| IntroductionThyroid cancer is one of the most common malignant tumors in the world,especially in China,Japan,South Korea and other regions,seriously affecting people’s health.Papillary thyroid carcinoma(PTC)is the most common type of thyroid cancer,accounting for 50%to 70%of all thyroid cancers.In PTC,more than 50%of patients have regional lymph node infiltration or metastasis.For PTC,the only traditional treatment is radical surgery.However,for advanced PTC or extensive distant metastasis,patients are often unable to be operated,and are often accompanied by iodine refractory characteristics,or lose the opportunity of radical surgery and can only be used to estimate the rate of surgery,the prognosis is poor.In recent years,genetic research on thyroid cancer has included the study of mutated/non-mutated gene loci,including BRAF/MAPK,TERT PROMeter,PI3K,mTOR and/or tyrosine kinase receptor RET,EGFR,HGF/SF receptor,insulin\insulin-like receptor,etc.However,no highly effective and sensitive molecular diagnostic targets for PTC have been identified to guide clinical practice.At present,there is a lack of reliable molecular targets and targeted drugs for the diagnosis and treatment of PTC in international guidelines.Therefore,further through fully sequenced genome,transcriptome and other technical analysis,mining characteristics of PTC and genome-wide key abnormal expression of oncogenes and tumor suppressor genes and analyze the mechanism of PTC,will be to the pathogenesis of PTC,discover new target to provide the basis of experimental study of molecular diagnosis and treatment,has important scientific significance and clinical application value.Centrosome is an important microtubule organization center(MTOC)in animal cells,which provides the basis for the function of many organelles.Its structure consists of a pair of centriole and pericentriolar material(PCM).The ultrastructure of centrioles showed that there were 9 groups of micro-tubules arranged at a certain Angle and formed a cylindrical structure.The two centrosomes are arranged proximally and vertically to form the basic structure of the centrosomes.In the intercellular phase,the two centrioles are different in structure.Among them,the centriole with subsidiary structure in the distal end is called mother centriole(MC),and the other is called daughter centriole/cradle.Physiological conditions,the centriole copy mainly in mother centriole dependent manner(mother centriole dependent,MCD)replicate synthesis,as a mother centriole precursor for synthesis of new centriole,and Sorokin and Anderson,two team found "from scratch" is not dependent on the MC(DE novo)ring structure of amplification,the cradle of body depend on(deutersome-dependent,DD).The synthesis method of MCD is that CEP63 and CEP 152 form a ring structure at the base of the parent centriole,recruit PLK4,determine the replication site of the daughter centriole,and then recruit the replication of the SAS-6 initiator centriole.Khodjakov and Huijie Zhao et al.found that the ring structure formed by CEP63 and CEP 152 had some structural similarities with the cradles observed by electron microscopy,and speculated that the centriole replication of MCD and DD shared some molecular mechanisms and key genes.Some or several key genes involved in the regulation of centriole replication have paralog,and different homologs mediate centriole amplification of MCD and DD pathways respectively.The phenomenon of centriole excess,centriole disordered structure,abnormal centriole length and centriole associated protein phosphorylation are related to the occurrence and development of many tumors,and may be directly related to the high mitotic index and high invasion of cancer cells.Lingle et al.for the first time found abnormal centrosomal amplification and abnormal phosphorylation of centrosomal proteins in breast cancer.Evidence of abnormal centriole amplification has been found in HeLa cell line and osteosarcoma cell U2OS.The currently widely accepted mechanism is that during the intermitotic phase,abnormal concentrations of proteins recruited to the surrounding centrioles,such as