| Part One The expression of SNHG5 and EMT markers in gastric cancer and peripheral blood and their correlationObjective: To investigate the expression of SNHG5 and EMT markers in gastric cancer tissues and peripheral blood and analyze their correlation.Methods:1 The expression of SNHG5,CDH1,N-cadherin and Vimentin in gastric cancer tissues and corresponding paracancerous tissues was detected by RT-q PCR.The analyzed the correlation between SNHG5 and EMT markers expression.2 The expression of SNHG5,CDH1,N-cadherin and Vimentin in peripheral blood of patients with gastric cancer was detected by RT-qPCR.The analyzed the correlation between SNHG5 and EMT markers expression.3 The expression of E-cadherin,N-cadherin and Vimentin in gastric cancer tissues and corresponding paracancerous tissues was detected by immunohistochemistry.4 Through the analysis of RT-q PCR data and immunohistochemical data,the correlation between SNHG5 and EMT-related proteins was analyzed.Results:1.RT-qPCR results showed that compared with the corresponding paracancerous tissues,SNHG5 and CDH1 were significantly lower in gastric cancer(P<0.001;P<0.001),while N-cadherin and Vimentin were significantly higher in gastric cancer(P<0.001;P<0.001).There was a significant positive correlation between SNHG5 and CDH1(r=0.630,P<0.001),but negatively correlated with the expressions of N-cadherin and Vimentin(r=-0.577,P<0.001;r =-0.525,P<0.001).2.The results of RT-qPCR showed that there was a positive correlation between the expression of SNHG5 and CDH1(r=0.834,P<0.001),but negatively correlated with the expression of N-cadherin and Vimentin in gastric cancer patients(r =-0.427,P<0.001;r =-0.642,P<0.001).3.Immunohistochemistry showed that the expression of E-cadherin in gastric cancer tissues was significantly lower than that in paracancerous tissues(P<0.001),while the expression of N-cadherin and Vimentin in gastric cancer tissues was significantly higher than that in paracancerous tissues(P <0.001;P <0.001).4.Analysis of RT-qPCR data and immunohistochemical data showed that SNHG5 expression was positively correlated with E-cadherin protein expression(P<0.001),but negatively correlated with N-cadherin and Vimentin protein expression(P<0.001;P<0.001).Part Two The role of SNHG5 in the regulation of EMT and its biological behavior in gastric cancer cellsObjective: To investigate the effect of SNHG5 on proliferation,migration and invasion of gastric cancer cells in vitro and its effect on EMT of gastric cancer cells.Methods:1 The expression of EMT-related protein in gastric cancer cell lines was detected by Western blot and the cells were screened.2 MTS,scratch healing,Transwell migration and invasion assays were used to detect the effect of SNHG5 overexpression or knockdown on proliferation,migration and invasion of gastric cancer cells.3 The effect of SNHG5 overexpression or knockdown on EMT-related protein expression in gastric cancer cells was detected by Western blot and immunofluorescence.Results:1.The expression of EMT-related protein in gastric cancer cell lines was detected by Western blot,and the gastric cancer cells SGC-7901 and MGC-803 were selected for in vitro experiments.2.MTS results showed that SNHG5 overexpression or knockdown did not affect the proliferation of SGC-7901 and MGC-803 cells(P>0.05;P>0.05).3.Scratch healing experiments showed that SNHG5 overexpression significantly reduced the migration ability of SGC-7901 and MGC-803 gastric cancer cells(P<0.001;P<0.001),whereas knockdown of SNHG5 significantly enhanced the migration ability of SGC-7901 and MGC-803(P<0.001;P<0.001).4.Transwell chamber migration experiments showed that overexpression of SNHG5 significantly reduced the migration ability of gastric cancer cells SGC-7901 and MGC-803(P<0.001;P<0.001),while knockdown of SNHG5 significantly enhanced the migration ability of SGC-7901 and MGC-803(P<0.001;P<0.001).5.Transwell invasion assay showed that overexpression of SNHG5 significantly attenuated the invasiveness of gastric cancer cells SGC-7901 and MGC-803(P<0.001;P<0.001),while knockdown of SNHG5 significantly enhanced the invasion ability of gastric cancer cells SGC-7901 and MGC-803(P<0.001;P<0.001).6.Western blot showed that overexpression of SNHG5 increased the expression of E-cadherin(P<0.001,P<0.01)and decreased the expression of N-cadherin and Vimentin in SGC-7901 and MGC-803 gastric cancer