| Part One To investigate the expression of MALAT1,miR-22-3p and ErbB3 in gastric cancer and paracancer tissues and analyze their effects on the prognosis of patients.Objective: To study the expression of MALAT1,miR-22-3p and ErbB3 in gastric cancer(GC)tissues and paracancer tissues and explore their effects on the prognosis of patients.Methods: The relative expression levels of MALAT1,miR-22-3p and ErbB3 m RNA in cancer tissues and adjacent normal tissues were detected by RT-PCR in 37 confirmed gastric cancer patients.The relative expression level of MALAT1 in GC patients in different pathological stages were detected;the correlation between MALAT1,miR-22-3p and ErbB3 was analyzed by Pearson method.The effect of up-regulation or down-regulation of MALAT1 on the survival of patients was confirmed.Results:1.RT-PCR detection results: compared with adjacent normal tissues,the relative expression levels of MALAT1 and ErbB3 were significantly up-regulated while miR-22-3p was significantly down-regulated in GC tissues(P<0.05).2.The expression of MALAT1 in GC tissues of Ⅲ+Ⅳ pathological stage was higher than that of I+II pathological stage,and the difference was statistically significant(P<0.05).3.MiR-22-3p was significantly negatively correlated with MALAT1 and ErbB3 respectively,and MALAT1 was significantly positively correlated with ErbB3(P<0.05).4.The effect of up-regulation and down-regulation of MALAT1 on the survival of GC patients: the five-year survival ratio of patients with up-regulation of MALAT1 was significantly lower than that of patients with down-regulation of MALAT1(P<0.0041).Conclusions:1.In the pathological development of GC,MALAT1 and ErbB3 showed significant tumor-promoting activity,and miR-22-3p showed significant tumor-suppressor activity;there was significantly correlation among the three indicators.2.With the increased expression of MALAT1 in GC tissues,the survival of patients was gradually shortened,which provided the basis for the following studies.Part Two The mechanism study of MALAT1 targeting miR-22-3p to regulate the proliferation and apoptosis of gastric cancer cells.Objective: To explore the differential expression of MALAT1 and its effect on the proliferation and apoptosis of GC cells by targeting miR-22-3p.Methods: GC cell lines SGC-7901 and BGC-823 were selected as the research objects,and GES-1 was selected as the control group.Subculture was carried out,and the third generation monolay cells were selected as the study subjects;MALAT1 and miR-22-3p were over-expressed and silenced separately.The transfection efficiency was verified by RT-PCR;the targeting relationship of MALAT1 to miR-22-3p was verified by DLR assay;the effect of MALAT1 expression on miR-22-3p was detected by RT-PCR;CCK-8 and cloning assay were used to detect the proliferation ability of cells in each group;the apoptosis of each group was detected by Annexin V-FITC;the protein levels of Ki-67,PCNA,Cleaved caspase-3,Bcl-2 and Bax were detected by WB.Results:1.Study on the mechanism of over-expression or silencing of MALAT1 in regulating the proliferation and apoptosis of GC cells: the expression of MALAT1 in GC cell lines SGC-7901 and BGC-823 were both significantly higher than that in GES-1(P<0.05).MALAT1 was successfully over-expressed and silenced in GC cell lines of the two groups separately,with statistically differences(P<0.05).Over-expression of MALAT1 significantly promoted the proliferation and inhibited the apoptosis of SGC-7901 cells,while silencing of MALAT1 exerted contrary effect on BGC-823 cells(P<0.05).When MALAT1 was over-expressed in GC cells,the protein expressions of Ki-67,PCNA,and Bcl-2 were significantly increased,while Cleaved Caspase-3 and Bax were significantly decreased;one the contrary,when MALAT1 was silenced,the protein expressions of the above indicators showed the opposite results(P<0.05).2.Study on the mechanism of MALAT1 in regulating the proliferation and apoptosis of GC cells by targeting miR-22-3p: compared with normal gastric cells GES-1,the expression of miR-22-3p in both SGC-7901 and BGC-823 cells were significantly decreased(P<0.05).MiR-22-3p was successfully over-expressed and silenced in SGC-7901 and BGC-823 cells respectively,with statistically significant(P<0.05).MALAT1 targeting miR-22-3p was verified by DLR assay: a binding site of miR-22-3p was predicted at 3’UTR of MALAT1.The wild-type(WT-MALAT1)and mutant(MUT-MALAT1)reporter vectors of MALAT1 were fully constructed and cotransfected into gastric cancer cells with miR-22-3p mimics respectively,and