The Mechanism Of PIWIL2 Regulating Autophagy And Apoptosis In Esophageal Squamous Cell Carcinoma Via Competitively Binding IKK With TSC

Objective:PIWIL2 is a cancerous testicular protein newly discovered in recent years.It is mainly expressed in testicular tissues in adults,and expressed in various tumors.In addition to playing an important role in spermatogenesis,PIWIL2 also plays an important role in the regulation of tumor cell proliferation and apoptosis.Our previous series of studies found that the expression of PIWIL2 was elevated in some tumor tissues,and revealed the relevant molecular regulatory mechanisms.Previous studies have shown that inhibition of autophagy and apoptosis is an important condition for the survival of tumor cells,but the mechanism of autophagy and apoptosis regulation of tumor cells remains to be further studied.Our research group preliminary verified that PIWIL2 is highly expressed in esophageal squamous carcinoma tissues and cells,and PIWIL2 interacted with IKK and related to autophagy of esophageal squamous cancer cells,but the specific mechanism is not clear yet.This topic will be on the basis of existing research further clarify PIWIL2 in esophageal squamous carcinoma cells and the molecular mechanism of autophagy and apoptosis,and for the clinical treatment of esophageal squamous carcinoma research provides new theoretical basis and direction.The focus is on achieving the following research objectives:1.To determine the molecular mechanism by which PIWIL2 interacts with IKK and regulates the activity of IκB kinase in human esophageal squamous cell carcinoma cells.2.To elucidate the molecular mechanism by which PIWIL2 regulates mTOR and Beclin1 pathways and promotes autophagy in human esophageal squamous cell carcinoma cells by interacting with IKK.3.To elucidate the molecular mechanism by which PIWIL2 activates NF-κB pathway and inhibits cell apoptosis in human esophageal squamous cell carcinoma cells by interacting with IKK.4.The molecular mechanism of PIWIL2 regulating autophagy and apoptosis in esophageal squamous cell carcinoma by competitively binding IKK with TSC was preliminarily elucidated.This study will clarify the molecular mechanism of PIWIL2 combined with IKK in the regulation of autophagy,cell proliferation and apoptosis in esophageal squamous cell carcinoma,and provide theoretical research basis and a new direction of diagnosis and treatment for clinical treatment,prognosis or early diagnosis of esophageal cancer.Materials and Methods:1.The expression of PIWIL2 in esophageal squamous cell carcinoma and paratumor tissues was analyzed by Western Blot and RT-q PCR.The interaction between PIWIL2 and IKK was determined by Quick Coupled Transcription/Translation Systems,coimmunoprecipitation and immunofluorescence fluorescence.RT-q PCR,Western Blot and other methods were used to detect the effect of PIWIL2 on IKK expression,and further to detect the effect of PIWIL2 on IKK kinase activity.2.Immunohistochemistry,Western Blot,RT-q PCR and other methods were used to analyze the correlation between the expression level of PIWIL2 in tissues and cells of patients with esophageal squamous cell carcinoma and autophagy.The expression vector of PIWIL2 and sh RNA were transfected into KYSE150,KYSE180 and KYSE510 esophageal squamous cell carcinoma cell lines,respectively.The expression of autophagy marker LC3 I/II and p62 were detected by RT-q PCR and Western Blot,and the regulation of autophagy level by PIWIL2 interaction with IKKα/β was analyzed.3.KYSE150,KYSE180,KYSE510 esophageal squamous cell cell lines were transfected with PIWIL2 expression vector and sh RNA,and treated with IKK inhibitor group.Nucleo /cytoplasmic separation and Western Blot were used to detect p65 nucleation.Hoechst33342/PI double-staining assay and flow cytometry were used to detect apoptosis level.The regulation of PIWIL2 on apoptosis level of esophageal squamous cell carcinoma cells through IKK-NF-κB pathway and its molecular mechanism were analyzed.4.PIWIL2 expression vector and sh RNA were