Regulation and mechanism of Myosin X on radiosensitivity of non-small cell lung cancer cell line H1975 in vitro
Shen Hui, Ou Haibin, Shao Jin, Jiang Yaofei, Liu Yu, Zhang Junhong, Xie Conghua
Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Hubei Cancer Clinical Study Center, Hubei Cancer Radiation Therapy Quality Control Center, Zhongnan Hospital of Wuhan University, Wuhan 430071, China
Abstract：Objective To investigate the effect and mechanism of Myosin X on the radiosensitivity of non-small cell lung cancer (NSCLC) cell line H1975 in vitro. Methods Western blot was applied to detect the expression level of Myosin X expression. The H1975 cell line with stable knockout of Myosin X (KO group) and infected with control virus (NC group) were constucted by using CRISPR/Cas9 technique. The knockout efficiency was validated. The radiosensitivity of two cell lines was measured by colony formation assay and single-hit multi-target model. γ-H2AX focus formation test and RNA sequencing (RNAseq) analysis were employed to identify the regulatory mechanism of the radiosensitivity of lung cancer cell lines mediated by Myosin X. Results The expression level of Myosin X in the H1975 cells was significantly up-regulated than those in other NSCLC cell lines (all P<0.01). The lentiviral vector of Myosin X sgRNA-Lenti-CRISPR v2 was successfully constructed. After the puromycin screening, H1975 cell lines with complete knockout of Myosin X and control cell lines (NC group) were obtained. Colony formation assay demonstrated that compared with the NC group, the radiosensitivity in the KO group was significantly higher (The D0 value was decreased from 1.28Gy to 1.03Gy, SF2decreased from 0.29 to 0.21, and the sensitization ratio was 1.24). The γ-H2AX focus formation test showed that the number of damage focus formed at 1h and 6h after irradiation in the KO group was significantly larger than that in the NC group (P<0.05. RNAseq analysis indicated that the expression level of ISLR in the KO group was significantly down-regulated than that IN the NC group (P<0.05). Conclusion Knockout of Myosin X can increase the radiosensitivity of H1975 cells probably by interfering the repair of DNA double-strand damage and down-regulating the expression level of ISLR.
Shen Hui,Ou Haibin,Shao Jin et al. Regulation and mechanism of Myosin X on radiosensitivity of non-small cell lung cancer cell line H1975 in vitro[J]. Chinese Journal of Radiation Oncology, 2021, 30(9): 949-955.
[1] Bray F, Ferlay J, Soerjomataram I, et al.Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6):394-424.DOI:10.3322/caac.21492. [2] Chen W, Zheng R, Baade PD, et al.Cancer statistics in China, 2015[J]. CA Cancer J Clin, 2016, 66(2):115-132.DOI:10.3322/caac.21338. [3] Liu Y, Peng Y, Dai PG, et al.Differential regulation of myosin X movements by its cargos, DCC and neogenin[J]. J Cell Sci, 2012, 125(Pt 3):751-762.DOI:10.1242/jcs.094946. [4] Cao R, Chen J, Zhang X, et al.Elevated expression of myosin X in tumours contributes to breast cancer aggressiveness and metastasis[J]. Br J Cancer, 2014, 111(3):539-550.DOI:10.1038/bjc.2014.298. [5] Dvornikov D, Schneider MA, Ohse S, et al.Expression ratio of the TGFβ-inducible gene MYO10 is prognostic for overall survival of squamous cell lung cancer patients and predicts chemotherapy response[J]. Sci Rep, 2018, 8(1):9517.DOI:10.1038/s41598-018-27912-1. [6] Sandquist JC, Larson ME, Hine KJ.Myosin-10 independently influences mitotic spindle structure and mitotic progression[J]. Cytoskeleton, 2016, 73(7):351-364.DOI:10.1002/cm.21311. [7] Li A, Ma Y, Yu X, et al.Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod-driven motility and cell-cycle progression[J]. Dev Cell, 201, 21(4):722-734.DOI:10.1016/j.devcel.2011.07.008. [8] Tokuo H, Bhawan J, Coluccio LM.Myosin X is required for efficient melanoblast migration and melanoma initiation and metastasis[J]. Sci Rep, 2018, 8(1):10449.DOI:10.1038/s41598-018-28717-y. [9] Meier JA, Zhang F, Sanjana NE.GUIDES:sgRNA design for loss-of-function screens[J]. Nat Methods, 2017, 14(9):831-832.DOI:10.1038/nmeth.4423. [10] 欧海斌,隋铭,邵晶,等.非传统型肌球蛋白Myo10对结肠癌细胞SW620生长的影响及其机制[J]. 武汉大学学报(医学版),2019, 40(4):552-557.DOI:10.14188/j.1671-8852.2019.0155. Ou HB, Sui M, Shao J, et al.Effects of non-traditional myosin Myo10 on SW620 growth of colon cancer cells and its mechanism[J]. Med J Wuhan Univ, 2019, 40(4):552-557.DOI:10.14188/j.1671-8852.2019.0155. [11] Buglione M, Jereczek-Fossa BA, Bonù ml, et al.Radiosurgery and fractionated stereotactic radiotherapy in oligometastatic/oligoprogressive non-small cell lung cancer patients:results of a multi-institutional series of 198 patients treated with “curative” intent[J]. Lung Cancer, 2020, 141:1-8.DOI:10.1016/j.lungcan.2019.12.019. [12] Berg JS, Cheney RE.Myosin-X is an unconventional myosin that undergoes intrafilopodial motility[J]. Nat Cell Biol, 2002, 4(3):246-250.DOI:10.1038/ncb762. [13] Courson DS, Cheney RE.Myosin-X and disease[J]. Exp Cell Res, 2015, 334(1):10-15.DOI:10.1016/j.yexcr.2015.03.014. [14] Sun Y, Ai X, Shen S, et al.NF-κB-mediated miR-124 suppresses metastasis of non-small-cell lung cancer by targeting MYO10[J]. Oncotarget, 2015, 6(10):8244-8254.DOI:10.18632/onco target.3135. [15] Ceccaldi R, Rondinelli B, D'Andrea AD.Repair pathway choices and consequences at the double-strand break[J]. Trends Cell Biol, 2016, 26(1):52-64.DOI:10.1016/j.tcb.2015.07.009. [16] Sofińska K, Wilkosz N, Szymoński M, et al.Molecular spectroscopic markers of DNA damage[J]. Molecules, 2020, 25(3):561.DOI:10.3390/molecules25030561. [17] Nagasawa A, Kubota R, Imamura Y, et al.Cloning of the cDNA for a new member of the immunoglobulin superfamily (ISLR) containing leucine-rich repeat (LRR)[J]. Genomics, 1997, 44(3):273-279.DOI:10.1006/geno.1997.4889. [18] Zhang K, Zhang Y, Gu L, et al.Islr regulates canonical Wnt signaling-mediated skeletal muscle regeneration by stabilizing Dishevelled-2 and preventing autophagy[J]. Nat Commun, 2018, 9(1):5129.DOI:10.1038/s41467-018-07638-4. [19] Jun S, Jung YS, Suh HN, et al.LIG4 mediates Wnt signalling-induced radioresistance[J]. Nat Commun, 2016, 7:10994.DOI:10.1038/ncomms10994.
2021, Vol. 30 
