Preliminary Study On The Effect Of SIRT5 In The In The Genesis And Development Of Hepatocellular Carcinoma

Background and objectiveHepatocellular carcinoma(HCC)is a common malignant tumor of the digestive system.The GLOBOCAN 2008 investigation program showed that HCC is the third leading cause of cancer deaths worldwide.Although the treatment style of curative resection combined with adjuvant chemotherapy displays a certain extent effect in improving the prognosis of HCC patients,it remains a lethal disease.The high metastatic capabilities of HCC cells are the reason for the poor prognosis and high mortality rates of primary HCC.As such,prevention and treatment of HCC migration and invasion is of great importance for improving the prognosis of HCC patients.It's therefore of significant importance to seek optimal mechanisms and biomarkers associated with tumor progression and metastasis.Silent information regulator is a protein family which functions as histone deacetylase proteins.Studies confirm SIRT by members of the family of histone and carried on a variety of the histone acetylation modification to regulate gene expression,thus then participated in the regulation of many kinds of important physiological activities including cell differentiation,apoptosis,aging,energy metabolism and stress tolerance.Research on the effect and role of SIRT family in malignant tumor biology function and related mechanism of exploration will help us more comprehensive understanding of the development mechanism of HCC,search for new markers of diagnosis and treatment and related targetsSIRT5,a silent information adjustment factor,is a member of the family.Recent studies reported that in breast cancer and lung cancer SIRT5 may play to the role of the cancer gene,but the specific biological function in early tumor,especially in the role of liver cancer,there is no relevant research reports.The present study will screen the dis-regulated mRNAs in HCC cases with poor prognosis and those with intrahepatic metastasis lesions by using high throughput gene microarray.Then further to detect SIRT5 expression in HCC specimens by using qRT-PCR and immunohistochemical technique,in order to clarify its clinical significance.At last,we well clarify the effect of SIRT5 in regulating HCC biological behaviors in vivo and in vitro.Aimed to further looking for early diagnosis of liver cancer and prognosis judgement,determine the treatment decision-making of new molecular markers and provides a theoretical basis to the treatment.Methods1.Identification of dis-regulated mRNAs in high risk HCC casesThrough the analysis of the mRNA gene chip technology in liver cancer tissue and its matching the difference of mRNA expression in the tissue adjacent to carcinoma,and using independent sample t-test,we preliminary screening and early recurrence and metastasis of liver cancer related mRNA of potential function,and then detected the candidate mRNAs levels in the large sample size of HCC and its matching organization through the qRT-PCR method to test the randomly selected mRNA expression level.By comparing with the results of the chip in order to validate the results of gene chip,the final screen may identify potential genes those play a role in the recurrence and metastasis of liver cancer of mRNA.2.The expression and clinical significance of SIRT5 in HCCUsing immunohistochemically,fluorescence quantitative PCR and protein immunoblot experimental methods,we detected and analyzed SIRT5 expression levels in HCC tissue and tissue adjacent to carcinoma.We used student 's t-test analysis to evaluate the SIRT5 expression in liver cancer tissues and adjacent tissues,and used Chi-square test and Spearman correlation analysis to identify the relationship between SIRT5 expression and clinical pathologic features and prognosis of HCC patients,in order to understand the rolf of SIRT5 in HCC procession and development.3.In vitro function experiment on the role of SIRT in HCC progressionWe first used the qRT-PCR and Western blot method to detect SIRT5 expression level in HCC cell lines,and selected the HCC cell lines with SIRT5 high expression.In SIRT5 high expression cell lines,we silenced SIRT5 expression using slow virus interference technology in,and further utilization of CCK 8,clone formation,Transwell,such as in vitro experiment technology proven SIRT5 rights to HCC cells,invasion and migration of influence.Then by using flow cytometry and Western blot techniques such as detection SIRT5 for HCC cell cycle between epithelial and mesenchymal markers related to conversion,with preliminary clear SIRT5 in regulating the HCC cell proliferation,invasion and migration of the possible mechanism.4.In vivo function experiment on the role of SIRT in HCC progressionUsing the silent SIRT5 HCC cell lines build in the above part,we established subcutaneous tumor mice model and hematogones metastasis model in nude mice.We record the subcutaneous tumor mice tumor volume,measuring the quality of tumor,and detection of nude mice subcutaneously into tumor cell cycle protein expression level;at the same time record the weight growth and liver cancer cells metastatic lesions form in hematogones metastasis model mice.Finally,by conducting in vivo experiments,we try to further clarify SIRT5 specific role in HCC progression,and to determine SIRT5 function in more complex environment in vivo.Results1.Screening and