Effect Of HOXB7 On Radiosensitivity Of Esophageal Cancer Cells And Its Mechanism Research Background

Research backgroundEsophageal cancer is a common gastrointestinal malignancy,ranking 8th and 6th in global incidence and mortality,respectively^[1,2].China is a high incidence area for esophageal cancer.Due to its occult onset,most patients have progressed to the advanced stage when they exhibit symptoms.For patients with advanced esophageal cancer,multidisciplinary comprehensive treatment is required.Radiotherapy is an important part of the comprehensive treatment of esophageal cancer.^[3,4].However,many patients have poor radiotherapy efficacy due to primary or secondary radiation resistance,so it is important to explore and clarify the potential mechanisms of radiation resistance in esophageal cancer to improve the efficacy of radiotherapy for esophageal cancer.As a member of the homeoboxgenes（HOX）family,HOXB7 is involved in various key processes in tumorigenesis and is involved in regulating treatment resistance in various solid tumors,including chemoresistance in esophageal cancer^[5].However,the relationship between HOXB7 and radiotherapy sensitivity in esophageal cancer is unclear,and we hypothesized that HOXB7 may also be involved in radiotherapy resistance in esophageal cancer.Therefore,in this study,we further explored the role of HOXB7 on the radiosensitivity of esophageal cancer cells and its potential mechanisms through cell biology experiments based on database search and clinical sample validation.Research method1.The significantly differentially expressed genes between esophageal cancer and normal tissues were searched in esophageal cancer gene expression profile database gse45670.R software was used to standardize the expression profile data,and log2foldchange>2.0 and P value<0.05 were used as thresholds to screen differentially expressed genes.Then,the heat map of differentially expressed genes was drawn.Further,the expression levels of differentially expressed genes were searched and verified in Cancer Genome Atlas（TCGA）and genotype tissue expression（GTEX）databases.2.From January 2015 to January 2018,92 specimens of esophageal cancer resected by thoracic surgery in our hospital were collected.The expression of HOXB7in esophageal cancer tissues and adjacent normal tissues was detected by PCR,Western blot and IHC.3.The expression of HOXB7 in human esophageal cancer cell lines（kyse30,kyse150 and kyse450）and normal esophageal epithelial cell lines（heec）was determined by PCR.4.Select the cell line with the highest expression of HOXB7,establish the radiation resistance cell line by fractional irradiation,obtain stable passage of radiation resistance cells,and further evaluate the expression of HOXB7 in the radiation resistance cell line,so as to confirm the correlation between HOXB7 and radiation resistance of esophageal cancer cells.5.Lentiviral vectors packaged with OE NC,oe-hoxb7,SH NC and sh-hoxb7infected esophageal cancer cells to regulate the expression of HOXB7 in the cells.The expression changes of HOXB7 in the cells after lentivirus transfection were verified by PCR,and the radiosensitivity changes of esophageal cancer cells after regulating the expression of HOXB7 in the cells were measured by clone formation test.6.The level of cell proliferation,cycle distribution and apoptosis after regulating the expression of HOXB7 in esophageal cancer cells were detected by edu method and flow cytometry.The protein expressions of proliferation related proteins（Ki67,PCNA,cyclin D1 and CDK4）and apoptosis related proteins（BCL2,Bax and caspase3）were measured by western blot,to further clarify the mechanism of the effect of HOXB7 expression on the radiosensitivity of esophageal cancer cells.Research results1.We searched and analyzed the database related to esophageal cancer.The results showed that there were differences in the expression of many genes in esophageal cancer and normal tissues,among which the expression difference of HOXB7 gene was the most significant,and it showed significant high expression in esophageal cancer tissues.PCR,Western blot and IHC also showed that HOXB7 was significantly overexpressed in esophageal cancer compared with adjacent normal tissues.2.The expression of HOXB7 in human esophageal cancer cell lines（kyse30,kyse150 and kyse450）and normal esophageal epithelial cell lines（heec）was determined by PCR.Compared with heec cell lines,kyse30,kyse150 and kyse450cell lines showed significantly higher expression of HOXB7,and kyse150 cell line had the highest expression.3.The kyse150 cell line with the highest expression of HOXB7 was selected,and the radiation resistant cell line（kyse150r）was established by fractional irradiation method to further evaluate the expression of HOXB7 in kyse150 and kyse150r cells.The results showed that the expression of HOXB7 in kyse150r cells was significantly higher than that in kyse150 cells（P<0.05）.4.Kyse150 and kyse150r cells were infected with lentiviral vectors packaged with OE NC,oe-hoxb7,SH NC and sh-hoxb7.PCR analysis showed that HOXB7expression increased in oe-hoxb7 cells and decreased after sh-hoxb7 treatment.Clonogenic assay showed that kyse150 or kyse150r cells overexpressing HOXB7 had decreased radiosensitivity,while cells silencing HOXB7 had increased radiosensitivity（P<0.05）.It is suggested that HOXB7 can regulate the radiosensitivity of esophageal cancer.5.The proliferation and apoptosis of esophageal cancer cells after overexpression of HOXB7 were detected by edu method and flow cytometry.The results showed that the proliferation level of esophageal cancer cells increased and the apoptosis level decreased after overexpression of HOXB7;Further western blot analysis showed that the protein expression of Ki67,PCNA,Cyclin D1,CDK4 and BCL2 increased in kyse150 and kyse150r cells overexpressing HOXB7,while the protein expression of Bax and caspase 3 decreased,while silencing HOXB7 showed the opposite result.Research conclusionWe searched and screened the gene HOXB7 with significant high expression in esophageal cancer tissues from the database based on esophageal cancer gene expression profile.Further,we used clinical tissue samples and cell experiments to verify the high expression of HOXB7 in esophageal cancer.Next,we elucidated the effect of HOXB7 on radiosensitivity of esophageal cancer cells and its potential mechanism through cell biology experiments.Our results reveal the key role and mechanism of HOXB7 in regulating radiosensitivity of esophageal cancer,which provides a new biomarker and potential therapeutic target for overcoming clinical radiotherapy resistance.