Study On The Effect Of Hsa_Circ₀054284 On The Biological Behavior Of NSCLC Cells And Its Mechanism

Background and purposeLung cancer is the greatest cancer in the world,its morbidity and mortality rank first,and the mortality rate is the highest among malignant tumors in China.According to the pathological classification of lung cancer,non-small cell lung cancer（NSCLC）accounts for a high proportion.The clinical treatment of lung cancer is mainly surgery combined with radiotherapy and chemotherapy in the early stage and conservative treatment in the late stage,but most of the clinical diagnosed cases are in the late stage,losing the opportunity of surgical operation,the prognosis of advanced lung cancer is very poor,and the 5-year survival rate is very low.The progress in the treatment of NSCLC benefits from a deeper understanding of the changes in the pathogenic genome of NSCLC,so it is necessary to explore the mechanism of NSCLC from the aspects of gene regulation and to find early diagnostic methods and therapeutic targets.Non-coding RNA plays an important role in tumorigenesis and development,forming complex regulatory networks.Circular RNA（CircRNAs）is a special non-coding RNA,that plays an important regulatory role in tumorigenesis in a variety of ways,affecting the occurrence and development of the disease.At this stage,we know that circular RNAs（CircRNAs）plays an important role in different types of cancer through sponge miRNAs（MicroRNAs）.However,the mechanism of their action in NSCLC is still unclear.In this study,we systematically studied the expression profile of CircRNA in NSCLC cancer tissues and matched paracancerous tissues.The results showed that the expression level of hsa_circ₀054284 in NSCLC tissues was significantly lower than that in adjacent normal tissues.Bioinformatics analysis showed that there was a base complementary pairing sequence that interacted with miR-18a-3p,which plays a regulatory role in many kinds of tumors.It is suggested that hsa_circ₀054284 may play an important role in NSCLC,but its specific role and mechanism in NSCLC have not been reported.In this study,we first confirmed the existence and low expression of hsa_circ₀054284 in NSCLC tissues and cells,and explored the effects of up-regulating the expression of hsa_circ₀054284 on the proliferation,invasion and apoptosis of NSCLC cells.Then the possible miRNA targets and downstream genes of hsa_circ₀054284 were detected by bioinformatics analysis and double luciferase reporter assay,and the effects of their interactions on the biological behavior of NSCLC cells were observed.To explore the role and mechanism of hsa_circ₀054284 in NSCLC,it is convenient to find a new way of early diagnosis and treatment.This paper is divided into two parts:the first part:to verify the existence form of hsa_circ₀054284 in NSCLC tissues and cell lines,to detect its expression level and to study its regulation on the proliferation,invasion and apoptosis of NSCLC cells;the second part:to explore the mechanism of hsa_circ₀054284 regulating the biological behavior of NSCLC cells.Part one:expression of hsa circ₀054284 in NSCLC and its regulation on cell proliferation,invasion and apoptosisMethod1.NSCLC tissue specimens and normal tissue specimens adjacent to cancer were collected according to the prescribed standards.2.Ordinary PCR and DNA sequencing confirmed the existence of hsa_circ₀054284 in NSCLC tissue samples and cell lines.3.The expression of hsa_circ₀054284 in 30 pairs of NSCLC tissues and normal tissues adjacent to cancer was detected by qRT-PCR.4.The overexpression vectors and unrelated sequences that up-regulated the expression of hsa_circ₀054284 were synthesized and transfected into NSCLC cell lines A549 and H520 with LipofectamineTM2000.The expression of hsa_circ₀054284 was detected by qRT-PCR.5.CCK-8 assay was used to detect the effect of up-regulation of hsa_circ₀054284 on the proliferation of A549 and H520 cells.6.Transwell assay was used to detect the effect of up-regulation of hsa_circ₀054284 on the invasive ability of A549 and H520 cells.7.The effect of up-regulation of hsa_circ₀054284 on apoptosis of A549 and H520 cells was detected by Flow cytometic assay.8.In order to study whether up-regulation of hsa_circ₀054284 could inhibit the proliferation of transplanted tumor in nude mice,the hsa_circ₀054284 lentivirus expression vector was constructed,and the hsa_circ₀054284 overexpression recombinant lentivirus was prepared and infected with A549 cells.Two groups of A549 cells（circ-EX,circ-NC）were injected subcutaneously into the dorsum of scapula of mice according to the number of 2 × 106.Two times a week for 3 weeks,the bioluminescence intensity of the transplanted tumor was detected by small animal imaging instrument,and the expression of Ki-67 in different treatments of the transplanted tumor in nude mice was detected by immunohistochemical staining,and the effect of hsa_circ₀054284 on the transplanted tumor in nude mice was evaluated.9.Detection of hsa_circ₀054284 expression in tumors of different treatment groups by qRT-PCR method.Result1.Hsa_circ₀054284 exists in NSCLC tissues and cells.The circular RNA is 375bp in length and is formed by the end-to-end connection of exon 13 and partial exon 14 of MTA3 gene.2.The expression of hsa_circ₀054284 in NSCLC was lower than that in paracancerous tissues,and lower than that in normal bronchial epithelial cells（NHBE）.3.CCK-8 results showed that the OD value of A549 and H520 cells decreased significantly in up-regulation of hsa_circ₀054284,circ-EX group,indicating that up-regulation of hsa_circ₀054284 could inhibit the proliferation of A549 and H520 cells.4.Transwell results showed that the number of transmembrane cells in up-regulated hsa_circ₀054284,circ-EX group was less,indicating that up-regulation of hsa_circ₀054284 could