Down-regulation of HCP5 inhibits cell proliferation, migration, and invasion through regulating EPHA7 by competitively binding miR-101 in osteosarcoma

Patients with osteosarcoma (OS) usually have poor overall survival because of frequent metastasis. Long non-coding RNAs (lncRNAs) have been reported to be associated with tumorigenesis and metastasis. In this study, we investigated the expression and roles of lncRNA human histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) in OS, aiming to provide a novel molecular mechanism for OS. HCP5 was up-regulated both in OS tissues and cell lines and high expression of HCP5 was associated to low survival in OS patients. Down-regulation of HCP5 inhibited cell proliferation, migration, and invasion, suggesting its carcinogenic role in OS. miR-101 was targeted by HCP5 and its expression was decreased in OS. The inhibitor of miR-101 reversed the impact of HCP5 down-regulation on cell proliferation, apoptosis, and metastasis in OS. Ephrin receptor 7 (EPHA7) was proved to be a target of miR-101 and had ability to recover the effects of miR-101 inhibitor in OS. In conclusion, lncRNA HCP5 knockdown suppressed cell proliferation, migration, and invasion, and induced apoptosis through depleting the expression of EPHA7 by binding to miR-101, providing a potential therapeutic strategy of HCP5 in OS.

lncRNA HCP5; Osteosarcoma; miR-101; EPHA7

Authorship

Yangmao Tu ^*Correspondence: Min Xu: <ngchongzhiguril@163.com>

Department of Orthopedics, Jingzhou Hospital of Traditional Chinese Medicine, Jingzhou, ChinaJingzhou Hospital of Traditional Chinese MedicineChinaJingzhou, ChinaDepartment of Orthopedics, Jingzhou Hospital of Traditional Chinese Medicine, Jingzhou, China

https://orcid.org/0000-0003-3289-5083

Qing Cai ^*Correspondence: Min Xu: <ngchongzhiguril@163.com>

https://orcid.org/0000-0003-2905-2368

Xuemei Zhu

Jingzhou Hospital of Traditional Chinese Medicine - Functional Section, Jingzhou, ChinaJingzhou Hospital of Traditional Chinese Medicine - Functional SectionChinaJingzhou, ChinaJingzhou Hospital of Traditional Chinese Medicine - Functional Section, Jingzhou, China

https://orcid.org/0000-0001-6181-8998

Min Xu Correspondence: Min Xu: <ngchongzhiguril@163.com>

Jingzhou Institute of Technology, College of Textile and Apparel Design and Art, Jingzhou, ChinaJingzhou Institute of Technology, College of Textile and Apparel Design and ArtChinaJingzhou, ChinaJingzhou Institute of Technology, College of Textile and Apparel Design and Art, Jingzhou, China

https://orcid.org/0000-0001-9556-1596

Correspondence: Min Xu: <ngchongzhiguril@163.com>

*These authors contributed equally to this work.

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Figures

Figures (6)

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Figure 1
HCP5 was highly expressed in osteosarcoma (OS) tissues and cell lines. A, QRT-PCR assay was performed to measure the expression of HCP5 in 40 pairs of OS tissues and adjacent normal tissues. B, The cumulative survival of OS patients with high HCP5 (n=19) expression or low HCP5 (n=21) expression was recorded. C, Relative expression of HCP5 in different OS cell lines and human osteoblast cell hFOB 1.19 was detected by qRT-PCR. Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

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Figure 2
HCP5 knockdown suppressed proliferation, migration, and invasion, and promoted apoptosis in osteosarcoma (OS) cells. A, The expression of HCP5 in MG-63 and U2OS cells transfected with si-NC, si-HCP5#1, si-HCP5#2, or si-HCP5#3 was measured by qRT-PCR. B and C, Cell proliferative capacity was evaluated by MTT assay. D, Cell apoptosis was detected 48 h after transfection by flow cytometry. E and F, Cell migration and invasion were confirmed by transwell assay in MG-63 and U2OS cells (100×, scale bar: 50 μm). Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

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Figure 3
HCP5 directly targeted miR-101. A, The online software miRcode predicted the putative binding sites between HCP5 and miR-101. B, The expression of miR-101 was exhibited in osteosarcoma (OS) tissues and adjacent normal tissues. C, Correlation between the expression of HCP5 and miR-101 in OS tissues. D, The expression of miR-101 was measured in OS cell lines MG-63 and U2OS and normal cells hFOB 1.19. E and F, Luciferase reporter assay was performed to confirm the interaction between HCP5 and miR-101 in MG-63 and U2OS cells. G and H, Relative expression of miR-101 was examined in MG-63 and U2OS cells transfected with si-NC (negative control), si-HCP5#2, vector NC, or HCP5. Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

