In the present study, an unbiased PCR array approach containing 84 cell cycle genes was applied on pooled RNA samples, to identify altered gene expression in HCV-associated liver disease. This analysis led to the identification of differentially expressed genes involved at cell cycle checkpoints, which may play a role in disease pathogenesis. Further investigation at protein level of four differentially expressed genes, selected on the basis of their crucial role in cell cycle regulation confirms the modulations in the expression of CDK inhibitor p27 and mitotic checkpoint genes MAD2L1 and KNTC1. It is important to note that these cell cycle proteins have not been implicated previously in chronic liver disease. Moreover, the association of p27 and KNTC1 expression with liver fibrosis suggests a role of these markers in the progression of liver disease.
Persistent inflammation and cell destruction during HCV infection has been suggested to induce proliferation of normally quiescent hepatocytes [17
], which helps in restoring liver mass and maintaining hepatic function. Moreover, previous reports including our own have shown an arrested cell cycle in hepatocytes, during HCV infection [12
]. Consistent with these data we found more than 3 fold increase in the mRNA levels of proliferation related genes Mcm-2, -3, -5, CDC2 and cyclin in early HCV group while the mRNA levels of cell cycle inhibitors p27, GADD45A, MAD2L1 and KNTC1 were also found to be significantly increased (Table ). These transcriptional changes suggest a mixed population of cells in the infected livers of chronic hepatitis C some of which are in proliferating state while others in an arrested state. In advanced HCV, cell cycle inhibitors p27 and MAD2L1 were found down-regulated while p15/CDKN2B and CDKN3 were up-regulated. More than four fold increase was also observed in the mRNA levels of DNA damage response genes ATR, HUS1, RAD1, RPA3 and RAD17 (Table ) suggesting increased DNA damage. These alterations were also accompanied by the down-regulation of tumor suppressor p53. Taken together these results are suggestive of deregulated cell cycle in chronic HCV infection that is considered as a consistent event in HCC [20
Association of CDK inhibitor p21 with impaired liver regeneration and progressive liver fibrosis has been reported earlier [22
]. In the current study, analysis at protein level by immunohistochemistry in HCV-infected hepatic biopsies showed a significant increase in the expression of p27 protein (Figure ) that belongs to the same KIP family as p21. This protein is involved in inhibiting cell cycle progression at both G1 and S phases and is also defined as a tumor suppressor protein [23
]. Interestingly, an inverse relationship between p27 protein expression and progression of liver fibrosis from F0 to F4 was found (Figure ). It would be pertinent to mention here that mouse models of chronic injury induced-liver tumorgenesis have shown that loss of p27 in advanced stages of disease promotes tumor cell proliferation [24
]. These observations signify the potential of p27 as a marker for prognostic implications in chronic HCV disease.
In contrast to p27, immunohistochemical evaluation of G1 phase inhibitor p15 revealed no significant difference in advanced HCV as compared to early HCV that conflicts with the changes observed at mRNA level. This inconsistency might have been due to decreased stability of p15 protein in advanced disease stages that increased the transcription through a regulatory feedback mechanism to maintain its levels. Another explanation for this inconsistency could be the use of pooled samples of whole liver lysates for mRNA expression while immunohistochemistry was done on individual formalin fixed paraffin embedded biopsies. Together, the expression of p27 and p15 in non-dividing hepatocytes might represent the population of cells which are arrested in cell cycle and not participating much in liver compensation, but are more active metabolically.
Defects in chromosomal segregation are a common feature of liver tumor cells suggesting a possible role of mitosis deregulation in the pathogenesis of HCC [25
]. While examining the alterations of cell cycle genes in early and advanced HCV, we found altered expression of mitotic checkpoint genes, MAD2L1, KNTC1, CDC16 and CDC34. KNTC1, also known as "rough deal protein" (ROD) is part of a complex involved in elaborating an inhibitory signal due to improper chromosomal alignment during cell division [26
], while member of Mad2 family, MAD2L1, participates in inhibiting Anaphase promoting complex (APC) [27
] from ubiquitinating securin, whose degradation is a prerequisite for sister chromatid separation and mitosis. Considering the crucial roles of KNTC1 and MAD2L1 in chromosomal segregation, we analyzed their respective proteins by western blotting. Results showed increased levels of both of these proteins in early HCV that is suggestive of mitotic checkpoint activation or mitotic arrest in the liver cells. This is consistent with the down-regulation of CDC16, which is a component of APC and promote chromosomal segregation. Importantly, KNTC1 expression significantly decreases in advanced stages of HCV group as compared to early HCV while the expression of MAD2L1 showed a modest reduction in advanced HCV group (Figure ). However, a possibility of error in assessing these changes could come about due to the fact that the expression of reference gene, β-actin also increases with the progression of liver fibrosis.
The role of KNTC1 in human cancers is not well documented, however, a single report describes mutation in this gene in colorectal carcinoma [28
]. On the other hand, depletion or reduced expression of MAD2 gene has been reported in mammalian cells with loss of mitotic checkpoint and subsequent chromosomal instability [29
]. Thus, low expression of these mitotic checkpoint regulators in advanced HCV might reflect loss of mitotic checkpoint control that could render cells to chromosomal instability. This hypothesis is also supported by a previous study which showed presence of near-aneuploidy DNA content in liver specimens from chronic hepatitis C patients [11
The altered expression of cell cycle regulators observed in present study might be specific to hepatitis C as it was rarely observed in non-viral hepatitis patients. Nevertheless, the low number of control liver samples, hold back in achieving statistically significant differences between the two groups, with varying degrees of fibrosis.