PLAC8 Regulates Biological Behavior Of Nasopharyngeal Carcinoma Cell

Background and objective: The incidence of nasopharyngeal carcinoma(NPC)is the highest among head and neck malignancies.Most of them are poorly differentiated squamous cell carcinoma with regional specificity.NPC is a complex and variable malignant disease.Genetic factors,Epstein-Barr virus(EBV)infection and environmental exposure are closely related to their development.At present,the more effective treatment for nasopharyngeal carcinoma is radiotherapy and chemotherapy.Despite the continuous development of medical technology in recent years and the emergence of new chemoradiotherapy methods,equipment and drugs,parts of the patients still have recurrence and metastasis after treatment.And The retreatment effect and prognosis of these patients are poor.Therefore,it is still of great significance to further study the pathogenesis of nasopharyngeal carcinoma and find some new and specific therapeutic approaches to save or prolong the lives of such patients.Placenta-specific 8(PLAC8),known as onzin or C15,contains 112 amino acids and is a cysteine-rich protein.It was found that PLAC8 was highly expressed in intestinal epithelial cells of zebra fish.And PLAC8 promoted epithelial mesenchymal transformation in colon cancer cells.PLAC8 had also been found to be associated with cell proliferation,apoptosis,carcinogenesis,immunity and autophagy.Our previous study found that the knockout of PLAC8 could increase the radiotherapy sensitivity of nasopharyngeal carcinoma CNE-2 cells.In order to further understand the relationship between PLAC8 and the malignant biological behavior of nasopharyngeal carcinoma,we investigated the effect of PLAC8 on the growth,proliferation,apoptosis and EMT of human nasopharyngeal carcinoma cells,and identified the molecular mechanism of PLAC8 in regulating the biological behavior of nasopharyngeal carcinoma cells.This study was divided into three parts:The first part: PLAC8 expression in nasopharyngeal carcinoma cells and its relationship with EMT marker proteinsObjective:To investigate the expression of PLAC8 in nasopharyngeal carcinoma tissues and cell lines and its relationship with EMT marker protein expression.Methods: 1.Immunohistochemical staining was used to detect the difference of PLAC8 expression in tissues of nasopharyngeal carcinoma and nasopharyngitis.2.Western blot was used to detect the expression differences of PLAC8 protein in immortalized epithelial cell line NP69 and nasopharyngeal carcinoma cell line C666-1,CNE-1,CNE-2,5-8F and 6-10 B.3.Immunofluorescence staining was used to detect the relationship between PLAC8 and EMT-related marker proteins(E-Cadherin,N-Cadherin and Vimentin)in tissues of nasopharyngeal carcinoma and nasopharyngitisResults: 1.Immunohistochemical results showed that,compared with nasopharyngitis,PLAC8 expression in tissues of nasopharyngeal carcinoma was significantly increased(P<0.01).2.Western blot showed that PLAC8 expression was significantly lower in NP69 cells and significantly higher in the nasopharyngeal carcinoma cells.3.Immunofluorescence results showed that,in contrast to nasopharyngeal carcinoma tissues,PLAC8 expression increased,E-Cadherin expression decreased,N-Cadherin expression increased,and Vimentin expression increased.Preliminary results suggested that PLAC8 expression may increase the invasion and metastasis ability of nasopharyngeal carcinoma cells.Conclu Sion: PLAC8 expression is significantly increased in nasopharyngeal carcinoma cells,which may be a contributing factor to the development of nasopharyngeal carcinoma.And PLAC8 is closely related to EMT protein expression in nasopharyngeal carcinoma,which may be an important factor affecting the invasion and metastasis ability of nasopharyngeal carcinoma cells.The second part: The effect of PLAC8 on the biological behavior of nasopharyngeal carcinoma cells and its molecular mechanismObjective:To explore the effect of PLAC8 expression on the biological behavior of nasopharyngeal carcinoma cells and its molecular mechanism.Methods: 1.The nasopharyngeal carcinoma cell line of PLAC8 knockout was constructed by CRISPR/CAS9 gene editing technology;Lentivirus lv-plac8 was used to infect and construct stable PLAC8-overexpressing nasopharyngeal carcinoma