The Influence Of Ammonia On Hepatocarcinoma Gene Expression Through High-throughput Trancriptome Analysis

BackgroundHepatocellular carcinoma,（HCC）is one of the most general malignant tumor in our country and it is the second leading cause of malignant tumor mortality in the world^[1].Hepatocellular carcinoma have the characteristics of concealed pathogenesis,rapid progress and high postoperative recurrence rate and so on.In recent years,although the liver cancer clinical research has made great progress,in terms of the treatment for liver cancer,especially for the intermediate and late primary hepatocarcinoma,treatment still need further explore.The liver,as an important digestive organs,has strong functions in metabolism,secretion,regulation and so on,and has powerful self-healing ability and synthesis.As we all know,a lot of toxic substances need to transport to liver to turn into non-toxic substances and be taken from the body or reused.Ammonia is the common cell metabolite,accumulation of excess which,through the blood brain barrier,may interfere with the energy metabolism of brain cells,resulting in brain edema and neurological unusual,and cause hepatic encephalopathy^[16].Thus ammonia-lowering therapies is important to the treatment of hepatic failure and liver cirrhosis^[17].As the study of pathogenesis of tumor in recent years,we found that there was a correlation between ammonia and tumorigenesis from that patients with cirrhosis and portal hypertension have incteased risk of liver cancer^[4][18].Recently,a reports about breast cancer suggest ammonia recycled in tumor cells as nitrogen source^[7],providing new perspectives to understanding of the mechanism of hepatocellular carcinom.High-throughput sequencing technology,a milestones for sequencing technology,is also referred to as deepsequencing or next-generation sequencing technology（NGS）^[19].The concept of the transcriptome was put forward for the first time by Velculescu etc in 1995 who obtained the first transcriptome data from the yeast cells^[20],then the transcriptome sequencing developed into RNA sequence analysis（RNA-seq）.RNA-seq as one important type of high flux measurement techniques,can get the gene sequence quickly and comprehensively form all of the cells or tissues,and used in study genetic differences and genetic function and variable shear,etc.Transcriptome sequencing can test the overall transcription activity in any species,without probe designed in advance,thus providing more precise digital signal,the higher detection flux as well as a broader scope.Shah researched in Granulosacell tumor（GCT）,successfully found a potential gene in the pathogenesis of tumor by transcriptome sequencing technologies^[21],then he found that single nucleotide mutations heterogeneity may result in breast cancer,and the result was published as the cover article in Nature^[22].Now as the huge advantage,the transcriptome sequencing technology is widely used in cancer research for example gastric cancer,prostate cancer,liver cancer,melanoma and so on,and become a significant experiment technology in the study of tumor biological informationThus,we use the transcriptome analysis technology in our experiment,to research the effect of ammonia on gene expression of hepatoma cell,contributing to illuminate the pathogenesis of hepatocellular carcinoma.ObjectiveTo study the effect of ammonia on gene expression of hepatocellular carcinoma by using high-throughput sequencing method,and explore the pathogenesis of liver cancer and provide the theoretical basis for clinical treatment of liver cancerMethods（1）Different concentrations of ammonia to cultivate liver cancer cells,we can study that the proliferation and apoptosis of liver cancer cell by CCK,and select suitable concentration of ammonia to cultivate 7721（2）the normal culture medium to culture 7721 cells as a control,whie collect suitable concentration of ammonia culture medium to culture cells to the 5 and 15generation as experimental groups,respectively to extract RNA and test the quality of RNA.（3）Using RNA-seq technology,we detected the gene expression of 7721 cells cultured in different medium condition and different time.Through HISAT,FeatureCounts,HTSeq and DESeq ect.software,we analysised the gene expression data and genetic diversity.Results1.The culture containing ammonia of concentration of 1mmol/L can significantly promote the growth of 7721 liver cancer cells,2.7721 cells cultured in contains ammonia medium to 5th generations,compared with 7721 normal cells,the number of up-regulation of gene expression is148,while downgrade expression is 58.3.7721 cells cultured in contains ammonia medium to 15th generations,compared with 7721 normal cells,the number of up-regulation of gene expression is770,while downgrade expression is 400.Conclusion1.Ammonia can promote the growth of 7721 liver cancer cells,2.ammonia can influence 7721 gene expression,and different cultivation time with ammonia can make the expression differences more significant.