LncRNA NUTM2A-AS1 Affects The Proliferation And Apoptosis In Pancreatic Cancer Cells Through The Mir-27a-3p/SSRP1

Background:Pancreatic cancer is one of the most common and deadly malignancies of the digestive system.In recent years,its morbidity and mortality have been increasing,the 5-year survival rate is less than 10%,and the prognosis is extremely poor.The main treatment methods for pancreatic cancer include surgery,radiotherapy and chemotherapy,and molecular targeted therapy.Due to its hidden clinical manifestations,high degree of malignancy,rapid development,and lack of early diagnosis strategies,most patients are diagnosed at an advanced stage and are not suitable for surgical resection,and radiotherapy and chemotherapy have extensive drug resistance and certain toxicity.The current emerging molecular targeted therapy has achieved satisfactory results in the treatment of various tumors with low adverse reactions.Therefore,it is of great social value to explore the molecular mechanism of pancreatic cancer development and develop new treatment strategies.With the advent of the era of big data,more and more studies have found that noncoding RNA is closely related to the occurrence and development of tumors.LncRNA(long non-coding RNA)is a type of RNA with a length of more than 200 nucleotides,which can It participates in biological processes such as chromatin modification,transcriptional regulation,translational regulation,and ceRNA regulation through a variety of molecular modes of action,thereby affecting the progression of the disease.In the study of pancreatic cancer,abnormal expression and mutation of various LncRNAs have been found to be closely related to cancer phenotype.The long-chain non-coding RNA NUTM2A-AS1(NUT family member 2A-antisense RNA 1)is the antisense RNA 1 of NUT family member 2A,and the biological function of NUTM2AAS1 in pancreatic cancer is still unclear.In this study,we aimed to explore its function in pancreatic cancer and find its possible target miRNAs and genes,clarify its mechanism of action,explore its possibility as a potential pancreatic cancer tumor marker,and lay a solid foundation for the future development of pancreatic cancer.Provide theoretical basis and experimental basis in diagnosis and treatment.MicroRNAs(miRNAs)are highly conserved small non-coding RNA molecules that play an important role in the development of tumors,affecting genes and pathways related to all cancer characteristics.MiRNAs can induce mRNA cleavage or repress translation by targeting the 3’UTR of the corresponding mRNA.In recent years,expression regulation of miRNAs has been found in various types of human cancers.More and more studies have shown that miRNA can regulate various processes such as tumor cell proliferation,apoptosis,metastasis and drug resistance.Structure-specific recognition protein 1(SSRP1),one of the histone subunits that can promote the synthesis of the chromatin transcription(FACT)complex,has been shown to be involved in almost all chromatin-related processes,including DNA replication,repair,and transcription.SSRP1 plays a tumor suppressor function in colon cancer,liver cancer,and gastric cancer,and can participate in the regulation of multiple tumor-related pathways,including AKT,WNT,and MAPK pathways,and plays an important role in the development of tumors.This article aims to explore the function and molecular mechanism of LncRNA NUTM2A-AS1/mir-27a-3p/SSRP1 axis in pancreatic cancer,and provide new ideas and new targets for exploring the occurrence and development of pancreatic cancer,preventing the occurrence of pancreatic cancer and improving prognosis.Objective:(1)To clarify the biological function of NUTM2A-AS1 in pancreatic cancer.(2)To explore the molecular mechanism of LncRNA NUTM2A-AS1/mir-27a3p/SSRP1 axis in pancreatic cancer.(3)To explore the biological function of SSRP1 in pancreatic cancer.Method:(1)Based on literature review and bioinformatics prediction websites and data mining websites such as TCGA and GEO,the expression level of NUTM2A-AS1 in pancreatic cancer tissues was explored using database information,and Kaplan-Meier test was used for survival analysis.(2)Design and synthesize siRNA of NUTM2A-AS1,use cell transfection technology to silence NUTM2A-AS1 in pancreatic cancer cells PANC-1 and AsPC-1,and detect the expression level by qPCR.