| Objective:Colorectal cancer(CRC)is one of the most common malignancies in humans,and it is the second leading cause of cancer death.In 2018,there were about 1.8million new cases of colorectal cancer and 860,000 deaths,and this number is expected to increase further to 2.2 million and 1.1 million in 2030.The death rate from colon cancer has declined slightly over the past decade as a result of increased awareness of health and the rapid development of early screening and treatment methods.However,the five-year survival rate for colon cancer patients is still only 64 percent,and for m CRC(metastatic colon cancer),the five-year survival rate is less than 12 percent.Therefore,it is particularly important to study the molecular mechanism and related regulatory processes of colon cancer metastasis.Human Leukocyte Antigen F Locus Adjacent Transcript 10(FAT10)is an important protein in the post-translational modification of proteins.Studies have found that FAT10 plays an important role in liver cancer,breast cancer,osteosarcoma,bladder cancer and other malignant tumors.Our previous studies have also shown that FAT10 can affect tumor progression through ubiquitin-like modification.However,the effect of FAT10 on colon cancer and its specific regulatory mechanism are still unclear.This study mainly elaborated the specific molecular mechanism and regulatory process of FAT10 regulation in colon cancer through the following contents: First,from a clinical perspective,the expression of FAT10 in colon cancer and its relationship with clinical prognosis were analyzed;Secondly,in vivo and in vitro experiments were conducted to confirm whether FAT10 can affect the metastatic ability of colon cancer.Finally,the specific biological mechanism of FATA10 affecting colon cancer metastasis was explored.Method:1.Quantitative real-time PCR(q RT-PCR),immunohistochemistry and Western blot analysis were used to detect the m RNA and protein expression levels of FAT10 in normal colon mucosal epithelial cells NCM460,colon cancer cells,colon cancer tissues and corresponding paracancerous tissues,and the differences in overall survival and clinicopathology between the high expression group and the low expression group of FAT10 were investigated.2.Stable cell lines with high and low expression of FAT10 were constructed,and the interference and overexpression effects were detected by q RT-PCR and Western blot.Real-time unlabeled dynamic cell analysis(Real Time Celi Anaiysis RTCA)and Transwell were used to detect the effects of overexpression and interference of FAT10 on the invasion and migration of colon cancer cells.At the same time,the constructed cell line was used to construct the orthotopic model of colon cancer tumor in nude mice to observe the liver metastasis.3.Through isobaric tags for relative and absolute quantification(i TRAQ)to detect the changes of downstream related target proteins after interference with FAT10 expression,Western blot and q RT-PCR were used to observe whether FAT10 has a regulatory effect on the protein and m RNA levels of downstream target gene ZO-1,at the same time by rescue experiments confirmed that ZO-1 is a key molecule FAT10 affect migration of colon cancer invasion.4.After adding autophagy lysosomal inhibitors and autophagy agonists to observe whether ZO-1 is degraded by the autophagy lysosomal pathway.Secondly,the effects of FAT10 on the level of autophagy in colon cancer cells were observed by Western blot,immunofluorescence and transmission electron microscopy.Moreover,in the case of adding actinomycetes CHX to block protein synthesis,it was observed whether changing FAT10 would affect the degradation rate of ZO-1.Then,autophagy lysosomal inhibitors were added to observe whether FAT10 still had a regulatory effect on ZO-1.Finally,immunoprecipitation technology was used to determine whether FAT10 directly binds to ZO-1 to play a regulatory role.5.The autophagy related proteins that may bind to FAT10 were detected by immunoprecipitation mass spectrometry(IP-MS),and further Western blot and immunofluorescence experiments were used to determine whether FAT10 affects the autophagy level of colon cancer cells and the specific regulatory mechanism by affecting the expression of this key protein.Finally,the relationship between FAT10 and related target proteins was verified by Western blot and immunohistochemistry.Result:1)Real-time quantitative PCR showed that the m RNA expression level of FAT10 in colon cancer cells was higher than that in normal colon epithelial cells NCM460,and the m RNA expression level of FAT10 in colon cancer tissues was also higher than that in adjacent tissues;Immunohistochemistry and Western blot confirmed FAT10 protein levels in colon cancer tissues was significantly higher by cancer,statistically significant difference(P < 0.05),and further through the survival analysis,we found that the FAT10 high expression of its overall survival in patients with colon cancer than FAT10 lower expression of colon cancer patients,patients with FAT10 high expression of the tumor infiltration depth,TNM stage and lymphatic metastasis number than FAT10 clinicopathological characteristics related to lower expression of patients is poor,statistically significant difference(P < 0.05).2)SW620 and Lo Vo cell lines were used to construct FAT10 cell lines with stable low expression,and HCT-116 cell lines with stable high expression of FAT10 were used to construct FAT10 cell lines.The