Screening And Verification Of Circular RNA Hsa_circ₀051488 And Its Biological Function And Mechanism In Lung Cancer Induced By Polycyclic Aromatic Hydrocarbons

Objective:Lung cancer is one of the fastest growing malignant tumors in China in recent years.Therefore,finding effective biological markers for early detection,diagnosis and treatment and prognosis evaluation is of great significance.Chromosome long arm 1region 3 band 3 subband 19q13.3 has been considered as a susceptible region for lung cancer.GWAS and epidemiological studies have shown that the genetic polymorphism and gene expression of 19q13.3 gene are closely related to the development and prognosis of lung cancer.This region contains three important genes,ERCC1,CD3EAP and PPP1R13L,which play important roles in cell DNA damage repair,cell proliferation and apoptosis,and play a role in the pathogenesis and progression of tumors represented by lung cancer.In recent years,non-coding RNA has become a hotspot in cancer research because of its important biological functions.Our previous studies have found that the ERCC1 gene located in the 19q13.3 region is mainly regulated by post-transcriptional regulation of microRNA,which is closely related to the occurrence and development of lung cancer.Another important member of the non-coding RNA family:circular RNA（CircRNA）has become one of the hotspots of tumor biomarkers because of its good stability in tissue and cells,tissue specificity and important tumor biological functions.It has been found that some circRNAs have been widely used as biomarkers affecting tumorigenesis and development.Whether 19q13.3,a susceptibility region of lung cancer,can produce circRNAs,whether these circRNAs are involved in the development of lung cancer,and its mechanism of action and as a good biomarker have not been reported so far.This study first screened the circRNAs produced in the 19q13.3 region by bioinformatics to detect whether it was expressed in lung cancer tissues and analyzed its association with clinical indicators of lung cancer.It was found that circRNA hsa_circ₀051488 is lowly expressed in lung squamous cell carcinoma tissues and cells and is associated with clinical indicators of poor lung cancer,and may have important biological functions and value as potential biomarkers.Therefore,in order to further explore whether hsa_circ₀051488 expression is related to the malignant degree of lung squamous cell carcinoma,clarify the mechanism by which hsa_circ₀051488 affects the biological characteristics of lung cancer and affect the development of lung cancer,and then the circRNA hsa_circ₀051488 originating from 19q13.3 as the inhibition of lung cancer.The biological markers of development provide scientific evidence.Methods:First,circRNAs generated from chromosome 19q13.3 region was screened according to the circRNA database（circBase）.Based on the expression of these circRNAs in lung adenocarcinoma cell line A549,a circRNA hsa_circ₀051488 highly expressed in A549 was screened.The circular RNA-specific primers were designed to detect hsa_circ₀051488 expression in normal lung epithelial cells 16HBE,lung squamous carcinoma cells LK₂ and A549.It was verified by RNase R hydrolysis linear RNA experiments whether hsa_circ₀051488 can resist the hydrolysis of exonuclease.Secondly,from October 2016 to October 2017,the study collected 64 pairs of cancer and paracancerous tissues from the First Clinical Hospital of China Medical University for the first time to diagnose lung cancer patients,and recorded the basic demographic characteristics of the patients.Smoking and drinking status and information on tumor-related cases.After RNA was extracted and reverse transcribed,the expression of hsa_circ₀051488 in tissues was detected by qPCR,and the difference of hsa_circ₀051488 expression in cancer tissues and adjacent tissues was analyzed.Subsequently,the relationship between hsa_circ₀05148 expression in tumor tissues and patient age,smoking and drinking,tumor size,lymph node metastasis and TNM staging was analyzed.Again,the carcinogen BPDE was used to induce malignant transformation of 16HBE cells,and soft agar colony formation and Transwell migration assays were used to determine the degree of malignancy.The nude mice were used to confirm the malignant phenotype of transformed cells,and HE staining and immunohistochemistry confirmed the transformed cells.Changes in the expression level of hsa_circ₀051488were detected during transformation and in the final transformed cells.The hsa_circ₀051488 overexpression plasmid was transfected into 16HBE-T and LK₂ cells after transformation,and its effect on tumor biological behavior was examined.Then,the