Genetic Profiling Of Papillary Thyroid Cancer By Third Generation Sequencing Technologies And The Correlation Between LAMB3 And DDB2 And Their Clinical Features

Objective We applied third-generation sequencing and bioinformatics technologies to screen and analyze the differentially expressed genes between papillary thyroid cancer and normal tissues adjacent to the cancer,and to explore the important genes related to the invasion and metastasis of papillary thyroid cancer.It will provide a new reference for the diagnosis of papillary thyroid cancer,the assessment of invasion degree and the selection of treatment plan.Methods1.Fresh samples of 15 cases of papillary thyroid carcinoma and para-carcinoma tissues were collected according to the inclusion criteria to conduct full-length transcriptome detection.After sequencing quality control,transcripts were quantified.The quantitative results of all transcripts of a gene were counted as the quantitative results of the gene to obtain the quantitative expression level of the gene.2.|log2(FC)|≥1 and p-value<0.01 were used as differential gene screening criteria.The functional annotations and pathway enrichment of these differentgenes were analyzed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Their genomes were analyzed based on literature review and genome genome analysis.Finally,the target gene was determined.3.Real time quantitative polymerase chain reaction(RT-q PCR)was applied to verify the difference in expression of LAMB3 and DDB2 between cancer and paracancer in 15 pairs of sequenced samples.4.Immunohistochemical methods were used to detect the semi-quantitative expressions of LAMB3 and DDB2 in tissue chips(including 58 cases of papillary thyroid carcinoma and corresponding paracarcinoma normal tissue),and Chi-square test and wilcoxon method were used to test whether the expression levels of the above two proteins in cancer tissues were statistically significant compared with those in paracarcinoma tissues.fisher test was used to analyze the correlation between LAMB3 and DDB2 protein expression and clinical indicators.P < 0.05 was statistically significant.5.Pearson correlation analysis showed a positive correlation between LAMB3 expression level and DDB2 expression level(P<0.001,R=0.7439).Results1.2147 differentially expressed genes were screened by transcriptome sequencing analysis of 15 cases of cancer and adjacent normal tissues,including 1047 up-regulated genes and 1100 down-regulated genes.2.KEGG enrichment analysis was performed on the significantly upregulated Top50 genes,and the results of the enriched pathways suggested significant enrichment in ECM-receptor interaction,local adhesion,PI3K-Akt signaling pathway,etc.The genes in the enriched signaling pathways were intersected with the upregulated Top50 differentially expressed genes in the sequencing results according to the screening criteria Log2 FC ≥ 4.9,P < 0.05,CPM values The LAMB3 gene was screened.KEGG enrichment analysis was performed on 1047 upregulated differential genes,and Top20 enrichment pathways showed significant enrichment in cell adhesion molecules,P53 signaling pathway,and S.aureus infection.After literature review and analysis,analysis of the significantly enriched pathways revealed that the P53 signaling pathway plays an important role in the occurrence of PTC with a P value of1.76E-05.28 genes were enriched on this pathway,and these 28 genes were intersected with 1047 upregulated differentially expressed genes from the sequencing results according to the screening criteria Log2 FC ≥ 1.3,P < 0.05,and CPM values DDB2 was selected as the target gene for the follow-up study.3.RT-q PCR results showed that the RNA expression of LAMB3 and DDB2 was higher in PTC cancer tissues than in normal tissues adjacent to the cancer,which was consistent with the trend of sequencing results.4.In 56 cases of PTC cancer tissues,there were 16 cases of high expression and 40 cases of low expression,and 0 cases of high expression and58 cases of low expression in 58 cases of paracancerous tissues,and the expression level of LAMB3 in PTC cancer tissues was significantly higher than that in paracancerous normal tissues(P<0.001);there were 27 cases of high expression and 28 cases of low expression of DDB2 in 55 cases of cancer tissues,and 57 cases of paracancerous normal tissues,with high expression of The expression level of LAMB3 in PTC was not related to age,gender,lymph node metastasis,tumor number and TNM stage,but correlated with tumor size and T stage.Compared with cancer tissues less than or equal to 2 cm,LAMB3 expression was higher in cancer tissues larger than 2 cm,and the expression level of LAMB3 was positively correlated with the size of PTC tumor tissues(P =0.001,r = 0.49).Compared with T1 stage,LAMB3 expression was higher in T2~T4 stage tissues,and the expression level of LAMB3 was positively correlated with T stage of PTC tumor tissues(P = 0.017,r = 0.339).the expression level of DDB2 in PTC was not related to age,gender,lymph node metastasis,tumor number,tumor size,lymph node metastasis,and TNM stage.5.Pearson correlation analysis showed a positive correlation between LAMB3 expression level and DDB2 expression level(P<0.001,R=0.7439).Conclusions The expression of LAMB3 and DDB2 in PTC cancer was significantly higher than that in normal tissues adjacent to the cancer,LAMB3 and was related to tumor size and T stage;the expression of DDB2 in PTC tissues was positively correlated with that of LAMB3.