| Background:Gastric cancer was one of the most common gastrointestinal malignancies in the world and China.The prognosis of early gastric cancer was good,but the diagnosis rate was low.In clinical practice,middle and advanced gastric cancer were still common,with poor prognosis and high mortality.Therefore,it was necessary to explore new diagnostic and treatment techniques and methods to improve the prognosis.Major breakthroughs have been made in molecular genetic studies,various molecular mechanisms in the process of occurrence and development of gastric cancer have been continuously revealed.the activation of oncogenes and the inactivation of tumor suppressor genes,the abnormal changes of growth factors and cell cycle regulation factors,and abnormalities in a variety of carcinogenic signaling pathways were involved in its development process.Previous studies and preliminary experiments in this project have found that BTG3(B-cell translocation gene 3)expression was lower in gastric cancer,and cell function experiments have proved that lower expression of BTG3 can promote proliferation,migration and invasion of gastric cancer cells,and inhibit apoptosis of gastric cancer cells.In addition,it was found that BTG3,miR106 b and Wnt pathways may be associated with gastric cancer,but the molecules involved and related regulatory mechanisms have not been elucidated,which requires further discussion.This topic early experiment also found ANRIL(antisense non-coding RNA in the INK4 locus)as a kind of long chain non-coding RNA,was over expression in gastric cancer,and can promote cell proliferation and malignant,and may have connection with miR-99 a.Moreover,miR-99 a maybe can regulate BMI1(B-cell-specificMoloney murine leukemia virus integration site-1),which had certain connection with the ANRIL on structure,the connection between the three factors was still not clear,and their role on occurrence and development of gastric cancer should be further confirmed.Objective:In this paper,the association of BTG3 with miR-106 b and Wnt/β-catenin signal pathway on proliferation of gastric cancer were discussed,and the association of ANRILwith miR-99 a and BMI1 on proliferation of gastric cancer were also discussed.The molecular regulation mechanism of the proliferation process of gastric cancer was clarified by multiple lines,providing theoretical basis for targeted treatment.Methods:Ⅰ The association of BTG3 with miR-106 b and Wnt/β-catenin signal pathway on proliferation of gastric cancer1.The expression of BTG3 protein was detected by immunohistochemistry,including 120 pairs of surgically resected gastric cancer tissue and precancerous tissues,116 cases of chronic atrophic gastritis and 110 cases of chronic superficial gastritis during the same period,and BTG3 protein expression and clinicopathological features of gastric cancer patients were Compared;2.The experssion levels of BTG3,miR-106 b,wnt1,β-catenin,c-myc and CyclinD1 in MKN-45 and SGC-7901 cells were measured using qPCR assay.3.MKN-45 and SGC-7901 cells were transfected with miR-106 b mimic and inhibitor.The transfection effect was detected by qPCR assay,and The effects on cell viability and BTG3 were measured using CCK-8 assay and Western blot.assays by miR-106 b in two gastric cancer cell lines was detected by luciferase reporter gene assay detected the targeted regulatory relationship between miR-106 b and BTG3.4.miR-106 b mimic and plasmid DNA of BTG3 co-transfected MKN-45 and SGC-7901 cells,The effects on cell viability and BTG3 were measured using CCK-8assay and Western blot.5.Two gastric cancer cell lines were transfected with plasmid DNA and shRNA targeting BTG3.The transfection effect was detected by qPCR and Western blot,the proliferation status of gastric cancer cells after transfection was detected by CCK8 assay,and the expression levels of wnt1,β-catenin,c-myc and CyclinD1 in gastric cancer cells before and after transfection were detected by Western blot.6.The expression of phosphorylated β-catenin in GES-1,SGC-7901 and MKN-45 and in gastric cancer cells transfected with BTG3 plasmid DNA were detected by Weston blot.Ⅱ The association of ANRIL with miR-99 a and BMI1 on proliferation of gastric cancer1.20 pairs of gastric cancer and corresponding paracancerous tissues were collected for surgical resection,and 20 cases of chronic superficial gastritis were selected as the control group by gastroscopy.Cell lines of SGC-7901,MKN-45 and GES-1 wereselected for cell culture.The expression levels of ANRIL in tissues and cells were detected by qPCR.2.SGC-7901,MKN-45 cell lines were cultured and transfected with shANRIL.qPCR was used to detect the transfection effect,the proliferation status of gastric cancer cells after transfection was detected by CCK8 assay.3.The expression levels of miR-99 a in tissues and in cells after ANRIL knockout were detected by qPCR.4.ANRIL-silenced cells were transfected by miR-99 a inhibitor again,the effects on the expression of miR-99 a and BMI1 were detected by qPCR,and the proliferation status of gastric cancer cells was detected by CCK8 assay.5.SGC-7901,MKN-45 cell lines were cultured and transfected with miR-99 a mimic and inhibitor,respectively.The expression of BMI1 before and after transfection was detected by qPCR.Luciferase reporter gene assay was used to detect the regulation of miR-99 a on BMI1 in