Anti-tumor Effects Of Knockdown Of Mir-210Combined With Radiotherapy On Human Hepatoma Xenograft In Nude Mice

Objective:SMMC-7721cells with stable transfection of the anti-sense miR-210or scrambled sequence were generated and implanted into nude mice followed by irradiation under normoxia and hypoxia conditions. The anti-tumor effects of knockdown of miR-210combined with radiotherapy under normoxia and hypoxia on human hepatoma xenograft in nude mice and its mechanism were investigated.Methods:SMMC-7721cells with stable transfection of the anti-sense miR-210or scrambled sequence were generated. Time courses of HIF-la mRNA expression and miR-210expression in SMMC-7721cells exposed to1%oxygen were analyzed by Real-time RT-PCR. SMMC-7721cells with stable transfection of the anti-sense miR-210or scrambled sequence were subcutaneously implanted into nude mice, when the diameter of tumor reached6-8mm, we assessed expression of HIF-la and miR-210target gene in tumor tissue. After irradiation of8Gy under normoxia and hypoxia conditions in24h, tumor volumes at different time points were observed, tumor growth delay time(TGD) and survival time were calculated. The cell proliferation and intratumoral microvessel density were detected by immunohistochemical staining while apoptotic cells in tumor tissue were detected by TUNEL method.Results:(1) The courses of miR-210expression in SMMC-7721and scrambled sequence transfection cells exposed to1%oxygen were increased by time, reaching the highest at48h, and no statistical differences at the same time point between the two groups, miR-210expression in anti-sense miR-210sequence transfection cells was lower than in SMMC-7721cells at each point in time.(2)14days were needed of SMMC-7721group and scrambled sequence transfection group for tumor diameter reaching about6-8mm, while21days were needed of anti-sense miR-210sequence transfection after inoculation.(3) The tumor volumes inhibition rate of scrambled sequence with irradiation under hypoxia, knockdown of miR-210, knockdown of miR-210with irradiation under hypoxia group, scrambled sequence and knockdown of miR-210with irradiation both under normoxia group30days after the beginning of therapy were19.93%,28.92%,37.24%,52.07%and71.51%; the average survival time were51.17±6.85d,53.83±6.85d,60.50±5.36d,72.50±5.58d and84.33±5.54d; TGD were2.8d,3.3d,4.8d,6.4d and11.9d. The radiosensitization ratio of knockdown of miR-210with irradiation under normoxia and hypoxia group were1.86and1.71.(4) Knockdown of miR-210decreased expression of HIF-1α, but increased the expression of proteins which are targets of miR-210in hepatoma xenograft. The cell proliferation and intratumoral microvessel density of knockdown of miR-210with irradiation group were significantly lower than those of scrambled sequence with irradiation group, respectively, while percentage of apoptotic cells in tumor tissue were higher1day after therapy.Conclusion:(1) Tumor growth was delayed in miR-210downregulated xenograft.(2) Knockdown of miR-210in combination with radiotherapy enhanced effect of radiotherapy under either normoxia or hypoxia conditions. The mechanism might be related to miR-210knockdown-induced inhibition of proliferation and angiogenesis and increase of apoptosis in xenograft.(3) MiR-210might be a new target of radiosensitization in hepatoma.