Differential Expression Analysis Of TsRNA In Hepatocellular Carcinoma And Preliminary Study Of Combined Diagnosis

Research Background and Purpose:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in digestive system.According to epidemiological studies,the incidence is significantly higher in males than in females,with a ratio of about 2.8:1.The current mainstream view is that HCC is related to liver cirrhosis,viral hepatitis,aflatoxin,alcohol,environment and other factors.The main treatment methods are radical surgery and liver transplantation.However,the prognosis of patients with HCC after surgery is still not optimistic due to tumor recurrence or metastasis and other factors.As a large country of hepatitis,China has a large patient base,and the most important marker for early diagnosis is alpha fetoprotein(AFP),but there are still about half of HCC patients with no abnormal AFP.Therefore,it is of great significance for HCC patients to search for new biomarkers for early diagnosis and treatment and prognosis,and explore the pathogenesis of HCC.Transform RNA(t RNA)is the most abundant small molecule non-coding RNA(small RNA)in living organisms,accounting for about 10% of the total RNA,and is an important tool for polypeptide chain translation.tsRNA(t RNA-derived small RNA,tsRNA)is derived by specific nucleases(such as Dicer and angiopoietin)in the ring,and generally 18-40 nucleotides in length which is subject to inflammation in specific tissues or cells under hypoxia or stress conditions.It mainly includes t RNA derived fragments(t RNA fragments,tRFs)and t RNA halves(t RNA halves,ti RNAs).In recent years,the technology of molecular biology has advanced by leaps and bounds,especially the maturity of transcriptomic high-throughput sequencing methods,a large number of studies have found that tsRNA,as a regulatory substance or signaling molecule of gene expression,is largely involved in tumor inhibition,development and other cellular activities.In order to further study the correlation between HCC and tsRNA,we plan to use high-throughput transcriptomic sequencing to obtain tsRNA expression profiles in HCC tissues,and compare and identify differentially expressed tsRNA molecules through bioinformatics methods.Further,quantification real-time fluorescence Polymerase Chain Reaction(QPCR)was used to verify whether tsRNA molecules are differentially expressed in HCC tissues.In addition,we will simultaneously analyze the correlation between the clinical data of HCC patients and tsRNA molecules,in order to find the value of tsRNA in the early clinical diagnosis of HCC and the assessment of the prognosis of patients.In order to further analyze the role of tsRNA in HCC,bioinformatics technology will also be used to predict the molecular targets of tsRNA and preliminarily explore the signaling pathway of tsRNA in HCC.Materials and Methods:A total of 108 patients who received radical resection of hepatocellular carcinoma in Western Theater General Hospital from September 2021 to September 2022 were selected and pathologically diagnosed as hepatocellular carcinoma.Hematoxylin-eosin(HE)staining was used to determine the differentiation degree of hepatocellular carcinoma through pathological review of hematoxylin-Eosin(HE).Immunohistochemical(IHC)staining is used to determine Ki-67 and P53 expression while clinicopathologic information of hepatocellular carcinoma patients is collected,including sex of the patient,age,tumor diameter,degree of capsule invasion,degree of cirrhosis and whether the patient is infected with hepatitis virus.The independent risk factors of poorly differentiated hepatocellular carcinoma were analyzed by Logistic multivariate analysis to compare the clinicopathologic features in different degrees of differentiation of hepatocellular carcinoma,so as to further clarify the clinical features that are highly correlated with the prognosis of hepatocellular carcinoma.Two patients who received radical resection of hepatocellular carcinoma in Western Theater General Hospital from September 2021 to September 2022 and were pathologically diagnosed as hepatocellular carcinoma were collected.The cancer tissues were used as the experimental group,and the adjacent normal liver tissues(> 5 cm)were selected as the control group.The expression profiles of small RNA in hepatocellular carcinoma tissues and adjacent tissues were analyzed by high-throughput sequencing technology.Small RNA with the highest expression abundance and most differential expression in hepatocellular carcinoma were selected