PLK4,SAS-6,SIL\STIL,etc.,are elevated,leading to abnormal amplification of the centrioles.Recent studies have shown that the number of centrosomes is regulated by autophagy.The specific mechanism of autophagy regulating centrosome number is still not fully understood.Shigeomi Shimizu et al.believed in a report in 2016 that CEP63 protein was engulfed by autophagy vesicules by binding to the zinc finger structure of p62,and then was mediated by the ubiquitination domain of p62 for ubiquitination degradation to maintain the normal centrosome number.At present,it is widely recognized by most researchers that autophagy is involved in the initiation,progression and metastasis of thyroid cancer,and the effects of autophagy on thyroid cancer cells are not identical at different stages.In the initial stage of thyroid cancer,autophagy scavenges reactive oxygen species and defective proteins and organelles in cells mainly through up-regulation of ATG and its corresponding protein family.At this stage,autophagy mainly inhibits the occurrence of tumor.In the progressive stage of thyroid cancer,autophagy mainly plays a role in maintaining the malignant degree of thyroid cancer and the proliferation of cancer cells.In addition,further experimental data are still needed to support the relationship between autophagy and thyroid cancer metastasis.Currently,existing studies have speculated that autophagy may affect the invasion and metastasis of thyroid cancer through autophagy regulatory gene(Beclin-1)and Epithelial mesenchymal transition(EMT).However,there is still a lack of research on how autophagy regulates the number of centrosomes in thyroid cancer to achieve sustainable and stable malignant biological behavior of tumor cells.The aim of this study was to investigate the molecular mechanism of CCDC67 gene’s antitumor effect and the biological function of the downstream key gene CEP63 in thyroid papillary carcinoma.This topic consists of the following four parts:The first part is the study of the regulatory effect of CCDC67 gene on PI3K/Akt/mTOR pathway;The second part,the effect of CCDC67 gene on autophagy;The third part discusses the biological function of CEP63 gene.In the fourth part,the regulation effect of CCDC67 gene on the expression of CEP63 gene was analyzed.Part Ⅰ Regulation effect of CCDC67 gene on PI3K/Akt/mTOR pathwayAims:Our previous study found that CCDC67 gene is a tumor suppressor gene in thyroid papillary carcinoma.The inactivation of this gene may be one of the important reasons for the development and development of thyroid papillary carcinoma,and it is also associated with BRAF V600E mutation.However,how CCDC67 gene exerts its tumor suppressive function in thyroid papillary carcinoma remains to be further studied.This part of the study aims to explore the changes in downstream gene expression caused by CCDC67 gene overexpression through proteomics and transcriptome sequencing technology,and to preliminarily analyze the anti-cancer mechanism of CCDC67 gene.Methods:1.The lentivirus Lenti-CCDC67-luciferase-puromycin plasmid was constructed.After cell transfection,the thyroid TPC-1 cell line with stable overexpression of CCDC67 gene was constructed by purinomycin screening.2.The TPC-1 cell lines before and after overexpression of CCDC67 gene were detected by isobaric tags for relative and absolute quantitation(iTRAQ)proteomics technique,and the changes of protein level expression before and after overexpression of CCDC67 gene were analyzed.Metascape was used to integrate and enrich the analysis data for Reactome,Corum,KEGG and GO databases.3.TPC-1 cell lines before and after overexpression of CCDC67 gene were detected by transcriptomics(RIP-RNA-seq)technique to analyze the changes of transcription level expression before and after overexpression of CCDC67 gene;Metascape was used to integrate and enrich the analysis data for Reactome,Corum,KEGG and GO databases.4.TPC-1 cells,CCDC67OE cells and NC cells in the period of exponential growth were collected and the total protein of the cells was extracted.The PI3K protein,Akt protein and mTOR protein in the results of proteomics and transcriptomics