Identification Of The Genes Associated With Growth And Metastasis In Human Hepatocellular Carcinoma

The two main characteristics of tumor cells are uncontrolled proliferation and aggressive behavior.It is a multigene-involved,multistep, and changing process.Defining genes that control growth and metastasis can provide new targets for interfering in this process.LASS2 and SLIT2 are two genes associated with growth and metastasis in HCC.However, their detailed molecular mechanisms for the development and progression of HCC are not very clear.We have identified LASS2,a previously unknown human homologue of the yeast longevity assurance gene LAG1.Yeast two-hybrid screening and glutathione S-transferase pull-down assays show that the LASS2 protein interacts with ATP6L,the 16 kDa subunit of proton pump V-ATPase, can provide proton hydrophilic transmembrane path.Furthermore,LASS2 protein is able to inhibit the colony formation of human hepatoma cells in vitro,which suggests that this gene may be involved in the regulation of cell growth.Bioinformatics analysis dedicate that LASS2 has an identical segment:TMSG-1.It is show that prostate cancer cell 1E8 of advanced metastatic potential strongerly express TMSG-1 when compared with 2B4 of non-metastatic potential.The results suggest that LASS2 genes maybe control metastasis.In our previous work,we also find that the inhibition of V-ATPase function via knockdown of ATP6L expression using RNA interfering technology can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity.So when we try to definitize a possible mechanism of LASS2 suppressing tumor metastasis,ATP6L is a interesting target.We want to determine the function of LASS2 and the correlation of LASS2 and ATP6L in tumor metastasis. Here,we precisely confirmed that LASS2 interacted with ATP6L by protein subcellular colocatlization and co-immunoprecipitation(co-IP) and LASS2(181-255) is the smallest binding motif which interacted with ATP6L.We construct the recombinant adenovirus vectors expressing human LASS2 gene and mutation of LASS2 gene including combined and uncombined with ATP6L.After LASS2 and LASS2(181-255) was overexpressed in HCCLM3 cells via recombinant adenovirus vectors,the secretion and activation of MMP-2 and the pHi recovery from NH4C1-prepulsed acidification were inhibited.Meantime,the growth, migration and invasion of HCCLM3 were suppressed in vitro when LASS2 and LASS2(181-255) was overexpressed in HCCLM3 cells,and the apoptosis of HCCLM3 was increasing.These results showed that LASS2 and LASS2(181-255) can inhibit growth of SMMC-7721 cell in vitro, which is through apoptosis o In conclusion,our data suggest that LASS2 is involved in the control of cancer growth and metastasis via interacting with ATP6L,a subunit of proton pump V- ATPase.SLIT2 are candidate ligand for repulsive guidance receptors,the ROBO gene family.Slit-Robo interactions mediate the repulsive cues on axons and growth cones during neural development.Recent findings have shown that SLIT2 appears to function as a novel tumor suppressor gene.In addition,hypermethylation of its promoter region has been detected in various cancers,including breast and lung cancer,colorectal carcinoma, and gliomas.Here,we report for the first time that there is epigenetic silencing of SLIT2 in human hepatocellular carcinoma(HCC).Downregulation of SLIT2 was detected in 6 of 8(75%) HCC cell lines by quantitative real-time RT-PCR(qRT-PCR),and the downregulation of SLIT2 was generally dependent on the degree of methylation at the promoter region. Furthermore,expression of SLIT2 was restored in relatively low-expressing cell lines after treatment with 5-aza-2-deoxycytidine (5-Aza-dC).Downregulation of SLIT2 expression was also detected in 45 of 54 primary HCC samples(83.3%),and the decrease in expression was significantly correlated with CpG island hypermethylation.This decrease of SLIT2 expression was also associated with lymph node metastasis in HCC.Moreover,overexpression of SLIT2 in SMMC-7721 cells induced by recombinant adenovirus suppressed cell growth,migration,and invasion, These results suggest that epigenetic inactivation of SLIT2 in HCC may be important in the development and progression of HCC.Thus,SLIT2 may be useful as a therapeutic target in the treatment of HCC.In conclusion,our experiments firstly reported that LASS2 is involved in the control of cancer growth and metastasis via interacting with ATP6L, a subunitof proton pump V- ATPase.Meanwhile we firstly confirmed that epigenetic inactivation of SLIT2 in HCC may be important in the development and progression of HCC.Our results implicate possible mechanisms of carcinogenisis and metastasis of tumor,and furthermore, the potential for cancer treatment.