Screening Of Differentially Expressed MRNA Related To Metastasis Of Canine Mammary Tumor

Mammary tumor is a tumor disease with high incidence of females.At present,surgical excision combined with chemical drug therapy is adopted in veterinary clinic of the most cases.The prognosis of benign tumors is generally good,but malignant tumors often lead to poor prognosis even lead to pets death.Invasion and metastasis are important characteristics of malignant tumors,and are also the main causes of poor prognosis and death of dogs.Therefore,looking for highly sensitive and specific markers for the prognosis of canine mammary tumors is of great clinical significance in evaluating the prognosis,determining the most effective treatment scheme in time,and controlling the metastasis of tumor.In this study,mRNA sequencing of 3 canine breast cancer tissues and their matched adjacent tissues was carried out by high-throughput sequencing technology.Then we selected some differentially expressed mRNAs related to metastasis of canine mammary tumors by GO functional analysis and KEGG Pathway analysis.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to verify the selected transfer related differentially expressed mRNAs.Meanwhile,We also detected the expression of these selected genes in the primary cell line(CHMp)and the metastatic cell line(CHMm)in vitro.In the same time,the expression of PTP1 B protein which is encoded by PTPN1 gene in canine breast cancer tissues and healthy breast tissues was detected by immunohistochemical technique.And the recombinant plasmid of PTPN1 eukaryotic expression was constructed to further explore the molecular mechanism of PTPN1 in tumor cell signal transduction.Transcriptome sequencing showed that 2208 differentially expressed mRNA werescreened between canine breast cancer and its' adjacent tissues,of which 1459 were up regulated and 749 were down regulated.PAI-1,PTPN1,VEGFC,VCAM1 and ICAM1 are mainly involved in ten most significant differently GO terms,including the process of cell migration,cell composition movement,cell adhesion,and these genes also play a role in HIF-1 signaling pathway,adherens junction signaling pathway,VEGF signaling pathway,and TNF signaling pathway,suggesting that these differentially expressed mRNA plays an important role in tumor invasion and metastasis.RT-qPCR results showed that the differentially expressed multiples and change trend of PAI-1,VEGFC,PTPN1,VCAM1,ICAM1 gene between canine breast cancer tissues and its' adjacent tissues were the same as that of transcriptome sequencing.The expression of PAI-1 and VEGFC were significantly different from that of the control group,but PTPN1,VCAM1 and ICAM1 were not significantly different expressed between the experimental group and the control group.The clinical samples of canine breast tumor were divided into malignant tumor group and benign tumor group through histopathological detection results.Then the diseased tissues and the corresponding surrounding tissues were taken and the express levels of PAI-1,PTPN1,VEGFC,VCAM1 and ICAM1 gene were detected by RT-qPCR.The results showed that PAI-1,PTPN1 and VEGFC gene were highly expressed in malignant tumor and benign tumor but there was no significant difference between the lesions and its' surrounding tissues;VCAM1 gene was significant low expressed in both malignant tumor group and benign tumor group,suggesting that VCAM1 is related to the occurrence and development of cannie mammary tumors;ICAM1 gene only showed significant low expression in malignant tumor group while there was no significant difference in benign tumor group,suggesting that ICAM1 might be a potential molecular marker for differentiating benign and malignant canine mammary tumors.In order to further clarify the role of these five genes in the metastasis of canine mammary tumor,RT-qPCR was used on the canine mammary tumor cell lines CHMp and CHMm.The results showed that the expression of ICAM1 gene in the metastatic cell CHMm was significantly different from that of the CHMp,while the expression of PAI-1,PTPN1,VEGFC and VCAM1 has no significantly difference between CHMm and CHMp.Thatfurther indicating that ICAM1 gene is involved in the process of canine mammary tumors' invasion and metastasis.In clinical,it is relatively cumbersome to detect the change of gene expression by molecular biology.Protein is the executor of various functions in the organism.Compared with gene,it can reflect the function and mechanism more directly,and it is more convenient and quicker to detect the change of protein content by immunological method.High-throughput sequencing technology can search for mRNA related to the metastasis of canine mammary tumor in the whole genome,and then find proteins that have changes and roles in the process of tumor metastasis through a significant change in the expression of mRNA.In this study,GO functional analysis and KEGG signaling pathway analysis showed that the PTPN1 gene is mainly used to participate in the adhesion process of canine mammary tumor cells through the dephosphorylation of its encoded protein.called PTP1 B.It can be speculated that PTP1 B was associated with the invasion and metastasis of canine mammary tumors.The PTPN1 gene was detected by RT-qPCR in our study and there is no significant high expression of PTPN1 gene between the tumor tissue and the non tumor tissue around the tumor.It is suggested that the role of PTP1 B is related to its tyrosine phosphatase activity and a small amount of tyrosine phosphatase 1B can affect the invasion and metastasis of the tumor.To explore the expression level of PTP1 B in canine mammary tumors and the specific molecular mechanism of protein tyrosine phosphatase 1B involved in the invasion and metastasis of canine mammary tumor,the collected canine breast cancer tissues and healthy tissues were analyzed by immunohistochemistry and the recombinant expression plasmid of PTPN1 was constructed.The results of immunohistochemical staining showed that PTP1 B was expressed weakly in both canine brest cancer tissues and healthy breast tumor tissues and the expression of PTP1 B in breast cancer tissues was higher than that in the healthy breast tissues,suggesting that tyrosine phosphatase activity is the main factor in the invasion and metastasis of PTP1 B in canine mammary tumors.In this experiment,we screened and analyzed the genes related to the metastasis of canine breast cancer,which provided a basis for the interpretation of the mechanism ofcanine mammary tumors' invasion and metastasis.And provided some reference for searching potential prognostic markers and the target of molecular therapy.