| Background and Objective Thyroid carcinoma of epithelial follicular cell origin is one of the most common malignant neoplasms in the global endocrine system.Globally,thyroid cancer is the most common cancer among South Korean women and the fifth most common cancer among American women.Papillary thyroid carcinoma(PTC)is reported to be the most common pathologic type of thyroid cancer,accounting for more than 80%.Differentiated thyroid cancers,which include papillary thyroid cancer and follicular thyroid cancer,have a high recurrence rate,which is 20-30% or even higher in some subtypes,although the mortality rate is low.Despite the high incidence and recurrence rates of PTC,most patients have a good prognosis after standard treatment such as surgical administration of radioactive iodine and regular review.However,inoperative and radio-iodine-refractory PTC may still lead to thyroid cancer-related death,and there is currently no effective treatment.The occurrence and development of thyroid tumors is a complex process involving multiple genes,including tumor suppressor genes,oncogenes and a large number of regulatory genes.It is widely believed that autophagy and metabolic systems help tumor cells survive by breaking down and redistributing proteins within cells.In our previous study,PCR showed that the expression of p62 in PTC tissues was higher than that in normal thyroid tissues.Therefore,we believed that p62 played an important role in PTC and realized accurate regulation of homeostasis by regulating the growth of PTC cells.The Sequestosome 1 gene(SQSTM1),also known as p62,located at 5q35,is an adaptor protein in the ubiquitination system and a van protein in selective autophagy,playing an important role in regulating intracellular protein degradation.However,it has also been reported that P62 is also involved in cell cycle,cell metabolism,the scavenging effect of selenium on peroxide free radicals and other cell biological activities.p62 gene mutation is closely related to Paget’s disease of bone,mouse myeloid leukemia progression,neurodegenerative disease,obesity,vascu lar aging,aging pathology and cancer.Composed of 440 amino acids,covering more than 10 domains and binding sites,p62 is a key center for regulating insulin signaling,energy balance,adipogenesis,BAT thermogenesis,inflammation,oxidative stress,apoptosis and other cellular activities.More and more evidence shows that p62 is involved in a variety of signal transduction pathways,including insulin signal transduction pathway,KEAP1-NRf2 pathway,ERK pathway and P38 /MAPK signal transduction pathway,which proves that p62 has important regulatory effects on cells.Therefore,we explored the effects of p62 on the physiology and function of PTC cells from multiple aspects,and further studied the pathogenesis of PTC.Material and Method 1.105 cases of tumor tissues and paracancer tissues resected by clinical thyroid surgery were collected.The expression of p62 gene in thyroid tissue was detected by RT-q PCR,and its correlation with clinicopathological features was analyzed.2.The extracted proteins from the normal thyroid cell line Nthy-ori 3-1 and the thyroid papillary cancer cell line TPC-1 were collected,and the expression of p62 protein in the cells was detected by Western Blot.3.CRISPR/Cas9 gene editing technology was used to knock out the p62 gene fragment of TPC-1 cell line,and the stable passage cell line p62-KO-TPC-1 was constructed.The knockout efficiency was verified by RT-q PCR and Western Blot experiments.4.The effect of P62 on the proliferation of tumor cells was evaluated by CCK-8 assay,colony formation assay,flow cytometry and RT-q PCR in TPC-1 and p62-KO-TPC-1 cells.Transwell assay was used to detect the effect of p62 on the invasion and migration of tumor cells.Apoptosis rate was detected by flow cytometry,and expression of apoptose-related proteins bax and Bcl-2 in TPC-1 and p62-Ko-TPC-1 were detected by Western Blot,so as to evaluate the effect of p62 on apoptosis of tumor cells.5.Fourteen BALB/C nude mice were inoculated with TPC-1 and p62-KO-TPC-1 cell lines,and the tumorigenesis model of TPC-1 nude mice was established to confirm the feasibility of TPC-1 and p62-KO-TPC-1 cell lines.Experimental cell lines(p62-KO-TPC-1),blank control cell lines(TPC-1)2 groups 7 female BALB/c nude mice,mice to observe the change of behavior,measure the weight and volume of tumors in mice on a regular basis,to vaccinate mice were euthanized at 20 days,many tumors weighing,according to the tumors had the weight,volume growth changes do figure and chart,analysis and calculation inhibitory rate and evaluate the results of two groups of cells into a tumor in mice.Hematoxylin-eosin staining(HE)was performed on the stripped nude mice tumor.The expression of P62 in the tumor