Study on the Expression of Focal Adhesion Kinase in Hepatocellular Carcinoma and Its Role in MHCC-97H Cell
|Keywords||hepatocellular carcinoma focal adhesion kinase prognosis adhesion invasion platelet-derived growth factor cytoskeleton RNA inteferrence|
Invasion and metastasis are the most crucial factors, which influences the prognosis of primary liver cancer. Focal adhesion kinase(FAK) over-expresses in many malignant tumors and correlates with tumor invasion and metastasis; however, the correlation between facal adhesion kinase and tumor prognosis is still unclear. The mechanism of how FAK is concerned with many signal transductions is very complicated. Besides, FAK might become a therapeutical target of primary liver cancer. RNA interference is an important research method in the era of late-gene. We investigated FAK expression in hepatocellular carcinoma (HCC) and its role in predicting HCC prognosis, studied the alternation of human HCC metastatic potential through the up-regulation by PDGF stimulation and down-regulation by RNAi in this study.PART ONEObjective: To investigate the distinction of focal adhesion kinase (FAK) expression in various HCC tissue and to explore the prognostic significance of FAK expression in HCC. Methods: We reviewed 50 consecutive patients who had undergone liver resection for HCC without preoperative treatment in our hospital. FAK mRNA expression in tumor tissue, corresponding non-cancerous liver tissues and tumor emboli was detected by real-time polymerase chain reaction (PCR) analysis and its protein expression was confirmed by immunohistochemical study and Western-blot analysis. We also statistically analyzed the correlation between FAK expression and clinnicopathological parameters. The correlation between FAK expression and HCC prognosis was investigated by univariate and Cox regression model analysis. Results: FAK mRNA was overexpressed in HCC tissue compared with corresponding non-cancerous liver tissues(0. 229 ± 0. 027 vs 0. 163±0. 019, P< 0.001), in tumor emboli with the comparison of tumor tissue(0. 506 ± 0. 155 vs 0. 377 ± 0. 176, P<0.05) 、 in tumor tissue with emboli compared with tumor tissue without emboli (0. 343 ± 0. 05 vs 0. 165 ± 0. 025, P=0.003) and in corresponding non-cancerous liver tissues with cirrhosis compared with tissue without cirrhosis(0. 191 ± 0. 029 vs 0. 141±0. 034, P>0.05) . The difference of FAK mRNA expression between high invasive group and low invasive group had statistic significance (0.283 ± 0.038 vs 0.161 ±0.032, P = 0.020) . FAK mRNA expression correlated significantly with embolism (P=0.003) and invasion (P<0.020). Univariate and Cox regression model analysis revealed that FAK expression was an independent prognostic factor for survival. Conclusion: FAK plays an important role in HCC progression, especially in vascular invasion and FAK expression is a prognostic factor of HCC.PART TWOObjective: To investigate the dynamic change of FAK mRNA expression in MHCC97-H cell after stimulated by different concentration of PDGF-BB and the correlation between FAK and cell adhesion and cell invasion. Method: After stimulated by different concentration of PDGF-BB, the dynamic change of FAK mRNA and MMP-2 mRNA expression in MHCC-97H cell was detected by real-time PCR analysis. Meanwhile, the change of FAK protein expression was studied by Western-blot analysis. The change of cell adhesive and invasive ability after stimulation was confirmed by cell adhesive assay and cell invasive assay respectively. Matrix metalloproteinase-2 secretion was measured by ABC-ELISA and gelatin zymography. Cytosekeleton rearrangement of MHCC97-H cell, which was labeled by immunofluorescent antibody, was supervised by Confocal Laser Scanning Microscope after stimulated by PDGF-BB . Result: FAK mRNA expression in MHCC-97H cell was upregulated after stimulated by different concentration of PDGF-BB, and raised to the culmination at the concentration of 10ng/ml with 112.6 folds increasing compared with the control group. Meanwhile, MMP-2 mRNA expression was found to be the similar change at the same time; it was upregulated by different concentration of PDGF-BB and it was about 56.9 folds in 10ng/ml group compared with the control group. We also found that FAK protein expression had been upregulated after stimulation. Moreover, the adhesive rate in each group of MHCC-97H after stimulation had significant difference (p<0.001), compared with the control group, and it was 54±2.08%、 66±1.84%、 69±1.41%、 69±2.42%、 71±1.37% and 66±3.28% in untreated, 1、 2.5、 5、 10 and 25ng/ml group respectively. The number of invasive cell was 26.63±1.67, 28.75±1.50 in 5ng/mlgroup and 10ng/ml group, respectively; and it had significant difference compared with the control group(P<0.05, P<0.001 respectively). Both the adhesive rate and the number of invasive cell reach the peak in 10ng/ml group . Matrix Metalloproteinase-2 secretion increased strikingly after stimulation detected by ABC-ELISA and zymography. We found that MHCC97-H cell had formed filopodium in 5minutes, lamellipodium in 15 minutes and focal adhesion in 30 minutes after stimulation by Confocal Laser Scanning Microscope. Conclusion: PDGF-BB upregulates FAK expression in MHCC-97H cell and the FAK upregulation promotes cell adhesion and invasion by remodeling cytoskeleton and enhanceing Matrix Matelloproteinase-2 secretion.PART THREEObjective: To study the role of knock-down FAK expression by FAK siRNA in MHCC97-H cell adhesion、 invasion and cytoskeleton rearrangement. Methods: FAK siRNA was transfected into MHCC97-H cell by Lipofectamine 2000, then, FAK expression was detected by real-time PCR and Western-blot analysis. The change of cell adhesive and invasive ability after FAK knock-down by RANi was checked by cell adhesive assay and cell invasive assay respectively. Meanwhile, Matrix metalloproteinase-2 secretion was checked by ABC-ELISA and gelatin zymography. Cytosekeleton rearrangement labeled by immunofluorescent antibody was examined by Confocal Laser Scanning Microscope. Result: FAK expression in MHCC97-H cell was obviously inhibited by specific FAK siRNA, both in mRNA and protein level; However, it was not inhibited by negative siRNA. Adhesion between MHCC97-H cell and Extracellular Matrix reduced for the role of RNA interference. Adhesive rate decreased from 57.3 % to 35.8 % after RNA interference (P < 0.05 ). Compared with untreated group,the number of cell penetrating matrigel also decreased from 31.3±2.6 to 14.5±3.1 after transfection ( P < 0.05 ). Besides, Matrix metalloproteinase-2 secretion was significantly reduced for FAK expression inhibited by FAK siRNA. FAK inhibition influenced Vinculin rearrangement, blocked the formation of lamellipodium, delayed the time of focal adhesion formation. Conclusion: Specific FAK siRNA knocks down FAK expression. Down-regulation FAK expression reduces adhesive rate and invasivenumber of MHCC97-H cell by influencing cytoskeleton rearrangement and descreasing Matrix metalloproteinase-2 secretion.