The Value Of Mirnas In Early Diagnosis Of Liver Cancer And Influence The Prognosis Of Liver Cancer Mechanism

Primary liver cancer is the sixth most frequent malignant neoplasm worldwide and more than half of the new cases happen in our country every year. Hepatocellular Carcinoma (HCC) constitutes90%of the primary liver cancer. Currently the major pathogen is the infection of hepatitis B virus (HBV) or hepatitis C virus (HCV), and in our country the leading cause for HCC incidence is HBV. The new trend in pathogenesis is that some non-infectious factors such as obesity, alcohol abusing which finally lead to HCC play a more important role. Those factor are attributed to be the main causes of increasing annual HCC incidence in western country. HCC was recognized as a insidious malignacy with very poor prognosis. Surgical measures including livr cancer resection and liver transplantation are considered as radical treatment of HCC. But unfortunately most of HCC patients enter to the late stage at their first diagnosis and lose the chance to have those radical treatments. Two weak points make the early diagnosis of HCC very difficulty:One is the early HCC is usually very small and hard to be detected by B ultrasound; The other is alpha fetal pretein(AFP) which is the only widely accepted serum biomarker for HCC diagnosis has a very low sensitivity. Besides, HCC is usually highly malignant and easily to replase or metastasize. The prognosis is very poor. It is clear that some clinical features including tumor diameter, nodule number, macro/micro invasion and pre-operative AFP often correlate with prognosis. And recently correlation between obesity, enducation status, living pattern (like somking and drinking status, exercise) and HCC prognosis was emphasized. If we want to have a deep understanding of how those factors affect the prognosis and set up the basis for personalized therapy for our patients, molecular study and interference is the key to open the door.MicroRNAs (miRNAs) are endogenous22nt RNAs that post-transcriptionally control the translation and stability of mRNAs and have been implicated with critical functions in cellular development, differentiation, proliferation, and apoptosis. Since a milestone study mapped hundreds of these miRNAs to regions of the human genome that are known to be altered in cancer, the relationship between miRNA and tumor became the hot topic in the area. Until now a large amount of evidences revealed the key role miRNA played in tumor development. Meanwhile some other studies showed miRNA present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. All these findings indicate us measurement of tumor-derived miRNAs in plasma can be an important approach for the blood-based detection of human cancer.Dur to the completely new manner miRNA controling gene expression, appearance of miRNA birng us new hope in tumorigenesis as well as complexity. It add another layer to the pathway already existed which itself is complicate. Thus the functioinal localization of most miRNA in pathway of tumor development remain unclear, especially in HCC.Regarding the shortage in HCC early diagnosis and insuffient functional study on the role of miRNA in pathway that affecting HCC prognosis, we performed microarray analysis on plasma miRNA from HBV related HCC patients and control group(including healthy volunteers, Chronic active hepatitis B patients and post HBV cirrhosis patients) and discovered differential expressed miRNA in HCC group. Validation for these miRNAs was done on a independent cohort and a diagnostic model was set up based on those miRNA passed validation. Further validation on the diagnostic model was done to test efficiency. Finally we found a miRNA panel that could work for diagnosis of HBV related HCC. We also tested the dynamic change of these miRNAs after surgery to judge if they can be used in therapy monitoring. Additionally, we also checked the expression level of these miRNA in HCC and non-tumor tissues and their correlation with HCC prognosis. Combining with prognosis related message RNA profiling, we initially mapped prognostic miRNAs into the addictive pathway for tumor development.Part One Screening and primary validation of differential expressed miRNAs in HBV-related HCC patients vs each control groupThis study aims to identify differential expressed plasma miRNAs in HBV-related HCC patients comparing to healthy control, Chronic hepatitis B (CHB) control and post HBV cirrhosis control and validate the interested miRNAs in another independent cohort. Those miRNAs pass the primary validation can be the candidates for next step.In this study, we enrolled two independent cohorts:the first one, plasma from healthy people, CHB patients, cirrhosis patients and HCC patients who diagnose or phsical check up at Zhong Shan hospital from Aug,2008to Dec,2008and meet the eligibility, totally137cases; the other, plasma from patients at Zhong Shan hospital or Shang hai public health center from Jan,2009to Feb,2009. The design and requitement are same as first one, totally102cases. After plasma miRNA isolation, we used Agilent Human MiRNA Microarray kit V2which contain723human miRNAs