The Effect And Mechanism Of ITGB3 In Radiosensitization Of Osteosarcoma

BackgroundsOsteosarcoma is a malignant bone tumor derived from multipotent mesenchymal precursor cells,which is more common in adolescents and children.Due to the lack of significant breakthroughs in treatment plans over the years,the prognosis of osteosarcoma has entered a new plateau period.Radiation therapy passed radiation from different sources such asγ-rays,X-rays,protons,and heavy ions is considered an important means of local cancer treatment.However,some malignant tumors such as osteosarcoma have high radiation resistance,and the serious side effects of high-dose radiation,such as nerve damage,bone marrow damage,and radiation-induced bone necrosis,limit the application of radiation therapy.Radiotherapy sensitization refers to the use of physical methods or drugs acting on targets to enhance the sensitivity of tumor cells to radiation and improve the efficacy of radiotherapy.Taking osteosarcoma as an example,the application of radiotherapy sensitizers to improve the radiotherapy sensitivity of osteosarcoma can reduce the dose,making radiotherapy suitable for preoperative tumor reduction,postoperative positive margin supplementary treatment,alternative treatment for patients who cannot undergo surgical treatment,and rescue treatment for tumor recurrence.ITGB3（integrin subunit beta 3）is a subunit of the integrin family that mainly binds with ITGA2B or ITGA5 to form complete integrinsα_2bβ₃ orα_Vβ₃,thus acting as a transmembrane glycoprotein for bidirectional signaling.Research suggests that overexpression of ITGB3 inhibits the mineralization of osteoblast precursor cells.Other studies have found that ITGB3 or integrinα_Vβ₃ promotes tumor progression and reduces treatment sensitivity.In terms of chemotherapy sensitivity,integrinα_Vβ₃overexpression causes cisplatin resistance in breast cancer and pancreatic cancer,and upregulation of ITGB3 expression can also cause cisplatin resistance in osteosarcoma.In terms of radiation sensitivity,inhibiting integrinα_Vβ₃ andα_Vβ₅ reduces the radiation resistance of melanoma and glioblastoma.However,no study has reported the regulatory effect of the ITGB3 subunit on radiation sensitivity in malignant tumors,including osteosarcoma.Due to osteosarcoma derived from multipotent mesenchymal precursor cells,the presence of tumor stem cell populations,and the presence of tumor bone in clinical pathology,it can be considered that osteosarcoma has osteogenic differentiation potential.Osteogenic markers such as RUNX2（RUNX family transcription factor 2）,OCN（osteocalcin）,and OPN（osteocalcin）are expressed at low levels in most human osteosarcoma cell lines,indicating that osteogenic differentiation deficiency is a characteristic of osteosarcoma.A study suggests that poor osteogenic differentiation is one of the reasons why osteosarcoma develops cisplatin resistance.Another study suggests that promoting the differentiation of liver cancer stem cells can play a role in enhancing the sensitivity of liver cancer radiotherapy.The above research suggests that regulating the degree of differentiation of malignant tumor cells may affect the sensitivity of malignant tumors to radiotherapy and chemotherapy.ITGB3 is the radiosensitizing target for osteosarcoma that we have screened in our preliminary work.In this study,the impact of ITGB3 on the radiotherapy sensitivity of osteosarcoma will be clarified through systematic in vitro and in vivo effects detection.Combined with the research background of osteosarcoma’s osteogenic differentiation potential,the possibility of different degrees of differentiation affecting the radiotherapy sensitivity of malignant tumors,and the influence of ITGB3 on osteogenic differentiation tendency,this study focuses on the mechanism of osteogenic differentiation-related pathways.To our knowledge,there is currently no literature exploring the impact of regulating the osteogenic differentiation pathway on the radiation sensitivity of malignant tumors,including osteosarcoma.Purpose（1）Through systematic in vitro validation of the target ITGB3 obtained through transcriptome sequencing screening,the in vitro effect of knocking down ITGB3 to promote radiotherapy sensitization in osteosarcoma was clarified.