| BackgroundThe incidence rate and mortality rate of cervical cancer are fourth in the world.Cisplatin is the first choice for neoadjuvant chemotherapy,concurrent radiotherapy and chemotherapy and late systemic treatment of cervical cancer.Although the clinical significance of neoadjuvant chemotherapy is still controversial,there are still many locally advanced cervical cancer patients who have received radical surgery through cisplatin neoadjuvant chemotherapy,and cisplatin tolerant patients are often unable to benefit from this;for synchronous radiotherapy and chemotherapy,cisplatin,in addition to its own killing effect on cervical cancer cells,also plays a sensitizing role in radiotherapy,Therefore,the response of cervical cancer patients to cisplatin directly affects the efficacy of concurrent radiotherapy and chemotherapy;for advanced cervical cancer patients with recurrence and metastasis,the prognosis is often poor,the survival period is short,and the prognosis of patients with cisplatin tolerance is even worse.Therefore,finding the key molecules that affect the sensitivity of cisplatin in cervical cancer may help to predict the therapeutic effect,better adjust the treatment plan,and may also help the development of cisplatin sensitizer or combination therapy drugs,and enhance the therapeutic effect of cisplatin in the treatment of cervical cancer.In recent years,the application of sequencing and bioinformatics technology has brought great convenience to the research of tumor development and drug sensitivity.Among them,the second generation sequencing technology has low cost and high sensitivity.It can obtain the whole genome information related to individuals or diseases in a short time through deep sequencing of millions of different DNA fragment mixtures.The second generation sequencing technology has been widely used in the research of Pan cancer,including cervical cancer.It is used to identify efficient,safe and specific molecular targets,to reveal the specific molecular mechanism of drug resistance,and to provide effective individualized treatment scheme for cancer patients.ObjectiveBased on the second-generation sequencing technology and bioinformatics methods,combined with cell and animal experiments,this paper elucidates the regulatory role and possible mechanism of BRCA1,a core gene related to cisplatin sensitivity of cervical cancer cells,in order to provide a new theoretical basis and direction for improving the sensitivity of cervical cancer cells to cisplatin.Methods 1.Screening and analysis of cisplatin resistance related genes in cervical cancer(1)The transcripts of cisplatin resistant cervical cancer cell SiHa/DDP and normal cervical cancer cell SiHa were sequenced by the second generation sequencing technology,and the differential expression and enrichment analysis were carried out.The protein interaction network was constructed by using the differentially expressed genes related to cisplatin resistance,and the core genes were screened by the cytohubba plug-in in the Cytoscape software.(2)The expression of core genes in cisplatin resistant cervical cancer tissues was verified by ROC curve and box chart,and further verified by qPCR and Western blot in cisplatin resistant cervical cancer cells.2.The regulatory role of BRCA1 in cisplatin sensitivity of cervical cancer(1)The BRCA1 overexpression plasmids(ov-BRCA1)and knockdown plasmids(shBRCA1)were transfected into the cells by liposome transfection technology.The cell models of shBRCA1-Si Ha and ov-BRCA1-SiHa and shBRCA1-SiHa/DDP were constructed respectively.The effect of BRCA1 on cisplatin sensitivity of cervical cancer cells was detected by MTS.(2)Flow cytometry,Hoechst staining and Western blot were used to detect the effect of inhibition of BRCA1 expression on apoptosis of Si Ha/DDP and SiHa cells induced by cisplatin.(3)The low expression of BRCA1(shBRCA1-SiHa/DDP)and negative control(NC-SiHa/DDP)Si Ha/DDP cells were injected into the subcutaneous of mice to construct cervical cancer animal model,and cisplatin was given regularly to observe the weight change and tumor volume change of tumor bearing mice.3.Mechanism of BRCA1 inhibition in cisplatin sensitivity enhancement of cervical cancer(1)The transcripts of shBRCA1-SiHa/DDP and NC-SiHa/DDP cells were sequenced by the second generation sequencing technology,and the results were enriched and analyzed to explore the possible downstream pathway of BRCA1 regulating cisplatin sensitivity in cervical cancer.(2)Mito tempo,a mitochondrial specific reductant,was used to detect the changes of cell viability after ROS inhibition by MTS.(3)The effect of NF-?B pathway inhibitor on cisplatin induced apoptosis of Si Ha/DDP cells and the change of ROS content were detected by MTS and flow cytometry.(4)Western blot was used to detect the expression of NF-?B p65 and I?B? in shBRCA1-SiHa/DDP and NC-SiHa/DDP cells,SiHa/DDP and SiHa cells.(5)The paraffin embedded tumor blocks of cervical cancer mice were prepared,and the morphological changes of tumor tissues were observed by HE staining.Immunohistochemical staining was used to detect the expressions of BRCA1,NF-?B p65 and I?B? in tumor tissues of the two groups.Results 1.Screening and analysis of cisplatin resistance related genes in cervical cancer(1)Morphological observation and drug resistance index of cisplatin resistant cell line SiHa/DDP in cervical cancerUnder the light microscope,SiHa/DDP cells were spindle shaped and more slender after induction of drug resistance.Binucleate cells could be seen.The adhesion between cells disappeared and there was a clear intracellular blank.IC50 results showed that SiHa/DDP had obvious resistance to cisplatin,and the resistance index was 5.57 times(> 5 times).