cells.2.CCK-8 assay was used to detect the effect of PLAC8 on the growth and proliferation of nasopharyngeal carcinoma cells in vitro.3.The effect of PLAC8 on growth and proliferation of nasopharyngeal carcinoma cells in vivo was detected in nude mouse xenograft model.4.The effect of PLAC8 on migration of nasopharyngeal carcinoma cells was detected by scratch test in vitro.5.Transwell assay was used to detect the effect of PLAC8 on invasion and metastasis of nasopharyngeal carcinoma cells in vitro.6.Immunofluorescence cell staining and western blot were used to verify the relationship between PLAC8 and the expression of EMT marker proteins E-Cadherin,N-Cadherin and Vimentin in nasopharyngeal carcinoma cells in vivo or in vitro.7.Western blot was used to verify whether PLAC8 regulates the biological behavior of nasopharyngeal carcinoma cells through the TGF-?/SMAD Signaling pathway.Results: 1.Nasopharyngeal carcinoma cells with PLAC8 gene knockout(ko PLAC8 CNE-2 cells)and nasopharyngeal carcinoma cells with PLAC8 gene overexpression(oe PLAC8 CNE-2 cells)were successfully constructed.2.Compared with the control group,the proliferation capacity of ko PLAC8 CNE-2 cells decreased significantly(P<0.05),while the proliferation capacity of oe PLAC8 CNE-2 cells increased significantly(P<0.05).3.Compared with the control group,the volume of nude mice in the ko PLAC8 cells group decreased significantly(P<0.05).4.Compared with the control group,the migration distance of ko PLAC8 CNE-2 cells was significantly shortened(P<0.05),while the migration distance of oe PLAC8 CNE-2cells was significantly increased(P<0.05).5.Compared with the control group,the number of membrane penetrating cells of ko PLAC8 CNE-2 cells significantly decreased(P < 0.05),while the number of membrane penetrating cells of oe PLAC8CNE-2 cells significantly increased(P < 0.05).6.Compared with the control group,E-Cadherin expression was increased,N-Cadherin expression was decreased and Vimentin expression was decreased in the ko PLAC8 group.And the expression of E-Cadherin decreased,N-Cadherin increased and Vimentin increased in the oe PLAC8 group.7.Compared with the control group,TGF-?1 expression,Smad2 phosphorylation level and Smad3 phosphorylation level of NPC cells in the ko PLAC8 group were decreased.TGF-?1 expression was increased in oe PLAC8 nasopharyngeal carcinoma cells,phosphorylation of Smad2 was increased,and phosphorylation of Smad3 was increased.Conclusion: PLAC8 can affect the malignant biological behavior of nasopharyngeal carcinoma cells by promoting their growth,proliferation,invasion and metastasis.This effect is related to the regulation of TGF-? /SMAD signaling pathway activity.The third part: PLAC8 affects the biological behavior of nasopharyngeal carcinoma cells by regulating autophagyObjective:To investigate the role of PLAC8 in regulating autophagy in affecting the growth,proliferation,apoptosis and EMT process of nasopharyngeal carcinoma cells.Methods: 1.Autophagy of nasopharyngeal carcinoma cell lines CNE-2 cells after PLAC8 knockout was observed by transmission electron microscopy.2.Autophagy double fluorescence system was used to detect the phenomenon of autophagy tide after CNE-2 cells PLAC8 knockout.3.Western blot experiment was used to observe the changes of autophagy related proteins in CNE-2 cells after PLAC8 knockout.4.Immunofluorescence and immunohistochemistry were used to detect the changes of autophagy-related proteins in xenograft tumors of nude mice with CNE-2cells.5.CCK-8 assay was used to detect whether the inhibition of proliferation in CNE-2 cells after PLAC8 knockout could be reversed after the use of autophagy inhibitors.6.Clone formation assay was used to observe whether the inhibition of colony formation ability after PLAC8 knockout in CNE-2 cells could be reversed after the use of autophagy inhibitors.7.Flow apoptotic cytometry was used to determine whether the apoptosis rate in CNE-2 cells after PLAC8 knockout could be reversed after the use of autophagy inhibitors.8.CCK-8 assay was used to detect whether the proliferation inhibition effect of PLAC8 knockout could be reversed after knockout of downstream autophagy related target gene Beclin-1 in CNE-2 cells.9.Clone formation assay was used to observe whether the inhibition of colony formation ability