| Thyroid hormone receptor interactor 4(TRIP4)is a subunit of the tetrameric nuclear activation signal co-integration 1(ASC-1)complex that exerts a pro-stimulatory effect.In recent years,studies have shown that TRIP4 is highly expressed in a variety of tumors and may be related to the occurrence and development of tumors.The gene chip database also records that the expression of TRIP4 in cervical cancer tissues is higher than that in normal cervical epithelial tissues.Its downstream may be related to the NF-?B pathway and regulate the proliferation and invasion of tumor cells.However,the role of TRIP4 in regulating the growth and radiation resistance of cervical cancer has not been reported,and its mechanism of tumor action has rarely been reported.The most common finding is an abnormality in the phosphatidylinositol 3-kinase(PI3K)pathway and a shorter survival time is observed in these patients with mutations.The proliferation and invasion ability of cervical cancer cells are closely related to the AKT/MAPK signaling pathway.Radiotherapy is the main treatment for modern cervical cancer treatment.The perfect radiation therapy mode has greatly improved the survival rate of patients with cervical cancer.However,the treatment of future tumors is not limited to a single treatment.There is increasing evidence that multidisciplinary treatment is an effective way to further improve the treatment effect.Therefore,finding comprehensive treatments that can reduce radiation resistance or assisted radiotherapy has become an important goal of modern medicine.Previous studies have shown that activation of the hTERT pathway is one of the causes of radiotherapy resistance in patients with cervical cancer.Finding a gene that inhibits the telomerase reverse transcriptase pathway may improve the radiosensitivity of cervical cancer.Therefore,this study identified TRIP4 as a potential target for cervical cancer and explored its role and molecular mechanisms in the growth and development of cervical cancer.To investigate the effect of TRIP4 gene on cervical cancer AKT/MAPK signaling pathway,and to find out why TRIP4 gene regulates tumor biological characteristics.At the same time,through various phenotypic experiments and mechanism experiments,the regulatory effect of TRIP4 gene on hTERT signaling pathway was discussed,and how to regulate its effect on cervical cancer radiotherapy.Finally,this study explored the relationship between TRIP4 and key genes in each pathway through radiotherapy in tumor-bearing nude mice,and further verified the above problems in animal experiments.Objective:1.To study the expression of TRIP4 gene in cervical cancer tissues and cells,and analyze its effects on proliferation,apoptosis and migration of cervical cancer cells.2.Study on the relationship between TRIP4 gene and AKT/MAPK signaling pathway and hTERT signaling pathway3.Study on the radiosensitivity of inhibiting TRIP4 gene to cervical cancer in tumor-bearing nude miceMethods:Part I:Expression of TRIP4 gene in cervical cancer and its effect on cervical cancer cells.1.The tumor tissues and adj acent tissues of 15 patients with cervical cancer were randomly selected.The total protein of the two groups was extracted.The expression of TRIP4 in different tissues was verified by Western Blot and immunohistochemistry.2.Six cervical cancer cell lines(HeLa,SiHa,Caski,DoTc2,HeLa S3,C33A)and one human normal cervical epithelial cell Ectl were selected for culture,and the corresponding tissue proteins were extracted.Western Blot experiments were performed to verify different cervical cancer cells.The expression of TRIP4.3.The expression of TRIP4 in HeLa and SiHa cells was knocked down by TRIP4-specific SiRNA.Two cell lines were observed after platelet colony formation assay,MTT assay,flow cytometry assay and AO/EB staining assay.Proliferative capacity,cell viability and apoptosis were observed,and the expression of key proteins related to the corresponding phenotype was observed by Western Blot.The Transwell experiment,scratch test and immunofluorescence experiment were used to observe the migration and invasion ability of HeLa and SiHa cells after TRIP4 knockdown.The expression of key proteins related to the corresponding phenotypes was observed by Western Blot.The expression of TRIP4 in HeLa and SiHa cells was overexpressed by TRIP4-specific plasmid.The above experiments were repeated to observe the effect of TRIP4 gene on cell biological characteristics after overexpression in two cell lines.Part II:Relationship between TRIP4 gene and AKT/MAPK signaling pathway and hTERT signaling pathway.1.HeLa and SiHa cells were cultured by cell culture technique,and the expression of TRIP4 in HeLa and SiHa cells was knocked down by TRIP4-specific SiRNA.The expression of TRIP4 was significantly knocked down by Western Blot.2.HeLa and SiHa cells were divided into untreated group(MOCK),no SiRNA control group(NC),which effectively knocked down Sil group(Sil)and effectively knocked down Si2 group(Si2).Knockdown transfection treatment was performed when the cells were cultured to 80%of the 10 cm culture dish.The cells were digested and collected after 48 hours of incubation.The total protein of the corresponding cells in each of the two cells was extracted.After BCA cells were quantified,the expression of key proteins in