to detect the intensity of each miRNA. Each microarray was loaded100ng. After nomalization, A Mann-Whitney test was performed to discover differentially expressed microRNAs in the three pairwise comparisons:HCC versus healthy, CHB, and cirrhosis, respectively. For those had more than two-fold change, we tested them with qRT-PCR in an independent cohort of plasma samples from102participants.The results showed that from the differentially expressed microRNAs in microarray analysis, seven detectable microRNAs with P value<0.01and fold expression change^2were identified between the HCC and healthy groups, seven detectable microRNAs with P value<10-9and fold expression change≥2were identified between theHCC and CHB groups, and two detectablemicroRNAs with P value<1010-9and fold expression change2were identified between theHCC and cirrhosis groups. Finally,15candidate microRNAs discovered viamicroarrays were selected for further testing by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). These15candidate microRNAs are miR-101, miR-122, miR-122*, miR-181d, miR-192, miR-194, miR-19a, miR-19b, miR-21, miR-223, miR-23b, miR-26a, miR-27a, miR-29c and miR-801. The15microRNAs discovered via microarray were first tested with qRT-PCR in an independent cohort of plasma samples from102participants.3miRNAs (hsa-miR-194, hsa-miR-23b and hsa-miR-29c) that showed CT values above35cycles in more than20%were excluded from further statistical analyses. Among the remain12miRNAs,7miRNA showed significant change between HCC group and general control group(P<0.05). These7miRNA are miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801. Addtionally, we use set miR-1228as endogenous control based on the microarray and qRT-PCT data.We isolated enough miRNA from human plasma and used microarray and qRT-PCR to detect the expression level. Results showed in same physical or pathological status the plasma miRNAs level were stable but would change drastically when the body condition changed. When comparing HCC with different control group the differentially expressed miRNAs were not the same(except miR-122appeared both in HCC vs healthy and HCC vs CHB as a differentially expressed miRNA). Part Two Foundation and validation of diagnostic model for HBV related HCCThis study aims to further validate the7miRNA candidates from part I and apply logistic regression model to estimate the risk of being diagnosed with HCC in the training set. Validate the diagnostic model in an independent cohort with same design to judge the general efficiency of diagnose HCC from other groups. Further we evaluate the diagnostic performance of the microRNA panel in different Barcelona Clinic Liver Cancer (BCLC) stages and investigate its power in sub-group of different serum AFP.In this study, for the cohort validating miRNA and modeling, we enrolled candidates and patients with the same design as in part I at Zhong Shan hospital or Shang hai public health center from Mar,2009to Aug,2009, totally305cases; for the cohort validating diagnostic model, candidates and patients with the same design were enrolled at Zhong Shan hospital or Shang hai public health center from Sep,2009to Jun,2010, totally390cases. In this phase, we continued validating7miRNA candidates and endogenous control miR-1228in the new independent cohort containing305cases. Taking data from previous validation in102case and in305this time together, we set up the training set and applied a stepwise logistic regression model to estimate the risk of being diagnosed with HCC. The parameters estimated from the training data set were used to predict the probability of being diagnosed with HCC for the independent validation data set (390plasma samples). Additionally, we evaluated the diagnostic performance of the microRNA panel in different Barcelona Clinic Liver Cancer (BCLC) stages and made comparison with AFP as further essessment.The results showed that among7miRNA candidates, low expression levels of miR-122, miR-223, miR-26a, and miR-27a were observed in patients with HCC compared with those in the control group (fold changes0.7,0.3,0.2, and0.3for miR-122, miR-223, miR-26a, and miR-27a, respectively). The diagnostic accuracy of these four microRNAs, measured by AUC, was0.553,0.643,0.665, and0.638, respectively. High expression levels of miR-192, miR-21, and miR-801were observed in patients with HCC compared with those in the control group (fold change1.4,1.9, and2.0for miR-192, miR-21, and miR-801, respectively). The corresponding AUCs were0.569,0.626, and0.629, respectively. The multivariate P values for all of seven miRNAs were P<0.05.A stepwise logistic regression model to estimate the risk of being diagnosed withHCCwas applied on the training data set (407plasma samples). All of the seven microRNAs turned out to be significant predictors. The predicted probability of being diagnosed with HCC from the logit model based on the seven miRNA panel, logit (p=HCC)=-1.424-0.292×miR-122+0.4511×miR-192+0.6112×miR-21-0.1796×miR-223-0.2487×miR-26a-0.3542×miR-27a+0.209×miR-801was used to construct the Receiver operating characteristic (ROC) curve. The diagnostic performance for the established microRNA panel