| Objective: To analyze the expression difference and epigenetic methylation level of PIK3 CA gene in nasopharyngeal carcinoma,and elaborate the relationship between PIK3 CA expression and clinical malignant phenotype and prognosis of NPC;then explore the effect of PIK3 CA expression pattern on proliferation,migration and invasion of nasopharyngeal carcinoma cells.On this basis,to further investigate the effect of PIK3 CA upstream regulation of PVT1/mi R-515-5p-PIK3 CA axis on proliferation,apoptosis and radiation resistance of NPC cells and its internal regulatory mechanism,so as to lay a scientific basis for revealing the molecular mechanism of the development and radiotherapy resistance of nasopharyngeal carcinoma and the screening of individualized therapeutic targets.Methods: Part one:(1)The expression of PIK3 CA in NPC and normal nasopharyngeal mucosa was detected by immunohistochemical SP staining,the association between PIK3 CA expression and clinicopathological parameters was analyzed,the effect of PIK3 CA expression on the prognosis of NPC was analyzed by Kaplan Meier method;(2)In vitro,the effects of PIK3 CA expression on proliferation,invasion and migration of nasopharyngeal carcinoma cells were detected by transfecting specific si RNA into NPC cells.(3)Pyrophosphate sequencing was used to detect the methylation level of PIK3 CA gene promoter region in nasopharyngeal carcinoma and normal nasopharyngeal mucosa.Part two:(1)The potential target sites between PIK3 CA and mi R-515-5p were predicted by star Base bioinformatics analysis,and the wild-type sequence containing PIK3 CA 3’-UTR binding site of mi R-515-5p was constructed and inserted into p GL3 vector,double luciferase reporter gene analysis was used to verify the binding site of PIK3 CA gene regulated by mi R-515-5p,and RIP detection was performed by anti-ago2 to analysis the interaction between mi R-515-5p and PIK3 CA.(2)The expression of PIK3 CA m RNA in NPC and normal nasopharyngeal mucosa was detected by q RT-PCR,and the protein expression of PIK3 CA gene in NPC and normal nasopharyngeal mucosa cell lines were analyzed by western blot;western blot was also used to detecting the regulation of PIK3 CA protein expression in NPC cells after mi R-515-5p knockdown.(3)The proliferation of PIK3 CA + mi R-515-5p transfected NPC cells was detected by CCK8 assay.The survival fraction and apoptosis of PIK3 CA + mi R-515-5p transfected NPC cells to PIK3 CA transfected NPC cells at different radiation doses(0,2,4,6,8 Gy)were analyzed by colony formation assay and flow cytometry,and western blot was used to detect the protein levels of cyclin D1 and Bax in the above two kinds of cells under different irradiation doses(0 and 8 Gy),so as to reveal the effect of mi R-515-5p on the radiation resistance of nasopharyngeal carcinoma cells.Part three:(1)The expression of PVT1 between NPC and normal tissues and in different TNM stages were detected by q RT-PCR,and the association between the expression of PVT1 and prognosis of NPC was analyzed;the difference of PVT1 level between normal mucosa and NPC cell lines were detected by q RT-PCR.(2)PVT1 was knocked down by lentivirus with sh RNA,the knockdown efficiency of PVT1 was verified by q RT-PCR;CCK8 method was used to evaluate the effect of PVT1 expression on cell proliferation.Cell clone formation ability test,detection of cyclin,flow cytometry and western blot were used to analyze the survival fraction,apoptosis and cyclin level of NPC cells at different irradiation doses(0,2,4,6,8 Gy).(3)The binding sites of PVT1 and mi R-515-5p were predicted by star Base,and the single target verification of mixed target positive candidate genes was carried out by double luciferase reporter gene method by using anti-ago2.Lentiviral vector was constructed,and q RT-PCR was used to evaluate the expression and its regulation of mi R-515-5p and PVT1 in NPC tissues and cells.