The Expression Of Hyaluronan In Human Non-Hodgkin-Lymphoma And Its Effects On CEM Cell Proliferation And Invasiveness

Objective: In this study, we first examined the expression and the clinical value of hyaluronan in non-Hodgkin-Lymphoma. Then we investigated the effect of proliferation and invasiveness of hyaluronan on CEM cells. Thus may offer a reference for the prognostic judgement and the clinical development of NHL, and provide a theoretical basis for developing a new anticancer target to NHL.Methods: 1. Collecting peripheral blood samples of 51 NHL patients, separation of plasma, using radioimmunoassay to detect the concentrations of hyaluronic acid in plasma, and using gelatin zymography to detect the activity of matrix metalloproteinase 2,9 in plasma. Another collection of 15 healthy people as control plasma samples. 2. Using 10%FBS of 1640 medium with concentration of 0ug/ml, 10ug/ml, 20ug/ml exogenous hyaluronan to culture CEM cells, and then determination the following indicators:⑴The proliferation of CEM cells after 24,48,72 hours of culturing with different concentrations of hyaluronan measured by MTT; The alive cells after 24,48,72,96,120 hours of culturing with different concentrations of hyaluronan counted by Trypan blue staining to draw a cellular growth curves;⑵The invasive ability of CEM cells after 24 hours of culturing with different concentrations of hyaluronan detected by millicell invasion chamber;⑶MMP2,MMP9 mRNA expression of CEM cells after 24 hours of culturing with different concentrations of hyaluronan detected by real-time quantitative PCR method;⑷The protein activity of MMP2,MMP9 of CEM cells after 24 hours of culturing with different concentrations of hyaluronan dectected by zymography.Results: 1. The expression of hyaluronan in the plasma of NHL patients: untreated group, relapsed or refractory group are significantly higher than control group(P<0.05); untreated group, relapsed or refractory group are significantly higher than completely remission group(P<0.05); completely remission group and the control group showed no significant difference(P>0.05); untreated group and relapsed or refractory group showed no significant difference(P>0.05). 2. In the untreated group, the expression of HA in plasma of the III,IV phase patients is higher than that of I,II phase patients(P<0.05). 3. In plasma, the expression of HA is positively correlated with the expression of MMP2, with the correlation coefficient r1=0.678,P<0.01; the expression of HA is also positively correlated with the expression of MMP9, correlation coefficient r2=0.654,P<0.01. 4. After treating with exogeous hyaluronan for 24h, the ability of proliferation of CEM cells are not obvious enhancement;after 48h, the ability of proliferation are enhanced remarkably. 5. After treating with exogeous hyaluronan for 24h, the ability of invasiveness of CEM cells are enhanced remarkably. 6. After treating with exogeous hyaluronan for 24h, the expressions of MMP2,MMP9 mRNA in CEM cell line are improved, and the activity of MMP2,MMP9 protein in CEM cell line are enhanced. This effect is in a dose dependent manner.Conclusions: 1. Hyaluronan is highly expression in the plasma of NHL patients, the expression of hyaluronan is associated with the NHL pathogenetic condition progress and clinical stage; the expression of hyaluronan in NHL patients'plasma is in direct correlation with the activities of MMP2,MMP9 protein; it implies that the highly expression of hyaluronan in NHL patients'plasma is related to the abilities of invasiveness and metastasis of NHL. 2. The exogeous hyaluronan can enhance the abilities of invasiveness in CEM cell line in vitro. The mechanism may be associated with the hyaluronan which can be up-regulation the expressions of MMP2,MMP9 mRNA and enhance the activities of MMP2,MMP9 protein.