The Impact And Underlying Mechanism Of Negative Co-stimulatory Molecule B7-H3 On Inhibiting Autophagy Of Colorectal Carcinoma Cells

Colorectal cancer(CRC)is one of the ten most predominant malignant malignancies that seriously threaten human health.The morbidity and mortality of CRC has been increasing rapidly,making it the third most common cancers worldwide in recent years.In China,the total cases and death cases of CRC both rank the first throughout the world,with the number of CRC cases accounts for 18.6% and the number of deaths accounts for 20.1% in the world.Recently,the overall 5-year survival rate of CRC has improved by the multidisciplinary diagnosis and treatment.However,a number of advanced colorectal cancer patients still lack effective treatment with unfavorable prognosis.Since immunotherapy is the hottest area in tumor research,it is valuable to explore the mechanism of tumor immune regulation as well as to find new prognostic markers in CRC.B7-H3 is one member of negative co-stimulatory molecules of B7 family,which is highly expressed in many malignant tumors,and is closely related to poor prognosis and progression of patients.It is considered as an indicator for early diagnosis and prognosis of tumors as well as targeted molecule for tumor therapy.Moreover,B7-H3 is also known as a "tumor-associated antigen",which could mediate tumor progression through nonimmunological pathways.Therefore,the basic research and clinical application of B7-H3 are frontier topics in cancer research.Since its receptor or interacting molecule has not been identified,the function of B7-H3 and its regulatory mechanism remains to be elucidated.Autophagy is an evolutionarily conserved process that delivers cellular materials to lysosomes for degradation,and plays a wide variety of physiological and pathological roles.With self-homeostatic mechanism for cellular recycling and metabolic regulation,autophagy has emerged as lysosomal degradation pathway for the breakdown of intracellular proteins and organelles.The excessive or persistent autophagy will lead to autophagic cell death and dysfunction of organs.The aberration in autophagy has been closely related to the formation and development of some malignant tumors.Recent progress has demonstrated that many molecules were involved in the regulation of autophagy.Thus exploring the key molecules and clarifying the mechanism will provide a new approach for cancer therapy.In the present study,our aim was to investigate the clinical significance of B7-H3 and its mechanism on regulating autophagy in CRC via both in vivo and in vitro.The research will explore a new mechanism for B7-H3 function of promoting tumor progression,and will also shed light on the combination therapeutic strategy with B7-H3 blocking antibody and autophagy activators.Part? The relationship between the expression of B7-H3 and autophagy in CRC cellsObjective: To investigate the expression of B7-H3 in CRC cells during autophagy occurrence.And then to analyze the effect of different B7-H3 expression on autophagy level in CRC cells.Methods: After knocking down B7-H3 by small interfering RNA(si RNA)in HCT116 Cells,m RNA sequencing was performed to analyze the different expressions of autophagy associated genes(ATG)between si B7-H3 and control cells.The nutritional deficiency medium EBSS was given to two CRC cell lines(HCT116 and SW480)to induce autophagy.The expression of LC3 B was detected by Western blot at different time points of 0 h,1 h,2 h,4 h and 2 h+medium 2 h.On this basis,after transfection with LC3-GFP lentivirus,autophagy particles were observed under fluorescence microscope to confirm the occurrence of autophagy.The expression of B7-H3 was also detected by Western blot and Flow cytometry(FCM)at different time.Lentiviral vector was used to construct HCT116 stably transformed cell lines that interfered with B7-H3(sh B7-H3)and over-expressed with B7-H3(LVB7-H3).The expression of LC3 B in each group of cells was detected by Western blot after 2 h treatment of normal culture condition and EBSS.And the changes of LC3 B and B7-H3 fluorescent particles in HCT116 and SW480 cells after B7-H3 interference were observed by immunofluorescence staining.Results: RNA sequencing showed that multiple autophagy related genes were upregulated significantly in si B7-H3 CRC cells,including ATG13?ATG12?ATG16L1?ATG14?ATG2A?LC3B?ULK1?FIP200 and p150.LC3-GFP particles were increased after EBSS stimulation since 2 hours in two CRC cell lines.It was found that the level of LC3 B II/I gradually increased after adding EBSS for 1,2,4 hours,which suggest that the autophagy was successfully induced.While adding EBSS 2 h after recovering with serum culture for 2 h,LC3 B II/I returned to the level of 0 h,suggesting that the autophagy level was restored.Meanwhile,Western blot and FCM were used to analyze the B7-H3 protein expression level in the above experimental groups.The results showed that the expression of B7-H3 was negative related to the autophagy level.When autophagy occurred,the expression of B7-H3 was down-regulated;and both autophagy and the expression of B7-H3 restored to normal state after exchanging to normal medium for 2 h.The expression of LC3 B were detected in HCT116 cells with B7-H3 interfering and over-expression,and results showed that LC3 B increased significantly after B7-H3 interfered,while reduced significantly as B7-H3 overexpressed.Furthermore,the immunofluorescence showed that the expression of LC3 B particles increased significantly in HCT116 and SW480 cells after B7-H3 interfered.Conclusion: The expression of several autophagy associated genes was significantly up-regulated after B7-H3 knocking down.The expression of B7-H3 was down-regulated when autophagy occurred in CRC cells,and was restored when autophagy returns to normal.The autophagy level was obviously increased after B7-H3 overexpression.In conclusion,the expression of B7-H3 inhibited autophagy in CRC cells.Part? B7-H3 regulates the biological function and metabolism of by inhibiting autophagy in CRC cellsObjective: To observe the effect of autophagy level changes on tumor cell function including migration,proliferation,lipid metabolism,energy metabolism,and tumorigenesis in CRC cells with B7-H3 interfered in vitro and in vivo,and to explore the impaction of B7-H3 regulating on autophagy in CRC cells.Methods:(1)HCT116 cells interfered with B7-H3 were used to detect and compare the migration ability,proliferation ability,triglyceride(TG)and ATP levels of CRC cells in each groups after adding autophagy inhibitor 3-MA.