Establishment And Applicaton Of Fluorescent Ligation-Based Quantitative PCR Assay For Detection Of MiR-122

miRNAs are a class of single-stranded,endogenous,non-coding RNAs that are widely distributed in living organisms.By pairing with incomplete bases at the 3'-untranslated region of m RNA,miRNAs can induce m RNA post-transcriptional degradation and inhibit m RNA translation,regulate many pathological and physiological processes in the body,including cell metabolism,differentiation,and apoptosis.miR-122 has been found to be abnormally expressed in tissues and body fluids to varying degrees during the occurrence and development of many tumors as well as viral infection,the difference of expression level was consistent with the expression of chemokines or diagnostic markers.miR-122 provides a new way for studying the molecular mechanism of tumor development,the route of virus infection and pathogenicity,and is expected to be an important molecular marker for early screening and prognosis of some cancers.At present,microarrays,next generation sequencing(NGS)and reverse transcription real-time fluorescent quantitative PCR(RT-qPCR)are the main methods to detect miRNA.However,because of the short miRNA sequences and the great difference in the abundance of miRNA in vivo,the traditional miRNA detection methods still have some problems such as low sensitivity and poor specificity.Accurate quantification of miRNAs in living organisms is critical to study their biological functions and use them as biological markers for diseases.Thus,it is very important to establish a sensitive,specific and low cost method for the detection of miRNAs.The aim of this study was to develop a novel method for the detection of miR-122in sera and tissue samples by using Ligation-qPCR method to sensitively detect miR-122,study the potential regulatory mechanisms during virus infection and its application as a molecular marker for some diseases.Firstly,we established a Ligation-qPCR method to detect miR-122 and compared the sensitivity of Ligation-qPCR method with MGB probe(A novel probe with a minor groove binder was added to the 3'end of the conventional probe)and double quenching probe.The new Ligation-qPCR method has a limit of detection(LOD)of 10~3 copies,and the linearity of the standard curve was good(R~2=0.9922).In order to improve the accuracy of miR-122 detection in sera,a similar Ligation-qPCR system for cel-miR-39was also established as a reference control.By screening various miR-122 fragments,we found a pair of fragments(A08,B14)of cel-miR-39 which could be susessfully used for Ligation-qPCR(LOD=10~3 copies).In order to verify the feasibility of Ligation-qPCR in detecting miR-122 in biological samples,RNA was extracted from the heart,liver,spleen and lung tissues of normal mice to detect miR-122.miR-122 was significantly overexpressed in mouse liver tissue(p<0.0001),which was consistent with the well-established liver specificity of miR-122.The results showed that the Ligation-qPCR method for the detection of miR-122 in the biological samples was feasible.In view of the significant difference in miR-122 expression in various diseases,we used the Ligation-qPCR assay to detect miR-122 in sera of liver cancer patients,transplanted breast cancer mice,influenza virus H1N1 infected and immunized mice,respectively,to explore the feasibility of its application as a molecular marker for cancers,virus infections and vaccine immunities.The expression of miR-122 in sera of24 healthy subjects,39 patients with liver cancer and 7 patients with liver cancer after operation was detected by Ligation-qPCR,the miR-122 levels in sera of hepatocellular carcinoma(HCC)patients was significantly decreased(p<0.0001),and the miR-122levels in sera of posr-operative HCC patients were higher than those in HCC patients(p<0.05).The results indicate that miR-122 has a potential to be used as a molecular marker for screening and prognosis of liver cancer.The area under the curve(ROC)curve was used to evaluate the feasibility of miR-122 to be used in liver cancer diagnosis.The results showed that the ROC of miR-122 was 0.8828(p<0.0001),which was larger than that of the traditional biomarkers(the AUC values of AST,ALT,and TBIL were 0.8260,0.7143,and 0.7108,respectively)in the diagnosis of liver cancer.On the other hand,the level of serum miR-122 in mice with transplanted breast cancer was not significantly different from that in healthy mice(p>0.05),there was also no significant difference in serum levels among H1N1 infected mice,H1N1 vaccine immunized mice and healthy mice(p>0.05).This suggestes that miR-122 may not used to diagnose breast cancer and H1N1 infection and vaccine immunity.We next used a mouse model infected with influenza A(H1N1)virus to study the relationship between miR-122 and histopathology.Lung,liver and spleen tissues from healthy mice,mice immunized with live attenuated H1N1 vaccine and mice infected with wild type H1N1 were collected and RNA were extracted for miR-122measurements.The results showed that the levels of miR-122 were significantly higher in liver tissues(p<0.01),but not in lung and spleen tissues(p>0.05).Compared with the level of miR-122 in immunized mice,miR-122 in lung and liver tissues of H1N1infected mice were lower(p<0.05)and higher(p<0.001),respectively,but there was no significant difference in spleen tissues(p>0.05).These results suggest that liver miR-122 may play an important role in some antiviral immune responses during the course of H1N1 infection,involve in regulating some H1N1 infection pathways in lung and liver tissues,but do not play a significant role in spleen.In conclusion,a Ligation-qPCR method for the detection of miR-122 has been established.The detection limit of this method is as low as 10~3copies/per reaction system.Therefore,this highly accruate Ligation-qPCR method can be used as a biomarker for liver cancer and to study histopathology of virus infection.