All trans retinoic acid-induced overexpression of Cx43 gene in RB cells and its inhibition on the growth of RB

Background One of the important machanisms of all trans retinoic acid (ATRA) is to regulate the expression of connexin (Cx) gene.ATRA inhibits the proliferation and differentiation of retinoblastoma (RB) cells,which is related to Cx43.However,the control site of ATRA and its effect on RB tumor in vivo have not been identified.Objective This study was to investigate the effect of ATRA on Cx43 expression in RB cells and its approach mechanisms.Methods ATRA solution of 1 × 10 2 mol/L was prepared with ethanol and formulated into 1×10 5,1×10-6and 1 × 10 7 mol/L of solution with culture medium further.Human RB cell line (HXO-RB44) was cultured and treated with different concentrations of ATRA for 2,4 and 6 days,respectively.The expressions of Cx43 protein and mRNA in RB cells were detected by Western blot and reverse transcription PCR (RT-PCR),respectively.RB models were established by injecting HXO-RB44 cell suspension into anterior chamber in the right eyes of 15 athymic mice.Eleven successful models were divided into the blank control group,negative control group and 1 × 10-5 mol/L ATRA group,and 0.5％ normal saline solution with athymic or 1 ×10-5 mol/L ATRA solution was injected into the anterior chamber in the negative control group and 1 × 10-5 mol/L ATRA group in the 3-day interval for 3 weeks.The model eyes were examined under the slit lamp microscope.The eyeballs were extracted at the end of the experiment for hematoxylin and eosin staining.Results Western blot assay showed that the absorbance values of Cx43 protein (ACx43/AGAPDH) were increased gradually as time lapse of ATRA treatment among the groups (Ftime =71.31,P =0.00; Fgroup =7.66,P =0.00).The expressions of Cx43 protein were significantly higher in the 1 × 10 5 mol/L ATRA group after 2 days,1 × 10-6 mol/L ATRA group after 4 days,1 × 10-7 mol/L ATRA group after 6 days than those in the blank control group at various time points (t =3.34,P＜0.01 ;t =2.33,P＜0.05;t =3.12,P＜ 0.01).RT-PCR showed that the absorbance values of Cx43 mRNA (ACx43mRXA/Aβ-actin) were significantly enhanced as the prolong of treatment time of ATRT among the groups (Ftime =90.90,P =0.00 ; Fgroup =6.86,P =0.00).The expressions of Cx43 mRNA were significantly higher in the 1 × 10-5 mol/L ATRA group after 2 days,1 × 10 6 mol/L ATRA group and 1 ×10-7 mol/L ATRA group after 4 days than those in the blank control group at various time points (t=3.57,P＜0.01 ;t=6.31,P＜0.01 ;t=2.22,P＜0.05).RB models were successfully created in 11 eyes on the 6-9 days following the intrachamber injection of RB cell suspension.The RB cells were filled with chamber in the blank control group 20 days after injection,and RB only occupied half of the anterior chamber in the 1 × 10-5 mol/L ATRA group.Histopathological examination exhibited that the RB cells were seen in the anterior and posterior chamber as well as vitreous in the blank control group,however,the cells were only found in the anterior chamber in the 1 × 10 5 mol/L ATRA group.Conclusions ATRA can inhibit the growth of RB in vitro and in vivo by inducing the expression of Cx43 gene in transcription process. Key words: All trans retinoic acid;  Retinoblastoma;  Connexin;  Animal model