The Role Of Endoplasmic Reticulum Stress Response IRE1α-XBP1s Signaling Pathway In Ovarian Cancer Cells

Objective:To study the role of endoplasmic reticulum stress response IRE1α-XBP1s signaling pathway in ovarian cancer cells,including the effect of IRE1α-XBP1s on the viability and proliferation of ovarian cancer cells in vitro,and on the growth of subcutaneously xenografted ovarian cancer cells in immunodeficient mice.Methods:Two ovarian cancer cell lines,OVCAR8 and HOC7,were used in this study.Immunodeficient BALB/c nude mice were used for in vivo experiments.1.The small interfering RNA combined with Lipofetamine 2000 was transferred into HOC7 cells in a good state of culture.After 2 days of culture,the cells were collected to extract RNA and protein,and q RT-PCR and Western blot experiments were performed to verify the silencing effect from m RNA and protein levels.HOC7 cells transfected with small interfering RNA were planted in a 6 cm culture dish in 1×10~4cells,and counted at 3 and 5 days,respectively,to observe the effect of knockdown of XBP1 on the proliferation ability of HOC7 cells.After that,the cells were planted in a96-well plate with 3×10~3 cells per well,and each cell had 8 different Wells.After 3days of culture,30 n M TG was added,and after 3 hours of culture at 37℃,CCK-8 was added in proportion to dilute the cells.The absorbance was measured at 450 n M and the corresponding cell viability was calculated.2.OVCAR8 and HOC7 cells were transfected with HBLV-H-XBP1sh RNA1-ZSGREEN-PURO lentivirus(sh XBP1).After 48 hours of virus transfection,purinmycin was used for screening for one week.Cells were collected to extract their RNA and protein,and q RT-PCR and Western blot experiments were performed.The expression of XBP1s was detected at m RNA and protein levels.After that,the lentivirus cells were planted in a 6cm culture dish in 1×10~4 cells and cultured at 37℃and 5%CO2.On the 3rd and 5th day of culture,the cells digested with trypsin were counted on a blood cell counting plate,and the corresponding data were recorded.The experiment was repeated for 3 times.The stable cell lines sh NC and sh XBP1 were planted in96-well plates with 3×10~3 cells per well,and each cell had 8 replicates of each kind.After 3 days of culture,30 n M TG was added.After 3 hours of culture at 37℃,CCK-8diluted in proportion was added and the absorbance was measured at 450 n M.And the corresponding cell viability value was calculated.Stable cells were planted in 6 Wells of a 6-well plate at a rate of 5×10~3 per well,and 3 re-wells were set.After 9 days of culture,the culture medium was removed,washed with PBS,fixed with 4%paraformaldehyde fixation solution,and stained with crystal violet.The results were observed,and the number of stained cell communities was measured with Image J software after taking photos and recording.Finally,add 1 m L of warm 0.4%agarose gel to each well in a 6-well plate,shake well and let stand until the surface is very smooth.Add a mixture of 0.2%agarose gel and cells,also 1 m L per well,shake well properly and put into the incubator.After that,complete medium should be supplemented every 2-3 days.After about 1 week of culture,the cells were observed under a microscope to see if there was significant colony formation,and the cells were photographed.Finally,the software Image J was used to quantitatively compare the area of cell clusters observed in the experiment.3.IRE1αendonuclease inhibitor MKC8866 was used to treat ovarian cancer cells MKC8866 was treated with 10μM of ovarian cancer cells,and the RNA of cells was extracted 24 hours later for q RT-PCR assay.The effect of this endonuclease inhibitor on the expression of XBP1s was detected at the m RNA level.MKC8866 was then treated with 0,0.5,1,5,10 and 20μM of ovarian cancer cells,respectively.After 48 h,the cells were collected to extract proteins for Western Blot assay.The effect of this endonuclide inhibitor on the expression of XBP1s was detected at the protein level.The cells were planted in a 6cm petri dish with 1×10~4 cells,and cultured at 37℃and 5%CO2.On the3rd and 5th day of culture,the cells digested with trypsin were counted using a blood cell counting plate,and the corresponding data were recorded.The experiment was repeated for 3 times.Cells were planted in a 96-well plate with 3×10~3 cells/well and each cell had 8 multiple Wells.MKC8866 cells were treated with 0,0.5,1,5 and 10μm respectively,and cultured for 3 days.CCK-8 diluted in proportion was added and cultured at 37℃for 3 hours.And the corresponding cell viability value was calculated.After that,the cells were planted in 6 Wells of 6-well plate with 5×10~3 cells per well,and divided into