| Objective: The incidence of head and neck squamous cellcarcinoma (HNSCCC) ranks sixth in systemic tumor. Because of thecentralization of vital organs and plenty of lymph nodes in the head and neck,the lymph node metastasis of HNSCC restricts the effect of operation andradio-chemotherapy. At present, chemotherapy has widely used in clinicaltreatment of HNSCC, especially the patients of cervical lymph node metastasis.However, primary or acquired resistance to chemotherapy has decreased thetherapeutic effect on the HNSCC patients with lymph node metastasis. Fanconianemia (FA) pathway dysfunction can change the sensitivity of tumor cells toDNA cross-linking agents, and FANCD2as a critical gene of the FA pathwaymay play an important role in affecting the tumor cell growth andchemosensitivity. In this experiment, by using the HNSCC lymph nodemetastasis HSC-4cell lines which has stability of FANCD2silent expression byshRNA interference, the possible role of FANCD2in HNSCC growing and theeffect of sensitivity to chemotherapy and the related mechanism by silencingFANCD2expression in HNSCC neck lymph node metastasis cells were studied.Methods: Used the HNSCC neck lymph node metastasis HSC-4cell linewhich had stably FANCD2silencing effect by shRNA interference in early experiment, experimental cells were divided into three groups: control group(wild-type HSC-4cells without any treatment), negative control group(FANCD2-shRNA-C, transfected invalid interference sequence of FANCD2inHSC-4cells), experimental group (FANCD2-shRNA, containing effectiveinterference sequence and stable silencing effect of FANCD2in HSC-4cells).After cell passage, puromycin selection, silencing effect of FANCD2protein was detected in Western Blot, and the silencing efficiency was alsocalculated. Detected the growth and proliferation of HSC-4cells byproliferation inhibition assay (CCK-8method) after FANCD2shRNAinterference, apoptosis and cell cycle of HSC-4cells was detected by FCM(Annexin V/PI staining method). In order to research the influence on thechemotherapy sensitivity by FANCD2shRNA in HSC-4cell, the inhibition rateof Cis-diamminedichloroplatinum (CDDP) effected on the HSC-4cells atdifferent time points and different concentration was detected by CCK-8method, and IC50value was also calculated, the changes of apoptosis and cellcycle about HSC-4cells after CDDP was detected by FCM. The expressionchanges of Bax、ERK1/2、P-ERK1/2and integrin β1protein were detected byWestern Blot after FANCD2shRNA interference so as to research the functionrelationship between FANCD2and above proteins. After DNA cross-linkingagent(CDDP) administration, the expression of these proteins in three groupscells were also dectected by Western Blot in order to study the possiblemechanisms about the chemosensitivity to DNA cross-linking agent by shRNA silencing FNACD2expression. Results:(1)Western Blot confirmedthat FANCD2protein existed in HNSCC neck lymph node metastasis HSC-4cell; the silencing effect of FANCD2could effectively and stablely exist by theinterference sequence designed in pre-experimental, which was significantlydifferent to the control groups (F=514.179, P<0.05), the silencing efficiencywas86%.(2)CCK-8analysis showed that FANCD2silent expression byshRNA interference could inhibit HSC-4cell growth in a time-dependentmanner. Different concentration of CDDP acting for48h, the inhibition rate ofthree groups cells also increased with the increase of concentration, differencewas statistically significant between each concentration groups(F=1247.992,P<0.05) and cell groups(F=118.724, P<0.05). Especially, the inhibition rate inexperimental group increased more obviously than in control groups. Afteradministration of16μg/ml CDDP cells at24,48and72h, the inhibition rate inthese three groups cells increased with the increase of interaction time,difference was statistically significant between each time groups(F=575.013,P<0.05) and cell groups(F=37.709, P<0.05). Similarly, the inhibition rate inexperimental group increased more obviously than in control groups. The IC50value of CDDP in the experimental group (16.633±1.137)μg/ml, compared withthe control group (34.567±1.193)μg/ml and negative control group(30.400±0.089)μg/ml, was significantly reduced (F=229.887, P<0.05).(3) Flowcytometry showed that the apoptosis rate (13.33%) in the experimental group,compared with the control group (1.69%) and negative control group (5.05%), increased significantly (F=403.277, P<0.05) after three groups of cells werecultured for72hours, decreasing the percentage of S phase cell and increasingthe G0/G1phase cells in the experimental group cells by FANCD2shRNAinterference (F=307.896, P<0.01). Administration of0、2、5μg/ml CDDP for48hours, the apoptosis rate of the experimental group was higher than that in thecontrol group and negative control group (F0μg/ml=137.335, P<0.05；F2μg/ml=581.633, P<0.05; F5μg/ml=289.719, P <0.05), furthermore, the apoptosisrate raised with the increasing of drug concentration and the proportion ofG0/G1phase of the cell cycle also raised with the increasing of CDDPconcentration, compared the proportion of G0/G1phase in the experimentalgroup with the control group and negative control group, difference wasstatistically significant(F0μg/ml=4178.897, P<0.05, F2μg/ml=33.864, P<0.05;F5μg/ml=27.243, P<0.05).(4)Western Blot results showed that Bax(F=15.168,P<0.05) and ERK1/2(F=14.888,P<0.05) protein increased in theexperimental group, P-ERK1/2(F=35.288, P<0.05) and integrin β1(F=2.583,P<0.05) protein decreaed in the experimental group after shRNA silencingFANCD2expression, compared with these control groups. Administration of2μg/ml CDDP for48hours, Bax (F=25.218,P<0.05) and ERK1/2(F=30.345,P<0.05) protein expression in the experimental group were higher than those inthe control groups, P-ERK1/2(F=50.201, P<0.05) and integrinβ1(F=22.227,P<0.05) protein expression were lower than those in the control groups.Conclusions:(1) FANCD2gene expression existed in HNSCC ncek lymph node metastasis HSC-4cells lines, the silencing effect of FANCD2by shRNAinterference existed stably in HSC-4cell.(2)FANCD2shRNA interferencecould suppress the growth and viability of HSC-4cells and increase apoptosis,causes cell cycle blockage, suggesting that FACND2may be associated withthe growth and activity of HNSCC, which provided the experimental basis forfurther research about FANCD2gene function and mechanism.(3)SilencingFANCD2expression by shRNA interference could improve the DNAcross-linking agent CDDP chemosensitivity of HNSCC neck lymph nodemetastasis HSC-4cell.(4)FANCD2gene may be involved in Bax-inducedapoptosis pathway so as to promote apoptosis of HSC-4cells; enhancedactivation of ERK1/2expression activated the phosphate pathway, jointing thedown-regulation of P-ERK1/2and integrin β1protein expression, mightcombined with FANCD2to inhibit HSC-4cell proliferation, promote apoptosisand lead to cell cycle arrest, thus achieve chemosensitizing.(5)This suggestsedthat FANCD2protein might affect the biological function and chemosensitivityof HNSCC cervical lymph node metastasis HSC-4cell lines, which mayprovide a new target and idea for HNSCC treatment, especially for cervicallymph node metastasis patients. |
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