The Effect Of The DNA Methylation Level Of LIM Homeobox Genes On The Development Of Cervical Cancer And Radiotherapy

Background and purpose of the study:Methylation of genome is an important factor which could affect onthe occurrence of tumor.Meanwhile radiotherapy could accurately thechange the DNA methylation state in cells.The use of genome-wide methylation high-through sequencing provides accurate and comprehensive informationon the methylation level ofgenome.In this experiment,DNA methylation level ofthewholegenomebefore and after radiotherapy in patients with cervical cancer was sequenced and analyzed,and genes significantly related to radiotherapy oncervical cancer were screened in combination with the clinical effects of radiotherapy.Among these DMG,the LHX2,LHX5 and LHX9 genes are related to the occurrence and development of cancer,unfortunately there are few studies on cervical cancer.The purpose of this study is to explore the occurrence,development,and function of LHX2,LHX5 and LHX9 with cervical cancer before and after radiotherapy.Method:1)Whole genome methylation sequencing analysis:BS-seq and differential DMR analysis on cervical cancer samples before and after radiotherapy,functional annotationwasperformed,and differential genes related to the prognosis of cervical cancer radiotherapy were screened.2)With the help ofBSP-PCR,qRT-PCR,WB,etc.to detect the genomic methylation level,mRNA expression level and protein level in cervical cancer tissues respectively andemploy Elisa to explore the expression level ofLHX2,LHX5 and LHX9 protein in the serum during radiotherapy.Combined with the clinical pathological characteristics,we could confirm the relationship between the methylation and multi-level expression characteristics of the target gene and the occurrence and development of cervical cancer.3)Change the methylation status and expression characteristics of the target gene in cervical cancer cell lines SiHa and C33A through radiotherapy,demethylation drugs,overexpression,silencing,etc.,and detect cell invasion,metastasis Growth,apoptosis characteristics,etc.,withone or in combination with different treatments,analyze the relationship between gene methylation and expression characteristics changes and cervical cancer.Result:1)The genome-wide DNA methylation profiles of three cervical cancer patients before and after radiation were constructed through BS-seq.mCG is the main method of genome methylation of the three patients;there are 2,666,2,873 and 1,411 differences DMR in the cancer tissues of the three patients methylated regions which were involving in 1,287,1,261 and 789 genes.2)Among the differential genes,LHX2,LHX5 and LHX9 showed the same methylation characteristics in patients who are sensitive to cervical cancer radiotherapy,and they are all hypomethylation after radiotherapy.3)Through the analysis of expression characteristics,it is found that the methylation level and gene expression level of LHX2,LHX5 and LHX9 could be used as marker genes to distinguish some FIGO stages.4)After 5-Aza-dC and radiotherapy treatment on SiHa and C33A cells,the methylation level oftarget gene was decreased,while theexpression levels of mRNA and protein wereincreased.The treated cervical cancer cells showed slower growth rate,decreased invasion and migration ability,as well as a significantapoptosis state.It was found that the expression level of invasion-related genes MMP2,MMP9,RacI decreased,and the expression level of apoptosis-related casp3,casp8 and casp12were up-regulated.5)After the construction of the target gene overexpression in cell line,it is found that the invasion,migration,and apoptosis functions of the LHX2-overexpression cell line are enhanced,showing a positive effect;while the LHX5 and LHX9-overexpression cell lines inhibit the invasion and metastasis of the cells,showing a negative direction influence.6)After the target gene expression is activated by radiotherapy and demethylation,siRNA is used to interfere the target gene to construct the target gene silencing cell line.The growth rate of the LHX2 gene silencing cell line increases,whilethe invasion and metastasis increase,and the apoptotic gene expression decreases.LHX2 is a cancer-promoting gene for cervical cancer.The LHX5 and LHX9 gene silencing cellshave opposite characteristics,which are genes that inhibit cervical cancer.Conclusion:Radiotherapy induced a decrease in methylation of the key genes LHX2,LHX5 and LHX9 of the LIM-homeobox family,and it was discovered that LHX2 is a cancer-promoting gene that inhibits cancer cell apoptosis in cervical cancer after radiotherapy.LHX5 and LHX9 are tumor suppressor genes,which are beneficial to the treatment of cervical cancer after radiotherapy is activated.