Hsa＿circ＿0072088/miR-1270/TOP2A Axis Regulates The Molecular Mechanism Of Non-small Cell Lung Cancer And The Relationship Between Its Associated Polymorphisms And Susceptibility To Non-small Cell Lung Cancer

Objective: According to the 2020 statistics of the International Agency for Research on Cancer,lung cancer ranks the second in new malignant tumors.Among the new death cases of cancer,lung cancer is the main cause of cancer death.Because the incidence of lung cancer is relatively hidden and not easy to be detected by patients,it affects the diagnosis and treatment of lung cancer.The exploration of early diagnosis methods of lung cancer has also been enriched and improved with the development of science and technology.The in-depth exploration of biomarkers related to lung cancer has important clinical value and significance in the auxiliary diagnosis,curative effect monitoring,prognosis evaluation and follow-up of lung cancer.As a special class of non-coding RNA,circ RNA has become a new hotspot in the field of biomarker research because of its unique biological characteristics: abundance,conservation,stability and specificity.With the deepening of molecular research,the mechanism of circ RNA has been gradually revealed.Its main biological functions include the regulation of transcription,the competitive endogenous RNA mechanism,and the interaction mechanism with RNA-binding proteins.One of the more extensive mechanisms is the competitive endogenous RNA mechanism of circ RNA.By exploring the mechanism of circ RNA,we can provide important clues for the diagnosis,progression and prognosis evaluation of cancer.Circ RNA is the product of selective splicing.To explore the relationship between the single nucleotide polymorphisms with the splicing function from the circ RNA-derived m RNA and disease,which provides a way to find disease-related SNPs from massive SNPs.Moreover,the samples needed for SNP detection are mainly peripheral blood.Because it is easier to obtain,SNP is a crucial marker for clinical early diagnosis and treatment.Therefore,this study identified the circ RNAs related to non-small cell lung cancer from a large number of circ RNAs obtained by gene sequencing technology in the GEO public database through bioinformatic analysis,combined with the differentially expressed m RNAs in non-small cell lung cancer in the TCGA database,predicted the potential target binding relationship through the online prediction database,and constructed and verified the potential competitive endogenous RNA network.Moreover,the exploration of SNP with splicing function of its source m RNA provides important clues for early diagnosis and early treatment marker of non-small cell lung cancer.Methods: The first part of the study is to search the GEO database for the research related to the expression of circ RNA in non-small cell lung cancer,and use R software to carry out the statistic analysis.In order to obtain differentially expressed circ RNA more accurately,this study conducted bioinformatics analysis through two analysis methods.The first method is to perform differential analysis on the expression matrix through the "limma" package of R software.The second method is to construct a weighted gene coexpression network,and obtain the circ RNA of this study by intersection of the different circ RNAs obtained by the two methods.Apply online prediction databases to explore the potential ce RNA network of circ RNA.By exploring the differentially expressed m RNAs in the TCGA database,we obtained the differentially expressed m RNAs in lung adenocarcinoma and lung squamous cell carcinoma.The highly differentially expressed m RNAs in lung adenocarcinoma and lung squamous cell carcinoma were intersected with the predicted target genes in the online prediction databases,and the m RNA with the largest difference in the intersection was selected as the target gene for this study,and verified the expression in lung cancer and adjacent tissues.Further use the method of systematic evaluation to explore the relationship between the expression of circ＿0072088 and the prognosis of pan-carcinoma,systematically search the relevant literature on the relationship between circ＿0072088 and the prognosis and clinical characteristics of cancer patients in the Chinese and English databases,and extract the relevant data after evaluating and screening the literature,and conduct a meta-analysis of the relationship between circ＿0072088 and the prognosis and clinical characteristics of pan-carcinoma.The second part of the study was carried out around the validation of competitive endogenous RNA network obtained in the first part.First,q RT-PCR was used to detect the differential expression of circ＿0072088 obtained by bioinformatic analysis in the first part in non-small cell lung cancer cells.Knockdown the expression of circ＿0072088,and use MTS cell proliferation test,Transwell cell migration test and cell apoptosis test to explore the effect of circ＿0072088 on proliferation,migration and apoptosis of A549 cell and SKMES-1 cell.The expression of mi R-1270 was detected in non-small cell lung cancer cells.Detect the expression of mi R-1270 after knocking down circ＿0072088.The overexpression model of mi R-1270 was constructed.The regulation of mi R-1270 on the proliferation,migration and apoptosis of A549 cell and SK-MES-1 cell was investigated by MTS,Transwell and apoptosis experiments.Further verify the regulatory effect of circ＿0072088 on the biological function of lung adenocarcinoma cell and lung squamous cell carcinoma cell by acting on mi R-1270,through rescue experiment.The regulation of mi R-1270 overexpression on TOP2 A m RNA expression was detected by q RT-PCR.The regulatory effect of circ＿0072088 and mi R-1270 on the protein expression of TOP2 A was detected by Western blot.Finally,the targeted binding relationship between circ＿0072088and mi R-1270 and between mi R-1270 and TOP2 A was verified by double luciferase report experiment.The third part: circ＿0072088 is derived from the selective splicing of ZFR precursor RNA(pre-m RNA).By studying SNPs with splicing and other functions on ZFR,it provides a basis for screening SNPs related to susceptibility to non-small cell lung cancer.This part of the study is a hospital-based case-control study