The Roles And Molecular Mechanism Of Polarity Protein Pard3 On Migration And Invasion In Gastric Cancer

Part1 The expression and clinical pathological significance of Pard3 in gastric cancerObjective: To study the expression level of Pard3 in gastric cancer tissues and adjacent tissues,and to analyze the correlation between Pard3 expression level and clinicopathological features of gastric cancer.Methods: 35 cases of gastric cancer specimens from the Department of Gastrointestinal Surgery,Wuhan Union Hospital from 2016 to 2018(including gastric cancer tissues and adjacent tissues)were determined and by RT-PCR,Western Blot,and immunohistochemistry to detect the level of Pard3 in gastric cancer tissues and adjacent tissues.The expression of the Pard3 was combined with their clinical data to analysis the the correlation between the Pard3 expression and the patient’s age,gender,TNM stage and tumor differentiation.RESULTS: The results of immunohistochemistry(IHC)showed that the expression of Pard3 in gastric cancer tissues was significantly lower than that in normal mucosa tissues(P<0.01).The results of RT-PCR and Western Blot showed that the expression of Pard3 in tumor tissues was significantly lower than that in para-carcinoma mucosa tissues(P<0.01).The correlation analysis of clinicopathological features showed that the down-regulation of Pard3 expression was associated with T stage and N stage of the patients(P<0.05),but there was no statistically significant correlation with tumor differentiation,age and gender(P>0.05).Conclusion:1.The expression level of Pard3 in gastric cancer tissues was significantly lower than that in adjacent mucosa tissues,and it was related to the T stage and N stage of tumors.2.The down regulation of Pard3 was not conducive to the prognosis of patients with gastric cancer.Part 2 The effect of Pard3 silence on the malignant biological behavior of gastric cancer cellsObjective: To study the effect of Pard3 silence on migration,invasion and lung metastasis of gastric cancer cell linesMethods: The gastric cancer cell line SGC7901 cells and HGC27 cells were transfected with sh-Pard3 of lentiviral vector to establish a stably transfected Pard3 silencing cell line for subsequent experiments.The effects of Pard3 silence on gastric cancer cell migration,invasion and lung metastasis formation ability were studied by the scratch assays,transwell assays,invasion assays and lung metastasis formation assays.Results: Western blot results showed that Pard3 was higher in normal gastric mucosal epithelial cell line GES1 than other gastric cancer cell lines such as AGS cells,MGC-803 cells,BGC-823 cells,MKN45 cells,SGC7901 cells and HGC27 cells.Pard3 silence significantly reduced the expression of Pard3 m RNA and protein(P<0.01).The scratch assays and transwell assays showed that reducing the expression of Pard3 significantly enhanced the migration ability of gastric cancer cells(P<0.05).Invasion experiments showed that Pard3 silence significantly increased the invasive ability of gastric cancer cells(P<0.05).The lung metastasis formation assays showed that the decrease of Pard3 expression promoted the formation of lung metastases(P<0.05).Conclusion: Pard3 silence promoted the migration,invasion and formation of lung metastasis of gastric cancer cells.Part 3 The mechanism of Pard3 silence regulating the migration and invasion of gastric cancer cells by activating NFκB pathwayObjective: To explore the mechanism of Pard3 silence affecting the ability of migration and invasion of gastric cancer cellsMethods: The stable transfected SGC7901 cells and HGC27 cells were analyzed by Western Blot,RT-PCR to study the effect of Pard3 silence on the expression of Snail,Zeb1,Zeb2,slug,N-cadherin,Vimentin and E-cadherin which are the key transcription factors and important indicators of Epithelial-mesenchymal transformation(EMT).Transcriptome sequencing was used to screen