β-防御素-1通过环状RNA HAS-CIRCpedia-5280抑制小鼠结肠癌的研究OA

[Objective]To verify the role of β-defensin-1 in inhibiting the growth of colon cancer via circular RNA HAS-CIRCpedia-5280 by subcutaneous tumorigenesis experiments in nude mice.[Methods]Lentiviral transduction technology was performed to transduce human colon carcinoma HCT-116 cells with the overexpression lentivirus carrying HAS-CIRCpedia-5280 and empty-vector control lentivirus,respectively.After polybrene-assisted infection and puromycin screening for 2 weeks,HAS-CIRCpedia-5280 overexpression stably transduced HCT-116 cell line(HAS-CIRCpedia-5280 cells)and empty vector control stably transduced HCT-116 cell line(NC cells)were constructed.Thirty BALB/c-nu nude mice were divided into 6 groups[oe-NC group,subcutaneously injected with NC cells;oe-circRNA group,subcutaneously injected with HAS-CIRCpedia-5280 cells;oe-NC+PBS group,subcutaneously injected with NC cells and intraperitoneally injected with PBS;oe-circRNA+PBS group,subcutaneously injected with HAS-CIRCpedia-5280 cells and intraperitoneally injected with PBS;oe-NC+hBD-1(human β-defensin-1)group,subcutaneously injected with NC cells and intraperitoneally injected with hBD-1;oe-circRNA+hBD-1 group,subcutaneously injected with HAS-CIRCpedia-5280 cells and intraperitoneally injected with hBD-1],with 5 mice in each group.The BALB/c-nu nude mice subcutaneous xenograft tumor model was established by subcutaneous injection of the above two kinds of stably transduced cells.The oe-NC+hBD-1 group and the oe-circRNA+hBD-1 group,the oe-NC+PBS group and the oe-cireRNA+PBS group were intraperitoneally injected with 0.1 mg/(kg·BW)hBD-1 or an equal volume of PBS,respectively,for intervention,twice a week(Monday and Thursday),and tumor volume was recorded every 7 days.All injections were completed within 4 weeks from the first administration.The nude mice were euthanized 72 h after the last administration,and the tumor tissues were dissected to measure tumor volume and weight.[Results]HAS-CIRCpedia-5280 overexpression(oe-circRNA)and empty vector control(oe-NC)stably transduced HCT-116 cell lines were constructed by lentivirus transduction.Fluorescence microscopy showed that most cells in the oe-circRNA group and oe-NC group exhibited clear green fluorescence,and the positive rate of cells was higher.The results of qRT-PCR showed that compared with the oe-NC control group,the expression level of HAS-CIRCpedia-5280 in the oe-circRNA group was extremely significantly up-regulated(P＜0.01).A subcutaneous xenograft tumor model of colon cancer in nude mice was constructed,and tumor growth was regularly monitored.The results showed that compared with the oe-NC group,the weight of transplanted tumor in the oe-circRNA group was extremely significantly increased(P＜0.01),and the volume was significantly increased(P＜0.05).qRT-PCR detection confirmed that the expression level of HAS-CIRCpedia-5280 in the oe-circRNA group was extremely significantly up-regulated compared with the oe-NC group(P＜0.01).An hBD-1 intraperitoneal intervention experiment was conducted on the colon cancer xenograft tumor model in nude mice.Compared with the oe-NC+PBS group,the weight of transplanted tumors in the oe-circRNA+PBS group was extremely significantly increased(P＜0.01)and the volume was significantly enlarged(P＜0.05);the weight of transplanted tumors in the oe-NC+hBD-1 and oe-circRNA+hBD-1 groups was extremely significantly decreased(P＜0.01)and the volume was significantly reduced(P＜0.05).qRT-PCR detection showed that,compared with the oe-NC+PBS group,the expression level of HAS-CIRCpedia-5280 in the oe-NC+hBD-1 group was significantly down-regulated(P＜0.05);compared with the oe-circRNA+PBS group,the expression of HAS-CIRCpedia-5280 in the oe-circRNA+hBD-1 group was significantly down-regulated(P＜0.05).[Conclusion]The experimental results indicate that overexpression of HAS-CIRCpedia-5280 promotes the growth of colon cancer xenograft tumors.Further studies found that hBD-1 treatment can effectively down-regulate the expression of HAS-CIRCpedia-5280 and inhibit the growth of xenograft tumors.It is therefore speculated that hBD-1 inhibits the progression of colon cancer through HAS-CIRCpedia-5280.