were detected and verified by Western-blot analysis.5.Statistical software SPSS22.0 and GraphPad were used for data processing.The t test was used for the comparison of two groups of data.One-way ANOVA was used for the comparison of related indicators of multiple groups of data.LSD-t test was used for the pair-wise comparison between groups.P<0.05 was considered statistically significant.Results:1.Through the detection and analysis of TPC-1 cell lines before and after overexpression of CCDC67 by iTRAQ proteomics,we found a total of 6000 differentially expressed proteins and 31307 peptide fragment.According to KEGG pathway analysis,mTOR signaling pathway showed different expression changes.2.RIP-RNA-seq transcriptome sequencing technology was used to detect and analyze TPC-1 cell lines before and after overexpression of CCDC67.A total of 1865 circRNA differentially expressed were found,including 1008 up-regulated and 857 down-regulated circRNA.28213 differentially expressed lncRNAs were found,including 7554 up-regulated and 20659 down-regulated.There were 8457 mRNA differentially expressed,among which 3494 mRNA was up-regulated and 4963 mRNA was down-regulated.Metascape integration enrichment analysis showed that the functions of differentially expressed circRNAs were concentrated in micro tubule,cellular skeleton,cell cycle and cellular component,and differentially enrichment in PI3K signaling pathway,cell cycle pathway and MAPK cascade were also observed.3.Western blotting analysis of PI3K/Akt pathway showed that the expression of PI3K was significantly inhibited and the expression of mTOR was significantly up-regulated after CCDC67 overexpression,but Akt expression was not significantlychanged(PI3K:TPC-1 vs.NC,p=0.022;TPC-1 vs.CCDC67OE,p=0.010;CCDC67OE vs.NC,p=0.002.AKT:TPC-1 vs.NC,p=0.741;TPC-1 vs.CCDC67OE,p=0.899;CCDC67OE vs.NC,p=0.948.mTOR:TPC-1 vs.NC,p=0.915;TPC-1 vs.CCDC67OE,p=0.006;CCDC67OE vs.NC,p=0.009.).Conclusions:The function of overexpressed CCDC67 in thyroid papillae is diversified.Its functions may involve many aspects of cell life activities such as cell cycle regulation,organelle synthesis and microtubule tissue center formation.At the same time,the overexpression of CCDC67 has a certain influence on the PI3K/Akt/mTOR signaling pathway,but the non-consistent changes in the expression of PI3K and mTOR remain to be further explored to verify the biological effect of CCDC67 by further experiments.Part Ⅱ Rearch of the Regulation effect of CCDC67 gene on autophagyAims:In our preliminary experiment,we accidentally found that CCDC67 gene overexpression in TPC-1 cell line resulted in an increase in the counts of autophagy marker structures(autophagosomes and autophagosomes)in the cytoplasm.Therefore,we tried to explore the functional mechanism of CCDC67 gene through autophagy as another entry point,and to detect the effect of CCDC67 gene on autophagy through more complete experimental means and schemes.Methods:1.TPC-1,CCDC67OE and NC cell lines in the exponential growth period were transfected with lentivirus overexpressing SenSGFP-StuBRFP-LC3.24H later,purinomycin was used for cell screening.The screened cells were seeded into 96-well plates at 1 × 104/ml,stained with Hocheng-33342,and placed in CQ1 for three-channel fluorescence scanning(SengFP,StubrFP and Hochest).After counting the ratio of red and green dot counts,the level of autophagy flow was evaluated.2.PC-1 cells,CCDC67 cells and empty vector transfected cells in,NC,were harvested and rinsed with normal saline and fixed with 1%glutaral for 30 minutes.After that,glass slides were made for transmission electron microscopy observation and photo,organelles were labeled and Autophagosome(AP)and Autophagol ysosome(ASS)were counted.3.TPC-1 cells,CCDC67OE cells and NC cells in the period of exponential growth were collected to extract the total protein of the cells.The autophagy marker LC3 was detected by Western-blot method,and the results were analyzed by ImageJ gray scale analysis.4.Statistical software SPSS22.0 and GraphPad were used for data processing.One-way ANOVA was used to compare the related indicators of multiple