The Effects Of MiR-7 Overexpressed On EMT And Invasion In Human Gastric Cancer Cells Induced By Diallyl Disulfide

Objective: The aim of this study was to investigate the effects of miR-7 overexpressed on the proliferation,migration,invasion and EMT through blocking EGFR/Rac1/Pak1 pathway in human gastric cancer SGC-7901 cells induced by diallyl disulfide(DADS)and to explore its molecular mechanism.we built the miR-7 overexpressed of SGC-7901 cells in human gastric cancer.It confirmed that miR-7 is a target of migration and invation in human gastric cancer cells by DADS.It provided theoretical and experimental basis for the therapy of gastric cancer and development of DADS.Methods: The lentivirus vector of miR-7 overexpressed gene was construct,and transfected it into SGC-7901 cells to establish the stable cell line of over-expressed miR-7.q RT-PCR assay were performed to detect the expression of miR-7 m RNA level.Each group was observed the morphology changes of human gastric cancer SGC-7901 cells cultured in vitro under phase contrast microscope.We confirmed that EGFR is the target gene of miR-7 by the target gene prediction software.The MTT,scratch,transwell migration and invasion assays were performed to evaluate the effects of miR-7 overexpressed and DADS on proliferation,EMT,migration and invation of the human gastric cancer SGC-7901 cell.In addition,Western blot assay detected that the expression of EGFR,Rac1/Pak1 pathway and EMT-related moleculesβ-catenin and vimentin.The expression of Vimentin and E-cadherin were detected by immunofluorescence assay.q RT-PCR experiment confirmed that the expression of EGFR,PAK1,Rac1,E-cadherin in SGC-7901 cells by DADS.Results:1.The successful construction of miR-7 overexpressed SGC-7901 cell with stably miR-7 to overexpress.At first,we bought the lentiviral vector of miR-7 overexpressed and lentiviral vector of empty carried from Hanbio Biotechnology Company.The two lentiviral vector were transfected into SGC-7901 cell with polybrene and puromycin added.Then,we saw that a large number of green fluorescent cells by fluorescence microscopy.The transfected and selected cells were added puromycin for maintenance screening.After amplification,the expression of miR-7 was detected by q RT-PCR.The SGC-7901 cell line with miR-7 overexpressed and empty carried,which express moderate stable cell line,were selected,screened and passaged for 3 passages for use later.2.It predicted EGFR is the target gene of miR-7.The EGFR 3’-UTR was associated with miR-7 binding site GUCUUCC by Target Scan、Pic Tar and miRandathe prediction software.Morphological changes of SGC-7901 cells by DADS.The group of SGC-7901 cell without DADS were activity and formed a polygon and spindle,pleomorphism,highly irregular cell nuclear,stick a wall firmly,propagation speed by inverted microscope.The morphology of the SGC-7901 group was similar to that of the SGC-7901/empty vector group.The cell growth of SGC-7901/miR-7+DADS group was the most obvious,and the cell morphology became round and loose,filled with a large number of granular and vacuolar material.It indicated that DADS and miR-7 overexpressed can inhibit the EMT in SGC-7901 cells.4.Effects of DADS and miR-7 overexpressed on the proliferation,in SGC-7901 cells.MTT assay showed that the proliferation rate of miR-7 overexpressed group decreased by 86.54%,74.25%,65.65% and 86.54%,73.90%,65.39%,compared with SGC-7901 and vector groups respectively at 24 h,48h and 72h(p<0.05).The proliferation rate of SGC-7901 + DADS group was 82.26%,71.90% and 63.09%,82.57%,72.53% and 63.39%,respectively,compared with SGC-7901 cell group and vector group at24 h,48h and 72 h after treatment with DADS(p<0.05).The cell proliferation rate of miR-7 + DADS group was decreased by 74.92%,60.91%,51.97% and 74.92%,60.63%,51.76% compared with SGC-7901 cell group and vector group respectively(p<0.05).The proliferation rate of miR-7 + DADS group was significantly decreased by 86.57%,82.03%and 70.16% compared with miR-7 group(p<0.05).The results showed that DADS and miR-7 overexpressed could inhibit