The Impact Of Growth In Vitro And Chemotherapy Sensitivity On ShRNA Targeted Silencing FANCD2in Head And Neck Squamous Cell Carcinoma SIHN-005A Cell

Objective: Head and neck squamous cell carcinoma (HNSCC),as clinically common malignant tumor, its incidence is gradually rising trend inrecent years. Existing local comprehensive treatment including surgery,radiation and chemotherapy, however, treatment effects and the5-year survivalrate is still not high. The breakthrough progresses urgently need on thedevelopment and treatment of sensitivity in the HNSCC theoretical studies,devote to providing experimental basis for chemotherapy and gene therapy.This study based on the technology of short hairpin RNA (shRNA) targetedsilencing FANCD2, human HNSCC SIHN-005A cells was observed onproliferation, apoptosis and cell cycle in vitro and studied on the influence ofchemotherapy sensitivity. The related mechanism of affecting chemotherapysensitivity were initially studied for exploring the possibility of FANCD2inHNSCC research and treatment, in order to improve the sensitivity and curativeeffect of chemotherapy in HNSCC and explore something new. This study wasdevoted to make a feasible application outlook for connection and feasibilitybetween FA pathway and chemotherapy sensitivity of head and neck tumor.Methods:(1) The cells were divided into three groups, including experimentalgroup (FANCD2shRNA), the invalid sequence control group (FANCD2 shRNA-C) as well as the blank (non-transfected) control group (Control), cellsabove were recovered and subcultured. The silencing effect of FANCD2shRNA cells was detected by Western Blot to proved the stable existence andthe silencing efficiency was also calculated.(2) OD values of groups cells weredetected by cell proliferation inhibition test (CCK-8) and the inhibition rates atdifferent times was calculated, after that drawing the inhibit cell growth curves.(3) Apoptosis and morphology changes in groups of cells were detected andobserved by fluorescent Hoechst staining, comparing the influence ofSIHN-005A cell apoptosis with FANCD2silenced.(4) The cell cycle D1protein (cyclin D1) was detected by Western Blot to comparing the influence ofSIHN-005A cell cell cycle with FANCD2silenced.(5) Technologies includingCCK-8, Hoechst staining and Western Blot were chosen to exam the cellproliferation, apoptosis and cell cycle changes after intervention combined withCis-diamminedichloroplatinum(CDDP), besides comparing the chemotherapysensitivity changes in each groups.(6) Bax, Bcl-2and HIF-1alpha weredetected by Western Blot aim to the protein expression changes of SIHN-005Acell with or without CDDP, discussing the related mechanism of chemotherapysensitivity affected by FANCD2shRNA silencing. Results:(1) The FANCD2protein expression in experimental group SIHN-005A cell (0.53±0.09),decreased significantly by comparing with invalid sequences controlgroup(1.15±0.11)(F=78.823, P<0.05) and blank control group(1.34±0.07)(F=104.113, P<0.05) after FANCD2shRNA silencing, besides the silencing effect of60.5%.(2)Three groups of cell cultivation and continuous observationfor72hours, the proliferation of experimental group was not as good as the restof the groups, OD value and the inhibition rate increased along with theextension of time, respectively, was significantly different from the blankcontrol group (F=48.647, P<0.05) and invalid sequence control group(F=22.768, P<0.05).(3) Hoechst staining results showed that the apoptosis rate(13.19±1.38)%in experimental group was higher than invalid sequencecontrol group (2.22±2.01)%and blank control group(1.18±0.05)%, comparedFANCD2-shRNA group with invalid sequence group (F=35.732, P<35.732)and blank control group (F=42.387, P<0.05), respectively, the difference ofapoptosis rate was statistically significant.(4) Gray values of cell cycle proteinD1expression (cyclin D1) in FANCD2-shRNA group (0.73±0.11) wassignificantly cut down by comparison the invalid sequence control group(0.96±0.14) and blank control group (1.04±0.07), statistically different wasshown both in invalid sequence controls (F=8.317, P<8.317) and blank controlgroup (F=10.759, P<0.05).