Study On The Role And Mechanism Of PTEN Y68 Frame Shift In The Tolerance Of Endometrial Cancer To Chemo-radiotherapy

Endometrial cancer is one of the malignant tumors that endanger women’s life and health,and the incidence is increasing year by year and showing a younger trend.In my country,with the change of living habits and the increase of average life expectancy,the incidence of endometrial cancer continues to rise in some developed cities,accounting for about 20% to 30% of gynecological malignancies.Many factors are known to increase the incidence of endometrial cancer(including endocrine disorders,obesity,hypertension,diabetes,infertility,and exogenous estrogen stimulation,etc.).According to the differences in biological characteristics and pathogenesis,endometrial cancer is divided into type I(estrogen-dependent)and type II(non-estrogen-dependent);according to the different types of pathological tissues,endometrial cancer can be divided into uterine Endometrioid adenocarcinoma,serous carcinoma,clear cell carcinoma,mixed cell adenocarcinoma and carcinosarcoma,etc.[1].The histopathological classification of endometrial cancer plays an important role in the diagnosis and evaluation of treatment methods.However,the recurrence of high-grade endometrial cancer and the increasing mortality rate year by year are still quite difficult problems.With the deepening of research on the genetics of endometrial cancer,researchers have found that some fixed gene mutations are very common in endometrial cancer patients.With the completion of the Cancer Genome Project,molecular typing of endometrial cancer based on genome sequencing was first proposed by the Cancer Genome Atlas Project team in 2013,dividing endometrial cancers of different genotypes into: DNA polymerase ε hypermutation,Microsatellite unstable type,somatic low copy number variant/microsatellite stable type and high copy number variant/serous type [2].Among them,about 10% of patients with endometrial cancer have mismatch mutations in the POLE sequence,leading to missing corrections in the DNA replication process,which is manifested as an ultra-high mutation rate in somatic cells,including PTEN(94%),KRAS(53 %),PIK3CA(71%),etc.With the answers to these scientific questions,clinical attention has begun to develop personalized treatment plans for endometrial cancer based on genotype differences.At present,drug therapy and radiotherapy are still the most widely used methods in cancer treatment.However,due to the heterogeneity of malignant tumors,there are many clinical tumors that show resistance to chemotherapy and resistance to radiotherapy.Therefore,research on gene mutations related to radiotherapy and chemotherapy resistance is very important for the design of malignant tumor treatment programs.Research Background:PTEN is a recognized tumor suppressor and one of the most commonly mutated genes in human tumors.The PTEN gene is located on chromosome 10q23.3,with a total length of about 200 kb,including 9 exons and 8 introns.Mainly divided into 4 domains: N-terminal phosphatidylinositol 4,5 diphosphate binding domain(amino acids 1-15),phosphatase catalytic domain(amino acids 16-185),C2 functional domain(186-351)Amino acids)and C-terminal(amino acids 352-403).Numerous studies have shown that PTEN mainly regulates the G1-S phase transition of the cell cycle to ensure the accuracy of DNA replication and the stability of chromosome structure,and it can also block the abnormal proliferation of tumor cells.Mutations in the PTEN gene can be detected in a variety of tumor tissues,including endometrial cancer.Screening of oncogene mutations related to endometrial cancer at home and abroad shows that PTEN is one of the important candidate genes.The mutation rate of PTEN in endometrial cancer is the highest compared with other tumors.37%~61% of endometrial cancer patients have PTEN gene mutation,and 40% of endometrial cancer patients have PTEN gene loss of heterozygosity.The common mutation sites of PTEN are mainly located in the 5th exon(between the 122-132 amino acids),and the phosphatase function is usually lost after mutation.A large number of studies have shown that mutations of PTEN lead to the loss of its tumor suppressor function,including inhibition of cell survival,reduction of proliferation,and regulation of G1-S phase transition.At present,there are still many specific contents that have not been clarified clearly