groups of data,and LSD-t test was used for pair-wise comparison between groups.P<0.05 was considered statistically significant.Results:1.Transmission electron microscopy(TEM)showed that the number of autophagosomes and autophagosomes in CCDC67OE group was significantly increased compared with TPC-1 group and negative control group(TPC-1 vs.NC,p=0.9148;TPC-1 vs.CCDC67OE,p=0183;CCDC67OE vs.NC,***p<0.048).2.The results of autophagy flow monitoring showed that the overexpression of CCDC67 resulted in the enhancement of autophagy flux,and the relative red-green dot count of autophagy indicated the dynamic transformation process of autophagosome to autophagosolysosome(TPC-1 vs.NC,p=0.891;TPC-1 vs.CCDC67OE,p=0.001;CCDC67OE vs.NC,***p<0.001.).3.Western blotting showed that LC3 protein in CCDC67OE group was significantly higher than that in TPC-1 group,NC group and NTHY-ROI 3-1 group(TPC-1 vs.NC,p=0.9148;TPC-1 vs.CCDC67OE,p=0183;CCDC67OE vs.NC,p=0.027,TPC-1 vs.Nthy-ori 3-1,p=0.341;NC vs.Nthy-ori 3-1,p=0.472;CCDC67OE vs.Nthy-ori 3-1,p=0.223).Conclusions:In thyroid papillary carcinoma TPC-1 cell line,overexpression of CCDC67 gene resulted in increased autophagy.Referring to the high level of autophagy in normal thyroid follicular epithelial cells,we speculated that the elevated level of autophagy is a crux in the reversal of malignant phenotype of thyroid papillary carcinoma by CCDC67 gene.Part Ⅲ Rearch of the Clinical Expression and Tumor Biological Function of CEP63 GeneAims:In the first part of transcriptome sequencing,we focused on the differential expression of CEP63 gene.Subsequently,CCDC67 and CEP63 were found to be paralogue genes with similar structure and to dominate two respective pathways of centrosome replication,namely CCDC67-Deutersome Dependent(DD)and CEP63-Mother Centrosome Dependent(MCD).Therefore,in this part,we focused on the expression and biological function of CEP63 in thyroid papillary carcinoma,and tried to explore the possible regulatory mechanism between CCDC67 gene and CEP63 gene directly.Methods:1.Tumor tissues of 24 patients with papillary thyroid carcinoma in the Department of Thyroid Surgery,The First Affiliated Hospital of Zhengzhou University from March to May 2018 were collected.Total RNA was extracted and RT-PCR was performed.2.Using Cas9/CRISPR technology,the thyroid papillary cancer cell lines knocked out by CEP63 were constructed.After gene knockout,purinomycin was screened for monoclonal culture amplification.Finally,KO knockout sites were verified by Sanger sequencing technology,and the knockout efficiency of CEP63 gene was verified by Realtime-PCR and Western blotting.3.GeneChip Primeview Human microarray chip of Affymetrix was used to analyze the differential expression genes after CEP63KO,and Metascape was used to perform Reactome,Corum,KEGG and GO database integration and enrichment analysis on the basis of the original analysis,to explore the biological functions of CEP63 and potential downstream regulatory targets.4.According to the results of microarray chip analysis,the biological function of CEP63 gene was preliminarily analyzed by Transwell migration experiment and flow cycle detection.5.The thyroid papillary cancer cell line knocked out by CEP63 and the blank control cell line TPC-1 were used to carry out subcutaneous tumor-bearing experiment in nude mice,and the biological analysis of the proliferation ability of CEP63 gene was conducted.After the tumor was visible,the tumor volume was measured the next day and the body weight of the nude mice was recorded.The nude mice were sacrificed after the tumor reached 20mm,and the weight and final volume of the tumor were measured.6.The subcutaneous tumor-bearing tumor of nude mice was collected and fixed in formalin solution,then the immunohistochip was made.Then,immunohi stochemical staining of PI3K,CyclIND,DDI3T,CREB,BAD and mTOR proteins was performed on the chip,and the results of staining were statistically analyzed by calculating the Integrated Optical Density(IOD)using ImageJ.7.Statistical software SPSS22.0 and GraphPad were used for data processing.One-way ANOVA was used to compare the related