| Gastric cancer is one of the digestive malignant tumors that seriously threaten human health.The molecular mechanism of gastric cancer development is complex and may involve chronic H.pylori infection,activation of signaling pathway and oncogenes,epigenetic mutations,etc.The mechanism is still not well understood.Long non-coding RNA(lnc RNA)is a class of endogenous non-coding RNAs with a length greater than 200 nt,which plays an important role in biological processes such as tumor proliferation,differentiation,invasion,inflammatory response,angiogenesis,and metabolism.Recent studies have reported that lnc RNA SNHG3 is highly expressed in gastric cancer tissues and is closely associated with survival prognosis of gastric cancer patients,but the functions of lnc RNA SNHG3 in gastric cancer and its molecular mechanisms are still unclear.In this study,we investigated the role of lnc RNA SNHG3 on the proliferation,cell cycle progression,migration and invasive in gastric cancer cells and elucidated its molecular mechanism.Part Ⅰ.The expression of lnc RNA SNHG3 in gastric cancer and its clinical parametersObjective: To detect prognostic lnc RNAs from TCGA database,verify the expression of lnc RNA SNHG3 in gastric cancer tissues and gastric cancer cells,and analyze its correlation with the clinical parameters of gastric cancer patients.Methods: Sequencing and clinical data of gastric cancer tissues and paraneoplastic tissues were downloaded from TCGA database.The relationship between the lnc RNAs expression levels and survival prognosis of gastric cancer patients was analyzed by Kaplan-Meier Plotter.26 pairs of clinical gastric cancer and paraneoplastic tissues were collected,and q RT-PCR was performed to detect the expression of lnc RNA SNHG3 in gastric epithelial cells GES-1 and gastric cancer cell lines AGS,MGC-803,HGC-27,BGC823,as well as 26 pairs of gastric cancer tissues and corresponding paraneoplastic tissues.Then,the 26 gastric cancer patients were divided into lnc RNA SNHG3 low expression group and high expression groups for correlation analysis between the clinical pathological characteristics and lnc RNA SNHG3 expression.Results: Bioinformatics analysis showed that the expression of lnc RNA SNHG3 was significantly upregulated in gastric cancer tissues(P<0.01),and its expression level was significantly negatively correlated with the survival prognosis of gastric cancer patients(P<0.01).q RT-PCR results showed that compared with gastric epithelial cells GES-1,lnc RNA SNHG3 expression levels were significantly higher in AGS,MGC-803,HGC-27,and BGC-823 cells(P<0.01).Lnc RNA SNHG3 expression was significantly higher in 26 pairs of gastric cancer tissues than in paraneoplastic tissues(P<0.01),and the analysis of clinical pathological characteristics showed that the expression of lnc RNA SNHG3 closely correlated with the clinical stage of gastric cancer patients(P<0.05).Conclusions: Lnc RNA SNHG3 was significantly upregulated in gastric cancer cells and tissues,and its expression level was negatively correlated with the survival prognosis of gastric cancer patients and correlated with the clinical stage of gastric cancer patients.Part Ⅱ.Functions of Lnc RNA SNHG3 on the proliferation,cell cycle progression,migration and invasive in gastric cancer cellsObjective: To investigate the effect of lnc RNA SNHG3 expression level on the proliferation,cell cycle progression,migration and invasive in gastric cancer cells and the effect of knocking down lnc RNA SNHG3 on tumorigenesis in vivo.Methods: FISH was used to detect the subcellular localization of lnc RNA SNHG3 in gastric cancer cell lines,AGS,HGC-27,MGC-803,and BGC-823.Si RNA and pc DNA plasmid transfection were used to construct lnc RNA SNHG3 knockdown and overexpression gastric cancer cell models in MGC-803,HGC-27 cells,BGC-823 and AGS cells,respectively.CCK-8and cell clone formation assays were used to detect changes in the proliferation ability of gastric cancer cells.Flow cytometry was used to detect the cell cycle progression of gastric cancer cells;Transwell and stromal gel invasion assays were used to detect changes in the migration and invasion ability of gastric cancer cells.Nude mice tumorigenesis assay was performed to observe the proliferation ability of gastric cancer cells in nude mice after knocking down lnc RNA SNHG3.Results: FISH assay showed that lnc RNA SNHG3 was found to primarily localize to the cytoplasm rather than the nucleus in gastric cancer cells AGS,HGC-27,MGC-803 and BGC-823.Compared with the control group,the proliferation and clone formation of gastric cancer cells in the lnc RNA SNHG3 knockdown group were significantly inhibited(P<0.01).Conversely,after overexpression of lnc RNA SNHG3,the proliferation and clone formation of gastric cancer cells were significantly enhanced(P<0.01).Flow cytometry analyses further confirmed that the cell cycle progression was blocked in G0/G1 phase,and the number of cells in S and G2 phases was