the proliferation of SGC-7901 cells in a time-dependent manner.Mi R-7 could enhance the proliferation of DADS.5.Effects of DADS and miR-7 overexpressed on the migration and invasion in SGC-7901 cells.Transwell migration assay showed,the migration cells of SGC-7901 /miR-7 group(84.6±7.3)was significantly lower than that of SGC-7901group(186.4±6.8)and SGC-7901/vector group(184.6±6.7)(p<0.05);SGC-7901+DADS group(105.0±7.2)and SGC-7901+DADS group(95.1±6.2)were significantly lower than those in untreated DADS group(p<0.05).The migration cells of SGC-7901/miR-7 + DADS group(61.0±6.3)are least than others.The scratch woud healing assay showed that SGC-7901/miR-7 group(28.0%)had decreased on migtation rate compared with SGC-7901 group(42.1%)and SGC-7901/vector group(41.9%)(p<0.05).SGC-7901+DADS group(31.9%)and vector+DADS(30.7%)decreased compared with the untreated DADS groups(p<0.05),SGC-7901/miR-7+DADS group(10.6%)has the lowest migration rate.The results showed that miR-7 overexpressed and DADS could inhibit the migration of SGC-7901 cells,and miR-7 overexpressed could enhance the ability of DADS to inhibit migration.DADS could up-regulate the migration of SGC-7901 cells.Transwell invasion assay showed,the number of cells in SGC-7901/miR-7 group(24.0±3.8)was significantly lower than that in SGC-7901group(60.4±6.5)and SGC-7901/vector group(61.2±4.3)(p<0.05).SGC-7901+DADS group(26.8±3.3)and SGC-7901+DADS group(25.6±4.0)were significantly lower than those in untreated group(p<0.05)SGC-7901/miR-7+DADS group(8.6±2.1)were least.The results showed that miR-7 overexpressed and DADS could inhibit the invasion of SGC-7901 cells,and miR-7 overexpressed could enhance the ability of DADS to inhibit the invasion.DADS could up-regulate the invasion of miR-7 overexpressed in SGC-7901 cells.6.Effects of DADS and miR-7 overexpressed on EGFR,Rac1,Pak1.q RT-PCR and Western Blot assays showed that the expressions of EGFR,Pak1 and Rac1 were down-regulated by DADS in untreated group(p <0.05).The expression of EGFR,Pak1 and Rac1 in SGC-7901/miR-7was significantly lower(p<0.05)than the control group.The SGC-7901/miR-7+DADS group were least.The results indicated that DADS and miR-7 overexpressed can regulate EGFR/Rac1/Pak1 pathway.Immunofluorescence assay showed that the EGFR,Pak1 and Rac1 fluorescence in the DADS treated group were lower than those in the untreated group,and the fluorescence were intensity of the miR-7 +DADS group was the weakest in the miR-7+DADS group.The results were consistent with q RT-PCR and Western blot.8.Effects of DADS and miR-7 overexpressed on EMT.Western blot showed that the expression of β-catenin and Vimentin was down-regulated by DADS in untreated group and the expression of E-cadherin was up-regulated(p<0.05).The expression of β-catenin and Vimentin was down-regulated and The expression of E-cadherin was up-regulated in miR-7 group(p<0.05).The expression of E-cadherin was significantly higher and the expression of β-catenin and Vimentinin were lower than others in miR-7+DADS group(p<0.05).The results showed that DADS and miR-7 overexpressed could inhibit the EMT of SGC-7901 cells.miR-7 overexpressed could enhance the ability of DADS to inhibit EMT.DADS could up-regulate the invasion of miR-7overexpressed in SGC-7901 cells.Immunofluorescence assay showed that the expression of Vimentin in the DADS treated group was lower and the expression of E-cadherin was higher than that in the untreated group.The Vimentin fluorescence was attenuated in the miR-7 group.The E-cadherin fluorescence was enhanced in the miR-7 group.The expression of E-cadherin was highest in the miR-7+DADS group and Vimentin fluorescence is the weakest.The results were consistent with those of Western blot.Conclusion1.Upregulation of miR-7 can inhibit the proliferation,migration,invasion and EMT through EGFR/Rac1/Pak1 pathway in human gastric cancer cells induced by diallyl disulfide.2.miR-7 overexpressed can enhance the effect of DADS on the proliferation,EMT,migration,and invasion through EGFR/Rac1/Pak1 pathway in human gastric cancer SGC-7901 cells.