ZC3H14 Inhibits HCC Progression By Deactivating Integrin Signaling

ObjectiveHepatocellular carcinoma?HCC?is one of the most common cancers in China,especially with high incidance and mortality.Genetic variations play important roles in the development of HCC.Genomic copy number deletion at chromosome 14q31.1-32.13 was frequently observed in HCC;however,the relevant functional target?s?at that locus is not well determined.Here,the aim of the research is to identify the major gene?s?in the 14q31.1-32.13 deletion and to investigate the effect and mechanism of the candidate gene ZC3H14 on HCC cells growth and metastasis through combing bioinformatics analyses and experimental investigation.MethodsThe genomic copy number data,mRNA expression data and survival information of a collection of 362 HCC patients were obtained from The Cancer Genome Atlas?TCGA?database.The relative copy number?log₂ transformed?greater than 0.3 or less than-0.3 were defined as genomic amplification or deletion,respectively.The differentially expressed genes were determined as ones with fold change>1.2 and multiple testing-corrected P value<0.00081?adjusted for 62 genes at chromosome 14q31.1-32.13?by Student's t test.To validate this genomic finding,we also analyzed the CNA-mRNA expression correlation in a retrospective cohort of 274 HCC patients.The correlation between genomic copy number and mRNA expression were calculated by Pearson correlation analysis.In addition to the TCGA dataset,gene expression datasets from other three HCC cohorts were obtained from the Gene Expression Omnibus?GEO;Accession No.GSE14520,GSE22058 and GSE69164?to verify the differential expression of ZC3H14 in HCC tissues,adjacent non-tumor liver tissues and portal vein thrombus.GSE14520,including 225 HCC tissues and 220 adjacent non-tumor liver tissues,were primarily analyzed using Affymetrix HT Human Genome U133A Array.GSE22058,consisting of 96pairs of HCC tissues and non-tumor liver tissues,were primarily analyzed using Rosetta/Merck Human RSTA Custom Affymetrix 1.0 microarray.GSE69164,including 11 HCC tissues,matched adjacent non-tumor liver tissues and portal vein tumor thrombus?PVTT?tissues,were primarily analyzed by RNA-sequencing.HepG2 and MHCC-97L cell lines,which exhibit higher mRNA expression levels of ZC3H14 than the other cell lines,were stably transfected with two independent shRNAs targeting ZC3H14.The proteins were extracted and the silencing effects were confirmed by Western blotting assays.ZC3H14 was also cloned into the lentivirus packing expression vector?pLV-Neo-Flag?and stably transfected into the SMMC-7721 and MHCC-97H cells that have relatively low expression levels of ZC3H14.The effects of knockdown or ove-rexpression of ZC3H14 on the cells growth and proliferation of HCC cell lines were detected by CCK8 assays,colony formation assays and BrdU assays.Transwell assay was used to detect the effect of knockdown or over-expression of ZC3H14 on the migration ability of HCC cell lines.In order to further verify whether ZC3H14 can also promote the growth and proliferation of HCC in vivo,we established a subcutaneous xenograft mice model to verify the effect of ZC3H14 on the growth and metastasis of HCC cells in nude mice.To delineate the mechanism of ZC3H14 in the suppression of tumorigenesis,we performed mRNA expression profiling to examine the gene expression changes in ZC3H14-depleted and control HepG2 cells.GSEA was used to explore the mechanism of ZC3H14's tumor suppressive role in HCC.qRT-PCR assays was used to verify the expression changes of downstream molecules of the pathway.We then assessed the effect of ZC3H14 on integrin pathway in HCC cells by biochemistry assays.Next,we used Cyclo?RGDyK?,an inhibitor of the integrin pathway,to assess whether ZC3H14 plays the tumor suppressor role in an integrin-dependent manner in HCC cells.ResultsIn this study,we further analyzed the expression levels of the 62 genes at14q31.1-32.13 locus using the TCGA mRNA expression dataset,and observed that 22 genes are significantly down-regulated in tumor tissues compared to the matched adjacent non-tumor tissues.Among these 22 down-regulated genes,17genes showed significant correlation between their mRNA expression levels and genomic copy number at 14q31.1-32.13.Notably,among these 17 genes,ZC3H14 ranked the first according to the correlation strength.Therefore,we speculated that ZC3H14 is the major gene?s?in the 14q31.1-32.13 deletions.In our HCC samples,we observed frequent copy number deletion?17.1%?and down-regulation of ZC3H14 in primary HCC tissues;mRNA expression was also significantly down-regulated and positively correlated with genomic copy number in HCC tissues.In the HCC samples of four independent public databases,ZC3H14 was significantly down-regulated in HCC tumor tissues compared with paired adjacent non-tumor tissues.Moreover,low expression levels of ZC3H14 correlate with poor clinical outcomes.ShRNA-mediated depletion of ZC3H14 promoted cells growth,proliferation,colony formation and migration in vitro.Consistently,over-expression of ZC3H14 could significantly inhibit the cells growth,proliferation,colony formation and migration.The subcutaneous xenograft mice model showed that knockdown of ZC3H14significantly promoted the growth of HCC cells.To further assess the tumor suppressor function of ZC3H14 indicated in subcutaneous xenograft model,we also established an orthotopic xenograft model.Knockdown of ZC3H14significantly promoted the growth and lung metastasis of HCC cells.ZC3H14knockdown enhanced the integrin pathway.Additionally,ZC3H14-depleted cells had increased mRNA expression levels of several known oncogenes,including fibronectin 1?FN1?,collagen type III alpha 1 chain?COL3A1?,laminin subunit beta 3?LAMB3?and myosin light chain 9?MYL9?.Western blotting detection revealed that knockdown of ZC3H14 significantly promoted the levels of p-Src and p-Fak in the Integrin signaling pathway;Over-expression of ZC3H14 significantly inhibited the levels of p-Src and p-Fak.Subsequently,we found that Integrin inhibitor could eliminate ZC3H14 knockdown induced ability to promote cell growth and migration,and weaken the promotion effect of ZC3H14 knockdown on p-Src and p-Fak.Taken together,these results indicated that the integrin signaling cascade is required for the tumor-suppressive role of ZC3H14 in HCC cells.ConclusionsThis study uncovered the underlying mechanism by which the duplication of ZC3H14 plays a tumor suppressive role of HCC.By using numerous biochemistry assays,we clarified that the presence of ZC3H14 is a crucial player that inhibits integrin pathway.Take together,our findings shed light on the critical role of ZC3H14 as a tumor suppressor in the development of HCC.