(3)After silencing NUTM2A-AS1 in pancreatic cancer cells PANC-1 and AsPC-1,CCK-8 cell viability experiments,colony formation experiments,cell cycle experiments,cell apoptosis experiments,and cell scratch experiments were performed.(4)Predict miRNAs that NUTM2A-AS1 may bind to using Diana and Targetscan databases.(5)The expression level of miR-27a-3p in pancreatic cancer was analyzed by TCGA database.(6)Construct miR-27a-3p mimics and inhibitors,and use cell transfection technology to observe the effect on the cell viability of pancreatic cancer cells through CCK8 experiments.And whether miR-27a-3p inhibitor can reverse the effect of siNUTM2AAS1 on cell viability.(7)The expression level of miR-27a-3p was detected by qPCR after silencing NUTM2A-AS1.(8)The interaction between NUTM2A-AS1 and miR-27a-3p was verified by dual luciferase reporter assay.(9)Using miRDB and Targetscan databases to predict the target genes that miR-27a-3p may bind to.(10)Use the TCGA database to explore the expression level of SSRP1,and use KaplanMeier test for survival analysis and use Western-blot to detect the expression level of SSRP1 in normal human pancreatic cells and pancreatic cancer cell lines.(11)The expression level of SSRP1 after overexpression of miR-27a-3p in pancreatic cancer cells was detected by qPCR experiment and Western-blot experiment.(12)Dual luciferase reporter assay was used to detect the luciferase activity of SSRP1 WT reporter plasmid and Mut reporter plasmid,and to verify the relationship between miR-27a-3p and SSRP1.(13)Using the expression levels of NUTM2A-AS1 and SSRP1 in the TCGA database,a correlation analysis was performed to analyze the linear relationship between the two.And use qPCR and Western-blot experiments to verify.(14)The siRNA of SSRP1 was artificially constructed,and the knockdown level was verified by qPCR and Western-blot in pancreatic cancer cells.(15)After silencing SSRP1,observe the effects of CCK8 experiments,cell counting experiments,colony formation experiments,cell cycle experiments,and apoptosis experiments on pancreatic cancer cells.(16)Tumor formation experiment in nude mice to observe the survival period and tumor growth changes of mice after SSRP1 gene interference.The expression of related proteins in animal tissues was detected by IHC.Result:LncRNA NUTM2A-AS1 expression was upregulated in PD AC,miR-27a-3p was low expression,and SSRP1 was highly expressed.In human pancreatic cancer cells PANC-1 and AsPC-1,knocking down NUTM2A-AS 1 can inhibit pancreatic cancer cell viability,promote apoptosis,inhibit cell migration ability,and develop cell cycle arrest,while further cell transfection of miR-27a-3p inhibitor can reverse this phenomenon.The correlation between qPCR experiment,Western blot experiment,and diluciferase reporter experiment was verified.According to further prediction results of bioinformatics,SSRP1 was the direct target gene of miR-27a-3p,and qPCR and Western blot results showed that the expression level of SSRP1 decreased after overexpression of miR-27a-3p,and the correlation between the two was verified by diluciferase experiments.SSRP1 is highly expressed in pancreatic cancer cells,and knocking down SSRP1 can inhibit cell viability,promote apoptosis,inhibit cell migration,and inhibit the growth of pancreatic cancer transplanted tumors in mice in vivo experiments.In PD AC,interactions between LncRNA NUTM2A-AS1,mir-27a3p,SSRP1 regulate the proliferation and apoptosis capacity of pancreatic cancer cells as well as cell migration capacity.Conclusion:(1)LncRNA NUTM2A-AS1 can regulate the proliferation,apoptosis and migration of pancreatic cancer cells.(2)LncRNA NUTM2A-AS1 negatively regulates the expression of miR-27a-3p.(3)LncRNANUTM2A-AS 1 can positively regulate the expression of SSRP1,and miR27a-3p can bind to the 3’-UTR end of SSRP1 and negatively regulate the expression of SSRP1.(4)The LncRNANUTM2A-AS1/mir-27a-3p/SSRP1 axis can regulate the proliferation,apoptosis and migration of pancreatic cancer cells.(5)SSRP 1 can regulate the proliferation and apoptosis and migration ability of pancreatic cancer cells,and can inhibit the growth of pancreatic cancer transplanted tumors in mice.