involving 391 patients with non-small cell lung cancer and 391 healthy controls.All the subjects were non-smoking women.Genotyping was performed using Taqman probes by extracting DNA from the peripheral blood of the study subjects.In this study,SPSS software was used for statistical analysis,and unconditional logistic regression was used to analyze the association between ZFR single nucleotide polymorphism and non small cell lung cancer.The interaction between genotype and environment was explored by combining the interaction evaluation index relative excess risk due to interaction(RERI),attributable proportion of interaction(AP),and synergy index(SI)proposed by Anderson et al.Results: In the first part,three data sets:GSE101586,GSE101684,and GSE112214 were obtained by searching the GEO database,including 12 lung cancer tissue samples and 12 matched paracancerous control samples,which can be used to analyze differentially expressed circ RNA in non small cell lung cancer.Intersect the difference analysis results of "limma" package with the analysis results of gene co-expression network to obtain hsa＿circ RNA＿103809(hsa＿circ＿0072088).The potential ce RNA mechanism was predicted through the online prediction databases,and intersected with the first 100 m RNAs with high differential expression in lung adenocarcinoma and lung squamous cell carcinoma obtained from the difference analysis in the TCGA database.The TOP2 A gene with large difference was selected as the target gene for this study.In this study,29 lung cancer tissue samples were obtained from lung cancer patients attending Shengjing Hospital affiliated to China Medical University.Further validation in lung cancer tissues and its adjacent tissues revealed that TOP2 A was differentially overexpressed in lung cancer tissues.Therefore,the ce RNA network of this study is determined as circ＿0072088/mi R-1270/TOP2 A.According to the inclusion and exclusion criteria of the literature,20 articles were retrieved for meta-analysis,including 13 articles containing information related to prognosis and 14 articles containing information related to clinical characteristics.After the combined analysis of effect values,it was found that the high expression of circ＿0072088 was associated with poor prognosis of patients(HR and its 95% confidence interval: 2.067,1.558-2.742;P=0.000),and the high expression of circ＿0072088 was associated with lymph node metastasis of patients(OR and its 95% confidence interval:3.025,2.028-4.512;P=0.000).In the second part,compared with the normal lung epithelial cell BEAS-2B,circ＿0072088 was found to be highly expressed in lung adenocarcinoma cell A549 and lung squamous cell carcinoma cell SK-MES-1(A549:P<0.01;SK-MES-1:P<0.01).In its functional experiments,knockdown circ＿0072088 has the effect of inhibiting the proliferation and migration of A549 cell and SK-MES-1 cell and promoting cell apoptosis.The experiment found that mi R-1270 was differentially low expressed in A549 cell and SK-MES-1 cell(A549:P<0.001;SK-MES-1:P<0.001),and overexpression of mi R-1270 could inhibit cell proliferation and migration and promote cell apoptosis.The experiment found that the expression of mi R-1270 increased after knocking down circ＿0072088(A549:P<0.001;SK-MES-1:P<0.01),and further verified that circ＿0072088can regulate the biological function of cell by affecting mi R-1270 through the rescue experiment.In addition,the experiment found that the expression of TOP2 A gene decreased after the overexpression of mi R-1270(A549:P<0.001;SK-MES-1:P<0.001),and the expression of TOP2 A protein increased after the overexpression of circ＿0072088(A549:P<0.05;SK-MES-1:P<0.01),while the expression of TOP2 A protein decreased after the overexpression of mi R-1270(A549:P<0.001;SK-MES-1:P<0.001).Finally,the target-binding relationship between circ＿0072088 and mi R-1270 and between mi R-1270 and TOP2 A was verified by double luciferase report experiment.In the third part,we found rs1051489 with splicing function of ZFR gene is associated with the susceptibility of non-small cell lung cancer.Compared with AA genotype,GG genotype is a protective factor for non-small cell lung cancer(age adjusted OR is 0.512,95% confidence interval is 0.273-0.960,P value is 0.037),and its recessive model also plays a protective role in non-small cell lung cancer(age adjusted OR is 0.473,95%confidence interval is 0.255-0.877,P value is 0.018).In the stratified analysis of age,it was found that when the age is more than 60 years old,the rs4867440 single nucleotide polymorphism is associated with the susceptibility of non-small cell lung cancer.The dominant model is a protective factor for non-small cell lung cancer(OR:0.572,95%confidence interval 0.333-0.982,P=0.043),and compared to the T genotype,the C genotype is a protective factor for non-small cell lung cancer(OR:0.618,95% confidence interval 0.385-0.991,P=0.046).In the analysis of the interaction between ZFR single nucleotide polymorphisms and oil exposure and the susceptibility to non-small cell lung cancer,crossover analysis found that in the rs4867440,the risk of non-small cell lung cancer in the CT+CC genotype oil exposed individuals(P=0.042)and the TT genotype oil exposed individuals(P=0.001)was higher than the CT+CC genotype non-oil exposed individuals.In the analysis of rs1051489,rs430522 and rs368410,it was found that the risk of non-small cell lung cancer in high-risk genotype oil exposed individuals was higher than that in protective genotype non-oil exposed individuals(rs1051489: P=0.015;rs430522: P=0.005;rs368410: P=0.002).Conclusion: 1.Circ＿0072088 is highly differentially expressed in non-small cell lung cancer.Knocking down circ＿0072088 has the biological function of inhibiting the proliferation and migration of non-small cell lung cancer cell and promoting cell apoptosis.2.The high expression of circ＿0072088 is related to the poor prognosis and lymph node metastasis of cancer patients,and can be used as a biomarker for the prognosis and lymph node metastasis evaluation of cancer patients.3.Circ＿0072088 can affect the progression of non-small cell lung cancer through circ＿0072088/mi R-1270/TOP2 A axis.4.The SNP site rs1051489 with splicing function on ZFR gene is associated with susceptibility to nonsmall cell lung cancer.