Screening, Validation And Functional Studies On Novel Molecular Markers Of Hepatocellular Carcinoma Based On Transcriptomics

Background:Hepatocellular carcinoma(HCC)is a kind of primary malignant tumor of liver that derived from liver parenchyma cells.HCC is the 5th most common cancer worldwide and the 2nd-leading cause of death from cancer every year.There is a high incidence of HCC in our country,every year,both the morbidity and mortality of HCC accounting for more than half of the world.Among the males under 60 years of age suffering from a malignant tumor in our country,both the incidence and fatality rate of HCC are ranking first.In recent years,as the development of medical image,surgery and so on,the diagnosis and treatment of HCC has already made great progress.But,there are still many problems such as the lack of effective diagnostic markers,low survival rate and high recurrence rate in HCC patients.AFP(Alpha-Fetoprotein)is a traditional HCC molecular marker,however,as a HCC serological marker its positive rate is only 60%-70%.Many HCC patients are diagnosed at the advanced stage of the disease,and have lost the chance to be treated.Recently,the treatment of HCC also tends to be diverse,except for traditional surgical resection,radiotherapy and chemotherapy,drug targeting therapy are becoming more and more common,but the effect of chemotherapy and drug targeting therapy is always poor.One of the reasons is multidrug resistance.In view of this phenomenon,we find that ubiquitin-specific protease 22(USP22)is a molecular marker of hepatocellular multidrug resistance and also reveals related mechanisms.By detecting the expression of USP22,it provides a new idea for the individualized selection of drug targeted therapy for HCC patients.At present,researches on omics are moving ahead incuding transcriptomics,genomics,and proteomics and so on.Transcriptomics is an important kind of omics that studies the expression of genes at the level of RNA.By researches on transcriptomics,more and more key molecular markers of tumor have been found.These key molecular markers play important roles in studies on the molecular mechanism of tumor development and progress,screening of diagnostic markers of HCC,prediction of prognosis,the screening of therapeutic targets of HCC and therapeutic evaluation of HCC.Objectives:Based on HCC clinical samples and transcriptome technology,this study was to screen the potential molecular markers of HCC that were related with the diagnosis of HCC,malignant biological features of HCC,and prognosis of HCC and so on.By up-regulating or down-regulating these potential molecular markers in HCC cells,we studies their functions to make sure the roles they were palying in HCC and related mechanisms.According to both the clinical and cytological results,we wanted to make sure that these molecules were the the novel molecular markers of HCC and could be used to help the diagnosis,prognosis evaluation or therapeutic target screening of HCCMethods:1.An Agilent expression microarray including 39303 lncRNA and 32205 mRNA was used to detect and analyze the expression spectrum difference between 5 pairs of HCC tissues and corresponding adjacent tissues.According to the size of P value and fold change and literature reading,these differentially expressed genes(including mRNA and lncRNA)which could be the potential molecular markers of HCC were found.2.The expression of these differentially expressed genes were detected in clinical samples by means of immunohistochemistry,western blotting,and enzyme-linked immunosorbent assay and so on.The relationship between the expression of differentially expressed genes and clinicopathological parameters and prognosis of HCC patients was analyzed respectively.The potential application value of differentially expressed genes as a diagnostic marker,prognostic marker or therapeutic target of HCC was preparatory determined.3.The expression of differentially expressed genes were up-regulated or down-regulated in HCC cells.Cell function experiments including Cell Counting Kit-8(CCK-8)assay,cell invasion and migration assay,cell cycle and apoptosis assay and so on were done to detect the influences of differentially expressed genes on the biological behaviors of HCC cells.HCC cells stably transfected with differentially expressed were constructed by Lentivirus.The assay of subcutaneous tumor growth in nude mice was done to detect the influences of differentially expressed genes on tumorigenicity in vivo.4.According to the results of cell function experiments or further use of Agilent expression microarray,the specific mechanisms of differentially expressed genes in the