Study On The M Echanisms Of The Up-regulated Expression Of COX2 And Its Mechanisms Of Anti-apoptosis In Endoplasmuc Reticulum Stress Of HepG2 Cell Stem

Objective: To investigate the mechanisms of the up-regulated expression of COX-2 and its mechanisms of anti-apoptosis in endoplasmic reticulum stress of Hep G2 cell stem.Methods: Immunohistochemistry was used to detect the expressions of COX2, ATF6, IRE1, PERK and to analyze their relevance; si-RNA were transfect to inhibit the expression of UPR pathway proteins; RT-q-PCR was used to detect the expression of COX2 m RNA; TUNEL was used to observe the morphological changes of apoptosis; Annexin V + PI double staining flow cytometry was used to detect apoptosis rate; and Western blot assay was used to detect the expression of COX2, CHOP, Bcl2 and Bax.Results: 1. The expressions of COX2, ATF6, IRE1, PERK in HCC tissue and Correlationship between COX2 and ATF6, IRE1,PERK Immunohistochemistry was used to detect the expressions of COX2, ATF6, IRE1, PERK and analyze the correlationship between COX2 and ATF6, IRE1,PERK. The results are as follows: In 164 cases of liver cancer patients, there are 109 cases of COX2 positive patients, 55 cases of negative patients; 134 cases of ATF6 positive patients, 30 cases of negative patients; 90 cases of IRE1 positive patients, 74 cases of negative patients; 91 cases of PERK positive patients, 73 cases of negative. The expression of COX2 is positively correlated with ATF6(r = 0.198, P = 0.011). 2. The detection of transfection efficiency of si-ATF6, si-IRE1, si-PERK Inverted fluorescence microscope was used to observe the intensity of fluorescence after transfection. Additionally, Western blot was used to detect the expressions of ATF6,IRE1,PERK. si-ATF6-2, si-IRE1-3 and si-PERK-3 were selected according to it’s high transfection efficiency, which can effectively inhibit the expression of ATF6, IRE1, PERK protein respectively. 3. The influence of COX2 expression in ERS of Hep G2 cells after transfected with si-RNA to inhibit UPR. 3.1 RT-q-PCR was used to detect the changes of expression of COX2 m RNA level Use RT-q PCR assay quantificationally detect the expression of COX2 m RNA, the results are as follows: the expression of COX2 m RNA in TM group significantly increased compared with the control group(P <0.01). After transfected si-RNA to inhibit UPR pathway, the expression of COX2 m RNA in TM+si-ATF6 group decreased significantly than the TM group(P <0.01), whereas the expression of COX2 m RNA in TM + si-IRE1 group, and TM + si-PERK group showed no changes. 3.2 Western blot was used to detect the expression of COX2 protein The expression of COX2 protein is detected by the Western blot. The results are as follows: the expression of COX2 protein in TM group significantly increased compared with the control group(P <0.01); The COX2 protein expression in TM+si-ATF6 group significantly decreased compared with the TM group(P <0.01). And COX2 protein expression in TM + si-IRE1 group, and TM + si-PERK group showed no changes. 4. The influence of cell apoptosis after transfected with si-ATF6 4.1 TUNEL assay was used to observe the morphological changes of apoptosis Morphological changes of apoptosis were observed by TUNEL, the results are as follows: The percentage of apoptotic cells in the control group: 4.10 ± 0.94%, the percentage of apoptotic cells in TM group: 15.85 ± 2.43%, the percentage of apoptotic cells in TM + NC group: 17.42 ± 2.15%; the percentage of apoptotic cells in TM + si-ATF6 group: 34.62 ± 3.28%. The percentage of apoptotic of TM + si-ATF6 is significantly higher than the TM group(P <0.01) and the control group(P <0.01). 4.2 FCM was used to detect the percentage of apoptotic of Hep G2 cellAnnexin V + PI double staining flow cytometry was used to detect cell apoptosis rate, the results are as follows: The apoptosis rate in the control group: 4.97±0.34%, the apoptosis rate in TM group: 10.87±1.30%, the apoptosis rate in TM + NC group : 11.49±2.22%; the apoptosis rate in TM + si-ATF6 group: 26.95±3.42%. The apoptosis rate in TM + si-ATF6 group is significantly higher than the TM group(P <0.01) and the control group(P <0.01). 5. To investigate the mechanism of anti-apoptosis with the overexpression of COX2 Western blot was used to detect the protein expression of CHOP, Bcl2, Bax, The results are as follows: the expression of CHOP in TM + si-ATF6 group is significantly higher than the TM group(P <0.01) and the control group(P <0.01),and the Bcl2/Bax ratio was significantly lower(P <0.01).Conclusion: In ERS of Hep G2 cells, the overexpression of COX2 depends on the activation ATF6 pathway in UPR. The mechanism of anti-apoptosis with the overexpression of COX2 may be related to the expression of CHOP and the regulation of Bcl2/Bax ratio.