ROCK2 Affects The Metastasis Of Hepatocellular Carcinoma By Regulating Ubiquitin-independent Proteasomal Degradation Of P53

Background and Objective:Epithelial mesenchymal transition（EMT）is the initial step for malignant tumor to invade into surrounding tissues and undegoes distant metastasis.Tumor suppressor gene p53 has been confirmed to be involved in the regulation of EMT in tumor cells.However,under unstressful conditions,p53 protein is extremely unstable.Ubiquitin-proteasome system is considered to be the main pathway for p53 protein degradation.Therefore,stabilizing the expression of p53 protein may be an effective strategy for preventing the invasion and metastasis of HCC.Rho associated coil forming protein kinase 2（ROCK2）,a key signal molecule in Rho/ROCK signal transduction pathway,is involved in the biological function of regulating cell adhesion,contraction,migration and division.A number of studies have shown that ROCK2 participates in cellular proliferation,migration and angiogenesis by regulating the expression of tumor suppressor.However,the role and mechanism of ROCK2 regulating tumor suppressor（especially p53）in the process of EMT and invasion and metastasis of HCC is still unclear,which needs further study.Therefore,this study aimed to explore the effect of ROCK2 regulating p53 expression on HCC metastasis.Methods:1.We used p53^+/+HCCLM3 cells and p53^-/-Hep3B cells to construct stable ROCK2 knockdown HCC cell lines.Then,the effects of ROCK2 on cancer metastasis and EMT of HCC in vivo and in vitro were explored by animal imaging system and H&E staining,Western blotting and immunofluorescence.2.The impact of ROCK2 on p53 protein expression was detected by quantitative PCR,Western blot and immunoprecipitation.3.The molecular biological mechanism of ROCK2 regulating p53 protein expression was explored by quantitative PCR,Western blotting,CRISPR/Cas9 and immunoprecipitation.4.Western blotting was used to explore the relationship between the expression of ROCK2 and p53 in p53^+/+HCC tissues.Results:1.Compared with HCCLM3-shNC group,the number of metastatic foci in HCCLM3-shROCK2 group were significantly reduced.However,there was no significant difference on lung metastasis between Hep3B-shROCK2 group and Hep3B sh NC group.Western blot showed that ROCK2 inhibition in HCCLM3increased the expression of E-cadherin,occludin and p53,and decreased the expression of N-cadherin and vimentin;however,in Hep3B cells,down-regulation of ROCK2 expression had no significant effect on the expression of EMT related molecular markers.Furthermore,Western blot showed that knockdown of ROCK2 in HCCLM3 cells increased the expression level of p53 protein and inhibited the EMT process,while down-regulation of p53 could abolish the changes caused by ROCK2knockdown.Overexpression of ROCK2 did not affect the expression of EMT related molecular markers in Hep3B cells,but ROCK2 overexpression could lead to the decline of p53,E-cadherin,occludin protein expression,and the increase of N-cadherin and vimentin expression when p53 was rescued.2.The results of real-time PCR showed that ROCK2 knockdown had no significant impact on p53 mRNA level.Western blot showed that down-regulation of ROCK2 expression promoted the increase of p53 protein level,while the up-regulation of ROCK2 expression promoted the decrease of p53 protein expression.The degradation dynamics assays showed that the half-life of p53 protein was extended in ROCK2-downregulating HCC cells but shorten in ROCK2-upregulating HCC cells.After MG132 was added to block the protein degradation by proteasome pathway,neither up-regulation nor down-regulation of ROCK2 expression had an impact on the level of p53 protein,suggesting that ROCK2 may affect the expression of p53 protein by promoting the proteasomal degradation of p53 protein.Furthermore,immunoprecipitation showed that the ubiquitination level of p53 protein was increased when ROCK2 was knocked down,and decreased when ROCK2 was overexpressed.In addition,there was no significant change in the expression of MDM2 and p-MDM2 regardless