transfected into KYSE150 and KYSE180 cell lines of esophageal squamous cell carcinoma cells,respectively.The effect of PIWIL2 on proliferation of esophageal squamous cell carcinoma cells was detected by crystal violet staining test and soft AGAR clone formation test.5.IKKβ kinase-dead variant and sh RNA transfection were used to transfect esophageal squamous cell carcinoma cell lines KYSE150 and KYSE180.The binding mode of PIWIL2 to IKKβ was detected by Western Blot assay,and the cells were treated with Rapamycin,an mTOR inhibitor,to analyze the molecular mechanism of PIWIL2 regulating autophagy level and Beclin1 pathway of esophageal squamous cell carcinoma cells through IKK-mTOR signaling pathway and promoting autophagy.6.The KYSE150 cell line with stable knockdown of PIWIL2 was constructed,and normal KYSE150 cells were used as negative control,and BALB/c nude mice inoculated to construct xenograft tumor model.Tumor forming animals were grouped and injected with the IKK inhibitor BAY11-7082,and DMSO was used as control.After the treatment,the animals were killed,the tumor bodies were stripped,weighed,fixed and embedded in paraffin.HE staining was performed on paraffin sections,and histological observation was performed under microscope.Cell apoptosis and autophagy were detected by immunohistochemical method.Results:The protein and m RNA expression levels of PIWIL2 in esophageal squamous cell carcinoma tissues were significantly higher than those in paratumor tissues by Western Blot and RT-q PCR.Quick Coupled Transcription/Translation Systems and co-immunoprecipitation results showed that PIWIL2 interacted with IKK,IKKβ interacted with TSC1,and the binding of PIWIL2 to IKKβ reduced the binding of TSC1 to IKKβ.In esophageal squamous cell carcinoma cells KYSE180 and KYSE510,cell proliferation rate was significantly increased after overexpression of PIWIL2 by crystal violet and soft AGAR clonal fomation assay.Cell proliferation rate was significantly reduced after PIWIL2 knockdown.Hoechst/PI 33342 and flow cytometry showed that apoptosis rate was significantly reduced after overexpression of PIWIL2.The apoptosis rate was significantly increased after PIWIL2 knockdown.The levels of autophagy markers p62 and LC3 in esophageal squamous cell carcinoma tissues were significantly higher than those in paratumor tissues by immunohistochemical assay.By RT-q PCR,LC3 m RNA expression was increased in KYSE150 cells overexpressing PIWIL2 compared with the control group,but there was no significant difference in p62.Western blot analysis showed that the levels of LC3II/I and Beclin1 and p62 increased in KYSE180 and KYSE510 cells overexpressing PIWIL2,and the LC3 II level further increased after overexpressing PIWIL2 and treated with chloroquine(CQ),a lysosomal inhibitorWestern blot and cell protein nucleus/mass separation assay showed that p65 distribution was transferred from cytoplasm to nucleus when PIWIL2 was overexpressed.When the IKKβ inhibitor BAY11-7082 was used simultaneously,p65 was blocked from cytoplasm to nucleus after overexpression of PIWIL2.In BALB/c nude mouse xenograft model,PIWIL2 was detected to affect the growth and autophagy levels of esophageal squamous cell carcinoma cells in vivo.Conclusion:1.This study confirmed that PIWIL2 was highly expressed in both tissues and cell lines of esophageal squamous cell carcinoma,and affected the level of autophagy in esophageal squamous cell carcinoma.2.The mechanism of IKK participating in autophagy and apoptosis of esophageal squamous cell cancer cells by the competitive binding of PIWIL2 and TSC in esophageal squamous cell cancer cells was firstly illustrated.3.PIWIL2 binds to IKK to promote autophagy of esophageal squamous cell carcinoma cells by inhibiting the mTOR signaling pathway and thereby affecting the downstream ULK1 and Beclin1.4.The combination of PIWIL2 and IKK activates the NF-κB signaling pathway,and inhibits the apoptosis of esophageal squamous cell carcinoma cells by promoting the entry of NF-κB into the nucleus to regulate downstream genes.