subcellular localization of hsa_circ₀051488 was analyzed by nuclear extraction and RNA to determine its possible function.Downstream target microRNAs were predicted by bioinformatics analysis.The target microRNA was confirmed by detecting a correlation analysis of downstream microRNAs caused by raising or decreasing the expression of hsa_circ₀051488,a dual luciferase reporter assay,and a tumor tissue expression amount.The effect of miR-6717-5p expression on the biological behavior of16HBE-T and LK₂ tumor cells was examined.Finally,bioinformatics methods were used to predict the miR-6717-5p downstream target genes by strictly limiting the screening conditions on the microRNA target gene prediction sites TargetScan,DIANA and miRwalk.By detecting the amount of microRNA and mRNA expression of downstream targets in tumor tissues,it was analyzed whether there was a correlation between hsa_circ₀051488,miR-6717-5p and target gene SATB2.The hsa_circ₀051488overexpression plasmid and/or miR-6717-5p mimic was transfected in vitro to detect the effect on the mRNA and protein expression of the downstream target gene SATB2,and finally confirmed whether hsa_circ₀051488 has endogenous competitive RNA,ie ceRNA,adsorbed miR-6717-5p,in turn,regulates SATB2 and plays an important role in the development of lung cancer.Results:1.The CircBase database screens the circRNAs produced in the 19q13.3 region,and screens the highest expression of circRNA hsa_circ₀051488 in A549.By sequence alignment,Hsa_circ₀051488 was cyclized by exons 8 and 9 of the ERCC1 gene（transcript number:NM₀01983.3）.The specific back-splicing primers were designed.qPCR detection showed that hsa_circ₀051488 was relatively high in A549 compared with 16HBE cells,which was consistent with the database results and low expression in LK₂ cells（P<0.05）.After RNA was extracted from 16HBE cells,the linear RNA was hydrolyzed by RNase R.The mRNA expression of GAPDH was significantly lower than that of the undigested group（P<0.05）,while the expression of hsa_circ₀051488 was not different from that of the undigested group.It is indicated that hsa_circ₀051488 is not easily degraded by exonuclease and has a closed loop structure,which is a true circRNA.RNA was extracted from cancer and adjacent tissues of lung cancer patients,and the expression of hsa_circ₀051488 was detected.Hsa_circ₀051488 was found to be lowly expressed in cancer tissues compared with matched adjacent tissues（P<0.05）.Further analysis showed that the expression of hsa_circ₀051488 was not different between adenocarcinoma and adjacent tissues,but was low in squamous cell carcinoma（P<0.05）.The low expression of hsa_circ₀051488 in squamous cell carcinoma was associated with larger tumor size（P<0.05）,positive lymph node metastasis（P<0.05）and late TNM（P<0.05）,and was not associated with age,sex,smoking or drinking.Hsa_circ₀051488 is suggested as a biomarker for predicting the development and progression of lung squamous cell carcinoma.The treatment of 16HBE cells with different concentrations of BPDE for 24h showed that the IC₅₀ of BPDE on 16HBE cells was about 2.5μmol/L.The malignant transformation of 16HBE cells induced by BPDE at a concentration of 1μmol/L was selected according to the literature.In the induction phase of transformation,with the increase of the number of exposures,the IC50 of BPDE decreased slightly,that is,the sensitivity of cells to BPDE decreased slightly.During the transformation,compared with 16HBE cells,the formation ability of the soft agar clones of the transformed cells was gradually enhanced,the migration ability of Transwell was gradually enhanced,and the cell doubling time of the 40th passage cells was significantly shortened（P<0.05）.The transformed 16HBE-T cells can form tumors in nude mice,and by staining the tumor tissue,HE staining results suggest that the transformed cells have smaller abnormal karyotypes and differentiate into squamous cell carcinoma.The results of immunohistochemistry showed that the transformed squamous cell carcinoma-specific proteins CK HMW and P40 were strongly positive,and the adenocarcinoma-specific proteins CK LMW and CK-7 were weakly positive or negative,suggesting that the transformed cells belong to adenosquamous carcinoma but more inclined.In squamous cell carcinoma.During the malignant transformation,the expression of hsa_circ₀051488 fluctuated in the first 20 passages,and gradually decreased in the latter20 passages.Compared with 16HBE cells,the expression of hsa_circ₀051488 was significantly decreased in 16HBE-T cells（P<0.05）,consistent with the results of human squamous cell carcinoma.After overexpression of hsa_circ₀051488 in 16HBE-T and LK₂ cells,it was found that