The Role And Mechanism Of Hedgehog Signaling Pathway In Natural Killer/T-cell Lymphoma

NKTCL,which is more common in Asian than Western countries,is an aggressive non-Hodgkin lymphoma type with poor clinical outcomes.There is no standard treatment strategy for NKTCL patients.Application of radiotherapy has greatly improved the prognosis of early-stage I/II NKTCL.Stage ?/IV NKTCL is not sensitive to traditional chemotherapy drugs.Some studies have reported that NKTCL patients benefit from L-asparaginase/Permenidase-containing regimens.But,refractory/relapse NKTCL patients have sub-optimal outcomes.Thus,novel treatments are needed for NKTCL.Hh signaling pathway is essential for embryonic development,tissue morphogenesis and stem cell generation.Statistics indicate that about 25% of all cancer-related mortality show signs of abnormal Hh signaling.Dysregulation of Hh signaling modulates various aspects of hematological malignancies.Meanwhile,Hh signaling modulates the functions of leukemia stem cells,which have been an important cause for chemo-resistance and relapse of hematological malignancies.For this reason,blockade of Hh signaling may prevent hematological malignancies.Therefore,our study intends to start with the Hh signaling pathway expression of NKTCL tissues and NKTCL cell lines,then carry out the research on the inhibition of Hh signaling by inhibitors or RNA interference(RNAi)in vivo and in vitro,so as to find a new method to target NKTCL.This study is divided into the following four parts:PART ? Expression of Hedgehog signaling pathway in Natural killer/T-cell lymphomaObjective: To explore the expression of Hh signaling in human NKTCL tissues and NKTCL cell lines.Material and Method: In this study,NKTCL clinical characteristics and tissues were harvested from 30 patients from 2011 to 2018 at our hospital.The WHO classification of lymphoid neoplasms was applied for patient selection.Determination of Smo expression and Gli1 expression in NKTCL tissues were scrutinized by immunohistochemistry(IHC).PBMC and two human NKTCL cell lines(SNK6 and SNT8)protein expression levels were quantified by Western Blot.Results:(1)We retrospectively evaluated Smo expression and Gli1 expression in30 NKTCL tissues.We found positive Smo expression and Gli1 expression in 66.7%(20/30),73.3%(22/30)NKTCL tissues respectively,while 0%(0/10)Smo expression,20%(2/10)Gli1 expression in LRH tissues.In addition,Smo expression levels were positively correlated with those of Gli1.(2)We analyzed the correlation between Smo/Gli1 expression and the clinical characteristics of NKTCL to explore the effect of Smo/Gli1 in NKTCL progression.Smo expression was not significantly correlated with clinical characteristics.However,Overexpression of Gli1 was significantly associated with EBV encoded RNA(EBER).(3)Shh,Ptch1,Smo,Sufu and Gli1 were all expressed in SNK6 cells and SNT8 cells,while PBMC were not.Conclusion:(1)Hh signaling pathway is dysregulated in human NKTCL tissues.(2)Gli1 expression is related to EBER in NKTCL patients.(3)Hh signaling pathway is dysregulated in NKTCL cell lines.PART ? The effects of Hedgehog signaling pathway inhibitors on proliferation and apoptosis of Natural killer/T-cell lymphoma cell linesObjective: To explore the effects of Hh signaling pathway inhibitors(Smo inhibitor Sonidegib,Gli1 inhibitor GANT 61)on NKTCL cell lines in vitro.Material and Method: Two human NKTCL cell lines,SNK6 and SNT8,were subjected to various doses of Sonidegib/GANT 61 and incubated for distinct durations.CCK8 assay was run to assess proliferation,cell apoptosis was examined by flow cytometry and protein expression levels were quantified by Western Blot.Results:(1)Sonidegib significantly suppressed proliferation in NKTCL cells and the effect was dose-dependent.Further analysis revealed that Sonidegib treatment elevated the number of apoptotic cells in a dose and time-dependent.Moreover,Sonidegib downregulated Smo expression and Gli1 expression in NKTCL cells.(2)GANT 61 significantly suppressed proliferation in NKTCL cells and the effect was dose-dependent.Further analysis revealed that GANT 61 treatment elevated the number of apoptotic cells in a dose and time-dependent.Moreover,GANT 61 downregulated Gli1 expression in NKTCL cells.Conclusion:(1)Sonidegib,a smo inhibitor,blockade of Hh signaling suppress proliferation and promote apoptosis of NKTCL cell lines.(2)GANT 61,a Gli1 inhibitor,blockade of Hh signaling suppress proliferation and promote apoptosis of NKTCL cell lines.PART ? The biological effects and mechanism of Smo on Natural killer/T-cell lymphoma cell lines in vitroObjective: To explore the effects and mechanism of Smo silencing by RNAi on NKTCL cell lines in vitro.Material and Method: LV-Smo-RNAi lentivirus infects NKTCL cell lines(SNK6and SNT8)and were screened for stable cell lines by puromycin;To verify whether the stable strains were successfully screened,the expression of EGFP protein was observed by fluorescence microscope,the expression of Smo m RNA was detected by Real-time PCR,and the expression of Smo protein was detected by Western Blot.CCK8 assay was run to assess proliferation,cell apoptosis was examined by flow cytometry,Gli1 and PD-L1 expression levels were quantified by Western Blot.Rusults:(1)Smo silencing SNT8 stable cell line sh Smo SNT8 was successfully established: EGFP expression was observed by fluorescence microscopy,Real-time PCR detected downregulation of Smo m RNA in sh Smo SNT8 cells,Western Blot detected downregulation of Smo expression in sh Smo SNT8 cells.(2)Compared with the SNT8 cells and the sh Control SNT8 cells,the proliferation of sh Smo SNT8 cells were inhibited and cell apoptosis increased.Meanwhile,the downregulation of Gli1 expression and PD-L1 expression was also observed.Conclusion: Silencing of Smo could blockade of Hh signaling suppress proliferation and promote apoptosis of NKTCL cell line,which may play an anti-NKTCL role by regulating PD-L1.PART ? The effects of Smo on the Natural killer/T-cell lymphoma xenografts in vivoObjective: To explore the effects of Smo on NKTCL xenograft model in vivo.Material and Method: Sh Control SNT8 cells and sh Smo SNT8 cells were injected subcutaneously into nude mices to establish the model of human NKTCL xenograft in vivo.At the end of the experiment,the mices were killed and the xenografts were separated.IHC was used to detect Smo expression levels.Rusults: In the control group,xenograft formation started on the 5th day of inoculation,and the xenograft formation rate was 100%(4/4).In the experimental group,xenograft formation started on the 12 th day of inoculation and 1 xenograft did not develop with a xenograft formation rate of 80%(4/5).The xenograft weight of the experimental group was significantly lower than that of the control group.The Smo expression in the experimental group was significantly lower than that in the control group.Conlusion: Silencing of Smo could effectively blockade of Hh signaling suppress the growth of NKTCL xenograft in vivo.In Conlusion,Hh signaling pathway is dysregulated in both human NKTCL tissues and NKTCL cell lines.Inhibitors or Smo silencing by RNAi could blockade of Hh signaling in vivo and in vitro,and it's anti-NKTCL effect may be realized by regulating PD-L1.Hh signaling pathway is crucial to the development of NKTCL and thus holds huge promise as a treatment for NKTCL.