regarding the effect of PTEN mutation on endometrial cancer resistance to radiotherapy and chemotherapy.This study selected 7 gene-mutant endometrial cancer cell lines(ARK1,ECC-1,HEC-1-A,ISHIKAWA,MFE319,MFE319,etc.)that covered the 3 main genes of endometrial cancer(PI3K,PTEN and KRAS).SNG-II and SPAC-1-L cells)and two control endometrial cancer cell lines(ARK2 and HEC155 cells),using the real-time cell viability analysis system,firstly compared and analyzed the mutation pairs of the key genes of the three seeds of endometrial cancer The effect of docetaxel treatment on the activity of cancer cells.Secondly,we further compared the effects of three different mutant PTEN genes on the activity of cancer cells treated with docetaxel combined with ionizing radiation.In addition,we also analyzed the effects of different doses of ionizing radiation on the activity of docetaxel in the treatment of endometrial cancer cells.Finally,we compared the effects of docetaxel combined with ionizing radiation on killing PTEN mutant and wild-type cell lines.The effect of simultaneous ionizing radiation treatment on the killing of endometrial cancer by docetaxel,and the comparison of the apoptosis rate and the expression of related proteins.Research method:This study selected 7 gene-mutated endometrial cancer cell lines and 2 control endometrial cancer cell lines that cover the three main gene mutations of endometrial cancer(PI3K,PTEN and KRAS mutations),using real-time analysis of cell viability A detection system to study whether mutations in key genes of endometrial cancer affect its docetaxel and docetaxel combined with radiotherapy.After that,the wild-type ARK2 cells(control),ISHIKAWA cells(V317 frameshift mutation),SPAC-1-L cells(Y68,E288 frameshift mutation)were subjected to docetaxel by flow cytometry.Apoptosis rate after combined with ionizing radiation;real-time PCR(RT-qPCR)method and Western blotting method were used to detect the transcription level and protein level change of apoptosis-related protein.Research result:1.The effect of PTEN protein Y68 frameshift mutation on docetaxel resistance in endometrial cancerIn this study,the IC50 value of each cell on the cytotoxicity of docetaxel was determined by MTT method.After determining the modeling treatment conditions,we used the MTT method and xCELLigence real-time cell analysis method to detect the cell viability of the PI3 K,PTEN,and KRAS mutant groups and the control group after 2 days and 5 days of docetaxel treatment.The results showed that PTEN mutant endometrial cancer cells were more resistant to docetaxel treatment than other mutant and control cells.Among PTEN mutant cells,SPAC-1-L cells with a frameshift mutation in Y68 are significantly more resistant to docetaxel than other mutant cell lines with PTEN,indicating that the frameshift mutation of PTEN protein Y68 is responsible for endometrial cancer cells A key factor in docetaxel resistance.2.The effect of PTEN protein Y68 frameshift mutation on the effect of docetaxel combined with radiationThrough preliminary experiments,it is determined that the most suitable X-ray radiation dose for the 2-day and 5-day experiments is 2 Gy.Subsequently,through the MTT method and xCELLigence real-time cell analysis method,the cell viability of 9 seeds of endometrial cancer cells treated with docetaxel combined with 2 Gy ionizing radiation was detected for 2 days and 5 days.The results showed that among the PTEN mutant cells,the Y68 frameshift mutation SPAC-1-L cells were significantly more resistant to docetaxel combined with ionizing radiation than other mutant cell lines of PTEN,indicating that the PTEN protein Y68 frameshift mutation was caused by The key factor of endometrial cancer cell docetaxel combined with radiotherapy tolerance.3.The effect of PTEN protein Y68 frameshift mutation on the effect of ionizing radiation combined with low-dose docetaxelThe results of the above studies suggest that the PTEN protein Y68 frameshift mutation plays an important role in the resistance of endometrial cancer to the combination of radiotherapy and chemotherapy.Next,through the xCELLigence real-time cell analysis method,the CI value(cell index)of the ionizing radiation and non-irradiated control groups of MFE319,SNG-II and SPAC-1-L cells was analyzed.In MFE319 and SNG-II cells,the CI value of the low-dose docetaxel combined with ionizing radiation treatment group was significantly lower than