The Role Of Bit1 In Drug Resistance Of Esophageal Squamous Cell Carcinoma And Its Preliminarily Molecular Mechanisms

Background and ObjectiveHenan is the region with the highest incidence and mortality of esophageal cancer in China and even in the world.Henan esophageal cancer is dominated by esophageal squamous cell carcinoma（ESCC）（over 90%）,and chemotherapy is used in the comprehensive treatment of ESCC.Occupy an important position,but because tumor cells are susceptible to chemotherapeutic drugs,tumors that undergo metastasis are more resistant to chemotherapy,and tumors that are resistant to chemotherapy are also prone to distant metastases,leading to treatment failure.Patients who are resistant to chemotherapy usually show resistance to a variety of structurally and functionally different cytotoxic drugs,ie,multi-drug resistance（MDR）.MDR involves a variety of mechanisms,such as increased expression of cell-surface drug resistance proteins,increased drug efflux,activation of oncogenes,and blockage of apoptosis pathways.However,due to the complexities of tumor drug resistance mechanisms,the signaling regulatory network of MDR is still not fully elucidated,and it is necessary to constantly explore new mechanisms of tumor drug resistance to improve the efficacy of chemotherapy.Therefore,it is of great theoretical and clinical significance to study genes or molecules related to ESCC resistance.Bit1（Bcl-2 inhibitor of transcription 1）is a peptidyl-tRNA hydrolase.The first study suggested that Bit1 mediates integrin-dependent anoikis and also mediates apoptosis through the ErK signaling pathway.Later,it was considered that Bit1mediates integrin-dependent cell survival by activating the NF-κB signaling pathway,and studies have shown that Bit1 activates ErK signal and enhances the anti-apoptotic ability of cells.At present,there is only a very small amount of literature reporting the relationship between Bit1 and tumors,most of which are believed to inhibit the metastasis of tumors,such as the down-regulation of Bit1 expression and the promotion of cell metastasis in advanced breast cancer tissues.In lung cancer,Bit1inhibits metastasis by inhibiting epithelial-mesenchymal transition（EMT）.Our previous research shows that Bit1 is over-expressed in ESCC with lymph node metastasis and is positively related to Bcl-2 expression.Down-regulation of Bit1 can induce ESCC cell apoptosis,inhibit its proliferation,migration and invasion ability,and significantly inhibit in the growth of nude mice with ESCC transplanted tumors,the complexes of Bit1 and focal adhesion kinase（FAK）were formed in ESCC cells.The mRNA expressions of PXN and ABCB1（coding P-gp protein）mRNA in the gene chip expression profile decreased with the down-regulation of Bit1 expression.This shows that Bit1 plays a two-way regulatory role in both apoptosis and tumor metastasis.As for the relationship between Bit1 and chemotherapy sensitivity of tumors,it has not been reported at home and abroad.In order to further explore the relationship and molecular mechanism between Bit1 and ESCC resistance,the present study uses（1）Comparison of ESCC cells with different levels of Bit1 expression in biological characteristics and chemosensitivity;（2）Establishment and identification of ESCC-resistant cell line EC1/PTX;（3）Dynamic changes of Bit1 and related protein expression during establishment of drug-resistant cell lines EC1/PTX;（4）Changes of apoptosis and related proteins expression after down-regulation of Bit1 protein in drug resistant cell lines.These four parts to study the role of Bit1 protein in drug resistance of esophageal squamous cell carcinoma and its molecular mechanism.In order to provide evidence that Bit1overexpression can promote the development of resistance to esophageal squamous cell carcinoma,it also provides new ideas for discovering molecular markers of clinical metastasis and prognosis of esophageal squamous cell carcinoma.Methods1 Comparison of biological characteristics and chemosensitivity of ESCC cells with different levels of Bit1 expression.CCK-8,Wound-healing and Transwell were used to detect the proliferation,migration and invasion of EC1 cells（high expression of Bit1）and TE1 cells（low expression of Bit1）,and the differences were compared.CCK-8 was used to detect IC₅₀ values of paclitaxel（PTX）and cisplatin（CDDP）in EC1 and TE1 cells.2 Establishment and identification of ESCC-resistant cell line EC1/PTX.EC1 cells were induced by high-dose paclitaxel-shock-inducing binding time progressive increase.Observing the difference in morphology between EC1/PTX and parent EC1cells.Drawing the growth curve and calculating the doubling