Effect Of Bortezomib On Store-operated Calcium Channels In A549 Cells

[Background]Lung cancer is a common malignant tumor.It is characterized by high incidence rate and high mortality rate,which threatens human life to a great extent,and brings heavy financial burden to society and patients’ families.About 80% of lung cancer patients are non-small cell lung cancer(NSCLC).Therefore,it is an urgent problem to study the pathogenesis of lung cancer and improve the diagnosis and treatment of NSCLC patients.At present,many studies at home and abroad have shown that intracellular calcium plays the role of second messenger and plays a role in many biological stages such as cell proliferation,migration and apoptosis.Store operated calcium entry(soce)is the main mechanism of calcium influx in cells.Soce is associated with a variety of human malignant tumors,including lung cancer.Since most lung adenocarcinoma cells originated from bronchial epithelial cells,we selected human lung adenocarcinoma A549 cells for study.Ubiquitin proteasome system(UPS)is the main degradation system in cells.In cells,proteins that need to be degraded are sent to barrel proteasome for degradation after binding with ubiquitin.UPS can regulate intracellular homeostasis,cell growth,proliferation and migration.Proteasome inhibitor bortezomib has become the standard treatment for multiple myeloma and achieved obvious results.Moreover,bortezomib has also played an important role in the treatment of other malignant tumors,including the treatment of lung cancer.Therefore,UPS targeted therapy may become a selective and effective method for the treatment of lung cancer.However,the specific mechanism of bortezomib in the treatment of lung cancer remains to be studied.Recently,many literatures have reported that STIM1,Orai1 and TRPC1 are important proteins on store operated calcium channels(socc)and participate in soce.Therefore,in this study,we investigated the effect of bortezomib on reserve manipulated calcium channels in A549 cells and on A549 cells.[Research purposes]1.To study the effect of bortezomib on the proliferation and migration of lung cancer A549 cells;research method2.To study the effect of bortezomib on the protein expression of factors STIM1,Orai1 and TRPC1 on A549 cells;3.To study the effect of bortezomib on the manipulated calcium influx of calcium pool on A549 cells,so as to provide a theoretical basis and a new method for the treatment of lung cancer.[research methods]1.Detection of cell proliferation by CCK-8: A549 cells were inoculated into 96 well plates,3000 cells / well were inoculated respectively,and the cell proliferation was detected by CCK-8 method at 24 h,48 h and 72 h.2.Transwell was used to detect the migration ability of cells in each group: A549 cells were divided into normal group,solvent control group,bortezomib low-dose experimental group,bortezomib medium dose experimental group and bortezomib high-dose experimental group.Transwell was used to detect the migration ability of cells in each group.3.The effects of different concentrations of bortezomib on the m RNA expression level of A549 cells were detected by q PCR: 1)cell grouping: normal group,solvent control group,bortezomib low-dose experimental group,bortezomib medium dose experimental group and bortezomib high-dose experimental group.2)Cell RNA extraction.3)CDNA was prepared.4)The primer sequences of STIM1,Orai1,TRPC1 and GAPDH were amplified by fluorescence quantitative PCR.5)Conduct data processing and analysis and draw histogram.4.Western blot was used to detect the protein expression levels of STIM1,Orai1 and TRPC1 in different treatment groups: the Protoculture medium of each group was sucked and discarded,and 1 × After the PBS is soaked once,add the lysate,shake the shaker at 4 ℃ for30 min,blow the cells repeatedly with a pipette,and then move it to a 1.5 ml centrifuge tube for quantitative detection of protein.10% polyacrylamide gel was used to electrophoretic.The protein in the glue was transferred to the PVDF membrane,and was added to the TBST of skim milk powder(5%)to shake 2 h.Incubate primary anti shaking at 4 degrees overnight,1× In tbst solution,shake and rinse for 5 minutes,4 times in total.Add secondary antibody and act at room temperature for 1.5 h.in tbst solution,shake and rinse for 5 minutes,4 times in total,exposure and imaging.5.The level of intracellular calcium and the changes of soce were measured by immunofluorescence.The cells were cultured,stimulated by bortezomib when the fusion degree was about 70%,and the cell calcium was measured at 60 hours.[Results]1.Bortezomib can inhibit the proliferation and migration of A549 cells.2.Bortezomib can inhibit the m RNA and protein expression of STIM1,Orai1 and TRPC1 in A549 cells3、Bortezomib can inhibit the basal calcium influx and the manipulated calcium influx of calcium pool in A549 cells[Conclusions]Bortezomib can inhibit the proliferation and migration of A549 cells,which may be caused by the inhibition of calcium pool manipulation calcium influx on A549 cells by inhibiting the expression of STIM1,Orai1 and TRPC1 m RNA and protein.