was confirmed.The expression of proliferation related proteins PCNA,PTEN and P53 in the tumor model constructed by two cell lines was detected,and the difference was analyzed by Image J software.7.The extracted proteins from TPC-1 and P62-Ko-TPC-1 cells were collected,and the expression of proteins in the invasion and migration pathway related to proliferation,apoptosis and migration was detected by Western Blot,so as to explore the mechanism of p62 on TPC-1 cells.8.SPSS 21.0 series was used for statistical analysis of the experimental data,chi-square test of the four-grid table was used for comparison of the two sample rates,one-way an OVA was used for comparison of multiple mean values,and paired T-test was used for analysis of the two quantitative samples.P<0.05 was considered statistically significant.Result 1.The expression rate of P62 protein in 105 PTC tissues was 69.5%(73/105),and the difference was statistically significant(P < 0.05).The expression of p62 was correlated with tumor size(P=0.0066),but there was no statistical correlation between the expression of p62 gene and age,gender,TNM stage or lymph node metastasis(P >0.05).2.Compared with the normal thyroid cell line Nthy-ORI 3-1,the expression of p62 protein in the thyroid papillary cancer cell line TPC-1 was significantly increased.3.Cell growth was detected by cck-8 method and plate colony formation experiment,indicating that the ability of colony formation was weakened and the cell growth rate was significantly slowed down after p62 knockdown(P >0.05).4.Flow cytometry analysis showed that inhibition of p62 expression in tpc-1 cells reduced the proportion of G1 cells and increased the proportion of S cells(P >0.05).Combined with the experimental results of cck-8 and plate colony formation,the tpc-1 cell cycle was blocked in S phase after the knockout of p62.We evaluated the effect of p62 on apoptosis by flow cytometry,and the results showed that the apoptosis rate was significantly increased after the removal of p62(P >0.05).Western Blot was used to detect the expression of Bax and Bcl-2 proteins in TPC-1 and p62-KO-TPC-1 cells,indicating that the expression of Bax significantly increased and the expression of Bcl-2 significantly decreased after the knockout of p62.5.According to the transwell invasion and migration experiment,the number of cells passing through the transwell chamber was significantly reduced after p62 was knocked down,and the ability of cell invasion and migration was significantly reduced.6.The tumor growth curve was drawn by measuring tumor volume at regular intervals,which showed that the tumor growth of the inoculated p62-KO-TPC-1 cells was more gradual,the curve was downward,and the growth ability was significantly reduced.In the control group,the tumor growth curve was steeper and the growth ability was significantly faster after TPC-1 inoculation,and the difference was statistically significant(P<0.05).After tumor weight was measured after tumor removal,it was found that the tumor with TPC-1 cells inoculated was larger,with statistical difference(P<0.05).7.Immunohistochemical staining showed that p62 protein expression was low in the tumor tissues of p62-KO-TPC-1 in the experimental group,and the proliferation related proteins P53,PTEN and PCNA were deeply stained,and the expression level was significantly higher than that in the control group(P<0.05).8.Western Blot results showed that the expressions of ERK,AKT and CDK1 protein were significantly decreased after the knockout of P62.At the same time,p-Akt expression increased in p62-KO-TPC-cells,indicating that p62 lacks the function of promoting AKT phosphorylation and activating protein kinase.In TPC and p62-KO-TPC-1 cells,the expression of m TOR was not significantly changed,but decreased after treatment with the m TOR inhibitor rapamycin.The expression of autophagy-related protein LC3 in TPC-1 and p62-KO-TPC-1 cells before and after the autophagy inhibitor Bafilomycin A1 was detected,and the results showed that the knockout of p62 could induce the accumulation of LC3-II,showing autophagy activation.Conclusion 1.The expression of p62 gene was abnormally increased in the surgically removed thyroid papillary carcinoma specimens of clinical patients,and its high expression was correlated with the tumor size of corresponding patients,and p62 also showed abnormal accumulation in t PC-1 cells of thyroid papillary carcinoma.2.In TPC-1 cells,p62 significantly promoted cell proliferation,invasion and migration,and inhibited cell apoptosis.In vivo experiments also confirmed that p62 promoted tumor growth.3.p62 may regulate the proliferation of tumor cells through the ERK/AKT/AMPK pathway.4.p62 may inhibit cell apoptosis through bcl-2 mediated mitochondrial pathway... |
PDF Full Text Request