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miRNA-496 influences the malignant biological behaviors of cervical cancer HeLa cells and the growth of transplanted tumors in nude mice through mTOR
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Abstract:
Objective: To explore whether miR-496 affects the cycle, proliferation, migration, and invasion of cervical cancer HeLa cells and the growth of transplanted tumors in nude mice by targeting mTOR. Methods: The tumor specimens and para-cancerous tissue specimens of 74 patients with cervical cancer who underwent surgery at the Fourth Hospital of Hebei Medical University from December 2020 to December 2021 were selected. qPCR, WB, and immunofluorescence methods were used to detect the expression of miR-496 and mTOR at mRNA and protein levels in cervical cancer tissues. The TargetScan database was used to predict the target genes of miR-496, and the Dual-luciferase reporter gene experiment was further adopted to verify the targeting relationship. According to different transfectants, HeLa cells were divided into the control group, miR-496 mimic group, and miR-496 mimic+pMIR-mTOR group. CCK-8 assay, Flow cytometry, and Transwell experiment were used to detect the effects of miR-496 and mTOR on the proliferation, cell cycle, migration, and invasion of HeLa cells. The HeLa cells of the control group and miR-496 mimic group were inoculated subcutaneously into BALB/c nude mice to construct a transplanted tumor model of cervical cancer. After 21 d, the mice were sacrificed, and the tumor tissues of the mice were stripped and the tumor mass was weighed. Immunofluorescence and WB methods were used to detect the effect of miR-496 overexpression on the expression of mTOR and Ki67 in transplanted tumor tissues. Results: In cervical cancer tissues, miR-496 was lowly expressed, while mTOR was highly expressed at both mRNA and protein levels. miR- 496 could bind to the 3'-UTR sequence of mTOR mRNA. Compared with the control group and the miR-496 mimic+pMIR-mTOR group, the miR-496 level and S-phase cell ratio of HeLa cells in the miR-496 mimic group were significantly increased, while the proliferation level, the numbers of migrated cells and invaded cells were significantly reduced (all P<0.01). Nude mice transplanted tumor model of cervical cancer was successfully constructed. After 21 d of inoculation, the mass of transplanted tumors and the expression of mTOR and Ki67 in the transplanted tumor tissues in the miR-496 mimic group were significantly lower than those in the control group (all P<0.01). Conclusion: miR-496 is lowly expressed in cervical cancer tissues. Overexpression of miR-496 can inhibit the malignant biological behaviors of HeLa cells and the growth of transplanted tumors in nude mice through targeted regulation of mTOR.
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Project supported by the Key Projects of Medical Scientific Research Project of Hebei Province (No. 20210082)
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