Screening And Validate MicroRNA Associated With Early Recurrence After Radical Surgry In Primary Hepatocellar Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumor worldwide, seventh and fifth place respectively in the global cancer incidence and mortality, China’s Ministry of Health statistics derived liver cancer mortality is second place in total cancer mortality. HCC is group of primary hepatocellular carcinoma、cholangiocarcinoma and mixed cell carcinoma. More than90%of hepatocellular carcinoma is primary hepatocellular carcinoma. Surgical resection and liver transplantation is the chief treatment means of treating liver cancer, However,high recurrence rates seriously affect the prognosis of patients, who even undergoing radical liver cancer resection would relapse.which is related to biological characteristics of liver cancer. Extensive clinical studies confirmed that liver cancer has the highest relapse rate during the first year of operation. But the pr patients with recurrence got better than those patients with late recurrence. It is an important factor that affected the prognosis of patients about liver cancer early recurrence to. It is to identifying the key signaling pathways and target gene which affected HCC cuurence,which further take effective measures to improve resectable liver cancer and efficacy of surgery.The traditional method of treatment of liver cancer is late. Therefore, it is necessary to combine with the multi-factor resulted with HCCtofind out the mechanism of recurrence and metastases of HCC by microRNA gene profile. Finding out the sensitive gene about HCC recurrence as a treatment for liver cancer molecular target is bringing new hopes for clinical treatment of HCC recurrence.MicroRNA is a class of newly discovered non-coding small RNA molecules, which is abundance of mammalian regulatory genes, and predict that miRNA may regulate at least one-third of the human gene.However encoding miRNA itself is not protein-coding. It combined with the specific target mRNA to degradate or inhibit their translation,which reducing the corresponding target gene expression and regulation. So far,they have been discovered more than1000miRNAs, and confirmed over500. How did miRNA regulation of target genes and the target protein translation is unknown.With the study of miRNA function, more datas have shown that miRNAs have played a key role in human diseases, especially in the tumor, recurrence, metastasis, invasion. Now is one of the hotspots of tumor.The relationship between endogenous miRNA and tumors associated with HCC.Recently, studies have showed that different types of tumors have different expression profiling,,which suggested that the tumor miRNAs is tissue-specific expression. Some researchers found that HCC tissues of miR-18of precursor miR-18and of miR-224have higher levels comparing with normal tissue, expression of of miR-199a, miR-195, miR-200a, miR-125a have a low the level of expression than the normal tissue.However,In rat hepatoma tissues, let-7a, miR-21, miR-23, miR-130, miR-190, miR-17-92have a high expression level, but miR-122was significantly reduced inthe liver tissue. Abnormal expression level of the functional miRNA inHCC was associated with tumor cell differentiation, signal transduction.MiRNA is a completely natural endogenous antisense regulator,which may become an effective way to inhibit tumor progression. The liver cancer study reported that mainly the analog endogenous miRNA have enhanced the performance of target genes.Designing small molecules antagonistic miRNA, such as miRNA antisense oligo (deoxy) nucleotide (anti-miRNA oligonucleotides, AMOs), locked nucleic acid modified oligonucleotides and miRNA antagonist molecules and so on.Designed for the target gene targeting to specific miRNA in liver cancer cells, which has not been reported at home and abroad nowaday.Recently, some clinical data suggest that microRNAs of tumor over-expression or absence was ralated to tumor migration and invasiveness. Some showed that mir-144expression was out of contrlol in breast cancer, leukemia, liver cancer, keratinocyte tumors and other diseases. More and more data show that mir-144has played a key regulatory factor in tumor occurrence or development.It was reported that miR-200c and miR-200b the zinc finger E-box binding homeobox increased E-cadherin expression and inhibited epithelial-mesenchymal transdifferentiation.ZEB1and ZEB2binded miR-200family to transcription miR-200family upstream of the promoter E-box or the Z-box. miR-200family upstream of the promoter E-box or the Z-box binding inhibition of miR-200family of transcription of ZEB1and ZEB2. through negative feedback regulation of miR-200family target genes ZEB between the balance of expression. MiRNA may play a cancer-promoting effect may play a tumor suppressor role in tumors, even in the micro-environmental conditions change, both from the cancer gene role in erection of tumor suppressor genes. Let-7family, miR-206, miR-335was a tumor suppressor role in breast cancer, miR-378, miR-373, miR-21, miR-10b, etc. played a cancer-promoting role in breast cancer.A lot of reaserch ahout liver cancer related miRNA,but the specturm molecular mechanism of miRNA in postoperative recurrence is not clear.Domestic and international study found that mir-144is downregulated in the liver tumor, the abnormal expression was confirmed in a variety of tumors.whch suggested that mir-144may play a tumor suppressor gene in hepatocellular carcinoma. Mir-144would provide new