TBC1D23 Interacts With RAB11A To Promote The Poor Prognosis Of Non-small-cell Lung Cancer

Objective:The morbidity and mortality of lung caner remain high,which poses a great threat to human life and health.Lung cancer is divided into small cell lung cancer and non-small-cell lung cancer(NSCLC),of which NSCLC accounts for about 80%.Although there are various treatment methods for NSCLC,including surgery,radiotherapy,chemotherapy,neoadjuvant therapy and so on,it is still necessary to explore more accurate treatment.TBC1D23 is a member of TBC/RABGAP family with two domains including TBC domain and Rhodanese domain.Previous studies have reported that TBC1D23 plays a crucial role in intracellular material transportation,and that substance transportation plays an important role in tumor.This study explored the effect of TBC1D23 on the development of NSCLC through its regulation of substance transportation.RAB11A is a member of Rab small GTPase family and plays an important role in ligand and receptor transportation.Previous studies showed that RAB11A can transport integrin back to the cell membrane.Integrin,a member of the transmembrane receptor family,can mediate the adhesion between cells with cells and cells with extracellular matrix.After endocytosis,β1 integrin binds to FAK,and starts the phosphorylation of FAK,and then regulate the development of tumor.The main purpose of this study is to determine the relationship between TBC1D23 and NSCLC and the mechanism,so as to provide new ideas and directions for the treatment of NSCLC.Methods:1,Immunohistochemistry(IHC)detected the staining of TBC1D23 in the clinical samples of 173 cased with NSCLC and the staining of TBC1D23 and β1 integrin in two consecutinve sections of 28 blocks.None of these patients received chemotherapy or radiotherapy before operation.Specimens were scored according to IHC staining results:0(no staining),1(light yellow),2(yellow)and 3(dark brown).The specimens were also scored Waccording to IHC staining area:1(1%-25%),2(26%-50%),3(51%-75%)and 4(76%-100%).The highest score of all slices was 12 points,while the lowest score was 0 points.The study regarded scores≥6 as positive for staining,whereas scores<6 as negative for staining.The study analyzed the relationship between the expression of TBC1D23 and the age,gender,tumor size,tumor type,degree of differentiation,lymph node metastasis and TNM stage of patients with NSCLC.And the study also analyzed the relationship between the expression of TBC1D23 and β1 integrin.2,Western Blot detected the expression of TBC1D23 in 15 pairs of NSCLC tissues and normal tissues.β-actin content was used as an internal control.Western Blot detected the expression of TBC1D23 in HBE,H1299,A549,H460,H661,SK-MES-1,H226 and H292 cells.Western Blot also detected the effect of TBC1D23 on the expression of CyclinB1,CyclinD1,CDK2,CDK6,CDK1,RhoA,RhoC,MMP2,RAB11A,β1 integrin,FAK,P-FAK,MEK,P-MEK,ERK,P-ERK,P-JNK and P-p38.GAPDH was used as an internal control.The protein was separated by 10%gel SDS-PAGE and the electrophoresis solution was filled into the electrophoresis tank for electrophoresis.Then the protein was transferred on the polyvinylidene diflluoride(PVDF)membrane,and the primary antibody was added to the PVDF membrane for incubation.The next day,according to the different properties of the primary antibodies,Goat-anti-mouse or Goat-anti-rabbit secondary antibodies were added to PVDF membrane for incubation.Finally,after cleaning with TBST for three times,ECL luminous solution was added to PVDF membrane and luminous instrument was used to detect the results.3,Real-Time PCR detected the mRNA expression of TBC1D23 in 12 pairs of NSCLC tissues and normal tissues.Real-Time PCR also detected the effect of TBC1D23 on theβ1 integrin mRNA.