the AKT/MAPK signaling pathway was observed by Western Blot.HeLa and SiHa cells were subcultured in 6 cm culture dishes.The two cells were divided into four groups:Mock,Mock+IR,Si1 and Si1+IR.Each group consisted of 5 dishes,and about 1000 cells per plate were used for colony formation.In the experiment,the IR group was subjected to 2Gy,4Gy,6Gy,and 8Gy radiotherapy once after cell colony formation,and finally the cell colonies were photographed to prepare a radiation survival curve.The HeLa and SiHa cells of the well-grouped groups were separately processed,and the comet assay was applied.After fluorescence photography,the software used to measure the cell tail length after electrophoresis and statistics were performed.6.HeLa and SiHa cells were transfected with TRIP4-siRNA(si-TRIP4)or a plasmid expressing TRIP4,and the expression of TRIP4 and hTERT protein levels was detected by Western Blot.HeLa cells were cotransfected with TRIP4-siRNA and hTERT promoter-driven luciferase(-2053/+40)plasmid and assayed for luciferase activity.Chromatin immunoprecipitation assay was performed in HeLa cells using the hTERT promoter,and the TRIP4 protein in the nuclear protein-hTERT probe-streptavidin beads complex was detected by Western blotting in HeLa cells using an anti-TRIP4 antibody.The 5' DNA fragment(-2053/+40,-1655/+40,-902/?40,-321/+40,-234/+40,-144/+40)from position-2033 to+40 will human hTERT gene constructed as a promoter-driven luciferase expression vector pGL3.HeLa cells were co-transfected with TRIP4 and different hTERT promoter-driven luciferase plasmids,and luciferase activity was detected by a luciferase reporter assay kit.Part III:Study on the radiosensitivity of inhibiting TRIP4 gene to cervical cancer in nude mice.1.Analysis of expression of TRIP4 and hTERT protein expression in tissue microarrays from cervical cancer tissues and their corresponding adjacent normal tissues by immunohistochemical staining.2.Kaplan-Meier analysis of overall survival of cervical cancer patients with different TRIP4 expressions by log-rank test.Kaplan-Meier analysis of the overall survival of cervical cancer patients with different TRIP4 and hTERT expression was analyzed by log-rank test.3.Expression of TRIP4 and hTERT in HeLa cells was analyzed by Western blot after transfection with non-specific control shRNA or TRIP4 shRNA.Female nude mice were implanted with tumors under the left axilla,and tumor volume and mouse body weight were measured daily.After 21 days,the material was taken,cut into tumors,weighed,photographed,recorded,and counted.4.HeLa cell-derived xenografts were harvested on day 21 after treatment,tumor volume and weight were measured,and pictures of 4 groups of tumors were obtained.5.Each group was treated with paraffin sections for immunohistochemical staining,some tissues were extracted for total imprinting,and the remaining tissues were stored in formaldehyde fixative.6.The expression levels of TRIP4,hTERT,p-AKT,p-ERK and p-H2AX proteins in nude mouse tumor tissues were detected by Western blot.Immunohistochemistry and analysis of the expression of TRIP4,hTERT,p-AKT,p-ERK and p-H2AX in tumor tissues of nude mice were performed.Results:Part one:1.TRIP4 was highly expressed in cervical cancer cell lines and tumor tissues.15 patients with cervical cancer were randomly selected.There were 9 cases of high expression of TRIP4 in tumor tissues,6 cases of low expression,7 cases of high expression in adjacent tissues,and 8 cases of low expression.In one cervical epithelial cell line and six cervical cancer cell lines,the expression of TRIP4 in cervical cancer cells was significantly higher than that in normal cervical epithelial cells.The immunohistochemical results of 5 patients with cervical cancer were randomly selected.The expression of TRIP4 was higher in cancer tissues than in adjacent tissues(P<0.05).2.The TRIP4 protein of the knockdown HeLa cell line and SiHa cell line was transiently transfected.The plate cloning experiment and MTT assay showed that the proliferative ability of the knockdown group was weaker than that of the control group(P<0.05).3.FACS flow cytometry analysis of HeLa cell line and SiHa cell line with transient transfection knockdown of TRIP4 protein,knockdown of TRIP4 resulted in an increase of apoptotic cells of about 5%-20%,which was statistically significant compared with the control group(P<0.05).The degree of damage of cervical cancer cells in the TRIP4 knockdown group was compared by AO/EB staining.The brighter orange-red fluorescence was accumulated in the nuclear DNA of the TRIP4 knockdown group.The image analysis software found that the difference between the two groups was statistically significant(P<0.05).Apoptosis-related molecules were analyzed by immunoblotting.The knockdown of TRIP4 effectively increased the levels of Bax,casepase-3,casepase-9 and cleaved-PARP1,but decreased the level of Bcl-2(P<0.05).4.Transwell and scratch experiments showed that the invasion and migration ability of HeLa cell line and SiHa cell line knocking down TRIP4 protein were transiently transfected,and the degree of attenuation was statistically significant(P<0.05).Immunofluorescence showed that the expression of E-cadherin increased and the expression of N-cadherin decreased after transient transduction of TRIP4 in HeLa and SiHa cell lines.Immunoblotting showed that HeLa and SiHa cell lines were transiently knocked down and TRIP4 was detected.Detection of EMT-related protein expression revealed that knockdown of TRIP4 effectively increased E-cadherin levels,but decreased MMP-9,MMP-1,Slug,Snail,?