Circulating MiRNA, A New Biomarker For HCC And Acute Rejection After Liver Transplantation

The first miRNA, lin-4from Caenorhabditis elegans, was discovered by Ambros and coworkers in1993as an endogenous regulator of genes that control developmental timing. In2001, miRNAs were found to comprise a broad class of small RNA regulators, with at least dozens of representatives in each of several plant and animal species. During the last decade, more and more miRNA were found in plant and animals. miRNA have four characteristics:1) miRNA have a independent transcript with about22nt in length.2) miRNA is derived from precursor with secondary structure of stem loop.3) miRNA need to be processed by endonucleases Dicer before mature.4) miRNA and its precursor which have stem loop structure are conservative between species.Single-stranded forms of miRNA was found to be associated with effector assemblies known as RNA-induced silencing complexes (RISCs). Bartel et al found that miRNA direct Ago proteins to target mRNAs by interacting with sites of imperfect complementarity. Short "seed" sequences at the51ends of miRNA (nucleotides2-8) are most critical, and in some cases fully sufficient, for target selection. When directed to mRNAs via these interactions, Ago proteins perform a still incompletely defined activity that results in accelerated turnover and reduced translation of the targeted transcript.miRNA engage extensive biological significance, including cell growth, proliferation, apoptosis, stress through regulating gene expression. miRNA closely related to cancer and it may play the roles similar to oncogenes or tumor suppressor genes. Recently, scientists have mapped almost all the tissue miRNA expression profiles in human along with the development of the detection technique. The expression profiles showed that miRNA displayed tissue-specific characteristic and the expression spectrum or level would change with different various physiological or pathological conditions especially for cancer. These characteristics make miRNA become a promising class of new moleculars as tumor diagnosis biomarkers or therapeutic targets.Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. China is a country with high HCC incidence and more than half new cases are occurring in China each year. It has been clear that viral hepatitis and aflatoxin are carcinogenic factors for HCC. In China, the prevalence of hepatitis B is the main reason account for the high incidence of HCC. In the Western countries, the main risk is the hepatitis C. Although the Neonatal Vaccination Program has been on-going for many years in mainland of China, which reduced the amount of Hepatitis B carriers, Hepatitis C infection is on the rise recent years. In Europe and the United States, the incidience of HCC is also rising because of obesity, alcoholism and so on. Therefore, HCC control is still the importance and difficulty for the current study.The incidence of HCC ranked sixth worldwide, but the mortality is ranked third due to its high degree of malignancy. The onset of HCC is hidden and the5-year survival rate of HCC patients is only about7%. More than60%HCC patients access to advance stage at first diagnosis, thereby, they lost the opportunities for radical treatments. Alpha-fetoprotein (AFP) is the most commonly used diagnostic marker for HCC in the clinic. However, the sensitivity and specificity of AFP is not satisfactory. The abnormal AFP level can also be found in population other than HCC (pregnant women, acute and chronic hepatitis patients, germ cell tumors patients, gastrointestinal cancer patients). About30-40%HCC patients show normal AFP level. Disocvery of new accurate and effective early diagnosis biomarkers is the key to improve the the survival of HCC patients. The quantification of circulating miRNA need internal control. However, there have no accepted stable control for those kind of study, which led to huge variation of the results between different groups. The compatibility of the results from different study groups is poor. Therefore, what we need to do first is to screen stable control for the quantification of circulating miRNAHCC is the final outcome for many chronic end-stage liver diseases. The majority of HCC patients are associated with underlying liver disease. HBV-cirrhosis-HCC is referred to as "trilogy of liver cancer". Surgical resection only remove tumor and the residual diseased liver is the hotbed of future recurrence. Liver transplantation can remove the tumor and the cirrhotic liver, which eliminating the HCC and its "soil". Liver transplantation is the only definite treatment for end-stage liver diseases. Although the development of immunosuppressant is rapid, there are still20-40%of patients with postoperative rejection. Allograft rejection is an important factor that leading to the function loss of allograft. Early detection of acute rejection and timely treatment is critical for the function rescue of allograft. Thus discovery of accurate and effective biomarkers for acute rejection is an important mean to improve the survival of liver graft.According to the timeline model proposed by Anglicheau, in the development process of the disease, the earliest change will lies on molecular biology, and then pathomorphological change, clinical symptoms is the last thing to be observed. Molecular markers can monitor disease earlier than pathology, imaging examination. Serum/plasma biomarkers are clinically preferred for their easy access. miRNA can regulate gene expression (including the control of cell growth, proliferation, apoptosis, stress and other broad biological role) and its owe expression levels associated with disease state. Many studies have relevaled that miRNA could resist the degradation of the RNA enzyme and existed stably in the peripheral blood. In this study, we screened and validated circulating miRNA as biomarkers for liver cancer surgery by using high-throughput microarrays, quantitative RT-PCR and rat model. Chapter ISelection of Endogenous