（2）Clarify the promoting effect of knocking down ITGB3 on osteogenic differentiation tendency and the osteogenic differentiation-related pathways involved.（3）Clarify whether knocking down the pathways related to osteogenic differentiation activated by ITGB3and the high osteogenic differentiation tendency directly lead to the radiosensitization effect of osteosarcoma.（4）By constructing nude mouse models of subcutaneous osteosarcoma and tibial in situ implanted tumor,the in vivo effects and in vitro mechanisms of knocking down ITGB3 to promote radiotherapy sensitization of osteosarcoma were validated.Methods1.Molecular target screening affecting radiation sensitivity of osteosarcomaFirstly,by constructing non-irradiated and irradiated human osteosarcoma cell models,differential expression genes between the two cell models were identified through transcriptome sequencing.For the top 10 genes upregulated after irradiation intervention,literature searches were conducted to first exclude genes that have been extensively studied.Perform q PCR（real-time fluorescence quantitative polymerase chain reaction）on the remaining genes to verify m RNA（messenger RNA）expression trends and identify gene targets that correspond to the changing trends in transcriptome sequencing.The ITGB3 target obtained from initial screening was transfected with si RNA to construct ITGB3 knockdown osteosarcoma cells.After irradiation or non-irradiation intervention,cell viabilities were detected using the CCK8（Cell Counting Kit 8）method.The cell apoptosis rates were detected using Annexin V-FITC/PI（fluorescein isothiocyanate/propidium Iodide）flow cytometry.Finally,we confirmed that ITGB3 is a potential radiosensitizing target.2.Knocking down ITGB3 exerts radiosensitization effects on osteosarcoma in vitroFirstly,HOS and U-2 OS stably transfected human osteosarcoma cell lines were constructed using ITGB3 NC（negative control）and ITGB3 KD（knockdown）lentivirus.After non-irradiation or irradiation intervention,evaluate cell viability using CCK8 assay,evaluate cell apoptosis rate using Annexin V-FITC/PI flow cytometry,evaluate cell proliferation ability using clone formation assay,evaluate cell migration/invasion ability using Transwell migration/invasion assay,and evaluate cell cycle distribution using cell cycle flow cytometry.Evaluate the sensitization effect of knocking down ITGB3 in vitro radiotherapy for osteosarcoma by combining the above methods.3.Exploring the mechanism of knocking down ITGB3 leading to increased radiotherapy sensitivity in osteosarcomaAfter confirming the 5 main pathways of osteogenic differentiation to be selected through a literature search,the m RNA expression of key molecules in these pathways in ITGB3 NC and KD osteosarcoma cells was first detected by q PCR to identify differentially expressed osteogenic differentiation pathways.And JNK（c JUN N-terminal kinase）pathway was selected from q PCR initial screening.We validated the expression of key proteins JNK,c JUN（transcription factor jun）,and their phosphorylated proteins at the protein level through WB（western blot）.Using WB to investigate the effects of ITGB3 expression levels and irradiation factors on the expression levels of JNK pathway proteins,osteogenic differentiation-related proteins,and pro-apoptotic-related proteins after radiation intervention.By introducing the JNK pathway inhibitor SP600125 for rescue experiments,the regulatory effect of JNK pathway activation on the upregulation of osteogenic differentiation-related proteins and pro-apoptotic-related proteins,as well as the regulatory effect on improving cell apoptosis rate,was clarified.After constructing a short-acting knockdown cell model of RUNX2,OCN,and OPN using si RNA in ITGB3 KD HOS osteosarcoma cell,we conducted rescue experiments based on Annexin V-FITC/PI flow cytometry apoptosis detection to clarify the direct regulatory effect of osteogenic differentiation pathway on cell apoptosis.By promoting long-term