ideas for molecular typir、clinical diagnosised and treatment of HCC in future.MicroRNAs have played a cancer-promoting and tumor suppressor role by regulation of cell cycle proteins, Some scholars have suggested physiological concentrations of microRNA inhibited the expression of target proteins of the cell cycle in normal cells.If microRNA expression is too high to inhibit the regulation of cell cycle target proteins, It would prevent the differentiation of the cell cycle, cells in cancer poorly differentiated state. Related microRNAs in hepatocellular carcinoma research reports:miR-195inhibited the corresponding target genes cyclin CDK6and D1,which inhibited G1to S phase in liver cancer cells.However, MiR-195prevented the activation of downstream target genes by inhibiting the phosphorylation of RB protein. The molecular mechanism of miR-195has potential significance in cell cycle regulation in liver cancer.Because of specificity of tumor tissue, it is important for miRNA in tumor-related molecular typing in clinic diagnosis and treatment.With the discovery of tumor-associated miRNA and target genes about molecular mechanisms,Some question were thinked by more and more researchers.In the entire gene regulation network, miRNA is only indirect regulatory role, andit is not a single or specific gene expression products.A miRNA can regulate the expression of multiple target genes simultaneously or synchronized, if you just change a single miRNA may affect other unknown target genes. On the contrary, a single target gene may also be multiple miRNA regulation at the same time or synchronize. Therefore, miRNA used in clinical diagnosis and treatment is still too early, the exact mechanism of miRNA regulation of target genes need to be more detailed and in-depth research.The First Chapter Screening And Validate MicroRNA Associated with Early Recurrence after Radical Surgry in Primary Hepatocellar Carcinoma Objective:to select effectiveness of microRNAs and correlated with early recurrence after radical surgry in liver cancer by micro RNA array gene profiling, which analysising verified the results by tissue samples on the basis.Methods:10HCC cryopreservation tissue which were from liver carcinoma specimens data bank in our hospital after radical surgry were divided into2group:early recurrence group (5cases) and non-early recurrence group (5cases).total RNA was extracted for Cy3,RNA hybridization was executed on the chip with labeled miRNAs.microRNAs of significant and different expression was screened by SAM(version2.1) software statistical analysis.However,Results:Expressions of7miRNAs were different and significant in the HCC with early recurrence patients.Comparing with non-early recurrrence group,4miRNAs included mir-602、mir-45、mir-14、mir-486-5p were upregulated in eary recurrence group(mir-602increased2.29times;mir-451increased3.73times;mir-144increased5.28times;mir-486-5p increased2.5times). Comparing with early recurrrence group,3miRNAs included mir-551b、mir-96、 mir-502-3p were downregulated in non-eary recurrence group(mir-551b reduced0.21times;mir-96reduced0.12times;mir-502-3p reduced0.038times).Conclusions:The ananlysis of microRNA expression profile in primary hepatocellar carcinoma by microRNA genechip can screen some abnomal microRNAs correlated with early recurrence after radical surgry in liver cancer,which further find out some new potential therapeutic targets for HCC in future. The Second ChapterMir-144,Mir-502-3p Predicted by MiRNA Chip in HCC Tissues Relative Expression were Detected by Real-time Fluorescent Quantitative PCR (qRT-PCR) Object:More samples were validated mir-144, mir-502-3p miRNA predicted by gene chip miRNA expression levels between the group and the early non-relapse group differences in the recurrence of liver cancer by qRT-PCR.Method:82HCC cryopreservation tissue which were from liver carcinoma specimens data bank in our hospital after radical surgry were divided into2group:early recurrence group (50cases) and non-early recurrence group (32cases).total RNA was extracted by Trizol, andU6is reference gene.QRT-PCR real-time fluorescence detection of SYBR Green dye method for mir-144, mir-502-3p, U6-specific amplification primer.ABIsds2.1software was used for analysising results, drawing the amplification curve, solubility curve.Mir-144, mir-502-3p relative quantitative difference expression were computed between early recurrence group and the none-early recurrence group in hepatocellular carcinoma.Result:QRT-PCR experimental results are consistent with with the miRNA gene chip expression profiles predictions.Comparing with early recurrrence group,3miRNAs included mir-551、mir-96、mir-502-3p were downregulated in non-eary recurrence group(mir-551b reduced0.21times;mir-96reduced0.12times;mir-502-3p reduced0.038times).The result of mir-144、mir-502-3p expression shown on RT-PCR with liver cancer tissue cases in cryopreservation.Mir-144expression increased6.944times (43.893±107.890vs2.158±0.990P=0.018) in recurrence group than in non-early group, mir-502-3p expression reduced0.229times(6.702±9.775vs26.467+39.613P=0.009) in non-early recurrence than early recurrence group.Rbl may be a potential target gene of mir-144,and SET might be a potential target gene.Conclusions:mir-144, mir-502-3p were closely related to the recurrence of liver cancer early, of which expression might contribute to liver cancer recurrence, invasion, and recurrence,which is new target and molecular markers for early diagnosis and targeted therapy in HCC.