The Molecular Mechanism By Which MiR-326 Mediates The SIRT1/HIF1α/VEGFA Axis To Improve Chemotherapy Resistance In Non-small Cell Lung Cancer

Background/Aim: Lung cancer is one of the fastest growing tumors in the world in morbidity and mortality.LUAD is the leading cause of cancer-related death,with killing nearly 2 million people worldwide each year.Non-small cell lung cancer(NSCLC)accounts for 80% of lung cancers.With the progress of the aging of the world population,the change of the environment,the sharp increase of social and psychological pressure,the incidence and death rate of lung cancer are increasing year by year,and the trend is getting younger.Currently,there are many therapeutic options available in clinical practice,such as surgery,radiotherapy,chemotherapy,including targeted therapy and immunotherapy.Many targets have been explored at the genetic level of lung cancer,but there is still a large space to be explored.Chemotherapy remains the cornerstone of lung cancer treatment.Recent studies have shown that miRNAs have shown potential efficacy in the treatment of lung cancer.In the aspect of epigenetics,miRNAs may affect and participate in the occurrence and development of lung cancer by regulating histones.SIRT1 is a class III histone deacetylase(HDAC)that regulates many proteins involved in chromatin structure,apoptosis,autophagy,and mitochondrial transcriptional regulation.It is deeply involved in gene regulation,genomic stability maintenance,apoptosis,autophagy,proliferation,senescence and tumorigenesis,and plays a key role in multiple aspects of cancer drug resistance.It has been reported that targeting SIRT1 activity or gene expression may be one of the new strategies for the treatment of lung cancer.HIF1α(hypoxia-inducible factor 1α)has been recognized as an important anticancer drug target.HIF1α is strongly associated with tumor metastasis,angiogenesis,poor prognosis in patients,and the treatment of tumor resistance.The high redundancy and complexity of tumor angiogenesis driven by VEGFA(vascular endothelial growth factor-A)may explain why tumors often develop resistance to anti-angiogenic therapies that target VEGFA signal transduction.VEGFA is produced by most cells in the body but is upregulated in response to hypoxia.Methods:Bioinformatics analysis: Dig through the GEO database(https://www.ncbi.nlm.nih.gov/geo/)retrieval express chip GSE51853 miRNAs related lung cancer,using R language "limma" package,set | log FC | & gt;1,p value<0.05 was the threshold for difference analysis.Using the Star Base database(http://starbase.sysu.edu.cn/),RNAInter database(http://www.rna-society.org/rnainter/)and miRanda(http://www.micr Orna.org/microrna/home.do)to predict miRNA target genes,the three sites using different algorithms to take three predicted results of the intersection to increase the credibility of the results.The miRNA-targeted genes were predicted through the miRNA.org database.By UCSC(http://genome.ucsc.edu/)and JASPAR two website(http://jaspar.genereg.net/)analysis HIF1 alpha protein in VEGFA promoter could be binding sites;Propose theoretical hypotheses.Cytological experiments: Human lung cancer cell line H460 and human embryonic kidney cell line HEK293 were purchased from the Typical Cultures Preservation Committee Cell Bank,Chinese Academy of Sciences(Shanghai,China).H460 cells were cultured in RPMI-1640+10% FBS medium,and HEK293 was cultured in DMEM+10%FBS medium.Both cells were placed in an incubator at 37℃ and 5% CO2.Chemical-resistant cells H460-R were developed from the parent cell line H460 by gradient exposure of the cells with the chemotherapeutic drug DDP(cisplatin)from 0.05 μg/ m L to 2μg/ mL for about 12 months to obtain chemical resistance.Plasmid and siRNAs transfection:MiR-326 mimic,miR-326 inhibitor,overexpression of SIRT1(eo-SIRT1),anti-HIF1α(HIF1α)Si RNA,vascular endothelial growth factor A(VEGFA)si RNA and negative control(NCS)were synthesized by Gene Chem Co.,Ltd.