cultivation of osteogenic differentiation medium,we constructed osteosarcoma cells with high osteogenic differentiation tendency independent of ITGB3 expression and detected osteogenic differentiation-related protein levels through WB to clarify osteogenic differentiation tendency.Based on the results of Annexin V-FITC/PI flow cytometry apoptosis detection,evaluate the role of osteogenic differentiation culture in enhancing radiation sensitivity of osteosarcoma.4.Knockdown of ITGB3 exerts radiosensitizer effects on osteosarcoma in vivo and its mechanism explorationBy constructing nude mouse models of subcutaneous osteosarcoma and tibial in situ implanted tumors,the in vivo radiotherapy sensitization effect of osteosarcoma was evaluated after local irradiation or non-irradiation intervention in the model mice.In addition,the subcutaneous tumor group was set up with an additional group for the in vivo application of JNK inhibitor SP600125 for rescue experiments.The subcutaneous tumor model is used to plot the growth curve and quantitatively analyze the differences in tumor growth between groups based on the measurement results of tumor volume.The in situ implanted tumor model was used to quantitatively analyze the differences in tumor growth between groups based on the ratio of quantitative results of bioluminescence in vivo imaging before and after intervention in each mouse.For the subcutaneous tumor group,Ki67 immunohistochemistry was used to detect proliferation ability and TUNEL（terminal deoxyribonucleotidyl transfer mediated nick end labeling）staining was used to detect apoptosis levels for auxiliary verification of radiation therapy sensitization effect.Immunohistochemical staining of ITGB3,p-c JUN,and RUNX2 proteins was used to evaluate the expression levels of various proteins in subcutaneous osteosarcoma,and ARS（alizarin red staining）was used to evaluate calcium deposition.ResultsPart I:Molecular target screening affecting radiation sensitivity of osteosarcoma1.Preliminary screening of upregulated molecular targets in osteosarcoma cells after IR based on transcriptome sequencingBased on transcriptome sequencing results,the top 10 genes upregulated in HOS human osteosarcoma cells after irradiation were screened,including DDX19B（DEAD-Box helicase 19B）,THRB（thyroid hormone receptor beta）,ITGB3,etc.After literature searches,candidate molecular targets DDX19B,ITGB3,CHAC1（Cha C glutathione specific gamma-glutamylcyclotransferase 1）,and LPP（LIM domain containing preferred translocation partner in lipoma）were preliminarily selected,which were not or only had a few studies involved.2.Preliminary screening of m RNA expression of molecular targets based on q PCR and identification of ITGB3 as a potential molecular target for researchThrough q PCR detection,only the expression trend of ITGB3 m RNA in the four molecular targets mentioned above was consistent with the sequencing results,and it was included in the subsequent validation of the radiosensitization effect of osteosarcoma.3.Preliminary validation of knocking down ITGB3 to promote in vitro radiotherapy sensitization of osteosarcomaThe ITGB3 protein was significantly downregulated in the short-acting knockdown of HOS human osteosarcoma cells based on si RNA construction,which can be used for effect validation.Short-acting knockdown of ITGB3 combined with irradiation significantly reduced cell viability,and there was a positive interaction between the two factors.In addition,the short-acting knockdown of ITGB3 combined with irradiation significantly increased the level of cell apoptosis,and there was a positive interaction between the two factors.Part II:Knocking down ITGB3 exerts radiosensitization effects on osteosarcoma in vitro1.Construction of stable transfection of ITGB3 NC and KD human osteosarcoma cell linesBased on the ITGB3 NC/KD lentivirus,we constructed HOS and U-2 OS human osteosarcoma cell lines with stable translation of ITGB3 NC and KD for subsequent effect and mechanism studies.The effectiveness of knocking down ITGB3 at the m RNA expression and protein levels was confirmed through q PCR and WB