(2)The results showed that BRCA1 was closely related to cisplatin sensitivity in cervical cancer.The second-generation sequencing technology was used to sequence the transcripts of Si Ha/DDP and SiHa cells.A total of 526 differentially expressed genes related to cisplatin resistance were obtained,including 389 up-regulated genes and 137 down-regulated genes.On this basis,the protein interaction network was constructed,and 10 differential genes with the highest degree,including Myc,CDH1,CXCL10,oasl,BRCA1,CD274,CXCL11,hist1h2 ad,HLA-DRA,IFIT1,were selected as the core genes.By analyzing the expression profile of cisplatin resistant cervical cancer patients in geos56363,we found that only BRCA1 was highly expressed in cisplatin resistant cervical cancer patients,and had a high diagnostic value(AUC > 0.9).The results of qPCR and Western blot showed that the mRNA and protein levels of BRCA1 in Si Ha/DDP were significantly higher than those in SiHa cells.2.The regulatory role of BRCA1 in cisplatin sensitivity of cervical cancer(1)The model of BRCA1 overexpression and underexpression was successfully constructed to confirm the regulatory effect of BRCA1 on cisplatin sensitivity in cervical cancerThe results of qPCR and Western blot showed that shBRCA1-SiHa,ov-BRCA1-SiHa and shBRCA1-SiHa/DDP models were successfully constructed.MTS results showed that the sensitivity of BRCA1 cells in the low expression group was significantly higher than that in the negative control group and the blank control group,while the sensitivity of BRCA1 cells in the over expression group was significantly lower than that in the negative control group and the blank control group.(2)Inhibition of BRCA1 can promote cisplatin induced apoptosis of cervical cancer cellsBy flow cytometry,we found that the apoptosis rate of BRCA1 cells in the low expression group was significantly higher than that in the negative control group.Hoechst staining showed that SiHa and SiHa/DDP cells showed significant nuclear fragmentation and chromatin aggregation under the effect of cisplatin,and the degree of apoptosis in shBRCA1 group was higher than that in NC group.Western blot results showed that the expression of caspase 3 and Bax in shBRCA1-SiHa/DDP cells increased,while the expression of Bcl-2 decreased.(3)Effect of inhibition of BRCA1 expression on cisplatin sensitivity of cervical cancer in vivoThe antitumor effect of cisplatin on BRCA1 down regulated transplanted tumor mice(sh BRCA1)was significantly better than that of the control group(NC),indicating that the sensitivity of nude mice with low expression of BRCA1 to cisplatin increased.3.Mechanism of BRCA1 inhibition in cisplatin sensitivity enhancement of cervical cancer(1)Screening and enrichment of differentially expressed genes in shBRCA1-SiHa/DDP and NC-SiHa/DDP stable cellsBy sequencing the transcripts of shBRCA1-SiHa/DDP and NC-Si Ha/DDP cells,869 differentially expressed genes were found,including 254 up-regulated genes and 615 down-regulated genes.The results of KEGG pathway enrichment showed that the difference genes were mainly enriched in NF-?B signaling pathway,TNF signaling pathway,JAK-STAT signaling pathway,PI3 K Akt signaling pathway and other pathways related to drug resistance,and the enrichment degree of NF-?B signaling pathway was more significant.(2)Inhibition of NF-?B pathway can up regulate ROS level and enhance cisplatin induced apoptosis of Si Ha/DDP cellsThe results of MTS showed that the survival rate of SiHa/DDP cells treated with Mito tempo was significantly higher than that of the control group after 24 hours of cisplatin induction,and the survival rate of SiHa/DDP cells treated with NF-?B inhibitor combined with cisplatin was significantly lower than that of the control group after 24 hours of cisplatin induction.Flow cytometry showed that NF-?B inhibitor could significantly up regulate ROS content in SiHa/DDP cells.Western blot indicated that the expression of Caspase-3 and Bax in SiHa/DDP cells treated by cisplatin was increased,while the expression of Bcl-2 was decreased.(3)Inhibition of BRCA1 expression may enhance cisplatin sensitivity of cervical cancer cells by mediating NF-?B pathway inhibitionWestern blot results showed that compared with the control group,the expression of NF-?B p65 protein in the nucleus of shBRCA1-SiHa/DDP cells decreased significantly,the expression of NF-?B p65 protein in the cytoplasm increased significantly,and the expression of I?B? protein increased significantly.Meanwhile,the expression of NF-?B p65 in the nucleus of SiHa/DDP cells was higher than that of Si Ha cells,and the expression of I?B? was lower than that of SiHa cells.(4)Inhibition of BRCA1 expression on NF-?B pathway in vivoThe results of immunohistochemistry showed that the positive signal of NF-?B p65 was lower in the shBRCA1-SiHa/DDP group than in the negative control group,and the positive signal of I?B? was higher in the shBRCA1-SiHa/DDP group than in the negative control group.Conclusion1.Based on the second generation sequencing technology and bioinformatics method,BRCA1 was found to be the core molecule of cisplatin resistance regulatory network in cervical cancer,and up-regulated in cisplatin resistant tissues and cells.2.The down-regulation of BRCA1 is closely related to the increase of cisplatin sensitivity in cervical cancer.3.Inhibition of BRCA1 expression may enhance cisplatin sensitivity of cervical cancer by inhibiting NF-?B pathway activation and promoting cisplatin induced ROS accumulation. |
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