| Background:At present,lung cancer is one of the most common malignant tumors in the world.Its incidence rate ranks second in the total number of malignant tumors in the world,but its death case ranks first in the global death case of malignant tumors.Based on pathological classification,lung cancer is primarily categorized into two major types: small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).NSCLC comprises over 85% of all lung cancer cases.Most NSCLCs are already in the middle and late stages of the tumor upon initial detection,losing the optimal surgical opportunity.In recent years,although there has been significant progress in early screening and treatment of NSCLC,the 5-year survival rate of NSCLC patients is still less than 20%.The main reasons for this phenomenon are twofold: firstly,most NSCLC patients are already in the middle and late stages of the disease at the time of discovery,losing the best surgical opportunity;The second reason is that during the treatment process,tumor cells develop resistance to chemotherapy drugs,leading to the failure of clinical treatment.Therefore,exploring the molecular mechanisms underlying the occurrence and development of NSCLC and chemotherapy drug resistance,as well as finding new effective biomarkers for the treatment of NSCLC,is of great significance.In recent years,circular RNAs(circRNAs)have gradually become a focus of cancer research.CircRNAs are a type of non coding RNA with a special closed circular structure,formed by reverse splicing of precursor m RNA.They have characteristics such as diversity,high abundance,high stability,conservation,and cell tissue specificity.Numerous studies have shown that changes in circRNAs expression profile is closely related to various diseases,especially malignant tumors and tumor resistance.Therefore,differentially expressed circRNAs may serve as new tumor diagnostic and prognostic bio-markers,and may also become new therapeutic targets for malignant tumors.Objective:1.Perform high-throughput RNA sequencing on surgical samples of NSCLC patients(tumor tissue and adjacent normal tissue)to screen for differentially expressed circRNAs between the two,and analyze their potential biological functions in NSCLC through bioinformatics.2.Through in vitro experiments,explore the mechanism by which hsa＿circ＿0001098 participates in regulating the occurrence and development of NSCLC and the occurrence of cisplatin resistance,in order to reveal its potential as a diagnostic biomarker for NSCLC,and provide new clues for targeted treatment of NSCLC,especially for cisplatin resistant patients.Methods:1.Clarify the differentially expressed circRNAs in non-small cell lung cancer(1)Collect tumor tissues and adjacent normal tissues from NSCLC patients,and screen for different expression of circRNAs in NSCLC through high-throughput RNA sequencing and bioinformatics analysis;(2)Predicting the potential biological functions of circRNAs differentially expressed in NSCLC and the signaling pathways they may participate in regulation through GO and KEGG analysis;(3)Randomly select 3 downregulated circRNAs and 3 upregulated circRNAs,and validate the high-throughput sequencing results through RT-qPCR exprements.2.Exploring the pro-cancer effect of hsa＿circ＿0001098 in non-small cell lung cancer(1)Using RT-qPCR experiments to detect the relative expression levels of hsa＿circ＿0001098 in tumor tissues and adjacent normal tissues of 23 pairs of NSCLC patients,normal bronchial epithelial cells BEAS-2B,human lung adenocarcinoma cell lines(A549,H1299),and human lung squamous cell carcinoma cell lines(SK-MES-1).(2)Using RNase R digestion experiments to identify the circular structure of hsa＿circ＿0001098.(3)Using actinomycin D experiments to test the stability of the circular structure of hsa＿circ＿0001098.(4)Transfect the overexpression plasmid hsa＿circ＿0001098 and si-hsa＿circ＿0001098 to construct high and low expression models of hsa＿circ＿0001098.Useing RT-qPCR experiments to detect the transfection efficiency of hsa＿circ＿0001098.(5)Using CCK8 experiments and clone formation assays to detect the regulatory effect of hsa＿circ＿0001098 on NSCLC cell proliferation;Using wound healing assays and Transwell experiments to detect the regulatory effect of hsa＿circ＿0001098 on NSCLC cell migration.(6)Using Western Blot experiments to detect apoptosis-related proteins,including SOX2,TRAIL,Caspase-3,Caspase-9,Bax,Bcl-2,Cleaved-Caspase-3,Cleaved-Caspase-9 etc.