that of the low-dose docetaxel treatment group.In SPAC-1-L cells,there was no difference in the CI value of the cells in the low-dose docetaxel combined with ionizing radiation treatment group and the cells in the low-dose docetaxel treatment group.This indicates that the Y68 frameshift mutation of PTEN protein is more resistant to ionizing radiation combined with low-dose docetaxel treatment.4.The effect of different doses of ionizing radiation on the PTEN protein Y68 frameshift mutant endometrial cancer that is resistant to docetaxel combined with radiationTo further target the SPAC-1-L cells with the Y68 frameshift mutation of PTEN protein,a bivariate cell killing experiment with drug concentration gradient and radiation dose was designed [Docetaxel drug concentration is 0,10,20,40,80 and 100(ng/ml);Ionizing radiation doses are 0,2,4 and 8(Gy)];In-depth study of the effect of different doses of ionizing radiation on improving docetaxel-ionizing radiation combination therapy.The results show that the killing effect of the combined treatment on SPAC-1-L cells does not change with the increase of radiation dose,which suggests that the PTEN protein Y68 frameshift mutation has no effect on the tolerance of docetaxel combined with ionizing radiation therapy.The effect of ionizing radiation dose.5.The effect of simultaneous ionizing radiation treatment on the inhibition of endometrial cancer by docetaxelWe further studied whether ionizing radiation affects the ability of docetaxel to kill cells.We selected two chemotherapy-sensitive HEC155(PTEN normal)and ECC-1(PTEN mutant)cells,and used xCELLigence real-time cell analysis to detect the changes in CI values ??of cells treated with docetaxel combined with ionizing radiation.The results show that in both HEC155 and ECC-1 cells,the combined use of X-ray radiation can interfere with the cytotoxic effect of docetaxel.However,PTEN normal cell line HEC155 low-dose docetaxel combined with irradiation can improve the therapeutic effect to a certain extent,indicating that reasonable control of the treatment dose of the two treatment methods will help improve the clinical therapeutic effect.6.The effect of PTEN protein Y68 frameshift mutation on cell apoptosis rate and related protein expression after radiotherapy and chemotherapyIn order to further study the mechanism of PTEN and its related gene mutations affecting apoptosis in endometrial cancer cells,we selected three kinds of ECC cells,namely using wild-type ARK2 cells(control),ISHIKAWA cells(V317 frameshift mutation),and SPAC-1-L cells(Y68,E288 frameshift mutations)were treated with the following drugs,radiation and double-acting respectively.After 2 days,the cells were detected by flow cytometry for apoptosis,and RT-qPCR and Western Blot methods were used to detect apoptosis.Death rate and the expression of apoptosis-related proteins BAD,BIM and Casepase-3.The results showed that the apoptosis rate of ARK2 cell line was significantly higher than that of the other two types of cells.The ARK2 cell line was the most sensitive to drugs among the three types of cells.In contrast,the SPAC-1-L cell line was significantly lower than the other two types.cell.At the same time,it was found that the SPAC-1-L cell with the Y68 frameshift mutation of PTEN protein significantly reduced the expression of BIM protein compared with the other two cells,but the expression of BAD protein did not change significantly.Conclusion:The PTEN protein Y68 frameshift mutation enhances the drug resistance of endometrial cancer to docetaxel;weakens the effect of docetaxel combined with radiation therapy;the resistance to docetaxel combined with ionizing radiation therapy is not affected by ionization The effect of radiation dose;its effect on tumor cell apoptosis is significantly weakened,and the docetaxel resistance of endometrial cancer is enhanced by inhibiting the activation of BIM protein,and it is found that PTEN mutation reduces the radiation of endometrial cancer Sensitivity.Although there are still many detailed issues that need to be further studied and clarified,the results obtained in this study suggest that the pre-treatment screening of PTEN is a potential marker that is worthy of preclinical and clinical prognostic evaluation,and is designed for clinically targeted uterus.The treatment plan and prognostic evaluation methods of endometrial cancer provide new experimental data and theoretical basis.