Mechanism Of Action Of Long Non-coding RNA GMDS-AS1 In Lung Adenocarcinoma

BackgroundLung cancer is a serious threat to human health,About 700 000 people are diagnosed with lung cancer every year in China.The characteristics of easy metastasis,difficult surgical resection,and poor prognosis make it the most dangerous cancer,which leads to the highest mortality rate among all types of cancer.Non-small cell lung cancer(NSCLC)accounts for more than 85% of lung cancer.NSCLC is further divided into adenocarcinoma,squamous cell carcinoma,large cell carcinoma and so on,among which adenocarcinoma accounts for nearly 60%,thus becoming the main research object.The treatment of lung cancer in recent years such as against the epidermal growth factor receptor(EGFR),anaplastic lymphoma receptor tyrosine kinase(ALK)and the rapid development of the immune therapy.The 5-year survival rate of lung cancer is only about11%,because of the difficulty in early diagnosis of lung cancer and progress quickly.Therefore,to explore the pathogenesis of lung cancer and new targeted therapy has been a hot topic.In recent years,long noncoding RNAs(lncRNAs)have gradually come into the field of view and become one of the research hotspots,with the rapid development of bioinformatics and the wide application of whole genome sequencing technology.It has been reported that over 30,000 lncRNAs can be detected across the human genome.LncRNAs are defined as RNA molecules with length more than 200 nucleotides and little protein-coding potiential because of la cking open reading frames.Several studies have confirmed that lncRNAs are dysregulated in a variety of human tumors and are involved in tumor cell proliferation,apoptosis and metastasis.The relationship between lncRNA GMDS-AS1 and human diseases has not been reported yet,and the mechanism of tumor occurrence and development is still unclear.In our study,the expression level of lncRNA GMDS-AS1 were firstly detected in lung adenocarcinoma(LUAD)tissues and adjacent normal lung tissues,the expression level and its correlation with clinical and pathological data were analyzed.Then the effects of abnormal expression of lncRNA GMDS-AS1 on the proliferation and apoptosis of LUAD cells were investigated through in vitro cell experiments and in vivo animal experiments.Furthermore,the molecular mechanism of lncRNA GMDS-AS1 was explored through relevant experimental methods,which provided a new direction and theoretical basis for the targeted therapy of LUAD.MethodsIn the first part,a total of 20 cases of Luad tissues and their adjacent normal lung tissues were collected.RNA was extracted by Trizol method and then Reverse transcribed into c DNA.The expression level of lncRNA GMDS-AS1 in Luad tissues and adjacent tissues was detected by Reverse transcription and quantitative polymerase chain reaction(RT-q PCR).At the same time,the clinicopathological data of all patients were registered,and the correlation between the expression level of lncRNA GMDS-AS1 and the clinicopathological data was analyzed by statistical methods.The effects of lncRNA GMDS-AS1 on cell proliferation and apoptosis,as well as the inhibitory effect of lncRNA GMDS-AS1 on tumor were determined by CCK-8 assay,clone formation assay,flow-cytometric analysis,Western blot,luciferase assay detection and subcutaneous tumor transplantation animal experiments.The intracellular localization of lncRNA GMDS-AS1 was detected by nucleo-cytoplasmic isolation.In the second part,through bioinformatics prediction,we found that GMDS-AS1 contains the binding sequence of miR-96-5p.The wild-type and mutant-type reporter plasmid p GLO-GMDS-AS1 was further constructed and synthesized an miR-96-5p mimic.It was verified to determine whether GMDS-AS1 is the target gene of miR-96-5p in SPCA-1 and PC-9 lung adenocarcinoma cell lines.The miR-96-5p mimic and miR-96-5p inhibitors were transfected into SPCA-1 and PC-9 lung adenocarcinoma cells,and the m RNAs that were most significantly inhibited by miR-96-5p were identified by RT-q PCR.The wild-type and mutated reporter plasmids of this factor were constructed and verified to determine whether it is also the target gene of miR-96-5pin SPCA-1 and PC-9 lung adenocarcinoma cell lines.Through RT-q PCR and Western blot,we further determined whether the overexpression of GMDS-AS1 in SPCA-1 and PC-9 lung adenocarcinoma cell lines could reverse the inhibitory effect of miR-96-5p and explore its mechanism network.ResultsThe expression of lncRNA GMDS-AS1 was significantly decreased in LUAD tissues and cells.In vitro and in vivo,the up-regulation of GMDS-AS1 significantly inhibited the proliferation and promoted apoptosis of lung adenocarcinoma cells.The results showed that GMDS-AS1 is a tumor suppressor gene that can influence the development of LUAD.Further studies showed that GMDS-AS1 is the target gene of miR-96-5p,and GMDS-AS1 and miR-96-5p co-regulate the proliferation and apoptosis of LUAD cells.In the mechanism study,the biological information analysis showed that the expression of miR-96-5p was significantly increased in lung adenocarcinoma tissues.The miR-96-5p mimic and miR-96-5p inhibitors were transfected into SPCA-1 and PC-9LUAD cells,RT-q PCR results showed that miR-96-5p significantly inhibited the expression levels of CYLD,CDKN2 C,BRCA2,CDH1,TP53,and MAP2K4.Among them,miR-96-5p has the most obvious inhibitory effect on CYLD.Based on the predicted binding sequences,we constructed reporter plasmids p GLO-CYLD 3'UTR containing the wild-type binding sequence(WT)and the mutant binding sequence(Mut),respectively.In SPCA-1 and PC-9 cells,the activity of p GLO-CYLD 3'UTR WT was inhibited by miR-96-5p,while activated by anti-miR-96-5p,but miR-96-5p and anti-miR-96-5p both have no effect on the activity of p GLO-CYLD 3'UTR Mut.Western blot results indicated that the expression of CYLD was significantly inhibited by miR-96-5p,while promoted by anti-miR-96-5p.we also confirmed the expression of miR-96-5p was negatively related to CYLD in LUAD.We demonstrated that both GMDS-AS1 and CYLD are target genes of miR-96-5p.In SPCA-1 and PC-9 cells,RT-q PCR results showed that miR-96-5p can inhibit the expression of CYLD m RNA,while overexpression of GMDS-AS1 can reverse the inhibitory effect of miR-96-5p on CYLD expression.In SPCA-1 and PC-9 cells,RT-q PCR results showed that miR-96-5p can inhibit the expression of CYLD m RNA,while overexpression of GMDS-AS1 can reverse the inhibitory effect of miR-96-5p on CYLD expression.These findings suggest that the GMDS-AS1/miR-96-5p/CYLD network based on the ce RNA mechanism can regulate the proliferation and apoptosis of LUAD cells.It provides a new direction and theoretical basis for targeted therapy.Conclusion1.The expression of lncRNA GMDS-AS1 was significantly decreased in LUAD tissues and cells,and it was a tumor suppressor gene.2.GMDS-AS1/miR-96-5p/CYLD network based on the ce RNA mechanism can regulate the proliferation and apoptosis of LUAD cells.