control group(DMSO)and administration group(MKC886610μm),with 3 Wells in each group.After 9 days of culture,the culture medium was removed,washed with PBS,fixed with 4%formaldehyde fixation solution,and stained with crystal purple.The results were observed.After photographing and recording,the number of the stained cell community was measured using the Image J software.Finally,add 1 m L of warm 0.4%agarose gel to each well in a 6-well plate,shake well and let stand until the surface is very smooth.The mixture of 0.2%agarose gel and cells was added,also 1 m L per well,and divided into the control group and the administration group,with 3 rewells in each group.The mixture was shaken moderately and put into the incubator.After that,complete medium should be supplemented every 2-3 days.After about 1 week of culture,the cells were observed under a microscope to see if there was significant colony formation,and the cells were photographed.Finally,the software Image J was used to quantitatively compare the area of cell clusters observed in the experiment.4.OVCAR8 cells digested with trypsin were suspended in 50μL RPMI 1640medium and mixed with 50μL Matrigel in vitro.The mixture was then inoculated subcutaneously on the posterior side of the abdomen of female nude mice(BALB/C NU/NU,5 weeks of age)with 2×10~6 cells per side.After the presence of palpable tumors,they were randomized.The mice were divided into control group and administration group.The control group was given a mixture of sucrose and microcrystalline cellulose.MKC8866 in the administration group was given 150mg/kg/d(mixed solution of sucrose and microcrystalline cellulose).After 9 days of oral administration,the mice were killed by cervical dislocation.The subcutaneous tumor tissue was removed,fixed overnight with 10%formalin,and immunohistochemical staining was performed.Three distinct markers of XBP1s,Ki67,and Cleaved caspase-3were immunohistochemical staining(Ki67,associated with cell growth,and Cleaved caspase-3,associated with apoptosis).Results:1.After silencing XBP1 with si RNA,RT-q PCR and Western Blot experiments showed that si RNA could significantly inhibit the expression of XBP1s at m RNA and protein levels.Moreover,in the proliferation experiment,the growth rate of cells with lower XBP1s expression was significantly slower than that of cells with higher XBP1s expression,and the difference was statistically significant.In the determination of cell viability,it was also found that the viability of cells with low expression of XBP1s was significantly lower than that of cells with high expression of XBP1s,and the difference was statistically significant.2.After transfected into HBLV-H-XBP1 sh RNA1-ZSgreen-Purolentivirus,RT-q PCR and Western blot experiments showed that the transfection of the lentivirus could significantly inhibit the expression of XBP1s m RNA and XBP1s protein in ovarian cancer cells.In the subsequent cell function experiments,it was found that the proliferation rate and cell viability of the stable knockdown XBP1 cells were significantly lower than those of the control group,and the differences were statistically significant.From the comparison of the number of stained cells in different groups in the cloning experiment and the area of cell clusters in the suspension experiment,the colony formation ability of the stable knockdown cells of XBP1 was significantly weaker than that of the control group,and the difference was statistically significant.3.Treating ovarian cancer cells with 10μm MKC8866 inhibited the expression of XBP1s m RNA.MKC8866 was treated with 0,0.5,1,5,10 and 20μm.Western blot results showed that MKC8866 could significantly inhibit the expression of XBP1s,and there was a dose-response relationship between the concentration of MKC8866 and its ability to inhibit the expression of XBP1s.In subsequent cell function experiments,it was found that MKC8866 treated ovarian cancer cells were significantly weaker than the control group in terms of proliferation rate,cell viability and colony formation ability.4.After successful establishment of subcutaneous xenografting model in nude mice,the volume of tumor tissue growth was inhibited after oral administration of MKC8866,and the results of immunohistochemical experiments showed that the expression of XBP1s in tumor tissue was significantly reduced after a certain period of the administration of MKC8866.In addition,it decreased the expression of proliferation indicator Ki67 and enhanced the expression of apoptosis indicator Cleaved caspase-3.Conclusion:The endoplasmic reticulum stress response IRE1α-XBP1s signaling pathway plays an important role in the growth of ovarian cancer cells.