identification of dis-regulated mRNAs in high risk HCC casesWe use Roche mRNA chip detection of the 4 cases of early recurrence of HCC tumor(3 months or less)and its fresh pair(sex,age,stage)4 patients with no recurrence of early HCC tumor tissue(2 years)or higher;at the same time,also examined in four cases with multifocal intrahepatic metastasis in primary HCC focal organization and its tissue adjacent to carcinoma.Through clustering analysis screen differentially expressed mRNA in two groups of chip results.To rise,reduce 2 times as the cutoff value,in early recurrence of liver cancer tissues,found that compared with no early relapse cases there were 2665 differentially expressed mRNA,at the same time,another group of stove and tissue adjacent to carcinoma found there were 3065 differentially expressed mRNA,compared two groups of screening results in intersection,get specific high/low expression of mRNA expression.From chip screening differentially expressed by mRNA in 5 randomly selected,further the qRT-PCR method in 20 to liver cancer tissue and matching tissue adjacent to carcinoma,20 for early and late recurrence of HCC recurrence and expression levels in the organization,and found its expression level in HCC 20 on focal organization and 20 for early recurrence of HCC tissues were significantly increased,and the qRT-PCR detection by mRNA expression level of sorting and showed the same chip,proved the reliability of gene chip.2.SIRT5 was up-regulated in HCC tissues and correlated with prognosisWe first use qRT-PCR method to detected SIRT5 expression in 55 for fresh liver cancer tissue and its matching testing in tissue adjacent to carcinoma,at the same time we using the immunohistochemical technique(IHC)detected the SIR.T5 protein expression in 166 cases of HCC in paraffin sections.Results show that the SIRT5 mRNA expression in liver cancer tissue is significantly higher than tissue adjacent to carcinoma,difference have statistical significance(P = 0.0015);IHC testing found that most HCC cases SIRT5 said in liver cancer tissue to achieve obvious positive,mainly in the cytoplasm,and according to the IHC staining intensity,divided into completely negative,positive,and strong positive.By Spearman rank correlation analysis found SIRT5 mRNA expression and liver cancer patients with tumor diameter(P = 0.019),tumor number correlation(P = 0.027),and TNM stage(P=0.007),compared with no correlation with gender,age,pathological grading,portal vein tumor emboli,AFP,liver cirrhosis;and SIRT5 IHC staining scores and patients with liver cancer tumor diameter(P = 0.009),number of tumors(P = 0.015),and TNM staging(P = 0.007),and without correlation with gender,age,portal vein tumor emboli,AFP,liver cirrhosis,and degree of tumor differentiation.Kaplan and Meier survival curve analysis and log-rank test result was found that the expression level of SIRT5 HCC patients' overall survival and disease-free survival time correlation,SIRT5 high expression associated with poor outcome.Cox proportional hazards regression model confirmed SIRT5 high expression is independent risk factors of prognosis of HCC patients.3.In vitro experiments revealed that down-regulation of SIRT5 induced suppression the proliferation,invasion,and migration of HCC cellsWe established SIRT5 knock-down HepG2 and Huh7 cell lines through lentivirus transfection,and found HCC cells proliferation and clone formation and penetration Transwell Chambers ability were significantly decreased after SIRT5 suppression.Flow cytometry technique revealed that SIRT5 lower expression in HCC cells induced G/S phase retardation,and qRT PCR and Western blot test results found that cell cycle progression-promoting proteins including cyclin D1,CDK4,and CDK6 expression were decreased following SIRT5 silencing.In addition,the qRT-PCR and Western blot results also showed SIRT5 knock-down induced the increased expression of epithelial phenotype marker such as E-cadherin,and decreased mesenchymal phenotypic markers such as N-cadherin and Vimentin;in addition.4.In vivo experiments revealed that down-regulation of SIRT5 induced suppression of HCC progressionCompared with control group,subcutaneous tumor of the SIRT5 knock-down group showed slowed tumor growth significantly.After 5 weeks,unified execution,we found that the quality of tumor SIRT5 knock-down group was obviously lower than that of the control group.WB and qRT-PCR detection result shows that compared with the control group,in the silent SIRT5 subcutaneously into tumor tissue protein cyclinDl promote cell cycle progression of cycle,CDK4 and CDK6 mRNA and protein expression level decreased obviously;at the same time,silence of SIRT5 in subcutaneous tumor showed intermediated expression of phenotypic markers including N-cadherin and Vimentin mRNA and protein expression leves,in addition,the epithelial phenotype marker E-cadherin expression level corresponding increased.We found there was no visible liver metastatic lesions in the group of SIRT5 knock-down,and found seven mices in control cell group had visible liver metastasis lesions,the results were statistical difference significant.Conclusions1?Expression of SIRT5 is increased in HCC tissues,and it acts as an independent risk factor of the prognosis of HCC patients.2?SIRT5 might promote the proliferation and migration of HCC cells through regulation of cell-cycle and EMT.3?SIRT5 promotes HCC progression in vivo.