Tumor radiation-sensitizing and radiation bystander effect reducing function of miRNA, implementation method, and uses
A tumor radiation and application technology, applied in the fields of biotechnology and medicine, can solve the problems that there are no research reports on miR214
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Embodiment 1
[0044] Example 1 Construction and identification of HepG2 stable cell lines overexpressing miR214 and miR495
[0045] Stable cell lines overexpressing miR214 and miR495 were constructed using a lentiviral packaging system (purchased from SBI, USA). The components of the packaged virus were transfected into 293T cells with MegaTran1.0 transfection reagent, and the virus supernatant containing miR214 or miR495 precursor sequence (SEQ ID NO: 1 or SEQ ID NO: 2) was prepared, and then HepG2 cells were infected with the virus supernatant to obtain Stable cell lines incorporating miR214 or miR495 precursor sequences. These stable cell lines can utilize host cells to transcribe RNA precursors and cleavage to mature miR214 or miR495, so they can continuously overexpress miR214 or miR495. At the same time, an empty vector (control) was packaged as a cell line control. Stable cell lines use TRIzol (Invitrogen Company) to extract total cellular RNA, use SYBRQPCR reagent (Exiqon Company)...
Embodiment 2
[0046] Example 2 Verification of the radiosensitizing effect of miR214 and miR495 in vivo and in vitro
[0047] 2.1 In vitro radiation sensitization experiment of miR214 and miR495
[0048] HepG2 stable cells overexpressing miRNA214, miR495, and control group were used for colony formation experiments. The three types of cells were seeded in six-well plates with the same number, and each group was duplicated. After the cells were completely adhered to the wall, 60 The cells were irradiated with Co-γ rays once (dose 6Gy, dose rate 130cGy / min), then the plate was returned to the incubator for incubation, and the medium was replaced every two days. After the cells in the dish grow into cell groups, take out the plate for fixation and staining, and finally take pictures for counting. The results of clone formation experiments showed that the colony formation numbers of overexpressed miR214 group and miR495 group were significantly lower than those of the control group at the same...
Embodiment 3
[0051] Embodiment 3miR495 weakens the in vivo experiment of radiation bystander effect
[0052]At the end of the miR495 in vivo radiation sensitization experiment, the animals in the miR495 group were active and in good condition, while the animals in the control group were thin, sluggish and in poor condition. The two groups of mice were dissected to observe the morphological changes of the organs combined with HE staining. The results showed that multiple necrotic lesions were observed in the liver of the control group, and HE staining supported this phenomenon. are close to normal liver. In vivo experiments showed that overexpression of miR495 could effectively attenuate the liver damage caused by radiation side effects.
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