Study On The Effect Of Circ＿TMTC1 On Canine Mammary Gland Tumor Characteristics And Tamoxifen Resistance

Canine mammary tumors are the most common type of tumor in dogs.Mammary tumors account for 50% of all canine oncologic disease.In addition,as companion animals with close relationship with humans,dogs are in similar living environment as humans,and their spontaneous tumors are a good sample for human research on tumors.Therefore,the study of canine mammary tumors will not only contribute to the progress of animal treatment,but also provide new perspectives on human breast cancer research.Circular RNAs(circ RNAs)are a class of covalently closed,ring-like endogenous RNA that are widely present in mammalian cells and specifically expressed at tissue or developmental stages.Current studies have demonstrated that some circ RNAs play a role in promoting cancer development in human breast cancer,while circ RNA functions in canine mammary tumors have not been investigated.Therefore,in this study,the effect of circ＿TMTC1 on the function of cancer cells was investigated based on some circ RNAs that have been found in canine mammary tumor cells.Firstly,the loop structure of circ＿TMTC1 was identified by Sanger sequencing,agarose gel electrophoresis,actinomycin D treatment experiment,RNase R digestion experiment and nucleoplasm separation experiment.The expression of circ＿TMTC1 in tissues and cells was analyzed by RT-q PCR.After knockdown and overexpression of circ＿TMTC1,changes in the characteristics of canine mammary tumor cells were analyzed by CCK-8,Ed U proliferation assay,clone formation assay,migration and invasion assay.The tamoxifen resistance of the cells was assessed by IC50,Ed U proliferation assay,clone formation assay and cell apoptosis.The expression levels of EMT-related and target proteins were examined by Western blots.The regulatory roles of miRNAs with circ RNAs and target genes were verified by dual luciferase assays.Finally,tumorigenic,angiogenic ability and malignancy were analyzed by nude mice tumor formation analysis,HE staining and immunohistochemical analysis.The results of Sanger sequencing and agarose gel electrophoresis showed that circ＿TMTC1originated from the TMTC1 gene(XM＿005637011.4)on canine chromosome 27.A 636 nt circular RNA by reverse splicing of its exons 2-5.Within 24 h of actinomycin D treatment of cells,the linear TMTC1 gene intracellular abundance decreased sharply,while the circ＿TMTC1 abundance showed little change.The abundance of TMTC1 decreased significantly after 30 min of RNase R treatment,while circ＿TMTC1 resisted the shearing of RNase R.The results of nucleoplasmic separation experiments showed that circ＿TMTC1 was mainly present in the cytoplasm of canine mammary tumor cells.circ＿TMTC1 was highly expressed in tamoxifen-resistant canine mammary tumor cells(p<0.001)and canine mammary tumor tissues(p<0.001),increased with the tamoxifen treatment time.The experiments of overexpression and knockdown of circ＿TMTC1 in canine mammary tumor cell showed that knockdown of circ＿TMTC1 significantly inhibited the proliferation ability,invasive ability and tamoxifen resistance of tumor cells.During 5 days of culture,the OD450 values of the control group were gradually higher than those of the knockdown group,and the difference was significant(p<0.001).the percentage of Ed U-positive cells decreased from 41.3%to 30%(p<0.01).Clonogenic(p<0.01),migratory(p<0.001)and invasive(p<0.001)abilities were also dramatically reduced with knockdown of circ＿TMTC1.Knockdown of circ＿TMTC1 inhibited the EMT process in canine mammary tumor cells.The results of western blots showed that knockdown of circ＿TMTC1 significantly decreased the expression of mesenchymal molecular markers(N-cadherin,Vimentin)and increased the expression of epithelial molecular markers(E-cadherin,Cytokeratin8/18).The expression of EMT regulators Snail,Slug,and MMP2 was also significantly reduced.In terms of cellular resistance,knockdown of circ＿TMTC1 reduced the IC50 of tamoxifen-resistant canine mammary tumor cell lines from 12.5 μM to 6 μM(p<0.001).The proportion of Ed U-positive cells was also substantially reduced.The ability of cell clone formation was significantly reduced,and the rate of apoptosis was significantly increased.The results indicated that the tamoxifen resistance of canine mammary tumors was significantly reduced when circ＿TMTC1 knockdown.Circ＿TMTC1 overexpression significantly increased the proliferation,migration and invasion of canine mammary tumor cells as well as the tamoxifen resistance.It was found that miR-195-5p was regulated by circ＿TMTC1 and was highly and lowly expressed in circ＿TMTC1 knockdown and overexpressed tumor cells,respectively.The regulatory roles of circ＿TMTC1 and miR-195-5p were determined by dual luciferase assay.On the one hand,overexpression of miR-195-5p inhibited the proliferation,migration and invasion of canine mammary tumor cells and increased the sensitivity of cancer cells to tamoxifen.The inhibition of tumor progression caused by miR-195-5p could be partially attenuated when overexpression of circ＿TMTC1.On the other hand,knockdown of miR-195-5p promoted the proliferation,migration and invasion of canine mammary tumor cells and reduced the sensitivity of tumor cells to tamoxifen.These changes could partially alleviate these changes by knockdown of circ＿TMTC1.CDC42,the target gene of miR-195-5p,was regulated by circ＿TMTC1 and miR-195-5p at both protein and gene levels.Knockdown of CDC42 significantly reduced the proliferation,migration invasive capacity and tamoxifen resistance of canine mammary tumor cells.Finally,tumorigenic experiments showed that knockdown of circ＿TMTC1 caused growth inhibition,reduction of tumor malignancy,and inhibition of angiogenesis of canine mammary tumor cells in nude mice.The immunohistochemical results of Ki-67 showed that knockdown of circ＿TMTC1 had a significant inhibition effect on tumor proliferation.Meanwhile,CDC42 immunohistochemistry also demonstrated the trend of positive correlation between circ＿TMTC1and CDC42.In this study,we found that circ＿TMTC1 promoted canine mammary tumor progression and tamoxifen resistance through the effects on the miR-195-5p/CDC42 axis by examining the function of circ＿TMTC1 in canine mammary tumors in vitro and in vivo.