(Shanghai,China).Cells were inoculated overnight in a 6-well plate and mixed with 2 μg plasmid with X-treme Gene HP DNA transfection reagent(Roche Applied Science,Basel,Switzerland).According to the manufacturer’s instructions,the cells were transfected with the above plasmid using a Lipofectaminet M RNAIMAX transfection reagent(Thermo Fisher Scientific,Waltham,MA,USA).Cells were collected 48 hours after transfection for subsequent experiments.The expression of miR-326 and SIRT1 in non-small cell lung cancer cell line H460 and its drug-resistant cell line H460-R was detected by q RT-PCR assay.The dual luciferase reporter assay and RIP assay were used to verify and detect the binding and regulatory relationship between miR-326 and SIRT1,and SIRT1 inhibited HIF1α degradation through deacetylation.The regulatory relationship between SIRT1 and HIF1α was verified by Ch IP assay and q RT-PCR from multiple angles.The binding of transcription factor HIF1α to VEGFA promoter site was detected by Ch IP assay and double luciferase truncation assay.MTS method and flow cytometry were used to detect cell viability and apoptosis,respectively,to determine the improvement of drug resistance of H460-R cells.The protein expressions of SIRT1,HIF1α,VEGFA,C-PARP1,C-Caspase-3 and γ-H2 AX in the cells were analyzed by Western blot.The positive expression of γ-H2 AX was detected by luciferase.The survival and apoptosis of drug-resistant cells were monitored by MTS and flow cytometry.Animal experiment: Tumor formation in nude mice,BALB/c nude mice(NU/NU,female,4-6 weeks,n = 8 per group),transfected subcutaneous injection: NC Agomir group,miR-326 Agomir group,NC Agomir +DDP group,miR-326 Agomir +DDP group,all mice maintained life activities without pathogens;After one week,the mice were treated with DDP(3.0mg/kg body weight)every 3 days,and the tumor volume was measured for 4 weeks.Evaluate its tumorigenesis ability and evaluate the improvement of drug resistance.Results:1.MiR-326 was differentially expressed in non-small cell lung cancer;MiR-326 binds to the 3’UTR region of SIRT1 to negatively regulate SRIT1 and improve the chemotherapy resistance of NSCLC cells.2.MiR-326 improved cell drug resistance,while SIRT1 promoted cell drug resistance.3.SIRT1 regulates transcription factor HIF1α through deacetylation;Silencing SIRT1 promotes proteasomal degradation of HIF1α by inhibiting its deacetylation.HIF1α degradation improves NSCLC chemotherapeutic resistance.Silencing the expression of SIRT1 promotes the degradation of transcription factor HIF1α to improve chemotherapy resistance of NSCLC cells.4.HIF1α binds to VEGFA promoter,and SIRT1 promotes VEGFA expression through HIF1α.SIRT1 promoted the expression of VEGFA.Silencing VEGFA improved NSCLC chemotherapy resistance;SIRT1 promotes the expression of VEGFA through transcription factor HIF1α and regulates chemotherapy resistance.5.Overexpression of miR-326 improves chemical resistance of NSCLC cells in vivo.Conclusion:Chemotherapy remains the cornerstone of NSCLC treatment.In this paper,bioinformatics prediction combined with experiments confirmed that the research is reasonable and efficient research method.Through extensive literature review,it is found that miRNAs have been widely studied as important regulatory factors in the mechanism of tumor genesis and development,with mature technology and methods,and have also been extensively studied in epigenetics.The drug resistance of miR-326 in tumor chemotherapy has been studied,but the mechanism has not been studied and discussed.SIRT1,HIF1α and VEGFA have all been reported to be involved in the occurrence,development and drug resistance process of NSCLC,but whether they have a clear connection directly has not been reported.Therefore this article starts from this point carries on the discussion.In this study,a new pathway miR-326→SIRT1/HIF1α/VEGFA →NSCLC was found through the combined method of bioinformatics prediction,literature retrieval and experimental verification to explore the mechanism.However,miRNA is not in a one-to-one relationship with genes,and the study of single pathway is not the only pathway.Studies at the cell level are relatively limited,and animal experiments are relatively few.In future studies,further studies can be conducted in combination with clinical specimens to increase the credibility.MiR-326 mediated the expression of SIRT1/HIF1α/VEGFA axis to improve chemotherapy resistance in NSCLC.These results suggest that miR-326 is a promising therapeutic target for lung cancer.