time.Using CCK-8 to detect the IC₅₀ of two kinds of cells and calculating the resistance index.Using Transwell to compare the difference of two kinds of cell migration ability and invasive ability.Flow cytometry detects cell cycle and apoptosis differences.Plate colony formation assay comparing the colony forming ability of two types of cells.Western blot detection of drug-resistant cell resistance proteins P-gp,MRP,BCRP and apoptosis-related proteins Bcl-2 and Bax expression changes.3 Using western blot to detect the dynamic changes of Bit1 and related protein expression during establishment of drug-resistant cell lines EC1/PTX.Detecting the changes in expression of Bit1,FAK and p-FAK proteins in EC1/PTX cells.Detecting the dynamic changes in expression of Bit1,FAK,and p-FAK proteins during the establishment of drug-resistant cells and analyzing the connecting between P-gp and Bit1,FAK,and p-FAK proteins.4 Down-regulation of Bit1 protein in EC1/PTX cells induced by Bit1-siRNA.Flow cytometry for detection of apoptosis changes.Western blot was used to detect the expression of P-gp,MRP and BCRP,and the relationship with the change of Bit1 expression,the expression of FAK and p-FAK protein,and the relationship with the expression of resistance protein.Results1 The proliferation,migration and invasion ability of ESCC cell line EC1（high expression of Bit1）was significantly stronger than that of TE1（low expression of Bit1）（P（27）0.05）.The IC₅₀ values of chemotherapeutic drugs paclitaxel（PTX）and cisplatin（CDDP）in EC1 and TE1 cells were 0.033?0.001?g/mL,0.745?0.153?g/mL and 0.004?0.001?g/mL,0.274?0.032?g/mL,respectively,the former being 8.25times and 2.72 times higher than the latter,respectively.2 Establishment and identification of drug resistant cell line EC1/PTX:（1）Successful establishment of ESCC resistant cell line EC1/PTX.（2）Compared with parental cell EC1,the morphology of EC1/PTX tends to irregular long spindle shape and often shows agglomeration.（3）The proliferation rate of parental cell EC1 was higher than that of drug-resistant cell EC1/PTX,and showed significant difference after 96 hours.The EC1/PTX doubling time of drug-resistant cells was prolonged（P（27）0.05）.（4）The drug resistant cell line EC1/PTX has various degrees of resistance to various drugs,be also known as the multidrug resistance,among which the resistance to paclitaxel is the highest,the resistance index is 12.818（P（27）0.05）.（5）The migration ability and invasive ability of the resistant cell EC1/PTX were stronger than that of the parental cell EC1（P（27）0.05）.（6）Compared with parental cell EC1,the apoptosis rate of EC1/PTX cells was significantly decreased,the distribution of G₀/G₁and S phases increased,and the G₂/M phase decreased（P（27）0.05）.（7）The single cell proliferation ability and colony formation rate of the resistant cell EC1/PTX was significantly lower than that of the parental cell EC1（P（27）0.001）.（8）Compared with parental cell EC1,the expression of P-gp,MRP and BCRP protein in EC1/PTX cells increased significantly,the expression of apoptosis protein Bax decreased significantly,and the expression of Bcl-2 protein increased significantly（P（27）0.05）.3 Western blot to detect the dynamic changes of Bit1 and related protein expression during establishment of drug-resistant cell lines EC1/PTX:（1）The expression of Bit1 was significantly increased in the finally established drug-resistant cell line EC1/PTX,and the expression of FAK and p-FAK protein was also highest（P（27）0.01）.（2）During the establishment of drug-resistant cells,the expression of Bit1,FAK,and p-FAK proteins showed a gradually increasing trend and was positively correlated with the expression of P-gp（P（27）0.05）.4 After the down-regulation of Bit1 protein expression in EC1/PTX cells with Bit1-siRNA:（1）The apoptosis rate was increased after the down-regulation of Bit1protein expression in EC1/PTX cells with Bit1-siRNA.（2）The protein expressions of P-gp,MRP,and BCRP were decreased and correlated positively with the change of Bit1 expression（P（27）0.05）.（3）The expression of FAK、p-FAK protein also decreased at the same time and positive correlation with changes in the expression of resistance protein P-gp（P（27）0.05）.Conclusions1 Bit1 may be involved in the development of drug resistance in esophageal squamous cell carcinoma cells,and its high expression may promote esophageal squamous cell carcinoma cells to develop resistance to chemotherapeutic drugs,and downregulation of the expression may reverse the process.2 FAK may be activated as a downstream effector molecule of Bit1,thereby promoting the development of drug resistance in esophageal squamous cell carcinoma.