[Effect and mechanism of gene therapy of lentivirus mediated RhoA shRNA on ovarian cancer xenograft in vivo].

OBJECTIVE

To investigate treatment effects of lentivirus mediated RhoA short hairpin RNA (shRNA) on xenograft tumor of ovarian cancer in nude mice in vivo and the underlying mechanism.

METHODS

Human ovarian cancer cell line HO8910 were inoculated to establish subcutaneous xenograft model of human ovarian cancer. Tumor-bearing nude mice were assigned randomizely to three groups: Lenti-RhoA-sh group, Lenti- negative control (NC) group and phosphate buffered saline (PBS) group.lentivirus mediated RhoA shRNA, negative control lentivirus and PBS were respectively injected in the three groups. Effects of treatment were observed by tumor growth curve, tumor volume, tumor weight, and tumor inhibition rate. Xenograft tissues and liver, spleen, lung, and renal tissues were examined by hematoxylin and eosin (HE) staining or were detected by streptavidin-perosidase(SP)immunochemical method. The changes of RhoA gene expression in xenograft tissues after lentivirus mediated RhoA shRNA treated were also detected by real-time qPCR, immunochemistry and Western blot assay. Cell apoptosis in xenograft tissues were examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and apoptotic index (AI) were counted.

RESULTS

Compared with Lenti-NC group and PBS group, the growth speed of xenograft in Lenti-RhoA-sh group delayed significantly after injection 9 days (P < 0.01) . Tumor volume (338 ± 114) mm(3) decreased significantly in the Lenti-RhoA-sh group when compared with those in Lenti-NC group (1190 ± 332) mm(3) and PBS group (1101 ± 396) mm(3) (P < 0.01) . Tumor weight (0.23 ± 0.11) g decreased significantly in the Lenti-RhoA-sh group when compared with Lenti-NC group (0.79 ± 0.19) g and PBS group (0.74 ± 0.17) g (P < 0.01) . Real-time qPCR result shown that the expression of RhoA mRNA (0.30 ± 0.05) decreased significantly in the Lenti-RhoA-sh group compared with Lenti-NC group (0.95 ± 0.06) and PBS group(1.00 ± 0.11; P < 0.01) .Western blot result showed that the expression level of RhoA protein decreased significantly in the Lenti-RhoA-sh group (0.14 ± 0.06) compared with those in Lenti-NC group(0.78 ± 0.14) and PBS group (0.75 ± 0.13;P < 0.01). TUNEL staining displayed that AI significantly increased in the Lenti-RhoA-sh group (20.9 ± 3.4) % compared with those in Lenti-NC group (5.2 ± 2.0) % and PBS group (6.0 ± 2.1) % (P < 0.01).

CONCLUSION

Lentivirus mediated RhoA shRNA may be effectively down-regulate of the expression of RhoA, inhibit the growth of subcutaneous xenograft tumor of ovarian cancer in nude mice by increasing the cell apoptosis.