expression of FAT10 in SW620 and Lo Vo cells after interference was significantly decreased by Western blot detection,while the expression of FAT10 in HCT-116 cell line after overexpression was significantly increased.RTCA and Transwell experiments confirmed that the invasion and migration ability of SW620 and Lo Vo cells was significantly decreased after the interference of FAT10 expression,while the invasion and migration ability of HCT-116 cell line was significantly increased after the overexpression of FAT10,with statistical significance(P < 0.05).Finally,in vivo experiments confirmed that the number and area of liver metastasis in nude mice were significantly reduced after interference with the expression of FAT10,while the number and area of liver metastasis in nude mice were significantly increased after overexpression of FAT10,with statistical significance(P < 0.05).3)Through i TRAQ,we found that the tight junction-related proteins between cells changed the most after changing the expression of FAT10,and the expression of ZO-1 was the largest in tight junction-related proteins.After changing the expression of FAT10,ZO-1 was verified by Western blot.The expression of ZO-1 changed accordingly,and the two were negatively correlated.Real-time fluorescent quantitative PCR found that FAT10 did not affect the m RNA level of ZO-1.Furthermore,we found through RTCA and Transwell that after interfering with the expression of ZO-1 in SW620 colon cancer cells that interfered with FAT10,the decreased invasion and migration ability of the cells due to the decrease of FAT10 could be partially recovered,while the HCT-116 overexpressing FAT10 After overexpression of ZO-1 in the cells,the enhanced invasion and migration ability of cells due to the increase of FAT10 can also be partially recovered,indicating that ZO-1 is a key molecule for FAT10 to regulate invasion and migration of colon cancer.4)Previous studies have confirmed that ZO-1 is degraded by the autophagy-lysosomal pathway.To further clarify whether FAT10 affects the expression of ZO-1 through the autophagy-lysosomal pathway,first of all,we are adding an autophagy-lysosomal inhibitor 3-After MA and CQ,ZO-1 gradually increased over time,and after adding the autophagy agonist Rapamycin,it was found that ZO-1 gradually decreased over time.Secondly,transmission electron microscopy,Western blot and immunofluorescence showed that interference with FAT10 expression in SW620 colon cancer cell line could inhibit autophagy flow in colon cancer cells.Overexpression of FAT10 in HCT-116 cells can activate autophagy flow in colon cancer cells,suggesting that FAT10 can promote autophagy level in colon cancer cells.Then,when the protein synthesis inhibitor actomycin CHX was added,the overexpression of FAT10 could accelerate the degradation rate of ZO-1,while the degradation rate of ZO-1 was slowed down after interference with FAT10,indicating that FAT10 could affect the degradation of ZO-1.The expression of FAT10 was changed simultaneously by adding the autophagy lysosomal inhibitors 3-MA and Bafilomycin A1,and it was found that the autophagy lysosomal inhibitors 3-MA and Bafilomycin A1 could block the regulation of FAT10 on ZO-1.Finally,the immunoprecipitation result was found that FAT10 does not directly bind to ZO-1 in colon cancer cells.These results indicated that FAT10 affected the invasion and migration ability of colon cancer by affecting the degradation of ZO-1 through autophagy lysosomal pathway,but FAT10 did not affect its expression by directly binding to ZO-1.5)After it was confirmed that FAT10 could regulate the expression of ZO-1through autophagy lysosomal pathway,we further found through IP-MS that FAT10 may directly bind to autophagy related gene ATG3.Co-IP,immunofluorescence and Western blot further confirmed the results of IP mass spectrometry and the regulatory effect of FAT10 on ATG3.The protein expression of ATG3 decreased after the interference of FAT10 expression in SW620 colon cancer cells,while the protein expression of ATG3 increased after the overexpression of FAT10 in HCT-116 colon cancer cells.However,its m RNA level did not change,indicating that FAT10 could regulate the protein expression level of ATG3 without affecting its transcription.Secondly,through ubiquitination experiments,we confirmed that the molecular mechanism of FAT10 stabilizing ATG3 is that FAT10 and ubiquitins compete to bind ATG3,so as to stabilize ATG3 and promote the level of autophagy in colon cancer cells.Subsequently,Western blot and immunofluorescence related recovery experiments confirmed that ATG3 is a key molecule in FAT10’s regulation of autophagy level in colon cancer cells.Finally,we confirmed by Western blot and immunohistochemistry that FAT10 and ATG3 were highly expressed in colon cancer tissues,which were positively correlated,while ZO-1 was low expressed in colon cancer tissues,which was negatively correlated with FAT10.Conclusion:FAT10 is highly expressed in colon cancer cells and tissues,which is closely related to tumor metastasis and poor prognosis.Downregulation of FAT10 expression can inhibit the invasion and migration ability of colon cancer.Further mechanism studies showed that FAT10 could accelerate the autophagy degradation of ZO-1 and finally promote the metastatic ability of colon cancer through competing with ubiquitin to bind Atg3 and stabilize its expression. |
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