Stable Control for the Quantification of Circulating miRNA and Its ApplicationBackground and Purpose:Recently, increasing studies have focused on circulating miRNAs in cancer patients for its potential to be used as tumor markers. Proper and accurate normalization is critical to reveal the effect of dysregulated miRNAs in the carcinogenesis, progression and prognosis. Currently, there is no standard endogenous control for the circulating microRNA studies. This study is to identify and characterize appropriate endogenous stable control for the quantification of circulating miRNAs in hepatocellular carcinoma (HCC) patients and other cancer patients.Patient and Methods:The expression of global circulating miRNAs from control (34healthy,21chronic hepatitis B,25cirrhosis) and cancer patients (57HCC,41colon cancer,73lung cancer) were investigated using microarrays (cohort1). Candidate endogenous control miRNAs were selected according to the stability value. Another independent cohort (cohort2, n=184) was then adopted to estimate the candidate control miRNAs by using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) assay. The software programs geNorm and NormFinder were used as statistical tool to determine the stable endogenous circulating miRNAs. After the verification of the candidate circulating miRNAs, another group of cancer patients (cohort3,23esophagus cancers,21gastric cancers,24renal cancers,20prostatic cancers and21breast cancers) were used to further validate the most stable endogenous circulating miRNA. After getting the satisfying stable control for circulating miRNA, we use it as normalization for the quantification of circulating miRNA in HCC patients. ROC analysis was used to evaluate the performance of HCC related circulating miRNA as diagnostic biomarker for HCC. Results:Five candidate miRNAs (miR-1225-3p, miR-1228, miR-30d, miR-939and miR-640) from microarray analysis and4commonly used control miRNAs (miR-16, miR-223, let-7a and RNU6B from literature) were subjected to further evaluation. Data of independant cohort from qRT-PCR assay demonstrated that miR-1228have the lowest M value and present as the most stable circulating miRNA regardless of differences in tumor type and malignancy status. The stablity of circulating miR-1228remain to be satisfactory in the plasma form cohort3with CV (coefficient of variability) value of5.5%. By using miR-1228as internal control, the AUCs of the HCC related miRNA were0.888for early HCC (BCLC stage0and A) and0.879for AFP-negative HCC (AFP<400ng/ml).Conclusions:Accurate normalization is absolute prerequisite for circulating miRNA studies. Our study demonstrated miR-1228has sufficient stability for the evaluation of circulating miRNA expression on cancer patients and normal control especially for HCC patients. Chapter IICirculating miRNA as A Diagnostic Biomarker for Acute Rejection after Liver TransplantationBackground and Purpose:Liver transplantation is the only definite treatment for end-stage liver diseases. It can remove the tumor and the cirrhotic liver, which eliminating the HCC and its "soil". However, acute rejection (AR) of an organ transplant is a life-threatening complication. Currently, there are few diagnostic biomarkers suitable for the clinical application. Thus, discovery of accurate and effective biomarkers for acute rejection is an important mean to improve the survival of liver graft. In this study, we aim to determine the potential of plasma miRNA as a new biomarker for AR.Methods:We first established a rat orthotopic liver transplantation (OLT) model with Lewis rat as the donor and BN rat as the recipient (AR group). Control group (NR group) was adopted using BN rats as the donor and recipient. Specimens from AR group (n=4) and NR group (n=4) that collected at the7th day post OLT were used for microarray screening. We compared the differences in the spectrum and levels of miRNAs in both plasma and grafts between AR rats and control. AR related plasma miRNAs (FC>2, P<0.001) were selected and validated using qRT-PCR. Plasma from AR rats with or without tacrolimus treatment were used for miRNA dynamic monitoring. Circulating miRNA and serum ALT level was detected at3th,7th and10th day post OLT. To clarify the origin of AR-related circulating miRNAs, we compared the selected miRNA between AR group and NR group in brain, thymus, lung, heart, spleen, kidney and peripheral blood mononuclear cells (PMBCs). Drug-induced liver damage rat model were performed and In situ hybridization (ISH) was used to detect and localize the specific miRNA in allog rafts. Results:We found plasma miR-122, miR-192and miR-146a significantly up regulated when AR occur (FC>2, P<0.05) and the elevation could be repressed by immunosuppression. In liver injury rat model, upregulated plasma miR-122(FC=22.126, P=0.002) and miR-192(FC=8.833, P<0.001) rather than miR-146a (FC=1.181, P=0.594) were observed. To clarify the origin of AR-related circulating miRNAs, we compared the selected miRNA between AR group and NR group in organs and PMBCs. The results demonstrated that the only difference in the expression of the three microRNAs between AR rats (Lewis-to-BN) and NR rats (BN-to-BN) were found in the liver graft (P<0.001for miR-122, P=0.002for miR-192, and P=0.014for miR-146a). ISH revealed that the portal areas of the AR graft were brimming with lymphocytes which showed highly intense staining for miR-146a. The expression intensity of miR-146a in hepatocytes showed no difference between graft from the AR and NR groups. Further study on plasma microvesicles (MVs) demonstrated that miR-146a upregulated by six-fold in MVs isolated from AR plasma, while miR-122and miR-192showed no distinct change.Conclusions:Our study provides the global fingerprint of plasma miRNAs in AR rats and suggests plasma miR-122and miR-192reflect liver injury, while miR-146a may associate with cellular rejection. The combined biomarkers of circulating miR-122, miR-192and miR-146a may increase the sensitivity and specificity of diagnosing AR after OLT.