AIM:To investigate the role of osteopontin(OPN) and its splice variants in the proliferation of hepatocellular carcinoma(HCC).METHODS:The expression of OPN variants in HCC cell lines as well as HCC tissue samples and ...AIM:To investigate the role of osteopontin(OPN) and its splice variants in the proliferation of hepatocellular carcinoma(HCC).METHODS:The expression of OPN variants in HCC cell lines as well as HCC tissue samples and nontumour tissue was studied using polymerase chain reaction.OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines.OPN expression was studied in these cells using Western blotting,immunofluoresnce and enzyme linked immunosorbent assay.A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture.Huh-7 cells stably expressing either OPN-A,-B or-C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth.Expression of OPN mRNA and protein in these tumours was examined using reverse transcriptionpolymerase chain reaction and immunohistochemistry.RESULTS:OPN is expressed in HCC in 3 forms,the full length OPN-A and 2 splice variants OPN-B and-C.OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue.Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium.Transfer of OPN conditioned media to na ve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner.Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect.OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model,with Huh-7 cells expressing OPN-A showing the greatest proliferative effect.CONCLUSION:This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44.Modulation of this interaction could represent a novel strategy for the control of HCC.展开更多
AIM: To investigate over-expression of Osteopontin (OPN) pathway expression and mechanisms of action in human alcoholic liver disease (ALD), in vivo and in vitro acute alcohol models.
Hepatocellular carcinoma(HCC)is the most common primary liver malignancy,with increasing incidence worldwide.Alcohol-related cirrhosis(AC)accounts for 30%of the global incidence of HCC and HCC-related deaths.With the ...Hepatocellular carcinoma(HCC)is the most common primary liver malignancy,with increasing incidence worldwide.Alcohol-related cirrhosis(AC)accounts for 30%of the global incidence of HCC and HCC-related deaths.With the decline of hepatitis C virus(HCV)and decreasing HCV-related HCC,AC will soon become the leading cause of HCC.Excess alcohol consumption(>80 g per day for>10 years)increases the risk of HCC by 5-fold.However,only up to 35%of excessive drinkers develop cirrhosis and its associated HCC risk.Individual variation in susceptibility to HCC is known,but there is limited information to predict who among the patients is at high risk of progressing to HCC.Clinical risk factors for HCC include male gender,older age,severity of cirrhosis,obesity and presence of type 2 diabetes.In addition to ethnic variability in HCC risk,genetic variants are known to alter the risk of alcohol-related HCC.For example,single nucleotide polymorphisms in PNPLA3(rs738409,C>G)and TM6SF2(rs58542926,C>T)increase the risk of AC-related HCC,whereas HSD17B13(T>A)reduces the risk for HCC.Studies have also confirmed PNPLA3 and TM6SF2 to be independent risk factors for AC-related(but not HCV-related)HCC.Combining genetic risk factors with phenotypic/clinical risk factors has been explored for stratification of patients for HCC development.Risk allele rs378409-G in PNPLA3 when combined with phenotypic/clinical risk factors(BMI,age,sex)has enabled HCC risk stratification of AC patients into low-,intermediate-and high-risk subgroups.Similarly,a combination of the two genetic variants PNPLA3-G and TM6SF2-T has been independently associated with risk of HCC onset.Using a polygenic risk score approach of incorporating several genetic variants,prognostic performance of polygenic risk score that included PNPLA3 rs378409 and TM6SF2 rs58542926 improved HCC prediction better than with either variant alone.Incorporating new variants and risk factors has the potential to build better algorithms/models to predict onset,early diagnosis and treatments for AC-related HCC.However,clinical usefulness of these approaches is yet to be determined.展开更多
文摘AIM:To investigate the role of osteopontin(OPN) and its splice variants in the proliferation of hepatocellular carcinoma(HCC).METHODS:The expression of OPN variants in HCC cell lines as well as HCC tissue samples and nontumour tissue was studied using polymerase chain reaction.OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines.OPN expression was studied in these cells using Western blotting,immunofluoresnce and enzyme linked immunosorbent assay.A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture.Huh-7 cells stably expressing either OPN-A,-B or-C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth.Expression of OPN mRNA and protein in these tumours was examined using reverse transcriptionpolymerase chain reaction and immunohistochemistry.RESULTS:OPN is expressed in HCC in 3 forms,the full length OPN-A and 2 splice variants OPN-B and-C.OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue.Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium.Transfer of OPN conditioned media to na ve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner.Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect.OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model,with Huh-7 cells expressing OPN-A showing the greatest proliferative effect.CONCLUSION:This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44.Modulation of this interaction could represent a novel strategy for the control of HCC.
基金Supported by Philanthropic Anonymous Sourcethe University of Sydney Bridging Support Grant,in part for Honours ProjectSupported by the National Health and Medical Research Council,No.NHMRC Practitioner Research Fellowship for PH support
文摘AIM: To investigate over-expression of Osteopontin (OPN) pathway expression and mechanisms of action in human alcoholic liver disease (ALD), in vivo and in vitro acute alcohol models.
文摘Hepatocellular carcinoma(HCC)is the most common primary liver malignancy,with increasing incidence worldwide.Alcohol-related cirrhosis(AC)accounts for 30%of the global incidence of HCC and HCC-related deaths.With the decline of hepatitis C virus(HCV)and decreasing HCV-related HCC,AC will soon become the leading cause of HCC.Excess alcohol consumption(>80 g per day for>10 years)increases the risk of HCC by 5-fold.However,only up to 35%of excessive drinkers develop cirrhosis and its associated HCC risk.Individual variation in susceptibility to HCC is known,but there is limited information to predict who among the patients is at high risk of progressing to HCC.Clinical risk factors for HCC include male gender,older age,severity of cirrhosis,obesity and presence of type 2 diabetes.In addition to ethnic variability in HCC risk,genetic variants are known to alter the risk of alcohol-related HCC.For example,single nucleotide polymorphisms in PNPLA3(rs738409,C>G)and TM6SF2(rs58542926,C>T)increase the risk of AC-related HCC,whereas HSD17B13(T>A)reduces the risk for HCC.Studies have also confirmed PNPLA3 and TM6SF2 to be independent risk factors for AC-related(but not HCV-related)HCC.Combining genetic risk factors with phenotypic/clinical risk factors has been explored for stratification of patients for HCC development.Risk allele rs378409-G in PNPLA3 when combined with phenotypic/clinical risk factors(BMI,age,sex)has enabled HCC risk stratification of AC patients into low-,intermediate-and high-risk subgroups.Similarly,a combination of the two genetic variants PNPLA3-G and TM6SF2-T has been independently associated with risk of HCC onset.Using a polygenic risk score approach of incorporating several genetic variants,prognostic performance of polygenic risk score that included PNPLA3 rs378409 and TM6SF2 rs58542926 improved HCC prediction better than with either variant alone.Incorporating new variants and risk factors has the potential to build better algorithms/models to predict onset,early diagnosis and treatments for AC-related HCC.However,clinical usefulness of these approaches is yet to be determined.
