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| Molecular Mechanisms of LncRNA FAM225A/miR-205-5p/BRCA1 Mediated DNA Damage Repair in Cervical Cancer Radiotherapy Resistance |
| ZHEN Yulan, CHEN Yongfa, GUO Jingwen, et al |
| Songshan Lake Central Hospital, Guangdong Dongguan 523326, China |
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Abstract Objective: To investigate the effects of LncRNA FAM225A/miR-205-5p/BRCA1 axis on DNA repair, cell proliferation, and cell apoptosis rate of radiation resistant cells of human cervical cancer (CC). Methods: Cell Experiment Grouping 1: control group (HeLa cells) and radiation-resistant group (HeLa-229 cells). Bioinformatics analysis was performed to predict the direct sequence interactions between LncRNA FAM225A and miR-205-5p, as well as between miR-205-5p and the 3'-UTR of BRCA1. Dual-luciferase reporter gene assay was used to confirm the above direct sequence interactions. Cell Experiment Grouping 2 included: ① Control Group 2 (radiation-resistant HeLa-229 cells), ② Radiation group (radiation-resistant HeLa-229 cells irradiated with 6 Gy X-rays), ③ Radiation + Aden-LncRNA FAM225A shRNA group, ④ Radiation + Aden-shRNA NC group, ⑤ Radiation + Aden-LncRNA FAM225A-OE group, ⑥ Radiation + Aden-OE vector group. Comet assay was used to determine the repair of DNA repair in cells of each group. CCK-8 assay was used to measure the cell proliferation ability of each group. Flow cytometry was used to determine the apoptosis rate. RT-qPCR was used to detect the effect of knockdown/overexpression of LncRNA FAM225A on the expression of genes related to cell apoptosis, cell cycle, DNA damage repair, and cell survival promotion. Western blot was used to determine the protein expression levels of BRCA1, BRCA2, CDK1, p-PI3K, PI3K, p-AKT, and AKT in cells of each group. Results: Compared with the control group (HeLa cells), the relative expression levels of LncRNA FAM225A and BRCA1 mRNA in the radiation-resistant group (HeLa-229 cells) were upregulated (P<0.05), while the relative expression level of miR-205-5p was downregulated (P<0.05). LncRNA FAM225A inhibited the expression of miR-205-5p through direct sequence interaction, and miR-205-5p inhibited the expression of BRCA1 through direct sequence interaction with the 3'-UTR of BRCA1. Compared with Control Group 2, which had fewer and shorter comet tails, the radiation group had longer and more severe comet tails; the radiation + Aden-LncRNA FAM225A shRNA group had more severe comet tails, with the comet head and tail separated, making it difficult to interpret; the radiation + Aden-LncRNA FAM225A-OE group had fewer and shorter comet tails compared with the radiation group. Compared with Control Group 2, the apoptosis rate of the radiation group increased (P<0.05). Compared with the radiation group, the cell proliferation ability of the radiation+Aden-LncRNA FAM225A shRNA group decreased and the apoptosis rate increased (P<0.05); the cell proliferation ability of the radiation+Aden-LncRNA FAM225A-OE group enhanced and the apoptosis rate decreased (P<0.05). The results of mRNA level determination showed that compared with Control Group 2, the relative expression levels of apoptosis-related genes (Caspase-3, BAX, FADD), cell survival-promoting genes (PARP, SOX2, CKS1B, MEIS2), and DNA damage repair-related genes (BRCA1, BRCA2, ATM, RAD51) in the radiation group were increased (P<0.05); the relative expression levels of tumor suppressor transcription factors, proto-oncogenes (SPT6, p53, RNAP II, Rb), apoptosis-related genes (BCL2, BAD), cyclin-encoding genes (CDK1, CCNE1, CCNB1, CDC25A), and oncogenic signaling pathway protein factor-encoding genes (AKT, mTOR, PI3K) were decreased (P<0.05). Compared with the radiation group, the relative expression levels of PARP, CKS1B, Caspase-3, BAX, and FADD in the radiation+Aden-LncRNA FAM225A shRNA group were increased (P<0.05); the relative expression levels of SOX2, BCL2, BAD, SPT6, p53, RNAP II, Rb, BRCA1, BRCA2, ATM, CDK1, CCNE1, CCNB1, CDC25A, AKT, mTOR, and PI3K were decreased (P<0.05). In the radiation+Aden-LncRNA FAM225A-OE group, the relative expression levels of PARP, CKS1B, Caspase-3, BAX, and FADD were decreased (P<0.05); the relative expression levels of SOX2, BCL2, BAD, SPT6, p53, RNAP II, Rb, BRCA1, BRCA2, ATM, CDK1, CCNE1, CCNB1, CDC25A, AKT, mTOR, and PI3K were increased (P<0.05). The results of protein level determination showed that compared with Control Group 2, the relative expression level of BRCA2 in the radiation group was upregulated (P<0.05), while the relative expression levels of CDK1, p-PI3K, PI3K, p-AKT, and AKT were downregulated (P<0.05). Compared with the radiation group, the relative expression levels of BRCA2, CDK1, p-PI3K, PI3K, p-AKT, and AKT in the radiation+Aden-LncRNA FAM225A shRNA group were decreased (P<0.05), while those in the radiation+Aden-LncRNA FAM225A-OE group were upregulated (P<0.05).Conclusion: LncRNA FAM225A/miR-205-5p/BRCA1 axis promoted cervical cancer radiotherapy resistance by enhancing DNA damage repair.
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[1] 闫美辰,刘丽丽.宫颈癌流行病学及发病因素研究进展[J].锦州医科大学学报,2023,44(6):103-107,112.
[2] 张清伟,尹如铁.宫颈癌诊治热点与进展[J].实用医学杂志,2024,40(17):2357-2362.
[3] Chargari C,Peignaux K,Escande A,et al.Radiotherapy of cervical cancer[J].Cancer Radiother,2022,26(1-2):298-308.
[4] 李媚,刘琦.宫颈癌微小RNA与自噬的研究进展[J].沈阳医学院学报,2024,26(5):529-531,555.
[5] Liu C,Li Y.Hsa_circ_0000078 regulates miR-205-5p/EREG pathway to inhibit cervical cancer progression[J].Mol Biotechnol,2023,65(9):1453-1464.
[6] Chen N,Zhu X,Zhu Y,et al.The regulatory relationship and function of LncRNA FAM225A-miR-206-ADAM12 in gastric cancer[J].Am Transl Res,2021,13(8):8632-8652.
[7] Zhao H,Zheng GH,Li GC,et al.Long noncoding RNA LINC00958 regulates cell sensitivity to radiotherapy through RRM2 by binding to microRNA-5095 in cervical cancer[J].Cell Physiol,2019,234(12):23349-23359.
[8] Zhang C,Luo Y,Cao J,et al.Exosomal LncRNA FAM225A accelerates esophageal squamous cell carcinoma progression and angiogenesis via sponging miR-206 to upregulate NETO2 and FOXP1 expression[J].Cancer Med,2020,9(22):8600-8611.
[9] Zhang J,Zhang J,Pang X,et al.MiR-205-5p suppresses angiogenesis in gastric cancer by downregulating the expression of VEGFA and FGF1[J].Exp Cell Res,2021,404(2):112579.
[10] Li HN,Zhang HM,Li XR,et al.MiR-205-5p/GGCT attenuates growth and metastasis of papillary thyroid cancer by regulating CD44[J].Endocrinology,2022,163(4):22. |
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2025, Vol. 31 
