Experimental Study Of Invasion Mechanisms In Hepatocarcinoma Cells Due To Cortactin And Its Modulation On MMPs Secretion

The invasion of hepatocellular carcinoma (HCC) is the main reason of poorprognosis caused by high recurrence and metastasis rate after hepatectomy. HCC cellinvasion is based on the formation of invadopodia, in which cortactin, exo70andMMP-9are involved and related to each other. However, the exactly explanation oftheir interactions have not been attained. On the basis of previous research, wehypothesized that cortactin may promote HCC invasion by increasing MMPssecretion. And that exo70may work in coordination with cortactin in this process.In order to prove our point of view, we first detected the expression of CTTN(the coding gene of cortactin)、 exo70and MMP-9and assessed motility andinvasiveness of HCC cells. We found that compared with QSG-7701, a nonmalignanthepatocytes, the expression of CTTN, exo70and MMP-9was differences. There wasa covary relation between CTTN and exo70. But both CTTN and exo70wereindependent with MMP-9. The motility of SK-Hep-1, HepG2and LM3exceeded thatof QSG-7701, just as the outcome in the invasion detection. Significant differencewere not been found between MHCC97-L and QSG-7701. Although MMP-9expression was higher in HCC cells than that in nonmalignant hepatocytes, it wasindependent with cell invasion.We subsequently established CTTN knock down LM3cell models by usingRNAi technique. For stable transfection, we carried out antibiotics resistance genescreening. Lentivirus carrying CTTN and EGFP coding sequence were also packagedby which LM3was transduced to obtain cortactin overexpression cell lines. Followingthe establishment of other control cell lines, we also detected MMP-9expression,secretion and cell motility and invasion. The result showed that there was nostatistical difference in MMP-9expression between cell lines (P>0.05). On the contrary, the concentration of MMP-9in culture media had statistical difference and ispositive correlated with cell invasion (P<0.01). Also it had positive correlation withCTTN mRNA and cortactin expression (P<0.01). The higher CTTN expression thecell line had, the higher invasiveness the cell line possessed (P<0.01).Finally, we constructed shRNA vector to silence exo70in LM3which CTTNhad been already artificially changed. An inspiring discovery was made that CTTNsilenced LM3could not secrete MMP-9and lost its invasive phenotype after exo70RNAi.From all of these results we draw the following conclusions that there is acovariant relationship between the expression of CTTN and exo70in HCC cells. Upregulation of CTTN and exo70increases cell invasion which is closely related toMMP-9secretion rather than MMP-9expression. Simultaneously silencing CTTNand exo70will completely block MMP-9secretion, which effectively inhibit HCCinvasion. The results of this study deepen our understanding about the cooperativeaction between cytoskeleton system and intracellular transport system. Theapplication of gene transfer to simultaneously silence CTTN and exo70can depriveHCC invasive phenotype which would decrease HCC malignancy to the maximum.Hence the prognosis of HCC will be greatly improved as tumor progression slowingdown.