after PLAC8 knockout could be reversed after knockout of downstream autophagy related target gene Beclin-1 in CNE-2 cells.10.Flow apoptotic cytometry was used to detect whether the apoptosis rate of PLAC8 knockout could be reversed after knockdown of downstream autophagy related target gene Beclin-1 in CNE-2 cells.11.Transwell assay was used to observe whether inhibition of autophagy could reverse changes in migration and metastasis of CNE-2 cells after PLAC8 knockout.12.Western blot was used to detect whether autophagy inhibition could reverse the effects of autophagy related proteins and EMT marker proteins E-Cadherin and Vimentin expression in CNE-2 cells after PLAC8 knockout.13.The autophagy dual-fluorescence system was used to dynamically detect the fluorescence changes of CNE-2 cells after autophagy inhibition.14.Western blotting was used to detect changes in the activity of AKT/mTOR(autophagy signaling pathway)signaling pathway in ko PLAC8 CNE-2cells.15.The positions of PLAC8 and AKT were located by immunofluorescence double-label staining.16.The relationship between PLAC8 and AKT was verified by co-immunoprecipitation.17.Immunofluorescence and immunohistochemistry were used to detect the protein expression of AKT/mTOR pathway in xenograft tumor of nude mice with CNE-2 cells.Results: 1.Compared with CNE-2 cells in the control group,autophagosomes of nasopharyngeal carcinoma cells in the ko PLAC8 group increased significantly.2.Compared with the control group,the autophagy tide of CNE-2 cells in the ko PLAC8 group increased significantly.3.Compared with the control group,Beclin-1 expression was increased,LC3-II/LC3-I ratio was increased and P62 expression was decreased in CNE-2 cells in the ko PLAC8 group.4.Immunofluorescence and immunohistochemical experiments in vivo showed that,compared with the control group,Beclin-1 protein expression was increased,LC3-II protein expression was increased,and P62 protein expression was decreased in the ko PLAC8 group(P<0.05).5.Compared with the ko PLAC8 group,the proliferation of CNE-2 cells in the 3-MA +ko PLAC8 group was improved(P<0.05).6.Compared with the CNE-2 cells in the ko PLAC8 group,the colony formation ability of the 3-MA+ko PLAC8 group was improved(P<0.05).7.The apoptosis rate of 3-MA+ko PLAC8 group was lower than that of the ko PLAC8 group(P<0.05).8.Compared with CNE-2 cells in the ko PLAC8 group,the proliferation of CNE-2 cells in the Si-Beclin1+ko PLAC8 group was improved(P<0.05).9.Compared with CNE-2 cells in the ko PLAC8 group,the colony forming ability of CNE-2 cells in the Si-Beclin1+ko PLAC8 group was improved(P<0.05).10.Compared with CNE-2cells in the ko PLAC8 group,the apoptosis rate of CNE-2 cells in the Si-Beclin1+ko PLAC8 group was significantly reduced(P<0.05).11.After inhibiting autophagy,CNE-2 cell migration(without matrix glue)and transfer capacity(with matrix glue)of PLAC8 knockout cells were reversed(P<0.05).12.After the inhibition of autophagy,the expression levels of autophagy-related proteins and EMT marker proteins in CNE-2 cells after the knockout of PLAC8 were reversed(P<0.05).13.After inhibiting autophagy,compared with CNE-2 cells in the control group,autophagy tide in ko PLAC8 CNE-2 cells was significantly reduced(P<0.05).14.Compared with the control group,the phosphorylation level of AKT and mTOR decreased in the ko PLAC8 group(P<0.05).15.PLAC8 and AKT co-located in the cytoplasm of nasopharyngeal carcinoma cells.16.PLAC8 and AKT can bind to each other.17.Compared with the control group,the phosphorylation level of AKT and mTOR decreased in the ko PLAC8 in vivo.Conclusion: PLAC8 in nasopharyngeal carcinoma cells regulates autophagy through the AKT/mTOR signaling pathway.Inhibition of autophagy could reverse the changes of cell proliferation,colony formation ability,apoptosis rate and EMT after PLAC8 knockout in nasopharyngeal carcinoma.In summary,this study demonstrated that PLAC8 could increase the malignant biological behavior of nasopharyngeal carcinoma cells by promoting the TGF-?/SMAD signaling pathway.It can also inhibit autophagy of nasopharyngeal carcinoma cells by promoting the AKT/mTOR signaling pathway,thereby increasing their malignant biological behavior.PLAC8 is expected to be an effective target for targeted therapy of nasopharyngeal cancer in the future.