detection.2.Knockdown of ITGB3 combined with irradiation inhibited the viability of osteosarcoma cellsSimply knocking down ITGB3 or irradiation intervention both inhibited the cell viability of osteosarcoma.When knocking down ITGB3 in combination with irradiation,the cell viability was significantly weakened and had a positive interaction effect.3.Knockdown of ITGB3 combined with irradiation inhibited the proliferation ability of osteosarcoma cellsSimply knocking down ITGB3 or irradiation intervention both inhibited the cell proliferation ability of osteosarcoma.When knocking down ITGB3 in combination with irradiation,the cell proliferation ability was significantly weakened and had a positive interaction effect.4.Knockdown of ITGB3 combined with irradiation promoted apoptosis of osteosarcoma cellsSimply knocking down ITGB3 or irradiation intervention promoted cell apoptosis in osteosarcoma.When knocking down ITGB3 combined with irradiation,cell apoptosis was significantly increased and had a positive interaction effect.5.Knockdown of ITGB3 combined with irradiation inhibited the migration ability of osteosarcoma cellsSimply knocking down ITGB3 or irradiation intervention both inhibited the cell migration ability of osteosarcoma.When knocking down ITGB3 combined with irradiation,the cell migration ability was significantly weakened and had a positive interaction effect.6.Knockdown of ITGB3 combined with irradiation inhibited the invasive ability of osteosarcoma cellsSimply knocking down ITGB3 or irradiation intervention both inhibited the cell invasion ability of osteosarcoma.When knocking down ITGB3 in combination with irradiation,the invasive ability of cells was significantly weakened and had a positive interaction effect.7.Knocking down ITGB3 caused G1 blockade in osteosarcoma cellsWithout any intervention,osteosarcoma cells exhibited a mild fluctuation state.Simple irradiation intervention can cause a V-shaped trend in the proportion of G1 phase cells.Knocking down ITGB3,whether or not combined with irradiation,increased the proportion of G1 in osteosarcoma cells.Part III:The mechanism of radiosensitization in osteosarcoma induced by knocking down ITGB3.1.Knocking down ITGB3 upregulated osteogenic-related molecules in osteosarcoma cellsAccording to the WB detection results,the levels of RUNX2,OCN,and OPN proteins were upregulated after knocking down ITGB3.2.Knocking down ITGB3 enhanced osteogenic differentiation tendency and activated the apoptosis pathway in osteosarcoma cells by upregulating the JNK/c JUN pathway（1）The m RNA expression levels of JNK1 and JNK2 were upregulated after knocking down ITGB3We have completed the q PCR detection in osteogenic pathways including MAPK（mitogen-activated protein kinase）,BMP（bone morphogenetic protein）,NOTCH,and Wnt/β-Catenin and Hedgehog.The results showed that only the JNK1 and JNK2 m RNA expression levels were stably upregulated in HOS and U-2 OS cell lines after irradiation,indicating that the JNK pathway（a subpath of MAPK）was the key pathway for osteogenic differentiation induced by ITGB3 knocking down.（2）Knocking down ITGB3 and irradiation upregulated JNK/c JUN and their phosphorylated protein levelsKnocking down ITGB3 and irradiation both increased the levels of JNK,p-JNK,c JUN,and p-c JUN proteins and when these two factors were combined,they exerted a positive interaction,further amplifying the upregulation effect on the above proteins,namely the upregulation of the JNK pathway.（3）Knocking down ITGB3 and irradiation upregulated the levels of osteogenic-related molecular proteins,and this effect was antagonized by the JNK inhibitor.Knocking down ITGB3 and irradiation both increased the levels of RUNX2,OCN,and OPN proteins,and the use of the JNK inhibitor in rescue experiments downregulated the levels of these osteogenic-related proteins.