the results showed that the luciferase activity of GC cells in both groups was significantly decreased(P<0.05).In addition,over-expression of MALAT1 significantly down-regulated the expression of miR-22-3p,while silencing of MALAT1 significantly up-regulated the expression of miR-22-3p(P<0.05).The expression of MALAT1 was inhibited could suppressed cell proliferation and promoted cell apoptosis significantly,while the expression of miR-22-3p was inhibited showed the opposite result.When MALAT1over-expression was inhibited in GC cells,the expression of Cleaved caspase-3 and Bax were increased significantly,while the expression of Bcl-2was decreased significantly.When miR-22-3p expression was inhibited,the expression of Cleaved caspase-3 and Bax expression were decreased,and the expression of Bcl-2 was increased,with statistically significant differences.Conclusions: In the changing process of the biological activity of GC cells,MALAT1 negatively targeting miR-22-3p to regulate the cell proliferation and apoptosis;the over-expression of MALAT1 down-regulated the expression of miR-22-3p,ultimately inhibited cell apoptosis and promoted cell proliferation,which provided a basis for clinical research.Part Three Study on the mechanism of MALAT1 in regulating the proliferation and apoptosis of gastric cancer cells through miR-22-3p/ErbB3 axisObjective: To study the mechanism of MALAT1 in regulating the proliferation and apoptosis of GC cells through miR-22-3p/ErbB3 axis and providing theoretical basis for clinical research.Methods: GC cell lines SGC-7901 and BGC-823 were selected as the experimental groups,and GES-1 was selected as the control group.Subculture was carried out and the third generation monolay cells were selected as the research objects.In vitro experiment: MALAT1 and miR-22-3p were over-expressed and silenced separately.The expression of ErbB3 m RNA in GC cell lines SGC-7901 and BGC-823 was detected by RT-PCR;the targeted relationship between MALAT1 and miR-22-3p and which between MALAT1 and ErbB3 were identified by DLR assay;CCK-8 and cloning assay were used to detect the proliferation of SGC-7901 cell lines;Annexin V-FITC was used to detect the apoptosis of SGC-7901 cell lines;WB detected the expression levels of Cleaved caspase-3,Bcl-2 and Bax in each group.In vivo experiment: the nude mice model was established to investigate the influence of silencing of MALAT1 on tumor volume and weight.Results:1.In vitro experiment: the relative expression level of ErbB3 in GC cells was significantly higher than that in normal cells.A binding site of miR-22-3p was predicted at 3’UTR of ErbB3.When ERBB3-WT and ERBB3-MUT reporter vectors were cotransfected with miR-22-3p mimics,luciferase activity in GC cells of the two groups were significantly decreased compared with the control group(P<0.05).Silencing of MALAT1 significantly down-regulated ErbB3 and p-ErbB3,and silencing of miR-22-3p could reverse the above trends.Silencing of miR-22-3p alone significantly increased the expression of ErbB3 and p-ErbB3(P<0.05).Silencing of miR-22-3p significantly promoted cell proliferation and inhibited cell apoptosis,in addition,which significantly increased the expression level of Ki-67,PCNA and Bcl-2,decreased the expression level of Cleaved caspase-3 and Bax.Silencing of ErbB3 could reverse the above trend significantly;silencing of ErbB3 alone significantly inhibited cell proliferation and promoted cell apoptosis(P<0.05).2.In vivo experiment: SGC-7901 cells increased in a time-dependent manner in the injected nude mice.With the extension of postoperative time,the tumor volume and weight of the two groups both showed an increasing trend.The tumor volume and weight of MALAT1 inhibited group decreased significantly compared with the control group.MALAT1 could promote the proliferation of tumor in nude mice,that is,it is significantly carcinogenic,which is consistent with the conclusion of previous studies.Silencing of MALAT1 significantly up-regulated miR-22-3p and down-regulated ErbB3(P<0.05).Conclusions: In vitro and vivo animal model experiments,MALAT1 regulated the cell proliferation,apoptosis and tumor volume of GC by targeting miR-22-3p/ErbB3 axis.Silencing of MALAT1 could up-regulated miR-22-3p and down-regulated ErbB3,and then inhibited the proliferation and promoted the apoptosis of GC cells and ultimately inhibited the growth of tumor.The above research provided some inspiration for clinical research on the pathological mechanism of GC. |
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