as the important non-invasive biomarkers in liver cirrhosis study,but the application of these serum biomarkers has been limited for their low accuracy and specificity in cirrhosis diagnosis.Non-coding RNAs(nc RNAs)are a class of RNAs that can not be translated into protein,including short non-coding RNAs(snc RNAs)and long non-coding RNAs(lnc RNAs).Micro RNAs(mi RNAs),a kind of snc RNAs,are a class of endogenous,small(18–24nucleotides),non-coding single-stranded RNAs that negatively regulate gene expression through binding to the 3'-untranslatedregion(UTR)of target m RNAs.Lnc RNAs,which are defined as transcripts longer than 200 nucleotides,regulate expression of genes at transcriptional level,post-transcriptional level and epigenetic level.Recently,it has been confirmed that some nc RNAs exist not only in the tissues and cells,but also can be stably detected in serum and plasma,such as mi R-21,mi R-122,MALAT-1(metastasis associated in lung adenocarcinoma transcript 1)etc.It has been proved that,there are closely relationship of nc RNA expression both in the serum and the liver tissue,and nc RNAs might act as the important mediators in various chronic diseases,which could provide the great value for the diagnosis,treatment and prognosis evaluation in clinical investigation.In the current study,we investigated the serum expression levels of several target nc RNAs to explore the value of these nc RNAs as the non-invasive biomarkers for liver cirrhosis diagnosis.?Methods? Section 1: The value of serum mi R-192 and mi R-29 a as the non-invasive biomarkers for liver cirrhosis diagnosis 1.In our previous study,we have revealed the relationship between mi R-192,mi R-29 a and chronic liver disease,and mi R-192 and mi R-29 a were selected as the target biomarkers in our study.Serum samples from 80 liver cirrhosis patients and 36 healthy volunteer controls were collected respectively.Total RNA were extracted from the serum samples using the mir VanaTM PARISTM kit.The expression levels of mi R-192 and mi R-29 a were determined by q RT-PCR.Moreover,the new risk score model was established by the stepwise logistic regression.The area under the ROC curve(AUC)was defined as diagnostic value of liver cirrhosis.The diagnostic value of serum mi R-192,mi R-29 a and the new risk model,compared with the other united serum biomarkers,was analyzed by ROC curve.2.The Child-Pugh classification was used to evaluate the severity of liver cirrhosis.The relationship between the severity of liver cirrhosis and the expression levels of these mi RNAs and the risk scores was analyzed.Section 2: The value of lnc RNA uc.77+ and uc010 vfu.1 as non-invasive biomarkers for liver cirrhosis 1.The liver tissues of 5 patients with liver cirrhosis and 4 healthy volunteers were collected to determine the differential expression of lnc RNAs using lnc RNA expression microarray.10 up-regulated genes and 10 down-regulated genes were selected as candidates.Serum samples from 10 patients with liver cirrhosis and 10 healthy controls were collected to confirm the microarray results by q RT-PCR method.Two lnc RNAs(uc.77+ and uc010 vfu.1)were selected as non-invasive biomarkers candidates,which expression levels were stable and significantly different between two groups.2.Serum samples from 93 liver cirrhosis patients and 40 volunteer healthy controls were collected.The expression levels of those uc.77+ and uc010 vfu.1 were analyzed by q RT-PCR.The new risk score model was established by the stepwise logistic regression.The diagnostic value of uc.77+,uc010 vfu.1 and the new risk model,compared with the other united serum biomarkers,was analyzed by ROC curve.3.The Child-Pugh classification was used to evaluate the severity of liver cirrhosis.The relationship between the severity of liver cirrhosis and the expression levels of these lnc RNAs and the risk scores was analyzed.?Results? 