the effect of Pard3 silencing on downstream genes.The effect of Pard3 silencing on the NFκB pathway and the expression changes of Amotl2,Zo-1 and Myh9 were detected by Western Blot,RT-PCR and Immunofluorescence.The potential binding sites of NFκB/p-65 on Amotl2 promoter were predicted by the database.The Ch Ip assay was used to verify the binding of p-65 to the Amotl2 promoter.In addition,we also used immunofluorescence to detect nuclear translocation of NFκB/p-65 and expression changes of Zo-1 protein.Results: The silence of Pard3 in gastric cancer cell lines had no significant effect on the expression of Vimentin,E-cadherin and N-cadherin and other key EMT markers,as well as the expression of the key transcription factors such as Zeb1,Zeb2 and slug(P>0.05).The silence of Pard3 increased the expression of Amotl2 and Myh9(P<0.05)and decreased the expression of tight junction-associated protein Zo-1(P<0.01).The silence of Pard3 activated the NFκB pathway and promotes nuclear translocation of NFκB/p-65(P<0.05).The NFκB pathway inhibitor(PDTC)not only significantly inhibited the nuclear translocation of NFκB/p-65(P<0.05),but also reversed the expression of Amotl2(P<0.01),Myh9(P<0.05)and Zo-1(P<0.05)which were regulated by Pard3 silence.In functional assays,PDTC could attenuate the migration and invasion abilities induced by Pard3 deletion(P<0.05).The results of immunofluorescence assay showed that Pard3 silencing significantly promoted the nuclear translocation of NFκB/p-65 in gastric cancer cells,and PDTC could significantly inhibit the nuclear translocation of NFκB/p-65 in Pard3-silenced gastric cancer cells.Pard3 silence inhibited the expression of Zo-1 in gastric cancer cells,while PDTC reversed the inhibition of Zo-1 by Pard3 silence.Conclusion:1.The silence of Pard3 promoted the migration and invasion of gastric cancer cells without relying on the EMT pathway.2.The silence of Pard3 promoted the migration and invasion of gastric cancer cells by activating the NFκB pathway.3.The silence of Pard3 up-regulated Amotl2,MYH9 expression and down-regulated Zo-1expression by activating NFκB pathway.4.The silence of Pard3 activated the NFκB pathway to promote the transcription of Amotl2 and up-regulated the expression of Amotl2.5.In Pard3 silence cells,the NFκB pathway regulated Myh9 protein levels,but didn’t affect m RNA levels.Part 4 The mechanism of Pard3 deletion regulating the migration and invasion of gastric cancer by Amotl2Objective: To explore the mechanism of Pard3 silence promoting the migration and invasion of gastric cancer cells.Methods: The si-Amotl2 was used to knockdown the expression of Amotl2 in Pard3 silenced cells,and the expression of downstream genes Zo-1 and Myh9 were determined by RT-PCR,Western Blot and Co-Ip.The scratch assays,transwell assays and invasion assays were used to further study the effect of Amotl2 gene on the migration and invasion of Pard3 silenced gastric cancer cells.Immunoblotting experiments were used to detect the effect of Pard3 and Amotl2 co-silencing on tight junctions between cells.Result: In gastric cancer cells,the silence of Pard3 up-regulated the expression of Amotl2(P<0.05).After knocking down Amotl2 in Pard3-silenced cells,the migration and invasion of gastric cancer cells were significantly decreased(P<0.05).Knockdown of Amotl2 attenuated the up-regulation of Myh9(P<0.01)and the down-regulation of Zo-1(P<0.05)caused by Pard3 silence.The results of immunofluorescence showed that the knockdown of Amotl2 partially restored tight junctions which was disrupted by Pard3 silence.Conclusion:1.Pard3 silence promoted migration and invasion of gastric cancer cells by up-regulating Amotl22.Pard3 silence regulated the expression of Myh9 and Zo-1 by up-regulating Amotl23.Pard3,Amotl2 and NFκB pathways regulated the level of Myh9 protein but had no significant effects on m RNA levels.Amotl2 and Mhy9 combined to form a complex.