indicators of multiple groups of data,and LSD-t test was used for pair-wise comparison between groups.P<0.05 was considered statistically significant.Results:1.The mRNA expression of CCDC67 and CEP63 gene was detected in 24 cases of thyroid papillary carcinoma.2.The TPC-1 cell line CEP63KO was successfully constructed by Cas9/CRISPR,and stable cell lines confirmed with 2 and 14 bases were obtained by purinomycin screening and monoclonal culture.Subsequently,KO efficiency was verified by Western blotting.3.A total of 1301 differentially expressed genes were detected between CEP63KO and TPC-1 cells by GeneChip Primeview Human microarray microarray.In the classical Pathway enrichment analysis,the Endocannabinoid Cancer Inhibition Pathway(Endocannabinoid tumor Inhibition Pathway)was significantly activated,with a Z-score of 3.771.In the significant enrichment analysis of differential genes in disease and function,tumor,collective damage and abnormality,Cell movement and Cell death pathways were indicated to be significantly enriched,and the heat map showed that Cell movement of cancer cells was significantly inhibited after Cep63 gene knockout(Z-score=-2.994).Key molecules of the Endocannabinoid Cancer Inhibition Pathway,including PI3K,mTOR,CREB,CyclIND,etc.,were significantly inhibited,while CDH1(E-cadhrin),NUPR1,DDIT3,and TRIB3 were significantly activated.4.The results of Transwell migration assay showed that the migration ability of TPC-1 cells was significantly reduced after the knockout of CEP63 gene(Transwell experiment:TPC-1 vs.CEP63KO,p=0.0319).The results of flow cytometry showed that the cycle progression of TPC-1 cells showed significant S phase arrest and G2 phase decrease after CEP63 gene knockout(G1 phase:TPC-1 vs.CEP63KO,p=0.0810;S phase:TPC-1 vs.CEP63KO,****p<0.0001;G2/M phase:TPC-1 vs.CEP63KO,****p<0.0001).5.Xenograft model experiments in nude mice showed that the tumorigenesis and proliferation of TPC-1 cells were significantly inhibited,and the final volume and weight of TPC-1 cells were significantly decreased after the knockout of CEP63 gene(Xenograft model:tumor weight:TPC-1 vs.CEP63KO,****p<0.0001;tumor volume:TPC-1 vs.CEP63KO,****p<0.0001.).6.The immunohistochemical staining of the xenograft tissue showed a significant decrease in the expression of PI3K/cyclinD and a significant increase in that of DDIT3 in the CEP63KO group,whereas the expression change of CREB/BAD/mTOR remained nonsignificant between the TPC-1 and CEP63KO groups(PI3K:TPC-1 vs.CEP63KO,p=0.0116;cyclinD:TPC-1 vs.CEP63KO,p=0.0116;TPC-1 vs.CEP63KO,p=0.0002;DDIT3:TPC-1 vs.CEP63KO,p=0.0086;CREB:TPC-1 vs.CEP63KO,p=0.1612;BAD:TPC-1 vs.CEP63KO,p=0.7944;mTOR:TPC-1 vs.CEP63KO,p=0.3337).Conclusions:CEP63 plays an important role in the malignant biological maintenance of thyroid papillary carcinoma cell line TPC-1,and its knockout can significantly inhibit tumor cell cycle progression and cell activity.At this point,the biological function of CCDC67 is completely different from that of its paralogue gene.It is speculated that in normal thyroid papillary carcinoma cells,the mother centriole replication pathway dominated by CEP63 may have an inhibitory effect on the deuter centriole replication pathway dominated by CCDC67,and this inhibitory effect may be the joint link of thyroid papillary carcinoma cells to maintain malignant biological behavior.Part Ⅳ Rearch of the Clinical Expression and Tumor Biological Function of CEP63 GeneAims:In the first and second parts,we discussed the potential mechanism of CCDC67 gene’s anti-tumor function in thyroid papillary carcinoma from different perspectives,including the regulation of PI3K/Akt/mTOR pathway and the effect on autophagy.In the third part,we found that CCDC67 para-homologous genes have completely different biological effects in thyroid papillary carcinoma.On this basis,in the fourth part,we try to explore the relationship between the contents of the first three parts and analyze their internal relationship and the deeper mechanism,that is,the regulation of CCDC67 gene on the expression of CEP63 gene.Methods:1.Tumor and paracancerous normal tissues of 21 patients