(5) With the intervention of different concentrationCDDP, OD values in three groups detected by CCK-8experiment decreasedalong with the rise of CDDP concentration (F=48.647, P<0.05), while theinhibition rate increased (F=29.703, P<0.05), accordingly. The OD value inshRNA group decreased significantly comparing with invalid sequence group(F=78.642, P<0.05) and blank control group (F=82.521, P<0.05), and theinhibition ratio in experimental group increased statistically comparing with invalid sequence group (F=43.381, P<0.05) and blank controlgroup(F=67.928，P<0.05), respectively. After administration of16μg/ml CDDPin three groups cells, OD value (F=91.026, P<0.05) and inhibition ratio(F=29.703, P<0.05) increased with the extension of time, the OD value inshRNA group was significantly lower than that in invalid sequence group(F=47.384, P<0.05) and blank control group (F=57.275, P<0.05), and theinhibition ratio in experimental group was statistically higher than that ininvalid sequence group (F=56.464, P<0.05) and blank control group(F=69.022,P<0.05). IC50of CDDP concentration was detected to analyze thechemosensitivity changes in SIHN-005-A cell, shRNA experimental group(11.5±0.35μg/ml) was much lower than both invalid sequence group(18.4±1.11μg/ml)(F=46.324, P<0.05) and blank control group (19.9±1.42μg/ml)(F=77.722, P<0.05). In the Hoechst staining, the experimental group cellapoptosis rate(85.12±2.03)%emerged a more significant higher than invalidsequence control group(17.87±1.63)%(F=383.812, P <0.05) and blankcontrol group (11.94±2.42)%(F=454.839, P <0.05). Western Blot resultsshowed that after combined with CDDP, cyclin D1expression (0.55±0.09) inthe experimental group was significantly reduced than the invalid sequencesgroup (0.89±0.18)(F=22.482, P<0.05) and blank control group (0.97±0.24)(F=27.449, P<0.05). Comparing between with or without CDDP in shRNAgroup was statistically significant (F=19.867, P<0.05), either. Cell cycleretardation effect was more obvious in shRNA group cells combined with CDDP.(6) Related proteins of Bax, Bcl-2, HIF-1alpha were chosen to detectedby Western Blot with or without CDDP, results showed that after added withCDDP, Bax protein expression(2.78±0.17) in shRNA experimental group wasmarkedly improved than the invalid sequence control group(0.93±0.22)(F=193.272, P<0.05) and the blank control group(0.89±0.16)(F=211.736,P<0.05), difference of Bax protein expression in experimental group withCDDP (2.78±0.17) and without CDDP (1.69±0.24) was statisticallysignificant(F=98.744, P<0.05); Bcl-2protein expression(0.21±0.07) in shRNAexperimental group was inhibited apparently than the invalid sequence controlgroup (0.54±0.07)(F=117.452, P<0.05) and the blank control group(0.79±0.21)(F=139.229, P<0.05), difference of Bcl-2protein expression in experimentalgroup with CDDP (0.21±0.07) and without CDDP (0.63±0.06) was statisticallysignificant (F=144.738, P<0.05); differences of HIF-1α protein expressionbetween the experimental group(0.60±0.29) and the invalid sequence controlgroup (1.25±0.56)(F=93.733, P<0.05) and the blank control group(1.39±0.22)(F=105.832, P<0.05) was statistically significant, difference of HIF-1α proteinexpression in experimental group with CDDP (0.60±0.29) and without CDDP(1.13±0.32) was statistically significant (F=187.454, P<0.05). Conclusions:(1)The expression of FANCD2protein could be stable silent in human HNSCCSIHN-005A cell by shRNA interference technique;(2) The shRNA targetedsilence FANCD2in SIHN-005A had the ability of cell proliferation inhibition,accelerating and promoting cell apoptosis, arresting cell cycle in G1/S phase;(3) At the same time, with intervention of CDDP, FANCD2shRNA silencinghuman HNSCC SIHN-005-A cell was impressed both in cell growth inhibitionin vitro and chemotherapy sensitization, significant impacts on performances ofmore obvious cell proliferation inhibition, apoptosis promotion and cell cyclearrest, showed that shRNA inference silencing FANCD2expression couldimprove the sensitivity of HNSCC SIHN-005A cell to DNA cross-linking agentCDDP.(4) Silencing FANCD2expression by shRNA interference probablyworked by cutting down cyclin D1and HIF-1alpha, improving the ratio ofBax/Bcl-2, altered interaction influences, changed the growth in vitro andchemotherapy sensitivity of SIHN-005-A cell.(5) FANCD2was expected tobecome a new target in the HNSCC research and therapy, meanwhile, shRNAstable gene transfection technology could also provide better theoretical supportand technology platforms for tumor gene therapy.