was evaluated by using ROC analysis. The Area Under Curve (AUC) for the microRNA panel was0.864.The parameters estimated from the training data set were used to predict the probability of being diagnosed with HCC for the independent validation data set (390plasma samples). Similarly, the predicted probability was used to construct the ROC curve. The AUC of the miRNA panel was0.888(95%CI,0.852to0.917; sensitivity81.8%, specificity83.5%). The corresponding AUCs for patients with BCLC stages0, A, B, and C were0.888,0.888,0.901, and0.881, respectively. The diagnostic accuracy of the microRNA panel was then evaluated according to AFP level. In the low AFP (<400ng/mL) group, the AUC of the microRNA panel was0.879; in the elevated AFP (≥400ng/mL) group, the AUC of the microRNA panel was0.910. The performance of the microRNA panel in differentiating the HCC group from the healthy, CHB, and cirrhosis groups was also evaluated, respectively. The analysis demonstrated that in discriminating HCC from healthy AUC is0.941; CHB AUC is0.842and cirrhosis AUC is0.884.Combining the two validation set, in totally134healthy,147CHB,116cirrhosis and400HCC case, the mean CT value of miR-1228is28with CV4.5%,4.5%,3.6%,5.4%in each gourp.These results suggested7miRNA candidates were stably expressed in human plasma, and the expression level changed under different pathological condition. These points mede them qualified to be potential molecular biomarkers. The data showed that the accuracy of individual miRNA in discriminating HBV related HCC patients from control is good but still remain much room for improvment. When step-wise logistics regression model applied, it showed very high diagnostic performance for HCC and the further analysis indicated it is also very powerful to discriminate the early HCC (BCLC stage0, A) from the control group. In the subgroup analysis, the miRNA panel showed convincing accuracy of HCC diagnosis in both AFP negative and positive(400ng/ml as cutoff) patients. The evaluation of performance of the microRNA panel in differentiating the HCC group from the healthy, CHB, and cirrhosis groups demonstrated that the microRNA panel had high accuracy in discriminating HCC from each group. Generally speaking, this miRNA panel make substaintial progress in early HCC and AFP negative HCC diagnosis. Additionally, the data from large smaple size validation of miR-1228showed it can serve as a stable and reliable endogenous control in our study.Part Three Dynamic change of miRNA in panel from HCC patients after surgical treatment and assessment of its value in monitoring therapyThis study aims to investigate the dynamic change of miRNA in panel from HCC patients after surgical treatment and make assessment on its value in monitoring therapy. Additionally, check the expression stability of endogenous control miR-1228after surgery.We collected plasma from54patients in HBV related HCC group in model validation cohort and isolated miRNA under same standard as mentioned above. Compared the expression change of7miRNAs before and after surgery. And applied step-wide logistic regression model to indentify a monitoring model. ROC curve was performed to assess the monitoring effaciency of model. Meanwhile we compared post-operative expression of miR-1228with pre-operative to check its stability and reliability.In this study, we found the expression level of2miRNAs, miR-122and miR-801did not have significant change following surgery. The expression level of2miRNAs among7candidates, miR-21and miR-192were significantly decreased after HCC resection (median difference0.77, p=0.03;1.74, p<0.001respectively). The post operative expression level of3miRNAs, miR-223, miR-26a and miR-27a were elevated as compared to preoperative expression level (median difference:-0.61, p=0.02;-0.81, p=0.06and-0.55, p=0.06respectively). A model was set up by step-wise logistic regression Logit(p)=-0.138-0.6067X miR-192-0.5216X miR-21+0.6116X miR-223. AUC for this model was0.852in discriminate post-operative patients from pre-operative. Additionally, the mean CT value of endogenous control miR-1228in HCC patients after surgery tested by qRT-PCR is still28with CV5.6%.Our results in this part of study showed that there are5miRNAs among7in panel showed significant change after surgery which indicate sensitive response to surgical treatment. The initial investigation of monitoring model set up by step wise logistic regression including3miRNAs showed resonable performance but it need further validation in large cohort with prognosis data. The endogenous control still showed a stable expression under the impact from surgery. There is almost no change in pre and post operative reading which confirmed its reliability.Part Four Expression of7panel miRNAs in HCC and non-tumor tissue and its value in prognosis and role in pathwayIn this part of research aims to investigate the expression of7miRNAs in HCC and non-tumor tissue and its relationship with overall survival (OS) and disease free survival (DFS). We also aim to determine the gene profiling which related with miRNA expression and perform pathway analysis in order to map the miRNAs into the right functional position.We