of up-regulation or down-regulation of ROCK2,suggesting that ROCK2 may promote the degradation of p53 protein through ubiquitin-independent proteasomal pathway.3.The results of immunoprecipitation showed that there was no interaction between ROCK2 and p53 protein,suggesting that ROCK2 may promote the degradation of p53 protein by regulating the expression of an intermediate protein.Western blot showed that the level of p53 protein was decreased when FAT10 was overexpressed.At the same time,immunoprecipitation showed that FAT10 could interacted with p53 protein.Furthermore,we found that overexpression of ROCK2elevated the expression of FAT10 but reduced the expression of p53 protein,while knockdown of FAT10 abolished the decrease of p53 protein caused by ROCK2 overexpression.When ROCK2 was down-regulated,FAT10 protein level decreased and p53 protein expression increased.Overexpression of FAT10 abolished the increase of p53 protein level caused by ROCK2 knockdown.In FAT10 knockout HCCLM3 cells,knockdown of ROCK2 did not affect the changes of p53 protein and EMT related molecular markers,while knockdown of ROCK2 promoted the expression of p53 protein when FAT10 protein was restored.At the same time,immunoprecipitation results showed that overexpression of ROCK2 inhibited the ubiquitination of p53 protein,while knockdown of FAT10 abolished the decrease of ubiquitination of p53 protein caused by ROCK2 overexpression;knockdown of ROCK2 increased the level of ubiquitination of p53 protein,while overexpression of FAT10 abolished the increase of ubiquitination level of p53 caused by ROCK2knockdown.These results suggest that ROCK2 may affect p53 protein degradation and EMT by regulating the expression of FAT10 protein.4.We found that ROCK2 knockdown could lead to the reduction of FAT10mRNA.Western blot results showed that ROCK2 knockdown could result in the decrease of FAT10 protein expression and the increase of p53 protein expression,suggesting that ROCK2 affects the expression of FAT10 protein by regulating the transcription level of FAT10.Furthermore,we constructed a wild-type STAT3plasmid and two mutated STAT3 plasmids（tyrosine 705 mutated to phenylalanine,serine at 727 to alanine）.The results of real-time PCR showed that overexpression of wild-type or S727A plasmid increased FAT10 mRNA level,while overexpression of Y705F plasmid did not affect FAT10 mRNA level,suggesting that STAT3 protein affects FAT10 gene transcription through phosphorylation of STAT3 at tyrosine 705.Western blot results showed that the protein expression of p-STAT3（Y705）,FAT10and vimentin was decreased,while the expression of p53 and E-cadherin was increased when the expression of ROCK2 was knockdown.ROCK2 knockdown also downregulated the expression of p-STAT3（Y705）in nucleus and cytoplasm.5.We found that ROCK2 knockdown alone or combined with chemotherapy can elevated the mRNA levels of p21 and puma,and similar experimental results were obtained by Western blot.Flow cytometry showed that ROCK2 knockdown alone or combined with chemotherapy can increase the level of apoptosis.These results suggest that inhibition of ROCK2 expression could activate the function of p53protein in vitro.Furthermore,we found that the combination of fasudil,a ROCKs inhibitor,and chemotherapy could significantly inhibit the growth of tumor in nude mice.These results suggest that inhibiting the activation of ROCK2 could activate the function of p53 protein in vivo.Finally,we selected 30 HCC tissues with wild-type p53 by exon sequencing.Western blot showed that the expression of ROCK2 was positively correlated with the expression of FAT10,but negatively correlated with the expression of p53.Conclusion:In this study,we found that ROCK2 promotes the degradation of p53 through ubiquitin-independent proteasome pathway by regulating the expression of FAT10protein,thereby promoting EMT and metastasis of HCC cells.Our findings provide a new theoretical basis for the study of metastasis of liver cancer,and will provide a new research direction for the prevention and treatment for liver cancer.