(2)Nude mice were inoculated with HTC116 cells over-expressed with B7-H3,and autophagy inducer rapamycin was then given after subcutaneous tumorigenesis.The changes of the tumor size were measured,and statstic anylisis was performed.Results: The proliferation and migration ability of tumor cells with B7-H3 interfered was significantly reduced,and the addition of autophagy inhibitor 3-MA can reverse the effect.Moreover,B7-H3 can also regulate triglyceride and energy metabolism by the way of inhibiting autophagy.At last,the tumor animal model showed that B7-H3 promoted the tumorigenic ability of CRC cells by regulating autophagy.After adding the autophagy inducer rapamycin,the tumorigenic ability was significantly inhibited(P=0.0248).Conclusion: In vitro and in vivo function experiments showed that B7-H3 could mediate the changes of biological behavior and metabolism of CRC cells,and could promote the tumor progression by regulating autophagy.Part ? The molecular mechanism of B7-H3 in inhibiting autophagy in CRC cellsObjective: To investigate the molecular mechanism and related signal pathways of B7-H3 on inhibiting autophagy in CRC cells.Methods: Real-time PCR was used to verify the m RNA expression of autophagyrelated genes screened by m RNA Sequencing.HCT116 cells interfered with B7-H3 were used to verify that B7-H3 inhibits the formation of the ULK1-ATG13-FIP200 complex through Akt/m TOR pathway,resulting in negative regulation of autophagy by Western blot and Co-IP experiments.Results: By analyzing the sequencing chip data again,we found that several autophagyrelated genes have up-regulated.Among them,the important autophagy induction complex ULK1-ATG13-FIP200 gene expression had increased significantly,the results of m RNA expression were consistent with the chip data.Co-IP confirmed that the B7-H3 interference increased the expression of ULK1-ATG13-FIP200 complex.Furthermore,Western blot showed that B7-H3 regulates Ser757p-ULK1 inhibiting the formation of ULK1-ATG13-FIP200 complex through m TOR signal.The expression of autophagy inhibitors p-m TOR and p-Akt were both down-regulated after B7-H3 interfered.Akt activation experiments proved that the activation of m TOR by B7-H3 was regulated by Akt.Conclusion: The molecular mechanism study showed that the inhibitory effect of B7-H3 on autophagy in CRC cells was achieved by activating the Akt/m TOR signaling pathway and thereby inhibiting the formation of the ULK1-ATG13-FIP200 autophagy complex.Part ? The expression and clinical significance of B7-H3 and autophagy associated protein LC3 B in CRC tissuesObjective: To analyze the expression of B7-H3 and autophagy associated protein LC3 B in CRC tissues,and then to explore the relationship between B7-3/LC3 B expression and clinicopathological parameters,and to reveal the clinical significance of B7-H3 and LC3 B co-expression in CRC patients.Methods: Immunohistochemistry(IHC)was used to detect the expression of B7-H3 and LC3 B protein in paraffin tissue specimens of 197 cases of CRC.Statistic software SPSS23.0 was used to analyze the correlation and clinical significance between B7-H3 and LC3 B expression,as well as clinicopathological parameters and prognosis of patients.Results: IHC staining showed that,in CRC tissues,60.4% had B7-H3 high expression,39.6% had B7-H3 low expression.LC3 B high expression accounted for 26.9%,and LC3 B low expression was 71.3%.Statistical analysis showed that B7-H3 high expression significantly correlated with clinical prognosis(P < 0.001),while LC3 B did not significantly correlate with clinical pathological parameters and prognosis.Further analysis found that abnormal expression of B7-H3 negatively correlated with the expression of LC3 B in CRC tissues(r=?0.16,P=0.025).Survival data showed that patients with abnormally high expression of B7-H3 and low expression of LC3B(B7-H3highLC3Blow)had the worst prognosis,while B7-H3 low expression and LC3 B high expression(B7-H3lowLC3Bhigh)group had the best prognosis(P=0.001).Conclusion: B7-H3 was abnormally highly expressed in CRC tissues,which was significantly related to the clinical prognosis of patients,while LC3 B was lowly expressed in CRC tissues.There was a negative correlation between the expression of B7-H3 and LC3 B.The combined detection of B7-H3 and LC3 B would have great clinical value in predicting the prognosis of CRC patients.In summary,we explored the molecular function and clinical significance of the negative costimulatory molecule B7-H3 on the regulation of autophagy of CRC cells,and revealed a new mechanism of how B7-H3 regulated autophagy to promote tumor progression.Our research has not only provided an important theoretical basis for the poor prognosis of patients caused by the high expression of B7-H3 in CRC tissue,but also provided experimental basis and theoretical support for the treatment of B7-H3 blocking antibody combined with autophagy activator as well.