(4)CCK8 method was used to detect the proliferation of NPC cells transfected with mi R-515-5p and mi R-515-5p + PVT1,cell clone formation assay and flow cytometry were used to analyze the survival fraction and apoptosis of NPC cells transfected with PVT1+mi R-515-5p at different irradiation doses(0,2,4,6,8 Gy),and the protein levels of cyclin D1 and Bax were detected by western blot,in order to reveal the effect of mi R-515-5p regulated by PVT1 on radiation resistance of NPC cells.(5)Western blot was used to detect the protein levels of PIK3 CA,Akt and p-Akt in NPC tissues transfected with sh-PVT1 and sh-PVT1 + anti-mi R-515-5p to determine the regulatory relationship between PVT1/mi R-515-5p-PIK3 CA axis.(6)In vivo,in order to verify the function of PVT1,a xenograft mouse model was established by injecting C666-1 cells transfected with sh-PVT1 or sh-control,the tumor formation and growth in nude mice were dynamically monitored to verify the effect of PVT1 on the growth of NPC cells.Results: Part one:(1)The high expression rate of PIK3 CA protein in 71 NPC tissues was 81.69%,which was statistically significant higher than that in normal nasopharyngeal mucosa(31.25%).The expression of PIK3 CA in nasopharyngeal carcinoma was not correlated with gender,age,pathological type,Ki-67 protein,significantly correlated with T stage(P=0.000),N stage(P=0.008)and clinical stage(P=0.002),Kaplan Meier survival curve demonstrated that the survival rate of NPC patients with high expression of PIK3 CA protein was significantly lower than that of low and moderate expression(Log rank test: c2=7.995,P<0.05);(2)After transfection of PIK3 CA si RNA into NPC cells,MTT assay showed that the proliferation of NPC cells was significantly inhibited;cell scratch test and transwell experiment showed that the migration and invasion ability of NPC cells were significantly inhibited(P<0.05).(3)The promoter region methylation level of PIK3 CA gene was detected by pyrophosphate sequencing.Four methylation sites were found in the promoter region,among which 3 sites were significantly higher in NPC group than in normal nasopharyngeal mucosa group(P<0.05).Part two:(1)star Base found the potential target site between mi R-515-5p and PIK3 CA.Double luciferase reporter gene and RIP detection confirmed that mi R-515-5p regulated PIK3 CA gene expression in NPC cells through this binding site;western blotting analysis showed that the protein level of PIK3 CA in NPC cells transfected with mir-515-5p was lower than that in normal nasopharyngeal mucosa cells(P<0.05),suggesting that mi R-515-5p negatively regulates PIK3 CA expression in vitro.(2)CCK8 assay showed that the proliferation of NPC cells transfected with mi R-515-5p was significantly lower than that of transfected with PIK3 CA group(P<0.05),suggesting that mi R-515-5p reversed the effect of PIK3 CA on NPC cell proliferation.(3)After mi R-515-5p knockdown,the survival fraction of NPC cells reduced following with the increase of radiation dose.The decrease of survival fraction of PIK3 CA group was smaller than that of vector control group,and the difference was statistically significant(P<0.05).Demonstrated that PIK3 CA gene promoted the radiation resistance of nasopharyngeal carcinoma cells,and the up-regulation of mi R-515-5p significantly reversed the enhancement of radiation resistance induced by PIK3 CA.The results of flow cytometry and western blot showed that PIK3 CA overexpression could inhibit the apoptosis of NPC cells induced by radiotherapy(P < 0.05),and the inhibition effect of PIK3 CA on radiation-induced apoptosis was eliminated by mi R-515-5p transfection(P<0.05).In conclusion,PIK3 CA promotes the proliferation,and radiation resistance of NPC cells,and inhibits the apoptosis of NPC cells.At the same time,mi R-515-5p plays an opposite role in regulating the proliferation,apoptosis and radiation resistance of NPC cells.Part three:(1)At the tissue level,compared with the paried normal tissues,the expression of PVT1 in NPC tissues was significantly up-regulated,and the expression level of PVT1 in advanced stage(III + IV)NPC tissues was higher than that in early stage tumor tissues(P<0.05);at the cellular level,PVT1 was up-regulated in NPC cells than in nasopharyngeal epithelial cells,higher expression PVT1 resulted the cell survival rate decreased(P<0.05).The above results suggesting that PVT1 may be a oncogene of NPC,and its high expression leads the poor prognosis of NPC.