significantly reduced after knocking down lnc RNA SNHG3,while after overexpressing lnc RNA SNHG3,the cell cycle progression was converted from G0/G1 phase to S and G2 phases,and the number of cells in S and G2 phases was significantly increased.Transwell and stromal gel invasion assays showed that the migration and invasion ability of cells in the lnc RNA SNHG3 knockdown group was significantly decreased(P<0.01),while was significantly enhanced after overexpress of lnc RNA SNHG3(P<0.01).The xenograft experiments in nude mice showed that the growth rate and volume of tumors in the LV-sh-SNHG3 cell injection group were significantly smaller than those in the control group(P<0.01),and the weight of tumors was also significantly lower than that in the control group(P<0.01).Conclusions: Lnc RNA SNHG3 promoted gastric cancer cell proliferation,cell cycle progression,migration and invasion,as well as proliferation ability of gastric cancer cells in vivo.Part Ⅲ.The molecular mechanism of lnc RNA SNHG3/mi R-139-5p/MYB axis in gastric cancerObjective: To investigate the molecular mechanism of lnc RNA SNHG3/mi R-139-5p/MYB axis in the proliferation and cell cycle progression in gastric cancer.Methods: The lnc RNA-mi RNA-m RNA regulatory network of lnc RNA SNHG3 was predicted by bioinformatics approach.The expression of mi R-139-5p and MYB in TCGA gastric cancer database and its correlation with survival prognosis of gastric cancer patients were analyzed.The expression levels of mi R-139-5p and MYB in 26 pairs of gastric cancer tissues and gastric cancer cells were detected by q RT-PCR.The binding sites between lnc RNA SNHG3 and mi R-139-5p,mi R-139-5p and MYB sequences were validated using dual-luciferase reporter Assay and RIP assay.The mi R-139-5p level was measured by q RT-PCR after lnc RNA SNHG3 knockdown and overexpression.The expression level of MYB were detected by q RT-PCR and Western blot with mi R-139-5p mimic or mi R-139-5p inhibitor transfected.To design a rescue experiment,lnc RNA SNHG3 overexpression plasmid and mi R-139-5p mimics were co-transfected into gastric cancer cells,then the cell proliferation and ell cycle progression were detected,and the expression of MYB was detected by q RT-PCR and Western blot.Concurrently,the expression of MYB in nude mice xenograft tissues was detected by IHC,q RT-PCR and Western blot.Results: Bioinformatics predictions showed a ternary regulatory relationship between lnc RNA SNHG3/mi R-139-5p/MYB.TCGA data analysis showed that mi R-139-5p expression was significantly lower in gastric cancer tissues(P<0.01),while MYB expression was significantly higher in gastric cancer tissues(P<0.01).Meanwhile,mi R-139-5p level was significantly positively correlated with the survival rate of gastric cancer patients(P<0.01),and MYB expression level was significantly negatively correlated with the survival rate of gastric cancer patients(P<0.05).The dual-luciferase assay and RIP assay showed that the luciferase activity was significantly reduced in the GP-mir GLO-SNHG3 WT group compared with the GP-mir GLO-SNHG3 MUT group(P<0.01),and relative to the control Ig G group,lnc RNA SNHG3 and mi R-139-5p in the anti-Ago2 group were significantly enriched(P<0.01).The luciferase activity was significantly diminished in the GP-mir GLO-MYB WT group compared to the control group(P<0.01),confirming the presence of binding sites on mi R-139-5p sequence to lnc RNA SNHG3 and the 3’UTR of the MYB.The expression of mi R-139-5p was significantly increased in gastric cancer cells after knocking down lnc RNA SNHG3(P<0.01),while significantly decreased after overexpression of lnc RNA SNHG3(P<0.01).Correspondingly,MYB was significantly downregulated in the transfected mi R-139-5p mimics group(P<0.01),contrarily significantly upregulated in the transfected mi R-139-5p inhibitor group(P<0.01).The rescue experiment showed that the proliferation ability of cells in the pc DNA3.1SNHG3 transfected group was significantly enhanced(P<0.01),the cell cycle was converted from G0/G1 phase to S phase and G2 phase,and the number of cells in S phase and G2 phase was significantly increased(P<0.01).Meanwhile,MYB was significantly upregulated(P<0.01).In the pc DNA3.1 SNHG3 and mi R-139-5p mimic co-transfected group,the above effects were rescued(P<0.01).In the xenograft tissues,Ki67 and MYB expression was significantly lower in LV-sh-SNHG3 cell injection group by IHC,the m RNA expression of mi R-139-5p in LV-sh-SNHG3 group was significantly upregulated than that in the control group by q RT-PCR(P<0.01),and the protein expression of MYB in LV-sh-SNHG3 group was significantly downregulated than that in the control group by Western blot(P<0.01).Conclusions: Lnc RNA SNHG3 negatively regulates the repressive effect of mi R-139-5p on the target gene MYB through a ce RNA mechanism,thereby promoting proliferation,cell cycle progression,migration and invasion in gastric cancer. |
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