inhibit the invasive ability of A549 and H520 cells.5.Flow cytometry showed that the apoptosis rate in circ-EX group（29-42%）was significantly higher than that in circ-NC group（9-17%）（P<0.05）.The results showed that up-regulation of hsa_circ₀054284 could promote the apoptosis of A549 and H520 cells.6.the results of transplanted tumor in nude mice showed that the bioluminescence intensity of transplanted tumor decreased significantly in circ-EX group（P<0.05）.Immunohistochemical staining showed that the expression of Ki-67 in the cells of each group was consistent with the bioluminescence intensity of transplanted tumor,indicating that up-regulation of hsa_circ₀054284 could inhibit the growth of tumor in nude mice to some extent.Part two:the preliminary study on the mechanism of hsa circ 0054284Method1.Bioinformatics analysis of hsa_circ₀054284 sequences and prediction of related miRNA.2.Dual-Luciferase reporter assay confirmed that hsa_circ₀054284 and miR-18a-3p could interact with each other.3.Pearson correlation analysis was used to explore the relationship between intracellular miR-18a-3p and hsa_circ₀054284 expression levels.4.Detection of miR-18a-3p expression in NSCLC cells A549 and H520 after up-regulation of hsa_circ₀054284 by qRT-PCR method.5.MiR-18a-3p Inhibitor（miR-Inhibitor）and unrelated sequences（miR-NC）were synthesized and transfected into A549 and H520 cells with liposome LipofectamineTM2000.The expression of miR-18a-3p in each group was detected by qRT-PCR.6.To explore the effect of down-regulation of miR-18a-3p on the proliferation,invasion and apoptosis of NSCLC cells:the proliferation ability was detected by CCK-8,the invasion ability was detected by Transwell,and the apoptosis ability was detected by flow cytometry.7.Bioinformatics explores the target genes for predicting miR-18a-3p.Overlap primers were designed and wild type and mutant type target genes were amplified by PCR to construct recombinant reporter plasmids.Dual-Luciferase reporter assay was used to verify whether TIMP2 was the target of miR-18a-3p.The expression of TIMP2 protein was detected by Western blot.8.Transwell recovery test,synthetic si-TIMP2,designed different groups to treat A549 and H520,and Transwell test was used to detect the invasive ability of cells in each group.9.The data of each experiment were statistically analyzed with SPSS 25.0 software,and the difference was considered to be statistically significant when P<0.05.Result1.Bioinformatics analysis showed that there was a complementary binding region between hsa_circ₀054284 and miR-18a-3p.2.Dual-Luciferase reporter assay showed that the luciferase activity in miR-18a-3p mimic and pmirGLO-Wt circ₀054284 groups was significantly lower than that in the other three groups（P<0.05）.It is suggested that hsa circ₀054284 can act on the complementary binding region to regulate the expression of miR-18a-3p.3.The results of Pearson analysis showed that there was a negative correlation between the expression of hsa_circ₀054284 and miR-18a-3p mRNA（R2=0.431,P<0.05）.4.qRT-PCR results showed that overexpression of hsa_circ₀054284 in NSCLC cells decreased the expression of miR-18a-3p（P<0.05）.5.CCK-8 results showed that the OD value of miR-Inhibitor group was significantly lower than that of the other two groups,indicating that down-regulation of miR-18a-3p could inhibit the proliferation of A549 and H520 cells.6.Transwell results showed that the number of transmembrane cells in down-regulation miR-18a-3p,miR-Inhibitor group was less,indicating that down-regulation of miR-18a-3p could inhibit the invasive ability of A549 and H520 cells.7.Flow cytometry results showed that the apoptosis rate in miR-Inhibitor group（24-33%）was significantly higher than that in miR-NC group（11-18%）（P<0.05）.The results showed that down-regulation of miR-18a-3p could promote the apoptosis ability of A549 and H520 cells.8.There were three complementary binding regions between miR-18a-3p and target gene TIMP2 by bioinformatics analysis.9.Dual-Luciferase reporter assay showed that the first two complementary binding regions of miR-18a-3p and TIMP2 were significant,but the third complementary binding region seemed no significant difference.The luciferase activity of miR-18a-3p mimic and pmirGLO-Wtl/2 groups was significantly lower than that of other groups（P<0.05）.It is suggested that miR-18a-3p can regulate the expression of TIMP2 by acting on the first two complementary binding regions.10.The results of Western blot showed that the expression of TIMP2 protein decreased significantly when treated with miR-18a-3p mimic（P<0.05）,while the expression of TIMP2 protein in miR-18a-3p Inhibitor transfection group was significantly higher than that in miR-NC group（P<0.05）.The results showed that down-regulation of miR-18a-3p expression could up-regulate the expression of TIMP2 protein.11.The results of Transwell recovery assay showed that the invasion ability of cells treated with circ-EX alone was significantly inhibited,but the invasion inhibition ability of cells co-transfected with circ-EX and si-TIMP2 was not as strong as that of cells treated with circ-EX alone（P<0.05）.The results showed that si-TIMP2 could partially attenuate the effect of circ-EX on the target gene in the recovery experiment.Conclusions1.Hsa_circ₀054284 was found to be present and low expressed in NSCLC tissues and cells,and the annular RNA was full-length 375bp.2.Up-regulation of hsa_circ₀054284 can inhibit the proliferation and invasion of NSCLC cells and promote apoptosis.Down-regulation of miR-18a-3p can inhibit the proliferation and invasion of NSCLC cells and promote apoptosis.3.Hsa_circ₀054284/miR-18a-3p/TIMP2 axis may be one of the mechanisms by which hsa_circ₀054284 regulates NSCLC.4.Hsa_circ₀054284 plays an inhibitory role in cancer and may become a new therapeutic target.