Investigation Of DLC1 On Regulating Colon Cancer Cell Migration Via MANF Secretion

?Background and Objectives? With the change of living level and diet habit in China,the incidence and mortality of colorectal cancer continued to increase from 2000 to 2011.The 5-year survival rate of colorectal cancer,which was closely related to pathological type,was about 65%.Furthermore,invasion and metastasis of tumor are main causes of the high mortality.DLC1,also known as p122 Rho GAP,ARHGAP7 and STARD12,is a GAP of a class of Rho family protein,which can inhibit tumor genesis and metastasis.The mutation or deficiency of DLC1 is present in a good deal of cancers,such as breast cancer,colorectal cancer,lung cancer,etc.The frequency of mutation or deficiency of DLC1 could be comparable with p53.The aim of this study was to investigate the mechanism of DLC1 inhibiting the migration of colon cancer cells by regulating the secretion of cytokine MANF.?Methods?(1)Inhibitory effects of different isoforms of DLC1 on proliferation and migration of colon cancer cells Constructing the lentiviruses overexpressing different isoforms of DLC1.Through CCK8 and transwell to study the functions of different isoforms of DLC1 in colon cancer cell.(2)The effects of secretory proteins from SW1116 overexpressing isoform 2 DLC1(SW1116 DLC1 i2)on the proliferation and migration of colon cancer cells The conditioned medium of SW1116 DLC1 i2 was collected and used to culture SW1116 wild type(SW1116 wt)cell.CCK8 and transwell were used to study the effects of the conditioned medium.Differential proteins in SW1116 DLC1 i2 conditioned medium were further detected by silver staining and liquid chromatography–mass spectrometry(LC-MS).(3)The effects of secretory protein MANF on proliferation and migration of colon cancer cells Constructing the cell lines overexpressing MANF and MANF knockdown respectively,the supernatant of which was collected and concentrated.CCK8,transwell and wound healing test were used to study the influence of intracellular or extracellular MANF on the proliferation and migration of colon cancer cell.(4)DLC1 affecting the migration of colon cancer cells by promoting the secretion of MANF q RT-PCR was used to detect that DLC1 affected what genes related to the secretion of MANF.Transwell was used to study the changes of migration in the overexpressing MANF and DLC1 knockdown cell line.(5)Statistical method SPSS 21.0 software was used for statistical analysis.Pairing test or rank test was used in comparison between groups.Two-factor analysis of variance was used in comparison of multiple sets of sample mean.P <0.05 indicates that the difference was statistically significant.?Results?(1)Different isoforms of DLC1 were all down-regulated in colorectal cancer tissues.Isoform 1 DLC1(DLC1 i1)was down-regulated in 19 cases of colorectal cancer tissues(P=0.0091);isoform 2 DLC1(DLC1 i2)was down-regulated in 19 cases of colorectal cancer tissues(P=0.0094);isoform 3 DLC1(DLC1 i3)was down-regulated in 18 cases of colorectal cancer tissues(P=0.002).(2)Isoform 1 DLC1 and isoform 2 DLC1 had significant inhibitory effects on proliferation of SW1116 cell line comparing with vector group.Comparing with vector group,SW1116 overexpressing isoform 1 DLC1(SW1116 DLC1 i1)and SW1116 DLC1 i2 had a significant inhibitory effect on proliferation(P=0.002 and P=0.008 respectively).However,DLC1 i3 had not significant inhibitory effect on proliferation(P=0.15).(3)Isoform 2 DLC1 had a significant inhibitory effect on migration of SW1116 comparing with vector group.SW1116 DLC1 i2 had a significant inhibitory effect on migration(P=0.011)comparing with vector group.However,DLC1 i3 and DLC1 i4 had not significant inhibitory effect on migration(P=0.956 and P=0.234 respectively).Expressions of Snail and Vimentin were down-regulated in SW1116 DLC1 i2 and SW1116 DLC1 i4,which did not change significantly in SW1116 DLC1 i3.Meanwhile,the changes of expression of N-cadherin was not significant in all 3 cell lines. (4)The conditioned medium of SW1116 DLC1 i2 had an inhibitory effect on migration of colon cancer cells.The conditioned medium of SW1116 DLC1 i2 had a significant inhibitory effect on migration of HCT116 and SW1116(P=0.0017 and P=0.0002 respectively),but it had no significant effect on proliferation of these cell lines.(5)Secretory protein MANF was discovered in concentrated conditioned medium of SW1116 DLC1 i2 by LC-MS.Through the results of LC-MS,we found that the overexpression of DLC1 in SW1116 significantly up-regulated the expression of MANF in the supernatant.But further consequences of q RT-PCR showed that DLC1 did not regulate the transcription of MANF.(6)Intracellular overexpression of MANF had a significant inhibitory effect on proliferation of colon cancer cells.Intracellular overexpression of MANF had a significant inhibitory effect on proliferation of SW1116(P=0.0053),and intracellular MANF knock-down promoted the proliferation of cancer cells(P<0.05).Extracellular overexpression of MANF had no significant effect on proliferation of SW1116 and Hct116.(7)Both of intracellular and extracellular overexpression of MANF could significantly inhibit the migration of colon cancer cells.Intracellular overexpression of MANF could significantly inhibit the migration of SW1116(P<0.01).Extracellular overexpression of MANF(the conditioned medium of SW1116 with MANF overexpression or recombinant protein of MANF)could significantly inhibit the migration of SW1116 wt and Hct116 wt respectively(P<0.01 respectively).Intracellular or extracellular knock-down MANF could promote the migration of cancer cells(P<0.01 and P<0.05 respectively).(8)DLC1 affected the secretion of MANF by down-regulating the expression of GRP78 and SERCA1.In SW1116 DLC1 i2 cell line,the relative expression of GRP78 was down-regulated to 0.612±0.015(P<0.01),and the relative expression of SERCA1 was down-regulated to 0.543 ± 0.079(P = 0.015).After knock-down DLC1 in the SW1116 DLC1 i2 cell line(sh DLC1-1 and sh DLC1-4),the relative expression of GRP78 was up-regulated to 2.587±0.268(P<0.01)and 1.423±0.0705(P=0.0267)respectively;meanwhile the relative expression of SERCA1 was up-regulated to 1.656±0.184(P=0.0371)and 1.888±0.1482(P=0.0267)respectively.(9)DLC1 inhibited the migration of SW1116 by MANF.After overexpressing DLC1 i2 in knock-down MANF SW1116,the number of cell migrating was only slightly lower than that of the control group,but there was no significant statistical difference(P=0.0535 and P=0.0111 respectively).?Conclusion? The expression levels of different isoforms of DLC1 in colon cancer tissues were all lower than those in adjacent tissues,and isoform 2 DLC1 had the strongest inhibitory effect on migration and proliferation of colon cancer cell.DLC1 i2 affected the migration of colon cancer cells by promoting the secretion of MANF through down-regulating the expression of GRP78 and SERCA1.