(7)Using Western Blot experiments to detect cell proliferation-related proteins,including Cdc25 B,Cyclin B1,Cyclin B2,CDK1,Cyclin D1,etc.3.Hsa＿circ＿0001098 promotes the occurrence and development of non-small cell lung cancer by targeting miR-129-5p/SOX2(1)Predicting the target micro RNA of hsa＿circ＿0001098 through bioinformatics analysis,and verifying their direct targeting effect through dual luciferase reporter assays.(2)Using RT-qPCR experiments to detect the regulatory effect of hsa＿circ＿0001098 on miR-129-5p expression.(3)Using RT-qPCR experiments to detect the relative expression levels of miR-129-5p in normal bronchial epithelial cells and NSCLC cells.(4)Using CCK8 experiments and clone formation assays to detect the proliferation ability of NSCLC cells regulated by hsa＿circ＿0001098 targeting miR-129-5p.(5)Using wound healing assays and Transwell experiments to detect the migration and invasion ability of NSCLC cells regulated by targeting miR-129-5p by hsa＿circ＿0001098.(6)Predicting the target m RNA of miR-129-5p through bioinformatics analysis,and verifying their direct targeting effect through dual luciferase reporter assays.(7)Using Western Blot experiments to detect the expression level of SOX2 in NSCLC cells.(8)Using Western Blot experiments to detect the regulatory effect of miR-129-5p on SOX2 expression.(9)Using CCK8 experiments and clone formation assays to detect the proliferation ability of miR-129-5p in NSCLC cells regulated by targeting SOX2.(10)Using wound healing assays and Transwell experiments to detect the migration and invasion ability of NSCLC cells regulated by miR-129-5p targeting SOX2.4.Hsa＿circ＿0001098 regulates the occurrence,development,and cisplatin resistance of non-small cell lung cancer by targeting the miR-129-5p/SOX2 axis(1)Using Western Blot experiments to detect apoptosis-related proteins,including SOX2,TRAIL,Caspase-3,Caspase-9,Bax,Bcl-2,Cleared Caspase-3,Cleared Caspase-9,etc.(2)Using Western Blot experiments to detect cell proliferation-related proteins,including Cdc25 B,Cyclin B1,Cyclin B2,CDK1,Cyclin D1,etc.(3)Using CCK8 experiments to verify the resistance of cisplatin resistant NSCLC cells(A549/DDP).(4)Using RT-qPCR experiments to detect the expression level of hsa＿circ＿0001098 in cisplatin resistant NSCLC cells(A549/DDP)and their parental cells(A549).(5)Using CCK8 experiments to verify that hsa＿circ＿0001098 is involved in regulating the cisplatin sensitivity of NSCLC cells.(6)Using Flow cytometry to detect the effect of silencing hsa＿circ＿0001098 on the sensitivity of NSCLC cells to cisplatin.Results:1.Identification of DE circRNAs in non-small cell lung cancer(1)We collected tumor tissue and adjacent normal tissue from three non-small cell lung cancer patients who underwent surgery.Through high-throughput RNA sequencing,we detected 241 significantly differentially expressed circRNAs,of which 85 were upregulated and 156 were regulated.(2)GO and KEGG analysis predicted DE circRNAs,suggesting that these differentially expressed circRNAs may be involved in regulating enzyme activities such as histidine lysine methyltransferase,protein lysine methyltransferase,s-adenosylmethionine dependent methyltransferase,biological processes such as immunoglobulin binding,purine and purine nucleotide binding,as well as regulating m TOR,JAK-STAT,PI3K-Akt signaling pathway,etc.(3)Three downregulated circRNAs and three upregulated circRNAs were selected,and qPCR primers were designed for RT qPCR validation.The results showed that the RT qPCR results were consistent with the high-throughput RNA sequencing results,confirming the reliability of high-throughput RNA sequencing.2.Hsa＿circ＿0001098 plays a pro-cancer role in non-small cell lung cancer(1)The results RT-qPCR experiments showed that hsa＿circ＿0001098 was up-regulated in NSCLC tumor tissues and cells.(2)The results of RNase R digestion experiments confirmed the circular structure of hsa＿circ＿0001098.(3)The results of actinomycin D experiments confirmed that the stability of hsa＿circ＿0001098 is significantly higher than its parent gene BARD1 linear m RNA.(4)The results of CCK8 experiments showed that after hsa＿circ＿0001098konckdown,the cell viability of A549 decreased,while hsa＿circ＿0001098overexpression increased the cell viability of A549;The results of clone formation assays showed that after hsa＿circ＿0001098 konckdown,the number of clone formation in A549 cells decreased;After hsa＿circ＿0001098 overexpression,the number of clone formation in A549 cells increased,indicating that hsa＿circ＿0001098could promote the proliferation of NSCLC.