Study On The Effect Of Stromal Interaction Molecule 1 In Cisplatin-induced Ototoxicity

ObjectiveTo study the effect and mechanism of stromal interaction molecules 1（STIM1）in cisplatin-induced ototoxicity,and to provide a new evidence for clinical therapeutic target and treatment of cisplatin-induced hearing loss.Methods1.The localization and expression of STIM1 and Orai1 in the BALB/c mouse cochlea and The House Ear Institute-Organ of Corti 1（HEI-OC1）cells were shown by immunofluorescence,real-time PCR and Western blot.2.HEI-OC1 cells were incubated with 20μmo L/L cisplatin for 24 hours,to establish the ototoxicity model of cisplatin.The samples were then prepared and analyzed the expression levels of STIM1 and Orai1 by Real-time PCR,immunofluorescence and Western blot analysis.3.Different MOI of Scr-sh RNA（NC-LV3-GFP）were used to infect the HEI-OC1 cells,which for determine the optimal multiplicity of infection（MOI）and infection time.Then observe the number of GFP fluorescent cells per 24hours by fluorescence microscope.4.HEI-OC1 cells were stably transfected with 1×10⁷TU/m L of STIM1-sh RNA or NC-LV3 using lentivirus vector according to the manufacturer’s protocol.Then the transfected cells were treated with cisplatin and DMEM respectively.The interference of STIM1 expression level was confirmed by Real-time PCR.5.In order to increase the efficiency of lentivirus transfection,We have improved the technique of tympanum microinjection which was reported by langhainan^[1].BALB/c mice were randomly divided into two groups:Scr-sh RNA group（12 mice）and artificial external lymph group（6 mice）.Lentivirus and artificial lymph（1μL/min,5μL）were introduced into the cochlea of two groups of mice by modified intratympanic microinjection technique.Hearing threshold was measured using brainstem response（ABR）,which was measured to observe the effect of the operation on the hearing function,at 1day before operation,Postoperative day 1,3 and 7,respectively.And immunofluorescence staining was employed to localize LV3-GFP in cochlear slices.Laser confocal microscopy was used to observe the location and efficiency of transfection.6.Mice were divided into two groups:Scr-sh RNA group and STIM1-sh RNA group.The lentivirus（1×10⁶TU/ml,5μL,1μL/min）was administered to each group of mice by the modified injection method microinjection in the mouse cochlea.ABR test was carried out three days after the operation to confirm that the transfection had no effect on the auditory function of mice.Real-time PCR detection cochlear acknowledge successful silence STIM1.7.To establish the ototoxicity model of cisplatin in vivo,mice were divided into 4 groups:NS（Scr-sh RNA）group,NS（STIM1-sh RNA）group,CDDP（Scr-sh RNA）group and CDDP（STIM1-sh RNA）group.Three days in advance of sh RNA in infected,cisplatin（4.5 mg/kg/d,body weight）or normal saline were injected into the abdominal cavity of each group of mice for 5 consecutive days.After discontinuation of treatment,ABR and hair cell count were used to evaluate the hearing function of mice.Real-time PCR was used to evaluate the m RNA levels of STIM1 in cochlea.Apoptotic rate was determined by counting TUNEL-positive cells divided by total cells over a 400-?μm distance in the cochlea.And immunofluorescence staining was used to observe the Orai1protein levels in the organ of Corti（OC）,spiral ganglion（SG）,and striae vascular（SV）of the cochlea.Results1.Both STIM1 and Orai1 were widely expressed in the OC,SG and SV of the cochlea from normal mice and HEI-OC1 cells.Knockdown STIM1 gene expression can be reduced the level of STIM1 protein in the cochlea of mice（P<0.05,P<0.01）,but had no effect on the Orai1 protein level（P>0.05）.The low expression of STIM1 had no effect on the structure,function and apoptosis rate of hair cells.2.CCK8 proliferative activity test showed that the HEI-OC1 cells viability was dose-dependent with cisplatin concentration.The rate of cell viability decreased to 49.72±13.03 after 20μmo L/m L cisplatin treatment,so the above-mentioned dose were selected as the effective drug concentration.3.The level of STIM1 m RNA significantly increased by cisplatin in HEI-OC1 cells.And,the similar increase m RNA or protein levels of STIM1 and Orai1 appear in cochlea of cisplatin-induced ototoxic mice.4.After being transfected with lentivirus with MOI value of 10（titer of 1×10⁷TU/ml）,the structure of HEI-OC1 cells was clear,GFP fluorescence was obvious,and the transfection rate was about 60%.5.Using the improved microinjection technique in tympanum,ABR threshold of Scr-sh RNA group mice was significantly higher than that of pre operation（P<0.01）,and ABR threshold returned to normal level on the third day after operation（P>0.05）.6.The positive expression was observed in HC on the first day after the operation,and in hair cells,SG and SV on the third postoperative day.On the frist postoperative day,the average transfection efficiency was 99.0%,98.3%and 82.5%respectively in the basal,middle and apical turns of basement membrane.And it was 99%,98.1%and 87.2%on the third day after operation.The apical turn was slightly lower than that in the middle and basal turns（P<0.01）.7.Real time PCR results showed that STIM1-sh RNA significantly reduced the m RNA levels of STIM1in cochlea and HEI-OC1 cells（P<0.05,P<0.01）,and also decreased the STIM1 expression levels of were increased by cisplatin in mouse cochlea and HEI-OC1 cells（P<0.05）.8.ABR results showed that the threshold shift of ABR in CDDP（STIM1sh RNA）group was significantly lower than that in CDDP（Scr-sh RNA）group（P<0.01）,but there was no significant difference between NS（Scr-sh RNA）and NS（STIM1 sh RNA）group（P>0.05）.The results of cochlear hair cell count were consistent with ABR,and OHC deletion was the most significant in CDDP（Scr-sh RNA）group.STIM1 sh RNA could reduce OHC deletion induced by cisplatin.9.TUNEL results showed that apoptosis occurred in OC,SG and SV after cisplatin administration.The number of TUNEL-positive cells in CDDP（STIM1-sh RNA）group was significantly lower than that in CDDP（Scr-sh RNA）group（P<0.01）.10.Compared with the CDDP（Scr-sh RNA）group,the protein level of Orai1 decreased significantly in the cochlea of CDDP（STIM1-sh RNA）group mice（P<0.01）.ConclusionsSTIM1 and Orai 1 were expressed in normal mouse cochlea and HEI-OC1cells.STIM1 can induce apoptosis by activating Orai1 and mediate the ototoxicity of cisplatin.