with papillary thyroid carcinoma in the Department of Thyroid surgery of the First Affiliated Hospital of Zhengzhou University were collected from September to November 2018.Total RNA was extracted and realtime-PCR was performed to detect the expression levels of CCDC67 and Cep63 genes.At the same time,the expression of CCDC67 and CEP63 genes in carcinoma and paracancerous tissues of papillary thyroid carcinoma patients with different clinicopathological stages were analyzed.2.The expressions of CCDC67 and CEP63 genes in BRAF V600E mutant cell lines B-CPAP and TPC-1 in thyroid follicular epithelial cells Nthy-ORI 3-1 and thyroid papillary carcinoma were detected by Western-blot method.3.The expression of CEP63 in TPC-1 cell lines before and after CCDC67 gene overexpression was detected by Western-blot method,and the results were analyzed by ImageJ.4.The CEP63 protein,γ-tubulin and nucleus of the cells were labeled by fluorescence staining.The cells captured by laser confocal microscope in the stationary phase and the mitotic phase were used to analyze the protein expression,location,centrosomal morphology and mitotic state of the cells,especially the bound Cep63 protein bound with γ-tubulin and the free Cep63 protein in the cytoplasm.5.The autophagy agonist rapamycin(10nM)and the autophagy inhibitor Pavlomycin A1(10nM)were used to treat TPC-1 cells for 24H.Western blotting was used to detect the protein expression levels of CEP63,LC3,Beclinl and mTOR.ImageJ was used to analyze the results.6.TPC-1 cells,rapamycin-treated TPC-1 cells,and CEP63KO cells knocked out by CEP63 were used to conduct the plate colony formation experiment,respectively.After 48 hours,photos were taken,and the colony area was measured by ImageJ.7.TPC-1 cells,rapamycin-treated TPC-1 cells(TPC-1+ RAPA),CCDC67 overexpressed TPC-1 cells(CCDC67OE)and empty vector transfected cells(NC)were used to perform xenograft model in BALB/c nude mice,respectively.After the tumor was visible,the tumor volume was measured the next day and the body weight of the nude mice was recorded.The nude mice were sacrificed after the tumor reached 20mm,and the tumor weight and final volume were measured and the tumor inhibition rate was calculated.8.Statistical software SPSS22.0 and GraphPad were used for data processing.One-way ANOVA was used to compare the related indicators of multiple groups of data,and LSD-t test was used for pair-wise comparison between groups.P<0.05 was considered statistically significant.Results:1.A total of 24 PTC cases were tested for the presence of CEP63 and CCDC67.Subsequently,the expressions of CCDC67 and CEP63 in 21 PTC cases and adjacent normal tissues were analyzed.A significant increase in CEP63 expression was observed in PTC tissues,whereas a significant decrease in CEP63 expression was observed in the adjacent normal tissues.However,the expression of CCDC67 was just the opposite.Besides,the expression of CEP63 was higher than that of CCDC67 in PTC tissues,whereas in adjacent normal tissue the expression of CEP63 was relatively lower compared to that of CCDC67(CEP63:PTC vs.Normal tissue,p=0.0021;CCDC67:PTC vs.Normal tissue,p=0.0247;Normal tissues:CEP63 vs.CCDC67,p=0.0145;PTC:CEP63 vs.CCDC67,p=0.0047).2.The expression of CEP63 and CCDC67 in cell lines(TPC-1/B-CPAP/Nthy-ori 3-1)was also detected using western-blot analysis,which showed a higher expression level of CEP63 than that of CCDC67 in TPC-1 and B-CPAP cell lines,but only the change in TPC-1 was observed to be significant(TPC-1:CEP63 vs.CCDC67,p=0.001;B-CPAP:CEP63 vs.CCDC67,p=0.289;Nthy-ori 3-1:CEP63 vs.CCDC67,p=0.930).3.The CEP63 expression inhibition from CCDC67 was verified using westernblot analysis(CCDC67:TPC-1 vs.NC,p=0.008;TPC-1 vs.CCDC67OE,p=0.001;CCDC67OE vs.NC,p=0.048.)4.The results of laser confocal microscope detection showed that CEP63 is mainly localized in the cytoplasm and forms cytoplasmic aggregates.In the TPC-1 group,except for binding to y-tubulin,nonbinding CEP63 expression was mostly aberrantly accumulated and dispersed in the cytoplasm,whereas the nonbindingCEP63 fluorescence in the CCDC67OE group showed an obvious agglutination