cells(P<0.05,P<0.001),indicating that overexpression of SNHG5 can inhibit gastric cancer cells EMT.Knockdown of SNHG5 decreased the expression of E-cadherin(P<0.01,P<0.01)and increased the expression of N-cadherin and Vimentin(P<0.001,P<0.001;P<0.01,P<0.01),indicating that knockdown of SNHG5 can promote gastric cancer cells EMT.7.Immunofluorescence experiments showed that overexpression of SNHG5 increased the expression of E-cadherin,decreased the expression of N-cadherin and decreased the expression of Vimentin in gastric cancer cells SGC-7901 and MGC-803,indicating that overexpression of SNHG5 can inhibit the EMT of gastric cancer cells.Knockdown of SNHG5 reduced the expression of E-cadherin,increased the expression of N-cadherin and increased the expression of Vimentin in gastric cancer cells SGC-7901 and MGC-803,indicating that knockdown of SNHG5 can promote the EMT of gastric cancer cells.Part Three Study for the mechanism of SNHG5 regulating EMT and its effect on reversing EMT of gastric cancer cells in nude miceObjective: To investigate the mechanism of SNHG5 regulating the process of EMT and the formation of Twist / Mi2 / NuRD complex.Methods:1 The effects of SNHG5 overexpression on MTA2 and Twist subcellular localization in gastric cancer cell lines SGC-7901 and MGC-803 were examined by nuclear plasmapheresis and Western blot.2 The effect of SNHG5 overexpression on MTA2 and Twist subcellular localization and binding in gastric cancer cells SGC-7901 and MGC-803 was examined by immunofluorescence assay.3 Chromatin immunoprecipitation assay was used to detect the effect of overexpression of SNHG5 on the binding ability of Twist and E-cadherin promoter.4 The effect of over-expression of SNHG5 on the reversal of EMT in gastric cancer cells in nude mice was detected by nude mice tumor-bearing experiment.Results:1.Cytosolic plasma fractionation and Western blot showed that MTA2 and Twist were mainly expressed in the nucleus of gastric cancer cells SGC-7901 and MGC-803,and over-expression of SNHG5 reduced the expression of MTA2 and Twist in the nucleus(P<0.01)MTA2 and Twist expression increased(P<0.01),indicating that SNHG5 can prevent MTA2 and Twist into the nucleus.2.Immunocytochemistry showed that MTA2 and Twist were mainly expressed in the nucleus of gastric cancer cells SGC-7901 and MGC-803.Overexpression of SNHG5 reduced the expression of MTA2 and Twist in the nucleus and increased the expression of MTA2 and Twist in the cytoplasm,indicating that SNHG5 can prevent MTA2 and Twist from entering the nucleus.Co-localization experiments showed that the binding of MTA2 to Twist protein was decreased after SNHG5 overexpression,indicating that SNHG5 can inhibit the binding of MTA2 to Twist protein.3.Chromatin immunoprecipitation experiments showed that SNHG5 can inhibit the binding of Twist to the E-cadherin promoter region and reverse the inhibition of E-cadherin expression by Twist(P<0.01).4.Nude mice tumor experiments showed that SNHG5 overexpression can prevent gastric cancer cell tumor-bearing tumor in vivo EMT progress.Conclusion: E-cadherin was lowly expressed in tumor tissue and peripheral blood of patients with gastric cancer,while N-cadherin and Vimentin were highly expressed in tumor tissues of patients with gastric cancer,and the expression of SNHG5 was positively correlated with the expression of E-cadherin,But negatively correlated with the expression of N-cadherin and Vimentin,it shows that a certain degree of EMT occurs during the development of gastric cancer,and SNHG5 may negatively regulate the EMT process.Overexpression of SNHG5 can significantly inhibit gastric cancer cell migration and invasion ability,knockdown of SNHG5 can significantly enhance the ability of gastric cancer cell migration and invasion.Overexpression of SNHG5 can inhibit gastric cancer cells EMT,and knockdown of SNHG5 can promote gastric cancer cells EMT.SNHG5 blocks the entry of MTA2 and Twist into the nucleus,inhibits the binding of MTA2 to Twist protein and inhibits the binding of Twist to the E-cadherin promoter region,thereby preventing the Twist/Mi2/NuRD complex from binding to the promoter region of E-cadherin,Which in turn reversed the inhibition of E-cadherin expression by this complex.In vivo experiments show that SNHG5 overexpression can prevent gastric cancer cell tumor-bearing EMT tumor progression in vivo. |
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