(2)CCK8 assay showed that the down-regulation of PVT1 significantly inhibited the proliferation of NPC cells;cell clone formation assay showed that with the raising of irradiation dose(0,2,4,6,8 Gy),the survival fraction of sh-PVT1 group was significantly lower than that of sh-control group(P<0.05),indicated that the radiosensitivity of NPC cells was significantly promoted after PVT1 knockdown.Flow cytometry and western blot analysis showed that the apoptosis rate of NPC cells was significantly increased after PVT1 knockdown at different irradiation doses(0 and 8 Gy).Compared with sh-PVT1 group,the expression of cyclin D1 and Bax in sh-control group were significantly increased(P<0.05).knockdown of PVT1 significantly decreased the expression level of cyclin D1 and increased the level of Bax in NPC cells after radiotherapy.In conclusion,knockdown of PVT1 could inhibit the proliferation and radiation resistance of NPC cells and induce apoptosis in vitro.The target site of PVT1 was found by star Base analysis.Double luciferase reporter gene and RIP expriment showed that mi R-515-5p was the target gene of PVT1.Result of q RT-PCR confirmed that mi R-515-5p was negatively regulated by PVT1 in NPC cells in vitro.Clone formation test and flow cytometry also indicated that PVT1 could promote the proliferation and radiation resistance of NPC cells and inhibit the apoptosis through reverse regulation of mi R-515-5p.(5)Western blot analysis showed that the protein expression levels of PIK3 CA and p-Akt in sh-PVT1 group were lower than those in control group(P<0.05),and the down-regulation of PVT1 significantly reduced the levels of PIK3 CA and p-Akt,and the effect was reversed in sh-PTV1+anti-mi R-515-5p group than in sh-PTV1+ anti-mi R-515-5p group(P < 0.05).The results demostrated that PVT1 induced Akt pathway in vitro by interacting with mi R-515-5p in NPC cells.(6)The xenograft tumor model in nude mice confirmed that knockdown PVT1 inhibited the growth of tumor by interacting with PIK3 CA in vivo.Conclusion:(1)PIK3CA protein was highly expressed in NPC tissues and associated with the poor prognosis of NPC.Down regulation of PIK3 CA gene expression can inhibit the proliferation,migration and invasion of NPC cells;PIK3CA gene abnormal methylated in NPC,and its methylation level was significantly higher than that in normal nasopharyngeal mucosa.(2)Mi R-515-5p targeted PIK3 CA gene in vitro and negatively regulated PIK3 CA expression.PIK3 CA promoted the proliferation and radiation resistance of NPC cells and inhibited the apoptosis of NPC cells,and was reversed by mi R-515-5p.(3)PVT1 was highly expressed in NPC tissues and associated with poor prognosis of NPC.Mi R-515-5p was the target of PVT1 in NPC and negatively regulated by PVT1 in vitro.PVT1 promoted the proliferation and radiation resistance of NPC cells and inhibit the apoptosis through negative regulation of mi R-515-5p.At the cellular signal transduction level,PVT1 induced Akt pathway by inhibiting mi R-515-5p in NPC cells and promoting the proliferation and radiation resistance of NPC cells.Knockdown of PVT1 decreased the carcinogenic ability of PIK3 CA and inhibited the growth of tumor.PVT1/mi R-515-5p-PIK3 CA axis can promote the proliferation,invasion and migration of tumor cells,and induce radiation resistance of NPC cells,resulting poor prognosis of NPC.This study provides brand new targets and basis for the pathogenesis of nasopharyngeal carcinoma and key molecular screening of individualized targeted therapy. |
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