（4）Knocking down ITGB3 activated the apoptotic pathway and increased the rate of cell apoptosis,leading to increased radiation sensitivity in osteosarcoma,and this effect was antagonized by the JNK inhibitorKnocking down ITGB3 combined with irradiation significantly upregulated the expression of pro-apoptotic proteins BAX and cleaved Caspase-3,and increased the apoptosis rate of osteosarcoma cells.Regardless of whether combined with radiation intervention,the upregulation of pro-apoptotic proteins and increased cell apoptosis caused by knocking down ITGB3 was antagonized by the JNK inhibitor.3.Downregulation of osteogenic-related molecule RUNX2 expression alleviated cell apoptosis mediated by ITGB3 knockdown（1）Application of si RNA transfection downregulated the protein levels of RUNX2,OCN,and OPNAccording to the WB detection results,after transfection with RUNX2,OCN,and OPN si RNA,the expression of the aforementioned proteins was downregulated in ITGB3 KD HOS cells,and there was no significant off-target effect.Short-term knockdown models of osteogenic-related molecules based on ITGB3 KD stably transfected HOS cells were successfully constructed and were used for subsequent experiments.（2）Downregulation of RUNX2 protein levels antagonized the pro-apoptotic radiosensitization effect mediated by ITGB3 knockdownDownregulation of RUNX2 protein expression partially antagonized the pro-apoptotic radiotherapy sensitization effect mediated by ITGB3 KD,while downregulation of OCN and OPN protein expression had no significant effect on the pro-apoptotic radiosensitization effect mediated by ITGB3 KD.4.Osteogenic culture promoted apoptosis of osteosarcoma cells by upregulating the expression of osteogenic-related molecules,thereby exerting radiosensitization effects（1）Protein levels of osteogenic-related molecular upregulated by osteogenic cultureAccording to the WB detection results,after osteogenic culture,RUNX2,OCN,and OPN in ITGB3NC and ITGB3 KD HOS cells were upregulated to varying degrees.（2）Osteogenic culture exerted the radiosensitization effect of osteosarcomaThere was a positive second-order interaction between the three factors of osteogenic culture,knocking down ITGB3,and irradiation.Regardless of the expression level of ITGB3,the use of osteogenic culture alone slightly increased the apoptosis rate of osteosarcoma cells,to a lower extent than the use of irradiation alone.When osteogenic culture was combined with irradiation,it further increased the rate of cell apoptosis,and there was a positive first-order interaction between the two.Part IV:The effect and mechanism verification of radiosensitization induced by ITGB3 knockdown in osteosarcoma1.Knocking down ITGB3 exerted a radiosensitization effect on osteosarcoma in a subcutaneous tumor nude mouse model,and was antagonized by the JNK inhibitorThe growth ability of a subcutaneous tumor model constructed based on ITGB3 KD stably transformed HOS cells is weaker than that of a subcutaneous tumor constructed by ITGB3 NC stably transformed HOS cells.When combined with irradiation,knocking down ITGB3 had a more significant inhibitory effect on the growth of subcutaneous tumors,indicating that knocking down ITGB3 KD has a radiosensitization effect on osteosarcoma in vivo.The application of the JNK inhibitor partially antagonized the inhibitory effect of knocking down ITGB3 on tumor growth.2.Stable differential expression of ITGB3 in a nude mouse model of subcutaneous osteosarcoma constructed using ITGB3 NC or KD stable transfected cellsAccording to the immunohistochemical detection results of ITGB3,the subcutaneous tumor model constructed by ITGB3 NC stable transformation of HOS cells showed higher expression of ITGB3.In the subcutaneous tumor model constructed based on ITGB3 KD stable transfection of HOS cells,the expression of ITGB3 was significantly reduced.3.Knocking down ITGB3 radiosensitized osteosarcoma by inhibiting the proliferation level in a subcutaneous tumor nude mouse model,and this effect was antagonized by the JNK inhibitorAccording to the immunohistochemical detection results of the proliferation marker Ki67,knocking down ITGB3 inhibited the proliferation ability of subcutaneous osteosarcoma,and the effect was more pronounced after combined irradiation.The JNK inhibitor antagonized the inhibitory effect of knocking down ITGB3 on proliferation.4.Knocking down ITGB3 radiosensitized osteosarcoma by