β-actin content was used as an internal control.After RNA extraction and RNA reverse transcription,Real-Time PCR assays can be carried out.RNA reverse transcription follows:37℃ for 15min,85℃ for 5s,and 4℃ for preservation.Real-Time PCR follows:95℃ for 30s,40 cycles at 95℃ for 5s,and 60℃ for 30s.4,Immunofluorescence(IF)detected the localization of TBC1D23 in A549,H1299,SK-MES-1,H460 and H292 cells and detected the co-localization of TBC1D23 and RAB11A in cells.And IF also detected the effect of TBC1D23 on the co-localization of RAB11A and β1 integrin at perinuclear.In the assays,2%paraformaldehyde was used to fix,Triton X 100 was used to perforate and the primary antibodies were added to cells after 5%BSA blocks.The next day,FITC/TRITC-conjugated secondary antibodies were added according to the properties of primary antibodies.Finally,cell nuclei was stained using 4’,6-diamidino-2-phenylindole(DAPI).In IF of co-localization,after the first secondary antibody was incubated,the second primary antibody was incubated.And the third day,the second secondary antibody was incubated.Finally,the nuclei was stained by DAPI.5,A549,H1299,H460,H661,H226 and H292 cells were cultured by 1640 medium;SK-MES-1 cells were cultured by MEM medium;HBE cells were cultured by DMED medium.The medium contained 10%FBS.All cells were cultured in an incubator containing 5%carbon dioxide at 37℃.Lipo 3000 transfection kit was used for transfection.Si-NC,si-TBC1D23 and si-RAB11A were used to knockdown;pcDNA3.0-myc,pcDNA3.0-TBC1D23-myc and △TBC was used to over express.The medium was changed 6 hours after transfection,cells were collected for RNA extraction 24 hours after transfection,and cells were collected for protein extraction 48 hours after transfection.Lipo 3000 and G418 were used for stable transfection.Cells were inoculated into 6-well plate,one of which was used as the control group,and G418 was added to the cells of all the 6 wells every day.When the cells of control group died,the cells in the other 5 wells were collected and cultured in the culture flasks.Nocodazole(NZ)was used to synchronize integrin.And 4 hours after the incubated of NZ was 0-hour point.6,MTT assays detected the ability of proliferation of NSCLC cells.Firstly,100μl medium was added into the wells of 96-well plate and the number of cells was the same in it.Five multiple wells were prepared for each cell and cultured for 5 consecutive days.MTT solution and FBS-free medium was added into wells in the ratio of 9:1.And 100μl DMSO was added after 4 hours of dark culture.Finally,the wells were read by a microplate reader at a wavelength of 490nm.The assays were carried out continuously for 5 days,and the results were detected at the same time every day.7,Colony formation assays detected the ability of colony formation of NSCLC cells.Firstly,4ml medium was added into the wells of 6-well plate and there were 500 cells in it.The cells were cultured for several days.And then,it was fixed with methanol,and stained by hexamethylpararosaniline.8,Cell cycle assays detected the effect of TBC1D23 on the cell cycle progression of NSCLC cells.Firstly,the cells were stored in 70%alcohol overnight.The next day,propidium iodid(PI)and RNase A was added into the cells for 30 minutes in the dark at 37℃.Finally,the results were measured using the FACSCalibur flow cytometer.9,Cell scratch assays detected the migration of NSCLC cells.Cells were cultured in serum-free medium with Mitomycin for 2 hours,then four channels were marked in the cells and the width of the channels was recorded,when it is 0 hour point.After that,the width of the channels was recorded at 12 hour and 24 hour.10,Transwell assays detected the ability of migration and invasion of NSCLC cells.The invasion assays were conducted with 100μl Matrigel in advance.There was the 200μl medium with 2%FBS in upper chamber,600μl medium with 20%FBS in lower chamber.And the number of cells in control group and experiment group was the same.The cells were cultured for 24 hours.And then,it was fixed with methanol,and stained by hexamethylpararosaniline.11,Subcutaneous tumorigenesis assay in nude mice detected the effect of TBC1D23 on the progression of NSCLC in vivo.The number of cells injected into nude mice was 106,and these cells were concentrated in a 200μl medium without fetal bovine serum(FBS).The state of mice was observed every week.After 4 weeks,the mice were collected and the tumor was dissected from the mice for measurement.12,Immmunoprecipitation mass spectrometry(IP-MS)detected the protein that may interact with TBC1D23.Immunoprecipitation(IP)verified the results in mass spectrometry,and determined the interaction between RAB2A,RAB5C,RAB11A and TBC1D23.The study used lysis buffer and phenyl methyl sulfonyl fluoride(PMSF)to lyse cells and extract total protein.After that,80μl protein A+G agarose beads were added to the protein.After centrifugation,the supernatant was taken to measure the protein concentration,and the antibody was added according to