(5)The results of wound healing assays showed that after hsa＿circ＿0001098knockdown,the scratch width of A549 cells increased;after hsa＿circ＿0001098overexpression,the scratch width of A549 cells decreased,indicating that hsa＿circ＿0001098 could promote the migration of NSCLC cells;The results of Transwell experiments showed that after hsa＿circ＿0001098 knockdown,the number of migrating cells passing through the matrix gel was significantly reduced;after hsa＿circ＿0001098 overexpression,the number of migrating cells passing through the matrix gel significantly increased,indicating that hsa＿circ＿0001098 could promote the invasion of NSCLC.(6)The results of Western Blot experiments that hsa＿circ＿0001098 knockdown could upregulate pro-apoptotic proteins and downregulate anti-apoptotic proteins;hsa＿circ＿0001098 overexpression could downregulate pro-apoptotic proteins and upregulate anti-apoptotic proteins.(7)The results of Western Blot experiments showed that hsa＿circ＿0001098knockdown can downregulate cell proliferation-related proteins;hsa＿circ＿0001098overexpression could upregulate cell proliferation-related proteins.3.Hsa＿circ＿0001098 promotes the occurrence and development of non-small cell lung cancer by targeting miR-129-5p/SOX2.(1)Bioinformatics analysis predicted that the target micro RNA of hsa＿circ＿0001098 is miR-129-5p,and then confirmed through dual luciferase reporter assays that the two can directly bind.(2)The results of RT-qPCR experiments showed that miR-129-5p was down-regulated in NSCLC,over-expression of hsa＿circ＿0001098 could inhibit its expression,while knockdown hsa＿circ＿0001098 could promote its expression.(3)The results of CCK8 experiments and clone formation assays showed that miR-129-5p can reverse the effect of hsa＿circ＿0001098 on the proliferation ability of NSCLC cells.(4)The results of wound healing assays showed that miR-129-5p can reverse the effect of hsa＿circ＿0001098 on the migration ability of NSCLC cells;The results of Transwell experiments showed that miR-129-5p can reverse the effect of hsa＿circ＿0001098 on the invasive ability of NSCLC cells.(5)Bioinformatics analysis predicted that the target m RNA of miR-129-5p is SOX2,and then confirmed through dual luciferase reporter assays that the two can directly bind.(6)The results of Western blot experiments showed that SOX2 is highly expressed in NSCLC cells.(7)The results of Western Blot experiments showed that in NSCLC cells,knockdown miR-129-5p could promote SOX2 expression,while over-expression miR-129-5p could inhibit SOX2 expression.(8)The results of CCK8 experiments and clone formation assays showed that miR-129-5p can reverse the effect of SOX2 on the proliferation ability of NSCLC cells.(9)The results of wound healing assays showed that miR-129-5p can reverse the effect of SOX2 on the migration ability of NSCLC cells;The results of Transwell experiments showed that miR-129-5p can reverse the effect of SOX2 on the invasive ability of NSCLC cells.4.Hsa＿circ＿0001098 regulates the occurrence,development,and cisplatin resistance of non-small cell lung cancer through the miR-129-5p/SOX2 axis(1)The results of Western Blot experiments showed that miR-129-5p can reverse the effect of hsa＿circ＿0001098 on apoptosis-related proteins(SOX2,TRAIL,Caspase-3,Caspase-9,Bax,Bcl-2,Cleaved-Caspase-3,Cleaved-Caspase-9)and proliferation-related proteins(Cdc5B,Cyclin B1,Cyclin B2,CDK1,Cyclin D1).(3)The results of CCK8 experiments showed that A549/DDP cells have cisplatin resistance.(4)The results of the RT-qPCR experiments showed that hsa＿circ＿0001098expression was upregulated in A549/DDP cells.(5)The results of CCK8 experiments confirmed that knockdown hsa＿circ＿0001098 can increase the cisplatin sensitivity of A549/DDP cells,while overexpression of hsa＿circ:0001098 can reduce the cisplatin sensitivity of A549/DDP cells.(6)The results of flow cytometry showed that knockdown hsa＿circ＿0001098 can increase the sensitivity of A549/DDP cells to cisplatin.Conclusions:1.This study screened 241 differentially expressed circRNAs(85 upregulated and 156 downregulated)from NSCLC surgical samples,and analyzed the potential biological functions of these differentially expressed circRNAs through bioinformatics analysis.2.Hsa＿circ＿0001098 is upregulated in both NSCLC tumor tissue and cells.The overexpression of hsa＿circ＿0001098 can promote the proliferation,migration,and invasion of NSCLC cells,inhibit cell apoptosis,and further research has found that hsa＿circ＿0001098 exerts its biological function by targeting the miR-129-5p/SOX2 axis.3.Hsa＿circ＿0001098 is involved in regulating the occurrence of cisplatin resistance in NSCLC cells.Silencing hsa＿circ＿0001098 can increase the sensitivity of NSCLC cells to cisplatin,suggesting that hsa＿circ＿0001098 may become a new targeted therapeutic target for NSCLC. |
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