increasing the apoptosis level in a subcutaneous tumor nude mouse model,and this effect was antagonized by the JNK inhibitorAccording to the TUNEL test results,knocking down ITGB3 increased the apoptosis level of subcutaneous tumors in osteosarcoma,and further enhanced the apoptosis level of subcutaneous tumors after combined irradiation.The JNK inhibitor antagonized the effect of knocking down ITGB3 on promoting apoptosis.5.Knocking down ITGB3 increased the p-JUN protein level in a nude mouse model of subcutaneous tumors,and this effect was antagonized by the JNK inhibitorAccording to the immunohistochemical detection results of p-c JUN,knocking down ITGB3upregulated the level of p-c JUN protein in subcutaneous tumors of osteosarcoma,and it was more pronounced after combined irradiation.The JNK inhibitor antagonized the upregulation of p-c JUN protein levels caused by knocking down ITGB3.6.Knocking down ITGB3 increased the expression level of RUNX2 in osteosarcoma in a nude mouse model of subcutaneous tumors,but did not significantly increase calcium deposition,and the effect induced by upregulation of RUNX2 was antagonized by the JNK inhibitorAccording to the immunohistochemical results of RUNX2,knocking down ITGB3 upregulated the protein level of RUNX2 in subcutaneous tumors of osteosarcoma,improved osteogenic differentiation tendency,and became more pronounced after combined irradiation.The JNK inhibitor antagonized the upregulation of RUNX2 protein levels and osteogenic differentiation tendency caused by knocking down ITGB3.According to the results of alizarin red staining,the high osteogenic differentiation tendency caused by knocking down ITGB3 did not lead to a large amount of calcium deposition and mineralization in the subcutaneous tumor of osteosarcoma.7.Knocking down ITGB3 radiosensitized osteosarcoma in a nude mouse model of tibial in situ implanted tumorsKnocking down ITGB3 inhibited the growth of osteosarcoma in a tibial implant tumor model,to a lesser extent than irradiation.When knocking down ITGB3 combined with irradiation,it significantly inhibited the growth of osteosarcoma,and there was a positive interaction between the two in inhibiting the growth of in situ implanted osteosarcoma.Conclusions（1）ITGB3 is a potential target for osteosarcoma sensitization obtained through transcriptome sequencing screening and expression validation.Short-acting knockdown of ITGB3 using si RNA exerts an in vitro radiotherapy sensitization effect on osteosarcoma.（2）Simply knocking down ITGB3 exerts inhibitory effects on the vitality,proliferation,migration,and invasion of human osteosarcoma cells,and promotes apoptosis of human osteosarcoma cells.Although the effect is weaker than irradiation,it is considered an anti-tumor pathway for osteosarcoma.（3）Knocking down ITGB3 in combination with irradiation significantly increases the inhibition of human osteosarcoma cell viability,proliferation,migration,and invasion ability,as well as the promotion of apoptosis level.Moreover,there is a positive interaction between knocking down ITGB3 and irradiation in the above effects,indicating that knocking down ITGB3 has a good in vitro radiosensitization effect on osteosarcoma.（4）Knocking down ITGB3 activates the JNK/c JUN pathway by upregulating the levels of JNK/c JUN and their phosphorylated proteins,and has a positive interaction with irradiation in upregulating the JNK/c JUN pathway.（5）Knocking down ITGB3 upregulates the RUNX2 protein level and osteogenic differentiation tendency by activating the JNK/c JUN pathway,and subsequently induces the activation of the pro-apoptotic pathway,resulting in a radiosensitization effect on osteosarcoma.（6）Knocking down ITGB3 exerts an in vivo radiosensitization effect in human osteosarcoma subcutaneous implant tumor and tibial in situ implant tumor models.（7）Knocking down ITGB3 upregulates the expression of osteogenic-related molecule RUNX2 by activating the JNK/c JUN pathway in vivo,therefore improving the osteogenic differentiation tendency and apoptosis level of osteosarcoma,which is consistent with the results of in vitro mechanism research.