1.The value of serum mi R-192,mi R-29 a and the risk score as non-invasive biomarkers for liver cirrhosis We analyzed the expression levels of mi R-192 and mi R-29 a in 116 participants(80 liver cirrhosis patients and 36 volunteer healthy controls)by q RT-PCR method.Serum mi R-192 was significantly up-regulated in liver cirrhosis patients as compared to that of the control(P<0.0001).We also found that the expression level of serum mi R-29 a was significantly down-regulated in liver cirrhosis patients compared to that of the controls(P<0.0001).mi R-192 had an AUC of 0.8844(P<0.001,95% confidence interval(CI)=0.8175-0.9513)with sensitivity of 86.25% and specificity of 83.33% in separating the controls from liver cirrhosis patients.Meanwhile,mi R-29 a displayed an AUC of 0.8837(P<0.001,95% CI=0.8226-0.9448)with sensitivity of 76.25% and specificity of 86.11% in separating the controls from liver cirrhosis patients.In order to get a better diagnostic value of the combinational expression of two mi RNAs,a completely new classifier model was established by the stepwise logistic regression analysis.The formula of the classifier is as follow: Risk score =1.469-4.476×mi R29a+0.979×mi R192.The AUC of the new risk score was 0.9354(P<0.001,95% CI=0.8827-0.9881,sensitivity =86.25%,specificity=94.44%).The diagnostic values of the two mi RNAs and the new risk score were much higher than those of APRI(AUC=0.8080),FIB-4(AUC=0.8174),AAR(AUC=0.5941)and other clinical non-invasive indicators.2.The relationship between the severity of liver cirrhosis and mi R-192,mi R-29 a and the risk score The Child-Pugh classification was used to evaluate the severity of liver cirrhosis.There was also significant relationship between the severity of liver cirrhosis and mi R-192,mi R-29 a and the risk score.There was significant positive correlation between the serum level of mi R-192 and the severity stage of cirrhosis(P=0.026,Rho=0.248).In contrast,the serum levels of mi R-29 a decreased significantly along with the aggravation of cirrhosis stage(P=0.022,Rho=-0.334).The risk score positively correlated with the severity of liver cirrhosis(P=0.005,Rho=0.311).3.Lnc RNA uc.77+ and uc010 vfu.1 were selected by lnc RNA expression microarray 20 lnc RNAs were selected among the differently expressed lnc RNAs by lnc RNA expression microarray.Expression of uc.77+ and uc010 vfu.1 in liver tissues and serum was stable and significantly different between two groups by q RT-PCR detection,which validated the microarray findings.4.The value of serum uc.77+,uc010 vfu.1 and the risk score as non-invasive biomarkers for liver cirrhosis Both of the serum expression of uc.77+ and uc010 vfu.1 was significantly down-regulated in liver cirrhosis patients as compared to that of the healthy controls(P<0.001).uc.77+ expression had an AUC of 0.8188(P<0.001,95% CI=0.7413-0.8963)with sensitivity of 82.80% and specificity of 67.50% in separating the controls from liver cirrhosis patients.Meanwhile,uc010 vfu.1 expression displayed an AUC of 0.8446(P<0.001,95% CI=0.7754-0.9138)with sensitivity of 70.97% and specificity of 82.50% in separating the controls from liver cirrhosis patients.In order to get a better diagnostic value of the two lnc RNAs,a completely new classifier model was established by the stepwise logistic regression analysis.The formula of the classifier is as follow: Risk score=3.716-[4.007×uc.77+]-[2.445×uc010vfu.1].The AUC of the new risk score was 0.8849(P<0.001,95% CI=0.8224-0.9475,sensitivity =70.97%,specificity=90.00%).The diagnostic value of the two lnc RNAs and the new risk score were much higher than those of APRI(AUC=0.7637),FIB-4(AUC=0.7715),AAR(AUC=0.5575)and other clinical non-invasive indicators.5.The relationship between the severity of liver cirrhosis and uc.77+,uc010 vfu.1 and the risk score The Child-Pugh classification was also used to evaluate the severity of liver cirrhosis.There was significant relationship between the serum level of lnc RNAs and the severity of liver cirrhosis in cirrhotic patients.Negative correlation between the serum level of uc.77+ and the severity stage of cirrhosis(P<0.01,Rho=-0.621)has been confirmed.Meanwhile,serum uc010 vfu.1 level was also inversely correlated with the severity of cirrhosis(P<0.01,Rho=-0.582).Furthermore,the risk score positively correlated with the aggravation of cirrhosis stage(P<0.01,Rho=0.675).?Conclusion? 1.Expression of nc RNAs including mi R-192,mi R-29 a,uc.77+ and uc010 vfu.1 was stable and detectable in human serum,and the levels of these nc RNAs are significantly different between liver cirrhosis patients and the controls.2.The diagnostic values of mi R-192,mi R-29 a,uc.77+ and uc010 vfu.1 are much higher than the other clinical indicators,and the combined diagnostic value of the two nc RNAs has more advantage over that of single nc RNA.3.The significant relationship between the severity of liver cirrhosis and the serum levels of mi R-192,mi R-29 a,uc.77+,uc010 vfu.1 has been confirmed in the liver cirrhosis patients. |
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