| Perianal gland tumors (Also known as Hepatoid gland tumors), which arises from the sebaceous glands tissue surrounding the anus, accounts for 80% of all tumors that occur in the perianal area, particularly common in intact males. It ranks the top 3 among the all types of canine tumor types in male dogs. The correlative research of canine perianal gland tumors was mainly focused on characteristic of pathological shape and clinical treatment. The molecular mechanism of the occur and development progress of tumorsis still unclear. This study employed the high-throughout sequencing technology to analyze the difference of gene expression between perianal gland tumors and normal perianal gland tissue for the first time. The aim of the studies was to explore mechanism of tumor, to seek possible specific molecular diagnostic marker, and then to provide a basis for early diagnosis.The ten sample genes were extracted from older male dogs that suffered perianal gland tumors, the median age was 12.8yrs.There are 4 cases of well-differentiated perianal sebaceous adenoma,3 cases of moderately differentiated perianal adenoma,1 well differentiated perianal adenocarcinoma and 2 moderately differentiated perianal adenocarcinoma. These samples covered all the perianal gland tumor types except perianal gland epithelioma.The 6 normal perianal tissue samples were also extracted from older male dogs, the median age was 11.6 yrs. These tissues structure and cellular components of the above samples with routine paraffin section and H-E staining and were identified according to WHO tumors typing standard.In the studies, differentially expressed genes between Canine perianal gland tumors and normal perianal gland tissues were identified by IlluminaHiseq 2500 TMhigh throughput RNA sequencing. The 9 random differentially genes were validated by Real-time PCR, it found that the result of gene expression were consistent with reverse transcription groups, which proves Illumina sequencing reliability. The results show us, the comparison between the tumors experimental group and the control group, there were 588 differentlially expressed genes discovered, including 23 up-regulated and 565 down-regulated, the genes of significant difference were (FC≥2, FDR< 0.05= 94). In the Transcript isoforms that discovered, differentially display reverse transcription were 827, including 24 up-regulated and 803 down-regulated, the genes of significant difference were (FC≥2, FDR<0.05=26). Based on GO clustering analysis, there were 120 genes classified. The most clustering is Cellular Component cluster include 59 genes、the Biological Process cluster include 37 genes and the Molecular Function cluster include 24 genes. KEGG analysis found that 470 genes involves in 262 signal path ways,.Thereinto cellular process was involved 16 genes, the second were Olfactory transduction involved 12 genes, The Metabolism involved 8 genes,and each of the Pathways in cancers, SignalTransduction and PI3K-Akts are involved 7 genes.. In the date analysis, it found that some genes of up and down regulated obviously were possible correlation with formation and development of peranal gland tumors, e.g. MMP-1、FGF7、GST、ECM,etc. In the SNP and Indel, compared with control group, SNP and Indel in experimental group increased significantly, which suggests that there may be a large number of genetic mutations, at the same time, New SNP and InDel genes of perianal gland tumor were tissue-specific.In the studies, up-regulated MMP-1 and down-regulated FGF7 that significantly regulated were observed in immunohistochemical staining and analyzed by Imagepro Plus6.0 software of optical density, which found that MMP-1 expressed in normal perianal cell plasma and tumor cell plasma, OD were not obvious, so MMP-1 was not suitable to be tumor-specific markers. FGF7 expressed significantly in normal perianal cell plasma, but decreased in the tumor cells, the difference of OD was obvious, so FGF7 are expected to be specific molecular biomarker. |
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