gastric cancer cells.6.SGC-7901,MKN-45 cell lines were transfected with plasmid DNA and shRNA targeting BMI1.The transfection effect was detected by qPCR and Western blot,and the expression levels of Notch and mTOR signaling pathways after transfection were detected by Western blot assay.ResultsⅠ The association of BTG3 with miR-106 b and Wnt/β-catenin signal pathway on proliferation of gastric cancer1.Immunohistochemical results showed that the positive expression of BTG3 protein was light yellow to brown granules,located in the cytoplasm,and the expression of BTG3 in gastric cancer tissue were lower(P<0.001).The expression of BTG3 protein was not significantly correlated with the patients’ age,gender and lymph node metastasis,but was correlated with tumor stage(P=0.041)and degree of differentiation(P=0.032).the research analyzed the the relationship between H.pylori infection and BTG3 expression for the first time,and found that BTG3 protein expression in the H.pylori positive tissues was lower,difference was statistically significant(P=0.017).2.The results of qPCR showed higher expression level of miR-106(P<0.001),lower expression level of BTG3(P<0.001),and higher expression levels of wnt1,β-catenin,c-myc and CyclinD1(P<0.001)in gastric cancer cells.3.In SGC-7901,MKN-45 cell lines transfected with miR-106 b mimic,The expression level of BTG3 protein was significantly reduced(P<0.001),and theproliferation level of gastric cancer cells was accelerated(P<0.001).On this basis,BTG3 plasmid DNA was transfected again,and the above results were reversed.In SGC-7901,MKN-45 cell lines transfected with miR-106 b inhibitor,The expression level of BTG3 protein was significantly increased(P<0.001),and the proliferation level of gastric cancer cells was decreased(P<0.001).Luciferase reporter assays showed that wild-type BTG3-3’UTR reporter plasmid luciferase activity was significantly reduced after transfection with miR-106 b mimic(P<0.001).4.BTG3 expression was up-regulated in gastric cancer cells transfected with BTG3 plasmid DNA,the proliferation of gastric cancer cells was decreased(P<0.001),BTG3 expression was down-regulated in gastric cancer cells transfected with BTG3/shRNA,and the proliferation of gastric cancer cells was increased(P<0.001).5.Western blot showed higher expression of wnt1,β-catenin,c-myc and CyclinD1 in SGC-7901,MKN-45 cell lines compared with GES-1 cell(P<0.01).The expression of wnt1,β-catenin,c-myc and CyclinD1 decreased after the expression of BTG3 was up-regulated(P<0.001),and the expression of wnt1,β-catenin,c-myc and CyclinD1 increased after the expression of BTG3 was down-regulated(P<0.001).6.The expression levels of phosphorylated β-catenin in SGC-7901,MKN-45 cells were lower than in GES-1cell.After BTG3 plasmid DNA transfection,upregulation of BTG3 in gastric cancer cells revealed a corresponding increase in phosphorylatedβ-catenin,and the difference was statistically significant(P<0.001).Ⅱ The association of ANRIL with miR-99 a and BMI1 on proliferation of gastric cancer1.qPCR results showed that ANRIL expression was higher in gastric cancer tissues and gastric cancer cells(P<0.001),while miR-99 a expression was lower in gastric cancer tissues and gastric cancer cells(P<0.001).2.qPCR results of gastric cancer cells transfected with shANRIL showed a significant decrease in ANRIL expression,a significant increase in miR-99 a expression,and a corresponding decrease in BMI1 expression.And CCK8 showed a decreased proliferation level of gastric cancer cells after transfection(P<0.001).3.qPCR results showed that miR-99 a was highly expressed with ANRIL knockdown in gastric cancer cells,and the expression level of miR-99 a could be significantly reduced after transfection with miR-99 a inhibitors(P<0.001).4.Western blot analysis showed that the expression level of BMI1 protein was decreased after the transfection of miR-99 a mimics(P<0.01)and increased after thetransfection of miR-99 inhibitors(P<0.01).Luciferase reporter assays showed that wild-type BMI1-3’UTR reporter plasmid luciferase activity was significantly reduced after transfection with miR-99 mimics(P<0.001).5.The expression of BMI1 was increased in gastric cancer cells transfected with BMI1 plasmid DNA,and the expressions of Notch1,phosphorylated mTOR and phosphorylated p-70S6 K were increased correspondingly(P<0.01),and the expression of BMI1 was decreased after the transfection of BMI1/shRNA,and the expression levels of Notch1,phosphorylated mTOR and phosphorylated p-70S6 K were decreased correspondingly(P<0.001).Conclusions:1.The low expression of BTG3 in gastric cancer was related to the staging and differentiation of gastric cancer,and may be associated with H.pylori infection.2.BTG3 was targeted and regulated by miR-106 b in gastric cancer cells.Higher expression of miR-106 b and lower expression of BTG3 can lead to accelerated proliferation of gastric cancer cells,which may be related to the activation of Wnt/β-catenin signal pathway.3.The tumor suppressive effects of ANRIL knockdown in gastric cancer cells via cross-talk with miR-99 a.4.we provided a novel regulatory mechanism for ANRIL in gastric cancer,in which,ANRIL silence down-regulated BMI1 via miR-99 a,along with activation of the apoptotic pathway and inhibition of the Notch and mTOR pathways. |
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