hsa_circ₀051488 overexpression inhibited cell relative proliferation（P<0.05）,soft agar colony forming ability,migration ability（P<0.05）and invasion ability（P<0.05）.After nucleoplasmic separation,RNA was extracted and the nuclear RNA marker U6,cytoplasmic RNA marker MTRNR1 and nuclear distribution GAPDH and hsa_circ₀051488 were detected in the two components.It was found that U6 was mainly distributed in the nucleus and MTRNR1 was mainly distributed in the cytoplasm.The distribution of GAPDH nucleus was high,indicating that the extraction system was highly reliable.At this time,hsa_circ₀051488 was mainly distributed in the cytoplasm（P<0.05）.Therefore,considering hsa_circ₀051488 has ceRNA function,it may act as a microRNA sponge to inhibit downstream targets by inhibiting microRNA.Genes work.The bioinformatics sites miRanda and RNAhybird were used to calculate the minimum free energy of hsa_circ₀051488 binding to microRNAs,and the possible binding of microRNAs was initially screened by setting the free energy<-20 kCal/mol.The hsa_circ₀051488 overexpression plasmid and siRNA were transfected in vitro,and the expression of downstream microRNAs was detected by changing the expression of hsa_circ₀051488.Finally,the expression of miR-6717-5p and hsa_circ₀05148 was opposite,which was possible to bind to hsa_circ₀051488 microRNA（P<0.05）.There was a negative correlation between miR-6717-5p and hsa_circ₀051488 expression in patients with lung squamous cell carcinoma（r=-0.681,P<0.05）.The dual luciferase reporter assay showed that miR-6717-5p reduced the luciferase activity of the hsa_circ₀051488 reporter gene（P<0.05）,indicating that miR-6717-5p binds to hsa_circ₀051488.Bioinformation prediction,in vitro transfection model,population sample expression correlation and dual luciferase reporter gene assays were combined to determine that hsa_circ₀051488 has ceRNA function,ie,binds to miR-6717-5p.In order to verify the function of miR-6717-5p,in vitro transfection of miR-6717-5p mimics enabled relative cell proliferation of 16HBE-T and LK₂ cells（P<0.05）,soft agar colony forming ability,and migration ability（P<0.05）and invasive ability were enhanced（P<0.05）,but this enhancement could be countered when hsa_circ₀051488 was overexpressed,again verifying that hsa_circ₀051488 may act as a ceRNA to inhibit miR-6717-5p.Bioinformatics was used to predict the target mRNA of miR-6717-5p.The results of TargetScan（score<-0.2）,DIANA and miRwalk（score>0.9）were initially screened and the intersections were taken and combined with literature reports that SATB2 is a possible target.mRNA.In squamous cell carcinoma,SATB2 expression was positively correlated with hsa_circ₀051488 expression（r=0.542,P<0.05）,and negatively correlated with miR-6717-5p expression（r=-0.531,P<0.05）.When hsa_circ₀051488 was overexpressed,SATB2 was up-regulated at both mRNA and protein levels（P<0.05）.When miR-6717-5p was overexpressed,SATB2 was down-regulated at both mRNA and protein levels（P<0.05）,and this down-regulation was offset by hsa_circ₀051488 overexpression.The above results further illustrate that hsa_circ₀051488 acts as a ceRNA,binds to and inhibits miR-6717-5p,and prevents it from inhibiting the downstream target gene SATB2.Conclusion:1.This study found that hsa_circ₀051488 originated from the lung cancer susceptible region 19q13.3,which was formed by cyclization of exons 8 and 9 of ERCC1gene and existed in cells.Low expression in lung squamous cell carcinoma,associated with longer tumor diameter,positive lymph node metastasis and advanced TNM.It suggests that hsa_circ₀051488 may be a valuable biomarker for predicting the development of lung cancer.2.This study successfully constructed 16HBE malignant transformed cells 16HBE-T induced by BPDE.The transformed cells have the characteristics of tumor cells,and the transformed cell pathological type tends to squamous cell carcinoma.The low expression of Hsa_circ₀051488 in malignant transformed cells is related to the degree of malignancy,suggesting that hsa_circ₀051488 is closely related to the occurrence and development of lung cancer.3.This study identified hsa_circ₀051488 subcellular localization,cell transfection model and the expression of squamous cell carcinoma,revealing that hsa_circ₀051488 has endogenous competitive RNA,ie ceRNA function,ie,miR-6717-5p,as miR-6717-5p sponge inhibits its function and prevents its inhibition of the downstream target gene SATB2,thereby inhibiting cell proliferation,migration and invasion.Hsa_circ₀051488affects the malignant biological behavior of lung squamous cell carcinoma by affecting the regulation of miR-6717-5 on SATB2,and participates in the development of lung squamous cell carcinoma.