for further study.According to the |log FC| > 1 and P < 0.05 were used to analyze the differentially expressed tsRNAs.Three tsRNAs were selected to verify their expression levels in 26 cases of hepatocellular carcinoma and 23 cases of para-carcinoma normal liver tissues by q RT-PCR.Correlation analysis was conducted between the expression levels of differentially expressed tsRNA and clinical characteristics of patients.The prognostic value of tsRNA for HCC was analyzed.ROC curve was drawn to analyze the diagnostic value of tsRNA as biomarkers for HCC.Objective to explore the molecular mechanism of tsRNA in the development and development of hepatocellular carcinoma by using bioinformatics analysis,gene function enrichment(GO)and gene pathway biosignaling(KEGG)analysis of target molecules.Results:1.There were differences in the percentage of hepatocellular carcinoma cirrhosis with different degrees of differentiation,P53 positive rate and Ki67 expression level statistical significance(P < 0.05);There were no significant differences in age,sex,tumor diameter,hepatitis virus infection or tumor involvement in liver capsule between groups(P > 0.05).Logistic multivariate analysis showed that liver cirrhosis,high expression of Ki67 and positive P53 were independent risk factors for poorly differentiated hepatocellular carcinoma(P < 0.05).2.A total of 4116 tsRNAs were detected by high-throughput sequencing in hepatocellular carcinoma,and their expression abundance was much higher than that of the other 4 small RNAs;A total of 2411 differentially expressed genes were screened out,including 1400 up-regulated tsRNA and 1011 down-regulated tsRNA,accounting for 59% and higher than the other 5 small RNAs.Has the difference expression tsRNA altogether seven subtypes,according to the |log FC| > 1 and P < 0.05,among which 3expressions were up-regulated and 8 were down-regulated.3.The expression level of tsRNA was verified in hepatocellular carcinoma and paracancer liver tissues: The expression of tRF5-18-TyrGTA-8 in HCC group(0.026±0.004)was higher than that in control group(0.012±0.002),and the expression of tRF5-18-Trp CCA-1 in HCC group(2.14±2.57)was higher than that in control group(1.22±1.18),The expression of tRF5-29-LysCTT-4 in HCC group(127.50±18.22)was significantly higher than that in control group(35.80±5.94)(P < 0.05),which was consistent with high-throughput sequencing results.4.Correlation analysis of clinical features of tRF5-18-TyrGTA-8,tRF5-18-Trp CCA-1 and tRF5-29-LysCTT-4 was performed.The correlation between tRF5-18-Trp CCA-1 and age was linear and positive,and pearson correlation coefficient was 0.69.tRF5-29-LysCTT-4 was positively correlated with tumor diameter,HBV load,alanine aminotransferase and Ki-67 expression,and the pearson correlation coefficients were 0.68,0.76,0.64,0.71,respectively(P < 0.05).The area under ROC curve of the combined diagnosis of the three Ts Rnas was 0.82(P < 0.05),the specificity was 0.83,and the sensitivity was 0.73.The area under the curve,specificity and sensitivity of the three tsRNAs combined to predict poor prognosis of HCC were 0.81(P < 0.05),0.71 and 0.90.Ki-67 combined with three tsRNAs predicted the area under the curve of poor prognosis of HCC was 0.85(P < 0.05),the specificity was 0.86,and the sensitivity was0.79.5.Bioinformatics analysis was performed on tRF5-18-TyrGTA-8,tRF5-18-Trp CCA-1 and tRF5-29-LysCTT-4,and the target m RNA of tRF5-18-TyrGTA-8 was predicted to be 462 species.The number of targeted genes of tRF5-18-Trp CCA-1 was 716 species.The target genes of tRF5-29-LysCTT-4 were 2340 species.The results of GO and KEGG pathway analysis of target genes were concentrated in oncogenesis,viral infection and EGFR tyrosinase inhibitor resistance and other pathways related to cancer prognosis.Conclusion:1.tsRNA expression profiles were different between hepatocellular carcinoma tissues and adjacent normal liver tissues.2.tRF5-18-TyrGTA-8,tRF5-18-Trp CCA-1,tRF5-29-LysCTT-4 were up-regulated in hepatocellular carcinoma and correlated with clinicopathologic features.3.tsRNA has good specificity and sensitivity in the diagnosis and prediction of poor prognosis of HCC,and the combined Ki-67 index can better improve the ability of tsRNA in the diagnosis and prediction of poor prognosis of HCC.4.tRF5-29-LysCTT-4 may affect the occurrence and development of hepatocellular carcinoma by regulating PD-L1 and EGFR related pathways mediated by its target genes,providing new ideas for further research on the mechanism of tsRNA in hepatocellular carcinoma.