A Pilot Study On Molecular Regulating Mechanism Of MiR-21 In Human Cervical Squamous Carcinoma Both In Vitro And In Vivo

Objective The microRNA-21 (miR-21) has been identified over-expressed in HPV relevant cervical squamous carcinoma.To study the effect of antisense oligonucleotide(ASODN) targeting hsa-miR-21 on cell proliferation and apoptosis of cervical squamous carcinoma cell lines SiHa cells in vitro and in vivo the mechanism in cervical cancer.The studies suggest an important role of miR-21 in the molecular etiology of cervical cancer and implicate the potential application of miR-21 in cancer diagnosis and gene therapies.Methods Human cervical squamous carcinoma cell line SiHa cells were transfected with specific phosphorothioate antisense oligodeoxynucleotides (ASO) targeting hsa-miR-21. We investigated the change of the miR-21 expression in SiHa after the transfection by using real time RT-PCR. The cell proliferation following transfection was evaluated by MTT assay and colony formation experiment. The cell apoptosis following transfection was analyzed by Annexin V-FITC/PI analysis and TUNEL assay. The protein expression of PDCD4 was measured by Western blot analysis. The transplanted tumor model was established by injecting human cervical carcinoma cell line SiHa into subcutaneous tissue of nude mice.The inhibitory effect of miR-21 antisense oligonucleotide on tumor growth was evaluated by morphocytology and immunohistochemistry (IHC).Results After transfect of antisense miR-21, the expression of miR-21 was decreased. The growth of cells treated with antisense miR-21 was obviously inhibited compared with that in Blank control groups and Negative control groups (P<0.05). Colony formation of both cell lines decreased highly after transfected with antisense miR-21. Flow eytometry assay showed that transfection with antisense miR-21 promoted apoptosis greatly. Fluorometric TUNEL System showed that the apoptotic rate (11.7±0.62)% in miR-21 Inhibitors group higher than the negative control group (3.9±0.15)% and the blank control group (3.2±0.46)%.The nude mouse model of human cervical cancer xenograft tumor was constructed successfully. The tumor growth curves and the average tumor weight demonstrated that the growth of cells transfected with antisense miR-21 was significantly inhibited. The xenograft formation rate was 37.5%,75% and 100%, respectively. MiR-21 inhibitor group were positive, and the volume and the weight of tumor was statistically smaller than that of the control groups (P<0.05). IHC showed that the expression of Ki67 were decreased significantly compared with other groups. Extensive dead tumor cells were seen in the miR-21 inhibitor group, and remarkable apoptosis was shown by Fluorometric TUNEL System.The PDCD4 protein expression of miR-21 modulated group was increased. The miR-21-ASODN up-regulates the expression of PDCD4 and could suppress the cellular proliferation and induce apoptosis in SiHa cervical carcinoma cells.Conclusions Contrast with normal cervical tissue, the expression of miR-21 in cervical squamous carcinoma tissue and SiHa cell were significantly higher level and difference.The AS-miR-21 down-regulation the expression of miR-21 in cervical squamous carcinoma cell SiHa,could suppress the cellular proliferation, influence the Cells cycle and induce apoptosis in SiHa cells.These results suggest that miR-21 may play an oncogenic role in the cellular processes of HPV relevant cervical squamous carcinoma. Studies of in vivo experiment demonstrate that antisense miR-21 inhibites the expression of miR-21, increases the apoptosis of tumor cells and inhibites the tumor growth. The miR-21 may be a new target gene for cervical carcinoma treatment. The miR-21-ASODN upregulate the expression of PDCD4 and could suppress the cellular proliferation and induce apoptosis in SiHa cervical carcinoma cells. PDCD4 may play an tumor suppressor as targets of miR-21 in the cellular processes of human cervical squamous carcinoma.The evidence that upregulation of PDCD4 may suppress cell growth by blocking protein synthesis.