The Role And Mechanism Of LncRNA NR₀28 In The Polarization Of Macrophages Associated With Gastric Cancer Immune Microenvironment

Part One Screening and validating LncRNA NR₀28 and the induction of tumor-associated macrophagesObjective: To induce THP-1 cells into M0,M2 and SM2?Similar M2,SM2?macrophages and to verify the expression levels of molecular markers.After successfully induction,the macrophage-associated lncRNA was finally determined by screening differentially expressed lncRNA in M0 and M2 macrophages.Methods:1.RT-qPCR method was used to detect the expression levels of molecular markers of M0,M2 and SM2 macrophages.2.The RNA of M0 and M2 macrophages was extracted by RNA technology.The differentially expressed lncRNA was screened by lncRNA chip hybridization and verified by RT-qPCR.3.Knockdown the genes by si-RNA knockdown technique and verify knockdown efficiency with RT-qPCR.Results:1.RT-qPCR analysis showed that M1 type molecular markers: INOS,HLA-DR? and TNF-? were decreased in M2 and SM2 macrophages induced by THP-1 cells?P<0.05?and M2 type molecular markers: The expression levels of CD206,Arg-1,CCL22,CCL3,IL-10 and VEGF were significantly increased?P<0.05?.2.The results of RT-qPCR analysis showed that the expression of LncRNA NR₀28 gene was significantly changed in 11 lncRNAs with up-regulated expression and 12 lncRNAs with decreased expression,which were consistent with the trend of chip screening results.3.The efficiency of si-RNA knockdown gene showed that LncRNA NR₀28 knockdown efficiency was significant?P<0.05?,so LncRNA NR₀28 was used for subsequent experiments.Summary:M0,M2 and M2 type macrophages models were constructed.The expression of M2 markers CD206,Arg-1,CCl22,CCl3,IL-10 and VEGF-C were elevated and the M1 marker TNF-?,HLA-DR and INOS were decreased,indicating that the model was successfully constructed.LncRNA microarray hybridization was performed on the RNA of M2 and M0 macrophages,and differential genes were selected according to expression fold,length,expression stability and tissue expression level.LncRNA NR₀28 was finally used for subsequent experiments according to gene knockdown efficiency.Part Two The specific role and mechanism of LncRNA NR₀28 in macrophage polarizationObjective: To investigate the effects of LncRNA NR₀28 in M2 and similar M2 macrophages on the proliferation,migration and invasion of gastric cancer cells,and to explore the mechanism of LncRNA NR₀28 in M2 macrophage polarization.Methods:1.MTS,scratch healing experiment,transwell migration and invasion assay were used to detect the effects of M2 and SM2 macrophage supernatants on gastric cancer cell lines to detect the effects of proliferation,migration and invasion.2.Knockdown of LncRNA NR₀28 gene by si-RNA knockdown technique,the expression level of cytokines in M2 and SM2 macrophage supernatants was detected by ELISA method.3.Transwell migration assay was used to detect the effect while knockdown LncRNA NR₀28 gene in M2 macrophages on the migration of gastric cancer cells.4.The distribution of LncRNA NR₀28 gene in the nucleus and cytoplasm of M2 macrophages was detected by nuclear pulp fractionation and RT-qPCR.5.Using FISH technique to detect the localization of LncRNA NR₀28 gene in M2 macrophages.6.RNA-pulldown and mass spectrometry were used to screen and identify the proteins directly interacting with LncRNA NR₀28 gene in M2 macrophages,and verified by Western blotting.7.Whole-genome sequencing technology was used to detect the change of mRNA expression profile and the involved pathways in M2 macrophages knockdown of LncRNA NR₀28.Results:1.MTS experiment results showed that the medium plus 20% M2 supernatant group and medium plus 20% SM2 supernatant group promoted the proliferation of gastric cancer cell lines BGC-823,AGS and SGC-7901?P<0.05?.The results of scratch healing experiments showed that M2 and SM2 macrophage supernatants promote the migration ability of gastric cancer cell lines BGC-823,AGS and SGC-7901 in vitro?P<0.05?.Transwell chamber experiments showed that gastric cancer cells with medium plus 20% M2 supernatant group and medium plus 20% SM2 supernatant promote the migration and invasion of gastric cancer cells?P<0.05?.2.ELISA analysis