-catenin and N-cadherin levels(P<0.05).At the same time,overexpression of TRIP4 in HeLa cell line and SiHa cell line showed that Transwell and scratch phenotype showed enhanced invasion and migration ability(P<0.05).Part two:1.The TRIP4 protein of the knockdown HeLa cell line and the SiHa cell line was transiently transfected,and the key protein expressions of PI3K/AKT and MAPK/ERK signaling pathways in the knockdown group were significantly different from those in the control group(P<0.05).The expression of key proteins in PI3K/AKT and MAPK/ERK signaling pathways in overexpressing HeLa cell line and SiHa cell line was significantly different from that in the control group(P<0.05).It is suggested that PI3K/AKT and MAPK/ERK and signaling pathways are inhibited after inhibition of TRIP4.2.In the case of transient transfection of the TRIP4 protein of HeLa cell line and SiHa cell line,the cells in the experimental group and the control group were irradiated.The survival curve of the radiation showed that the survival rate of the TRIP4 knockdown group was lower than that of the control group.Statistical significance(P<0.05).The comet assay showed that the tail of the TRIP4 knockdown group was longer than that of the control group,suggesting that DNA damage increased and the ability of cells to repair DNA was weakened.The Rad51 and p-H2 AX proteins in the control and experimental groups were detected by immunoblotting.The expression of DNA repair protein was inhibited when TRIP4 was knocked down(P<0.05).3.The TRIP4 protein of the knockdown HeLa cell line and SiHa cell line was transiently transfected,the expression of hTERT protein was inhibited by immunoblotting,and the expression of hTERT protein was overexpressed in the HeLa cell line(P<0.05).When HeRa cells with stable TRIP4 knockdown or overexpression were transfected with the hTERT promoter-driven luciferase plasmid,knockdown was observed to significantly inhibit hTERT promoter-driven luciferase expression(P<0.05).Chromatin immunoprecipitation assays were performed using the hTERT promoter in HeLa cells,and the hTERT promoter was amplified,indicating that TRIP4 binds to the hTERT promoter.The DNA-protein pulldown assay further confirmed the binding of TRIP4 to the hTERT promoter.4.Six different lengths of hTERT promoter-driven luciferase reporter vector were constructed,and the plasmid expressing TRIP4 and the luciferase reporter gene driven by hTERT promoter were co-transfected into HeLa cells with TRIP4 and hTERT(-2053/+40,-1655/+10 and-321/+10)luciferase expression was higher in cells co-transfected with luciferase plasmid(P<0.05).Part three:1.The HeLa cell line stably transfecting the TRIP4 gene was constructed,and the nude mice xenograft model was established and irradiated.The growth curve and radiation survival curve of the experimental group and the control group were obtained according to the body weight of the nude mice(P<0.05).The emission factor(EF)was 1.66.2.The effect of TRIP4 knockdown on the expression of tumor-associated protein in transplanted tumors was examined by immunoblotting and immunohistochemical analysis.The results showed that TRIP4 knockdown attenuated hTERT,p-AKT,p-ERK and p-of transplanted tumors.H2AX expression(P<0.05).3.To investigate the tissue microarray of 128 patients with cervical cancer.Immunohistochemical staining was used to compare the expression of TRIP4 protein and hTERT protein in cancer tissues and adjacent tissues.65 patients expressed high expression of TRIP4 and hTERT,accounting for 51 cases of all test cases.%,the relationship between TRIP4 expression and clinicopathological variables was determined,and the expression of TRIP4 was correlated with tumor volume and TNM staging.Overall survival(OS)analysis showed that patients with low TRIP4 and hTERT expression had significantly higher OS and longer survival(P<0.05).Conclusions:Part one:1.TRIP4 is highly expressed in cervical cancer cell lines and tumor tissues.2.Inhibition of the expression of TRIP4 can inhibit the proliferation of cervical cancer cells.3.Knock down TRIP4 to promote apoptosis of cervical cancer cells.4.TRIP4 promotes the migration and invasion of cervical cancer cells.Part two:1.TRIP4 promotes the growth and survival of cervical cancer cells in vitro through MAPK and PI3K/AKT signaling pathways.2.Inhibition of TRIP4 can increase DNA damage and radiosensitivity in cervical cancer cells.3.TRIP4 regulates the transcriptional activity and expression of hTERT.4.TRIP4 regulates DNA damage and radiosensitivity in cervical cancer cells by modulating hTERT expression.Part three:1.Knockdown of TRIP4 can down-regulate hTERT expression in a mouse model to inhibit cervical cancer progression.2.The nude mouse xenograft model showed inhibition of proliferation,apoptosis,invasion and DNA damage-related protein expression after TRIP4 knockdown.3.TRIP4 was positively correlated with hTERT expression in clinical tissue samples.High expression of TRIP4/hTERT predicted poor prognosis of cervical cancer. |
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