The Effect Of Cep63 On Thyroid Papillary Cancer Cells And Related Mechanism

Background and purpose:Thyroid cancer(TC)accounts for about 1%of all human cancers,and up to 90%of endocrine malignancies.Papillary thyroid carcinoma(PTC)is the most common pathological type,accounting for about 70-80%of all thyroid cancers,and the incidence of PTC has been increasing in recent years.Most PTC can be treated with surgical resection,TSH inhibition or combined with radioactive iodine.However,there are still a few PTC malignant degree is very high,bringing more serious consequences.A study has shown that genetic changes in PTC are caused by activation of oncogenes and inactivation of tumor suppressor genes.Although many genetic variants have been found in thyroid cancer,the molecular mechanism of PTC remains unclear.Therefore,it is necessary to further study the mechanism of PTC occurrence and development.As the precursor of centrosomes,Cep63 is located on chromosome 3q22.2 and encodes a 63-kDa protein with six coiled-coil domains;it is known for its ability to bind Cep 152 to form the annular structural base of the centrosome.Previous study found that Cep63 is involved in spindle assembly and DNA damage after spindle inactivation,binding to centrosomes and regulating mitosis,and additionally causes chromosomal instability and aberrations that lead to poor prognosis and potentially cancer-related overexpression.Cep63 has been shown to promote the development and evolution of tumors;however,the relationship between Cep63 and PTC development is unclarified.In the present study,Cep63 was knocked out via CRISPR/Cas9 technology to clarify its influence on TPC-1 cell behavior.We further investigated the effect of Cep63 on cell signaling pathways in TPC-1 cells.Materials and methods:1.The expression level of Cep63 in human thyroid cancer cell lines Nthy-ori-3,FTC-133 and TPC-1 were detected by western blotting,and the transcriptional expression level of Cep63 in 140 papillary thyroid carcinoma tissues and corresponding normal thyroid tissues were detected by RT-PCR.The correlation between the Cep63 expression level and age,gender,lymph node metastasis and pathological stage was analyzed.2.Cep63 gene was knocked out in TPC-1 cells by CRISPR/Cas9 technology,and stable Cep63-KO cell line was constructed.Western blotting and qRT-PCR were used to test the knockout efficiency of Cep63.CCK-8 and plate colony formation assay were used to detect the effect of Cep63 gene knockout on the proliferation of TPC-1 cells.The effects of Cep63 on TPC-1 cell cycle and apoptosis were detected by flow cytometry.The effects of Cep63 on cell invasion and migration were tested by cell scratch test and transwell test.3.The expression levels of related pathway proteins in TPC-1 and Cep63-KO cells were detected by Western blotting,and the function of the pathway in cells was further detected by treatment with the JAK inhibitor LY2784544.4.Subcutaneous tumorigenesis of nude mice was performed to detect the difference in the proliferation capacity of TPC-1 and Cep63-KO cells in vivo,and the changes in tumor volume and mouse weight were recorded.Finally,tumor bodies were removed and weighed after sacrifice to evaluate the difference in the tumorigenesis effect of Cep63 gene on TPC-1 cells in vivo.Immunohistochemistry was used to detect the expression level of related proteins in the tumors and cells.Results:1.The Cep63 expression level in PTC tissue was higher than that in normal thyroid tissue significantly,and it was significantly up-regulated in TPC-1 cells compared with Nthy-ori-3.Through analyzing the relationship between the Cep63 expression level and the clinicopathological features,it was found that the high expression of Cep63 was significantly correlated with lymph node metastasis in PTC,while the age,gender,tumor size,TNM stage,BRAF mutation state of the patients were not significantly correlated with the expression level of Cep63.2.Cep63 gene expression was knocked out by CRISPR/Cas9,and Western blotting and RT-PCR results showed that Cep63 expression was absent in cells.CCK-8 and plate cloning experiments showed that the proliferation of Cep63-KO cells was significantly decreased.The results of flow cytometry showed that compared with TPC-1 cells,the G0/G1 phase ratio of Cep63-KO cells was significantly decreased,while the S phase ratio was significantly increased,and the transition from G0/G1 phase to S phase was significantly increased.The apoptosis rate of Cep63-KO cells was significantly higher than that of TPC-1 cells(20.1%,2.0%).The results of cell migration and invasion experiments showed that compared with TPC-1 cells,the cell migration rate after Cep63 gene knockout decreased by 25.31%,and the number of Cep63-KO cells penetrating the transwell membrane was significantly lower than that of TPC-1 cells(147.0±10.12 and 226.3±11.89).3.Compared with the TPC-1 group,the expression levels of p-JAK and p-STAT3 in the Cep63-KO group decreased by 19.3%and 39.4%,respectively.After treatment with the JAK inhibitor LY2784544,the levels of p-JAK and p-STAT3 protein in TPC-1 cells decreased by 20.0%and 15.6%,respectively.Compared with the TPC-1 group,the expression levels of AKT,ERK and CDK1 in the Cep63-KO group were significantly lower(77.8%,22.3%and 66.9%,respectively).The expression of apoptotic protein BAX was significantly up-regulated and the expression of Bcl-2 was significantly inhibited.4.The results of animal experiments showed that the growth rate of the Cep63-KO group was significantly slower than that of the TPC-1 group,and the tumor volume and weight of the Cep63-KO group were significantly lower than that of the TPC-1 group.In addition,with tumor growth,the weight of mice in the TPC-1 group decreased significantly,while the weight of mice in the Cep63-KO group increased gradually.IHC results showed that the expression of related proteins AKT,ERK decreased in the Cep63-KO group.While the expression of Bcl-2 protein in Cep63-KO cells decreased,the expression of pro-apoptotic protein BAX increased.Conclusion:Cep63 is highly expressed in PTC tissues and TPC-1 cells,and is a significant correlation with lymph node metastasis,which may be involved in the occurrence and progression of PTC.The JAK/STAT3 and AKT/ERK pathways are involved in mediating the function of TPC-1 cells.Cep63 knockout may block the JAK/STAT3 pathways,thus inhibiting the proliferation,invasion,migration and promoting apoptosis of TPC-1 cells.Cep63 knockout was inhibited the proliferation of TPC-1 cells in vivo and involved in regulating the tumorigenesis of TPC-1 cells,suggesting that Cep63 mediated the malignant proliferation of TPC-1 cells.Cep63 plays an important role in promoting the malignant biological behavior of TPC-1 cells and may be a potential target for the new treatment of PTC.