results showed that knockdown LncRNA NR₀28 promoted the expression of M1 cytokines HLA,TNF-?,INOS and IL-12 in M2 and SM2 macrophage culture supernatants?P<0.05?,while M2 cytokines CD206,VEGF-C,CCL22 and IL-10 decreased?P<0.05?,indicating that LncRNA NR₀28 is an important factor for the polarization of macrophages to M2 phenotype.After knocking down LncRNA NR₀28,macrophages are directed to M1.3.Transwell chamber experiment results showed that knockdown LncRNA NR₀28 in M2 macrophages inhibited the migration of gastric cancer cells BGC-823,AGS and SGC-7901?P<0.05?.4.RT-qPCR experiment results showed that LncRNA NR₀28 was mostly localized in the nucleus of M2 macrophages?P<0.05?.5.FISH experiment results showed that the LncRNA NR₀28-specific probe was localized in the nucleus,and the fluorescence intensity in the nucleus was weakened after knocking down LncRNA NR₀28 in M2 macrophages.The results showed that LncRNA NR₀28 was mostly localized in the nucleus in M2 macrophages..6.RNA-pulldown experiments combined with mass spectrometry analysis results showed the target proteins: CHI3L1 and ARG1 were screened according to the scores for subsequent studies.7.Analysis of whole genome sequencing technology results showed that knockdown LncRNA NR₀28,the mRNA changed affected multiple pathways and biological functions,and the NF-?B pathway was selected for validation.Summary:M2 and SM2 macrophages can provide gastric cancer cells BGC-823,AGS and SGC-7901 in vitro a micro-environment for tumor survival,which can promote the proliferation,migration and invasion of gastric cancer cells.Knocking down the LncRNA NR₀28 gene can inhibit biological behavior of gastric cancer cells such as migration.These phenomena may be related to the interaction of LncRNA NR₀28 gene with CHI3L1 and ARG1 proteins.Part Three The expression of LncRNA NR₀28 in human tissues and the effect on the tumorigenic ability of gastric cancer cells in nude mice while knocking down LncRNA NR₀28Objective: To investigate the expression of LncRNA NR₀28 in TAMs in human gastric cancer tissues and adjacent tissues,and the effect of LncRNA NR₀28 on the expression of tumor microenvironment macrophages in nude mice.Methods:1.RT-qPCR was used to detect the expression of LncRNA NR₀28 gene in TAMs of gastric cancer tissues and adjacent tissues.2.M2 macrophages which stably express low level of LncRNA NR₀28 were constructed and injected into the tail vein of nude mice to observe the metastasis.3.Hematoxylin-eosin staining was used to detect the metastasis of tumor cells in nude mice.Results:1.Compared with adjacent tissues,RT-qPCR showed that the expression of LncRNA NR₀28 was significantly increased in TAMs of gastric cancer tissues?P<0.05?.2.In vivo imaging of mice showed that the tumor metastasis was more significant in M2sh-nc+BGC-823 group than M2sh-LncRNA NR₀28+BGC-823 group?P<0.05?.3.Hematoxylin-eosin staining showed that in the M2sh-nc+BGC-823 group lung metastasis increased,but no metastatic tumor cells were found in the liver,spleen and kidney.M2 macrophages with low expression of LncRNA NR₀28 reduced the ability of gastric cancer cells to metastasize in nude mice.Summary:M2 macrophages were isolated from human gastric cancer tissues and adjacent normal tissues.The results of RT-qPCR showed that LncRNA NR₀28 was highly expressed in gastric cancer tissues.The degree and size of lung metastases in nude mice with LncRNA NR₀28 knockdown group were significantly lower than those in the control group,indicating that LncRNA NR₀28 not only promoted the proliferation of gastric cancer cells in vitro,but also promoted the proliferation and metastasis of gastric cancer cells in vivo,indicating that LncRNA NR₀28 plays a key role in the gastric cancer development process.Conclusion:M2 type macrophage regulates the growth of gastric cancer cells in tumor microenvironment,promotes the proliferation,migration and invasion of gastric cancer cells,and knockdown LncRNA NR₀28 gene in M2 macrophages reduce the promotion of gastric cancer cells and affect the metastasis of gastric cancer cells.LncRNA NR₀28 is localized in the nucleus mostly and may interact with ARG1 and CHI3L1 proteins to influence the progression of gastric cancer.