The Effect Of Hypoxia Induced Factor-1α On Multi-Drug Resistance In HepG₂ Cell Line Under Hypoxia

[OBJECTIVE] The aim of the project is to study the possible relationship and regulation mechanism between HIF-la (Hypoxia Induced Factor) and mdrl (Multi-drug resistance) on tissue and cell line level through examining the expression and co-expression of HIF-1α and P-gp in hepatocellular carcinoma (HCC) and normal liver tissues as well as examining the expression change of HIF-1α and mdrl in mRNA and protein levels in HepG₂ cell line under chemical hypoxia induced by CoCL₂.[ METHODS ] On tissue level, immunohistochemistry was used to detect the expression of HIF-1α and P-gp respectively in thirty two HCC samples and ten normal liver samples. If the expression of HIF-1α and P-gp is positive in two of the continuous slides, the third continuous slide was used to detect the co-expression of both HIF-1α and P-gp. In addition, on cell line level ,immunocytochemistry was used to detect the expression and location of HIF-1α in HepG₂ cell line in normoxia and hypoxia in order to understand the expression rule of it. RT-PCR and Western Blot were used to examine the change of HIF-1α and mdrl in mRNA and protein level in normoxia and hypoxia.[RESULTS)1, In the thirty two HCC samples, eighteen samples expressed HIF-1α, the expression rate was 56.2% , almost all the positive granules were in the plasma; twenty-six samples expressed P-gp, the expression rate was 81.25%, most of the positivegranules were in the membranes of HCC cells; there are some correlation between theexpression of HIF-la and P-gp, %2=4.65, P<0.05 ,r=0.26.Among them, there aresixteen samples expressing both of them, and the expression of HIF-la and P-gppresented the trend of one way ,that is the stronger expression ofHIF-la(+++)accompanied with the stronger expression of P-gp(+++); ten samplesco-expressed HIF-la and P-gp, the co-expression rate was 62.5%.2n In the ten normal liver samples, we can not observe the visible positive expressionof HIF-1 a(—) and only two samples expressed P-gp(+), the expression rate was 20%.Compared with the expression of P-gp in HCC, the difference is prominent ,X2=12.86, P<0.001.3> Immunohistochemistry demonstrated HIF-la began to express (+) at 4 hours inblank group; HIF-la expressed as (+) in hypoxia I group, most of the positivegranules lied in the cytoplasm; however, in hypoxia II group its expression can bedenoted as (++) and most of the positive granules began to emerge in the nucleolus, inhypoxia III group the expression of HIF-la can be denoted as (+++) and kept stayingin the nucleolus.4. RT-PCR demonstrated that the expression of HIF-la's and mdrl's mRNA increasedcontinuously with the growing of the time. The increasing folds of HIF-la 'smRNA in hypoxia groups over blank group were 2.5 s 4.12> 5.75, PO.05. Theincreasing folds of mdrl's mRNA in hypoxia groups over blank group were 1.54>5.09^ 7.3 ,P<0.05.5> Western Blot demonstrated that the expression of HIF-la's and mdrl's protein alsoincreased continuously with the growing of the time. The increasing folds ofHIF-la's protein in hypoxia groups over blank group were 2.7 n 5.8s 7.9 ,P<0.05.The increasing folds of P-gp in hypoxia groups over blank group were 1.61 s 5.59s 7.1,PO.05.6 s Analyze the expression of HIF-la in different groups on mRNA and protein level,we can get the equation of Y=—0.07+1 .IX, P <0.01;Analyze the expression of P-gpin different groups on mRNA and protein level , we can get the equation of Y=—0.075 + 1.5 X, P <0.01;Analyze the con-elation of HIF-la's and P-gp's mRNA expression,r=0.875, P <0.001; Analyze the correlation of HIF-la's and P-gp's protein expression, r=0.917,P O.001 ?[CONCLUSION]1 ?> The expression of HIF-la and P-gp in HCC has correlation in some extent and the co-expression of HIF-la and P-gp can be detected in HCC, which might suggest there is some relationship and even some regulation mechanism between the transport factor HIF-la and mdrl in HCC .2n In HepG2 cell line of hypoxia induced by 100 u mol/L C0CL2, it is at 24 hours that most of HIF-la begins to transfer into nucleolus and carry on its function as transport factor in hypoxia.3> In HepG? cell line of hypoxia, HIF-la takes the role of regulating the expression of mdrl,and HIF-la regulates the expression of mdrl mainly in translation level. 4* The multi-drug resistance gene might also be one of the downstream genes of HIF-la.