Screening And Function Study Of Aberrantly Expressed LncRNAs In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors and the mortality rates forefront of various types of tumors.At present,surgery,chemotherapy,radiation therapy,liver transplantation is the main means of treatment of hepatocellular carcinoma,due to its fast progress,high degree of malignancy,individual differences,and overall patient survival rates did not rise markedly after treatment and postoperative recurrence and metastasis rate is still high.However,our country is hepatitis B power and population base is big,the occurrence of HCC is closely related to hepatitis B,so the HCC brings serious burden for the national economy and threats people's health.Therefore,find new tumor markers,explore the exact mechanism of liver cancer,develops the new specific molecular targeted drugs is the key of breakthrough the bottleneck in the treatment of liver cancer.The long noncoding RNAs(lncRNAs)are a class of non-coding RNA that over 200 nucleotides with no protein-coding potential.Initially it was considered genome transcription of "noise" and do not have the biological function.Recent years lncRNAs have been shown to have crucial roles in the regulation of multiple biological processes such as cycle,apoptosis,proliferation,and differentiation and and specific lncRNAs are correlated with cancer recurrence,metastasis and poor prognosis in different kinds of cancer.LncRNAs in the development of HCC are also favored by the scholars,such as the current study of H19,HOTAIR,MEG3 and so on.Therefore,the discovery of the novel LncRNAs and the exploration of its mechanism for HCC development provides a new thinking and new direction for HCC treatment.Part IScreening the aberrantly expressed LncRNAs in hepatocellular carcinomaAimsScreening the aberrantly expressed LncRNAs in liver cancer tissues and paracancerous tissues.MethodsCollected 3 samples of HCC tissues compared with paired adjacent non-tumor tissues.The diagnosis of each specimen was confirmed histopathologically.RNA extraction and purification,microarray experiments and data analysis was finished by SHANGHAI BIOTECHNOLOGY CORPORATION.RusultsUse Fold Change(linear)>?2 and the t-test(Student's t-test)p-value<0.05 for the differential gene screening,all differential expressed LncRNAs in hepatocellular carcinoma is 2320,in which overexpressed is 1048,and low expressed is 1272.Part?Expression and clinical significance of the novel longnon-coding RNA ZNF674-AS1 in human hepatocellular carcinomaAimsTo determine the expression level of ZNF674-AS1 in HCC,and then to evaluate the relationship between its expression levels and clinical pathological characteristics of patients with HCC.MethodsThe expression of ZNF674-AS1 in 137 pairs of tumorous and adjacent normal tissues from patients with HCC was detected by quantitative real-time reverse transcription polymerase chain reaction.Additionally,the potential associations between its level in HCC tissue and clinicopathological features were analyzed.Overall survival curves were plotted according to the Kaplan-Meier method.A p-value less than 0.05 was deemed to indicate statistical significance.ResultsWe found that the expression level of ZNF674-AS1 in cancer tissues from patients with HCC was significantly lower than those in matched normal tissues.Furthermore,the expression of ZNF674-AS1 was decreased in 72%(99/137)of HCC tissues compared with that in matched normal tissues.The expression of ZNF674-AS1 in five HCC cell lines(HCCLM3,SK-Hep1,HuH7,Hep3B,and MHCC97H)was significantly downregulated compared with that in the normal liver cell line QSG-7701.the ZNF674-AS1 levels were associated with clinical stage(p =0.039),histopathologic grade(p=0.045),and cancer distal metastasis(p= 0.041).A lower expression of ZNF674-AS1 was correlated with the adverse survival of patients with HCC.ConclusionThese results suggest that the aberrant expression of ZNF674-AS1 might be involved in the biological characteristics of HCC and might be a novel biomarker for predicting the free survival of HCC.Part?The downregulation of NCRUPAR is associated with the clinical characteristics of hepatocellular carcinomaAimsTo investigate the expression of NCRUPAR in HCC specimens and adjacent normal tissues and the potential relationship between NCRUPAR expression levels and the clinicopathological factors of patients with HCC.MethodsWe collected 137 samples of HCC tissues compared with paired adjacent nontumor tissues and measured the NCRUPAR levels in tissues and cell lines using real-time reverse transcription-polymerase chain reaction,and then analyzes the associations between NCRUPAR expression and the clinicopathological features of HCC.The Kaplan-Meier method estimating the 5-year recurrence free survival rates.Statistical significance was accepted at p<0.05.ResultsThe expression of NCRUPAR in the HCC cell lines HCCLM3,HUH7,MHCC97H,SK-Hepl and Hep3B was significantly downregulated compared with the normal liver cell line QSG-7701.It was downregulated in 73.7%(101/137)of the HCC tissues compared with paired adjacent normal tissues(p<0.05).More importantly,our results proved that NCRUPAR expression was associated with portal vein tumor thrombus(P=0.046),cancer distal metastasis(P=0.046),and especially histopathological grade(p=0.006).The Kaplan-Meier curve revealed that patients with low expression of NCRUPAR has a worse overall survival compared to patient with high expression of NCRUPAR.ConclusionsOur data suggest that NCRUPAR may plays crucial roles during cancer occurrence and progression and is a potential new biomarker of hepatocellular carcinoma.Part?The function and mechanism of HNF1A-AS1 in the development of hepatocellular carcinomaAimsTo explore the function and mechanism of HNF1A-AS1 in hepatocellular carcinomaMethodsUsing real-time reverse transcription-polymerase chain reaction to confirm the expression level of HNF1A-AS1 in the liver cancer tissues and liver cancer cell lines and analyzes the relations between its expression and the clinical pathology characteristic of HCC.The browser's Kaplan-Meier plot was used to explore overall survival between the patients in the high HNF1A-AS1 expression subgroup and the low expression subgroup.We explored the impact of HNF1A-AS1 knock-down in variety of biological processes in the HCC cell lines by cell viability,colony formation,flow cytometry,Transwell assays and in vivo tumorigenicity assays.Finally,the adoption of Western blot and bioinformatics to explore the mechanism of HNF1A-AS1 in hepatocellular carcinoma.ResultsBy detecting the expression of HNF1A-AS1 in a total of 138 paired clinical HCC tissues and paracancerous tissues and found that HNF1A-AS1 expression was significantly over-regulated in 91 paired clinical HCC tissues.HNF1A-AS1 expression levels in HCC were significantly associated with hepatitis B(P=0.036),cirrhosis(P=0.009),metastasis(P=0.01),clinical stage(P=0.002).The patients in the high HNF1A-AS1 expression level have worse overall survival compared to the low expression level.The expression of HNF1A-AS1 in the HCC cell lines HepG2,HCCLM3,SK-Hep1,HUH7,Hep3B,SMMC7721 was significantly upregulated compared with the normal liver cell line QSG-7701.Knock-down of HNF1A-AS1 in HUH7 and SMMC-7721 cell lines resulted in a significant decrease in cell viability,colony formation,the invasion and migration and induced cell-cycle arrest and apoptosis.HNF1A-AS1 knock-down suppressed HCC tumor growth in vivo.HNF1A-AS1 promotes HCC migration and invasion by regulate the expression of PIGR in EMT pathway.ConclusionsThe effects of HNF1A-AS1 on cell proliferation,cell cycle regulation,invasion and migration suggest that it promotes tumorigenesis and may act as a prognostic factor in HCC.