development and progress of HCC was then detected via western blotting.Results:1.MFAP4 is a novel molecular marker for diagnosis and prognosis of HCC,palys an anti-cancer role in HCC? According to the results of Agilent expression microarray,the expression of mRNA MFAP4 was significantly lower in HCC tissues than in corresponding adjacent tissues(P<0.05).? The mRNA expression of MFAP4 was significantly lower in HCC tissues than in corresponding adjacent tissues via qRT-PCR(P<0.05).? The protein expression of MFAP4 was significantly lower in HCC tissues than in corresponding adjacent tissues via WestemBlot(P<0.05).? The expression of MFAP4 was lower in both liver cirrhosis patients and HCC patients then in normal persons via ELISA(P<0.05).The expression of MFAP4 in the sera of HCC patients was associated with the gender of patients(P=0.000).The serum concentration of MFAP4 in male HCC patients were significantly higher than that in female HCC patients.? Immunohistochemistry showed that the expression of MFAP4 was significantly lower in HCC tissues than in corresponding adjacent tissues(P<0.05).The expression of MFAP4 was gradually down-regulated with the progression of HCC(P<0.05).MFAP4 was almost undetectable in HCC tissues,and decreased expression of MFAP4 in corresponding adjacent tissues was associated with poor post-operative survival(P=0.027).? Up-regulation of MFAP4 inhibited the proliferation,migration and invasion of HCC cell line Bel-7402.Up-regulation of MFAP4 promoted apoptosis and induced S phase arrest in Bel-7402 cells.Up-regulation of MFAP4 also inhibited the tumorigenic ability of Bel-7402 cells in vivo.? After the up-regulation of MFAP4 in Bel-7402 cells,the expression of some relevant proteins was changed.The expression of p27,pRB,BCL-Xs,CDK4 and CDK6 were increased.2.RP11 is a potential therapeutic target for HCC and a novel molecular marker for prognosis prediction,can promote the development of HCC? According to the results of Agilent expression microarray,the expression of lncRNA RP11 was significantly higher in HCC tissues than in corresponding adjacent tissues(P<0.05).? The expression of RP11 was significantly higher in HCC tissues than in corresponding adjacent tissues via qRT-PCR(P<0.05).? The expression of RP11 in HCC tissues was associated with tumor size and histological differentiation(P=0.017 and P=0.020).The higher the expression of RP11 in HCC tissues,the bigger of tumor size or the poor of histological differentiation.The expression of RP11 in HCC tissues was also associated with the prognosis of HCC patients.The higher the expression of RP11 in HCC tissues,the lower the overall survival rate of HCC patients(P=0.019).? After the down-regulation of RP11 in HCC cells,the ability of cell proliferation,cell invasion and cell migration were significantly enhanced,the apoptosis of cell was significantly increased,the cell cycle was changed.After the up-regulation of RP11 in HCC cells,the ability of cell proliferation and tumorigenic in vivo were weaken.? After the down-regulation of RP11 in HCC cells,the expression of CyclinE was decreased,the expression of activated Caspase-3 and PARP were increased.The down-regulation of RP11 could activate the TGF-beta/SMAD signaling pathway.Conclusion:1.The expression of MFAP4 was significantly lower in both the sera of HCC patients and HCC tissues.MFAP4 could be used for the differential diagnosis between HCC patients and normal people,cirrhotic patients and normal people,HCC patients and cirrhotic patients;the lower the expression of MFAP4 in corresponding adjacent tissues,the lower the overall survival rate of HCC patients.MFAP4 is a novel molecular marker for diagnosis and prognosis of HCC.2.MFAP4 was a suppressor of HCC.Up-regulation the expression of MFAP4 could inhibit the biological behaviors and tumorigenicity in vivo of HCC cells.By regulating the expression of some key proteins associated with tumor such as p27,MFAP4 actted as an antioncogene of HCC.3.The expression of RP11 was significantly higher in HCC tissues than in corresponding adjacent tissues.The higher the expression of RP11 in HCC tissues,the lower the overall survival rate of HCC patients.RP11 was a potential prognostic marker of HCC.4.RP11 was a promoter of HCC.Down-regulation the expression of RP11 could inhibit the biological behaviors of HCC cells.Up-regulation the expression of RP11 could promote the biological behaviors and the tumorigenicity in vivo of HCC cells.RP11 was a potential therapeutic target for HCC.RP11 could regulate the expression of some key proteins associated with tumor.The down-regulation of RP11 could activate the TGF-beta/SMAD signaling pathway.