the protein concentration.The next day,25μl protein A+G agarose beads was added into the solution.After 6 hours,clean the beads and add 2×loading buffer to perform Western Blot.IP-MS required electrophoresis only.13,Flow cytometry detected the the amount of β1 integrin at the cell membrane at different time.Cells were incubated with FITC-conjugated anti-β1 integrin,and fixed with paraformaldehyde.Finally,after washing the paraformaldehyde with PBS,the results were detected by flow cytometry.14,SPSS v18.0 was used to analyze IHC results with χ2 test.Graphpad Prism 5 system was used to analyze the other results with t test.The assays were repeated three times.P<0.05 was considered statistically significant.Results:1,IHC assays of 173 cases of NSCLC showed that the expression of TBC1D23 in NSCLC tissues was higher than that in normal tissues.The high expression of TBC1D23 was correlated with tumor differentiation,tumor size,lymph node metastasis and TNM stage.Therefore,the high expression of TBC1D23 was associated with the poor prognosis of NSCLC.Western Blot of 15 pairs of NSCLC tissues and normal tissues showed the expression of TBC1D23 was higher in NSCLC tissues.RT-PCR of 12 pairs of NSCLC tissues and normal tissues showed the mRNA exprssion of TBC1D23 was higher in NSCLC tissues.The analysis of 494 patients with NSCLC in Human Protein Atlas showed that the high expression of TBC1D23 was associated with the poor prognosis of NSCLC.TBC1D23 may be a potential target the treatment of NSCLC.2,IHC assays and IF assays showed that TBC1D23 was mainly located in the cytoplasm.3,Western Blot assays in HBE,H1299,A549,H460,H661,SK-MES-1,H226 and H292 cells showed that low TBC1D23 expression in H1299 cells,high expression in SK-MES-1 cells and moderate expression in A549 cells.4,MTT assays and colony formation assays showed that TBC1D23 promoted the proliferation of NSCLC cells.Cell cycle assays showed that TBC1D23 promoted the transformation from G1 phase to S phase of NSCLC cells.Western Blot assays showed that TBC1D23 promoted the expression of CyclinB1,CyclinD1,CDK2 and CDK6.5,Cell scratch assays and Transwell assays showed that TBC1D23 promoted the migration and invasion of NSCLC cells.Western Blot assays showed that TBC1D23 promoted the expression of RhoA,RhoC and MMP2.6,Subcutaneous tumorigenesis assay in nude mice showed that TBC1D23 promoted the malignant progression of NSCLC and TBC1D23 may be a potential target for the treatment of NSCLC in vivo.7,IP-MS assays showed that TBC1D23 may interact with RAB2A,RAB5C and RAB11A.IP assays showed that TBC1D23 interacted with RAB11A.And Western Blot assays showed that TBC1D23 could not affect the expression of RAB11A.IF assays showed that TBC1D23 and RAB11A were co-located at paranuclear.8,The study employed si-RAB11A to knockdown RAB11A in TBC1D23-overexpress NSCLC cells.MTT assays,colony formation assays,Cell scratch assays and Transwell assays showed that TBC1D23 promoted proliferation,migration and invasion of NSCLC cells by interacting with RAB11A.Western Blot assays showed that TBC1D23 promoted the expression of CyclinB1,CyclinD1,CDK2,CDK6,RhoA,RhoC and MMP2 by interacting with RAB11A.And Western Blot also showed TBC1D23 interacted with RAB11A to activate β1 integrin/FAK/ERK pathway.9,The study constructed a mutation without TBC domain called △ TBC.IP assays showed that TBC1D23 interacted with RAB11A via TBC domain.MTT assays,Colony formation assays,Cell scratch assays and Transwell assays showed that TBC1D23 promoted the proliferation,migration and invasion of NSCLC cells via TBC domain.Western Blot assays showed that TBC1D23 promoted the expression of functional protein and TBC1D23 activated β1 integrin/FAK/ERK pathway via TBC domain.10,Real-Time PCR assays showed that TBC1D23 could not affect the mRNA level of β1 integrin.Flow cytometry assays and IF assays in NZ washout system showed that TBC1D23 was involved in the interaction of RAB11A and β1 integrin at paranuclear.11,IHC assays of two consecutive sections of block from 28 cases of NSCLC showed that TBC1D23 was positively correlated with β1 integrin.Conclusion:1,TBC1D23 promoted malignant progression of NSCLC,and was associated with poor prognosis of NSCLC.2,TBC1D23 interacted with RAB11A via TBC domain,which activated β1 integrin/FAK/ERK pathway.3,TBC1D23 was involved in the interaction of RAB11A and β1 integrin at paranuclear.