used the already existed MicroRNA microarray(Made at Microarray Shared Resource, Comprehensive Cancer Center at the Ohio State University) check the expression level of7plasma miRNAs in HCC and non-tumor tissue. Combing with gene expression data from Affymetrix GeneChip HG-U133A2.0arrays we determined the correlation of miRNA with genes. For MicroRNA microarrays, we had278HCC samples and286non-tumor samples. And for Affymetrix genechip, we had238HCC samples and249non-tumor samples. Most of the samples in two platform were overlapping which were from HCC patients visited Zhongshan hospital at2002or2003. We had the comphrehesive clinical information and almost10-year following-up for all the cases. Among validation on7plasma miRNAs in tissue, we found miR-21and mir-27a were upregulated in HCC tissue while miR-192、miR-122、 miR-26a and miR-223were down-regulated in HCC tissue. MiR-801was not included in the Affemetrix microarray. From the relationship between miRNAs and HCC patients’prognosis, we found expression level of miR-21and miR-223in HCC tissue were significantly associated with overall survival while expression level of miR-192in HCC tissue was significantly associated with disease-free survival; expression level of miR-21,miR-223and miR-26a in non-tumor tissue were significantly associated with overall survival while expression level of miR-21and miR-223in non-tumor tissue were significantly associated with disease-free survival. To initially investigate the funtional location of miR-192in pathway, we classfied the cohort into low miR-192expression and high miR-192expression group. Determined the genone-wide change in these two group and found1304differential expressed genes. We perform IPA to analyze the pathway these involved to map miR-192to a right position.These results implicated that all the miRNAs were differentially expressed in HCC vs non-tumor tissue according to the checking the expression of7plasma miRNAs in tissue. Among them, miR-21and miR-27a were upregulated in tumor which indicated their tumor promoting effect while miR-192, miR-122, miR-26and miR-223were downregulated in tumor which indicated their tumor suppressing effect. All the finding were in accordance with conclusion by previous study. In the survival analysis, we found the abbrent expression of miRNAs in HCC or non-tumor all correlating with different survival prognosis. It indicated no matter different prognosis caused by HCC or non-tumor tissue, miRNAs always played an important role. To initially discuss the function in miR-192, we analyzed the gene profiles in low miR-192and high miR-192sub-group and perform the pathway analysis on differentially expressed genes. We found most of these genes centralized to four major points NF-κ B, Erk1/2、JNK and histone h4which indicated these were probably the regulating sits of miR-192.CONCLUSIONS1. There are stable expressed miRNAs in human plasma and the expression level of many miRNAs will significantly change when the human body are in different conditions (such as disease). These characteristics make miRNAs qualified to serve as potential molecular biomarkers.2. We found a plasma microRNA panel by screening in high output microarray and validating in large sample size cohort that has considerable clinical value in diagnosing HCC especially early-stage HCC. It has excellent performance in discriminating HBV related HCC patients from healthy, chronic hepatitis B and post HBV cirrhosis individually as well. Besides, it can be used successfully in diagnosing HCC patients without AFP elevation.3. We perform qRT-PCR on plasma from large cohort consitituting healthy controls, Chronic Hepatitis B patients, post HBV cirrhosis patients, pre-and post-operative HBV real ted HCC patients and show that expression of miR-1228is very stable and reliable in each group.4. Most of7these miRNAs are differentially expressed in HCC tissue vs non-tumor tissue and many of them correlate with overall survial or disease free survial. The pathway analysis shows they are not only good diagnostic biomarkers but also important players in HCC development.POTENTIAL APPLICATION1. The miRNA panel including7miRNAs can be served as a diagnostic tool for HCC especially early HCC in clinic.2. We may interfere those functionally important miRNAs in HCC prognosis to benefit our patients.NOVELTY1. This is the first project that demonstrate plasma miRNA can serve as molecular biomarker in HCC diagnosis especially in early HCC diagnosis through the study on large sample size cohorts with scientific study design which meet the reqirement of clinical application.2. Plasma miR-1228was found stably expressed in different physiological or pathological condition (healthy candidate, chronic hepatitis B patient, post HBV cirrhosis patient, HBV related HCC patient and HCC patients after surgery).This finding may change the current situation that there is no raliable internal control in plasma or serum miRNA research.3. The expressions of those miRNAs in tissue are investigated besides plasma. The prognostic value and funcational location of these diagnostic miRNAs in pathway are also discussed. We are first one telling the whole story.