SIPA1 Inhibits The Recurrence And Metastasis Of HCC Through Down-regulateing The Activity Of Rap1-ERK Pathway

Hepatocellular carcinoma (HCC) is a main malignancy that threaten our people now. With the improvement of liver surgical technique and management level of perioperation, the resection rate of HCC is increasing and the operation mortality rate is approaching zero at large liver surgery centers. But the overall mortality of HCC patients remains high and the 5-year survial rate of patients is still low. The postoperation recurrence and metastasis of HCC is the main cause of high overall mortality. The 5-year recurrence rate of HCC after operation is more than 60%, even the small HCC the 5-year recurrence rate is above 40%. Obviously the recurrence and metastasis after HCC resection is the bottleneck problem that severely restricts the improvement of long survival rate of HCC patients. So deep study of the mechanism of recurrence and metastasis of HCC and explore the methods of anti-metastasis are focus of HCC research, which are very meaningful for improvement of long survival rate of HCC patients.Signal-induced Proliferation-associated Gene 1 (SIPA1) was found play an important role in the blood system tumors in the past decade. Recent research found that mouse sipal gene was an important gene that control the metastasis of mouse breast cancer. It is well-known that the liver is a hematogenic organ in embryonic period and blood cells (for example platelet) had close relationship with tumor metastasis. These facts urge us to explore the role of SIPA1 gene in the HCC which had high potential of metastasis. Up to now there is no report about this both at home and abroad. So in this study we first examine the expression of SIPA1 gene in HCC specimens and HCC cell lines, then overexpress the gene by cotransfection the recombinant plasmid (pcDNA3.1-SIPA1) with HCC cell line. Molecullar mechenism of the regulation of SIPA1 to HCC metastasis was studied by in vitro and in vivo. The results as follows was observed:1. The SIPA1 mRNA and protein in 30 fresh HCC specimens, which consisted of cancer tissue and corresponding ANLT(adjacent non-tumor liver tissue), was detected by Reverse Transcription PCR(Polymerase chain reaction) and Western blot.The expression of mRNA and protein of SIPA1 in fresh HCC tissues was lower than in ANLTs significantly(P<0.05). The expression of SIPA1 mRNA was positively correlated with SIPA1 protein(r=0.757, P<0.001). The real time PCR was used to detect the relative accurate exprssion of SIPA1 mRNA, and the expression of SIPA1 mRNA in ANLT was found to be 4.17 times of that in cancer tissue. The expression of SIPA1 in 130 paraffin HCC specimens was dectected by immunohistochemistry, the positive expression rate of SIPA1 protein in cancer tissue was 62.3% (81/130). Low expression of SIPA1 in cancer tissue of HCC had close relationship with multiple tumor nodules, poor differentiation and vein invasion (P<0.05). Patients with negative expression of SIPA1 in cancer tissue had a shorter overall survial (OS) than the positive group (41.0±6.6 months vs. 66.0±5.2 months, P=0.003), the disease-free survial (DFS) of negative group of SIPA1 was shorter than the positive group(35.0±6.1 months vs.61.1±5.4 months, P=0.002). Multiple cox regression showed negative expression of SIPA1 in cancer tissue was a independent risk factor for HCC patients. Thus results suggested that negative expression of SIPA1 gene had relationship with tumor invasion and metastasis.2. Through reverse transcriptase-PCR and Western blot the expression of SIPA1 mRNA and protein was detected in L02 cell line, CCL13 cell line and three HCC cell lines (HepG2, MHCC97-L, HCCLM3), their invasion ability increases in order. The expression of SIPA1 mRNA and protein in HCCLM3 was found to be lowest among the five cell lines (P<0.05).Thus results indicated that low expression of SIPA1 gene had relationship with invasion of HCC cells. In addition, HCCLM3 cell line was chosen as the target cell for overexpression of SIPA1.3. To analyze the clinical findings, overexpression SIPA1 plasmid was constructed with gene cloning and recombinant DNA technique. The HCCLM3SIPA1+cell line overexpressing SIPA1 stably was established successfully through Lipofectamine 2000 transfection and G418 screening. Through reverse transcriptase PCR and Western blot, the SIPA1 mRNA and protein was proved expressing higher in HCCLM3SIPA1+cell line than in HCCLM3vector(P< 0.05). MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to detect the proliferation and adhesion ability of HCCLM3SIPA1+cells, and overexpression of SIPA1 gene was found to inhibit the proliferation ability and adhesion ability to fibronectin of HCC cells (P<0.05). The abilities of Wound-healing, Transwell invasion, formation of cytoskeleton of HCCLM3SIPA1+cell line were all weaker than that of HCCLM3vector cell line (P<0.05). Thus results all showed that overexpression of SIPA1 gene could inhibit the proliferaton, adhesion, motility and invasion ability of HCC cells in vitro.4. We further examined the in vivo relevance of the potential role for SIPA1 in HCC tumorigenesis and metastasis by using a orthotopic nude mice implantation liver cancer model. The tumors of mouse livers were measured, we found that the average volume of primary tumors in HCCLM3SIPA1+group was dramatically smaller than that of HCCLM3vector group (1.57±0.08cm3 vs.6.28±0.13cm3), which suggested that up-regulation of SIPA1 in vivo could inhibit growth of HCC. The expression of SIPA1 in liver tumors was determined by immunohistochemistry to ensure the difference of SIPA1 expression in vivo. The pulmonary metastasis was observed in the lung tissue sections, and the ratio of pulmonary metastasis in HCCLM3SIPA1+cell group was found significantly less than in HCCLM3vector cell group (16.7% vs.83.3%, P=0.021). Together, these data supported that SIPA1 played an important role in inhibiting HCC tumorigenesis and metastasis in vivo.5. Many researches have proved that SIPA1 gene play an import role in bood system tumors through regulating the Rap1 small G protein in hematopoietic progenitor cells. So we speculate that SIPA1 may regulate the Rap1 protein in HCC cells. Pull-down assay showed the level of Rap1-GTP was lower in HCCLM3SIPA1+cells than in HCCLM3vector cells (P< 0.05). Next, co-immunoprecipitations confirmed the direct interaction of SIPA1 and Rap1 proteins in HCC cells. When HCCLM3SIPA1+cells were exposed in chemical factor 8CPT-2Me-cAMP (final concentration 100μM), the Rap1 activity recovered, and the motility, invasion ability and the formation of F-actin cytoskeletal protein of HCCLM3SIPA1+cells also restored. Western blot showed the expression of p-ERK decreased when the SIPA1 was up-regulated (P<0.05), and the expression of p-ERK restored when Rap1 activity was recoverd by adding chemical factor 8CPT-2Me-cAMP(final concentration 100μM) to the HCCLM3SIPA1+ cells.Thus results indicated that SIPA1 inhibited the invasion and metastasis of HCC cells probably through down-regulating the activity of Rap 1-ERK pathway.Conclusions:SIPA1 is firstly found significantly down-regulated in HCC tissue. Low expression of SIPA1 in HCC is related with multiple tumor nodules, poor differentiation, vein invasion and early recurrence and metastasis. SIPA1 is an independent risk factor for the prognosis of HCC and may became a molecular marker of survival and recurrence after operaton. In vitro and in vivo exprements prove that SIPA1 plays an important role in inhibiting the proliferation ability, invasion and metastasis of HCC cells through down-regulating Rap1-ERK pathway.