and aggregation in the cytoplasm of G1/S phase cell or the perinuclear cytoplasm of mitotic cells.In the early stages of the mitotic phase,the CEP63 protein binds toγ-tubulin to form centrosomes and provides the driving force for chromosome separation.In the CCDC67OE and CEP63 knockout(KO)group,the red fluorescence counts of CEP63 presented a significant decrease(TPC-1 vs.CCDC67OE,p=0.013;TPC-1 vs.CEP63KO,p=0.003;CCDC67OE vs.CEP63KO,p=0.724).In the CEP63KO group,especially,we observed an asymmetric chromosome division that resulted from centrosome absence or dysfunction.5.The results showed a significant increase in CEP63 expression in the bafilomycin A1-treated group and a significant decrease in the rapamycin-treated group.A significantly high expression of LC3 was observed in the bafilomycin A1-treated group compared with that in other groups;the CEP63KO group also showed a significantly higher level than that in the Nrhy-ori 3-1 and rapamycin-treated groups.Beclinl showed similar results as those of LC3.The protein expression of mTOR in Nthy-ori 3-1 and rapamycin-treated TPC-1 cells was significantly low and that in bafilomycin-treated TPC-1 cells was significantly high.Consequently,these obstructing phenomena enhance mTOR expression,which transmits an inhibiting signal to downregulate the autophagy flux.A similar situation may also be observed in CCDC67 overexpression,which causes a relatively high expression of LC3 and Beclin1(CEP63:TPC-1+Rapa vs.Nthy-ori 3-1,p=0.0759;TPC-1+Baf vs.Nthy-ori 3-1,p=0.0017;CEP63KO vs.Nthy-ori 3-1,p=0.0604;TPC-1+Baf vs.TPC-1+Rapa,p=0.0173;CEP63KO vs.TPC-1+Rapa,p=0.2217;CEP63KO vs.TPC-1+Baf,p=0.0182.LC3:TPC-1+Rapa vs.Nthy-ori 3-1,p=0.9729;TPC-1+Baf vs.Nthy-ori 3-1,p=0294;CEP63KO vs.Nthy-ori 3-1,p=0.0334;TPC-1+Baf vs.TPC-1+Rapa,p=0.0086;CEP63KO vs.TPC-1+Rapa,p=0.0397;CEP63KO vs.TPC-1+Baf,p=0.3333.Beclin1:TPC-1+Rapa vs.Nthy-ori 3-1,p=0.9999;TPC-1+Baf vs.Nthy-ori 3-1,p=0.0015;CEP63KO vs.Nthy-ori 3-1,p=0.0056;TPC-1+Baf vs.TPC-1+Rapa,p=0.0015;CEP63KO vs.TPC-1+Rapa,p=0.0060;CEP63KO vs.TPC-1+Baf,p=0.6700.mTOR:TPC-1+Rapa vs.Nthy-ori 3-1,p=0.9999;TPC-1+Baf vs.Nthy-ori 3-1,p=0.0040;CEP63KO vs.Nthy-ori 3-1,p=0.7283;TPC-1+Baf vs.TPC-1+Rapa,p=0.0038;CEP63KO vs.TPC-1+Rapa,p=0.7096;CEP63KO vs.TPC-1+Baf,p=0.0150)6.In colony-forming assay,the size and number of TPC-1 cell colonies in the CEP63KO and rapamycin-treated TPC-1 groups were clearly fewer than those in the TPC-1 group,and the average clone formation ratio was significantly low in the CEP63 and rapamycin-treated groups(TPC-1 vs.TPC-1+Rapa,****p<0.0001;TPC-1 vs.CEP63KO,****p<0.0001;TPC-1+Rapa vs.CEP63K,***p<0.001.)7.Results from in vitro models showed a significant tumor suppression effect in rapamycin-treated and CCDC67OE groups and both treatments resulted in a downregulation of the CEP63 expression(Tumor weight:TPC-1 vs.NC,p=0.9320;TPC-1 vs.CCDC670E,p=0.0061;TPC-1 vs.TPC-1+Rapa,***p<0.001;NC vs.CCDC67OE,p=0.0226;NC vs.TPC-1+Rapa,***p<0.001;CCDC67OE vs.TPC-1+Rapa,p=0.365.Tumor volume:TPC-1 vs.NC,p=0.7764;TPC-1 vs.CCDC670E,****p<0.0001;TPC-1 vs.TPC-1+Rapa,****p<0.0001;NC vs.CCDC67OE,***p<0.001;NC vs.TPC-1+Rapa,***p<0.001;CCDC67OE vs.TPC-1+Rapa,p=0.9999)Conclusions:In papillary thyroid carcinoma,the high expression of CEP63 is correlated with the low expression of CCDC67.Overexpression of CCDC67 gene can decrease the expression of CEP63 and reverse the biological characteristics of TPC-1 cells.After the autophagy level was interfered with by rapamycin and babfloamycin A1,the expression of CEP63 protein also showed opposite changes to the autophagy level,suggesting that the expression of CEP63 protein was regulated by autophagy.Combined with the above results,it is not difficult to find that CCDC67 regulates autophagy through PI3K/Akt/mTOR signaling pathway,and then affects the expression of CEP63.This indicates that in thyroid papillary carcinoma,there is interpathway inhibition of MCD and DD pathways,two centrosome replication pathways,and the occurrence of this inhibition effect is likely to be the key link in the occurrence of thyroid papillary carcinoma. |
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