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Figure 4
Depletion of miR-101 could reverse the impact on cell proliferation, apoptosis, migration, and invasion caused by HCP5 knockdown in osteosarcoma (OS) cells. A, The expression of miR-101 in MG-63 and U2OS cells transfected with si-NC (negative control), si-HCP5#2, si-HCP5#2+miR-NC, or si-HCP5#2+miR-101 inhibitor was measured by qRT-PCR. B and C, Cell proliferation was assessed at 24, 48, 72, and 96 h after transfection by MTT assay in MG-63 and U2OS cells. D, Cell apoptosis was analyzed by flow cytometry. E and F, Transwell assay was used for cell migration and invasion analysis in MG-63 and U2OS cells. Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

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Figure 5
EPHA7 was a target of miR-101. A, Predicted binding sites between miR-101 and EPHA7 3'UTR. B, The expression of EPHA7 was measured through qRT-PCR assay in osteosarcoma (OS) tissues and adjacent normal tissues. C, Correlation analysis between miR-101 expression and EPHA7 expression in OS tissues. D and F, Relative luciferase activity was measured in MG-63 and U2OS cells cotransfected with miR-101 mimics and EPHA7-WT (wild-type) or EPHA7-MUT (mutant). E and G, EPHA7 protein expression level was observed by western blot in MG-63 and U2OS cells with NC (negative control) mimics, miR-101 mimics, NC inhibitor, or miR-101 inhibitor transfection. Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

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Figure 6
HCP5 regulated cell proliferation, apoptosis, migration, and invasion in osteosarcoma (OS) cells by controlling the expression of EPHA7 via targeting miR-101. A and B, Relative protein expression level of EPHA7 was determined by western blot in MG-63 and U2OS cells transfected with si-NC (negative control), si-HCP5#2, si-HCP5#2+miR-101 inhibitor, miR-101 inhibitor+si-NC, and miR-101 inhibitor+si-EPHA7. C and D, Cell proliferation was measured by MTT assay in MG-63 and U2OS cells containing diverse transfection. E, Cell apoptosis was examined using flow cytometry. F and G, Cell migration and invasion were detected in MG-63 and U2OS cells after transfection by transwell assay. Data are reported as means±SD. **P<0.01 (ANOVA).

Figure 1 HCP5 was highly expressed in osteosarcoma (OS) tissues and cell lines. A, QRT-PCR assay was performed to measure the expression of HCP5 in 40 pairs of OS tissues and adjacent normal tissues. B, The cumulative survival of OS patients with high HCP5 (n=19) expression or low HCP5 (n=21) expression was recorded. C, Relative expression of HCP5 in different OS cell lines and human osteoblast cell hFOB 1.19 was detected by qRT-PCR. Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

Figure 2 HCP5 knockdown suppressed proliferation, migration, and invasion, and promoted apoptosis in osteosarcoma (OS) cells. A, The expression of HCP5 in MG-63 and U2OS cells transfected with si-NC, si-HCP5#1, si-HCP5#2, or si-HCP5#3 was measured by qRT-PCR. B and C, Cell proliferative capacity was evaluated by MTT assay. D, Cell apoptosis was detected 48 h after transfection by flow cytometry. E and F, Cell migration and invasion were confirmed by transwell assay in MG-63 and U2OS cells (100×, scale bar: 50 μm). Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

Figure 3 HCP5 directly targeted miR-101. A, The online software miRcode predicted the putative binding sites between HCP5 and miR-101. B, The expression of miR-101 was exhibited in osteosarcoma (OS) tissues and adjacent normal tissues. C, Correlation between the expression of HCP5 and miR-101 in OS tissues. D, The expression of miR-101 was measured in OS cell lines MG-63 and U2OS and normal cells hFOB 1.19. E and F, Luciferase reporter assay was performed to confirm the interaction between HCP5 and miR-101 in MG-63 and U2OS cells. G and H, Relative expression of miR-101 was examined in MG-63 and U2OS cells transfected with si-NC (negative control), si-HCP5#2, vector NC, or HCP5. Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

Figure 4 Depletion of miR-101 could reverse the impact on cell proliferation, apoptosis, migration, and invasion caused by HCP5 knockdown in osteosarcoma (OS) cells. A, The expression of miR-101 in MG-63 and U2OS cells transfected with si-NC (negative control), si-HCP5#2, si-HCP5#2+miR-NC, or si-HCP5#2+miR-101 inhibitor was measured by qRT-PCR. B and C, Cell proliferation was assessed at 24, 48, 72, and 96 h after transfection by MTT assay in MG-63 and U2OS cells. D, Cell apoptosis was analyzed by flow cytometry. E and F, Transwell assay was used for cell migration and invasion analysis in MG-63 and U2OS cells. Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

Figure 5 EPHA7 was a target of miR-101. A, Predicted binding sites between miR-101 and EPHA7 3'UTR. B, The expression of EPHA7 was measured through qRT-PCR assay in osteosarcoma (OS) tissues and adjacent normal tissues. C, Correlation analysis between miR-101 expression and EPHA7 expression in OS tissues. D and F, Relative luciferase activity was measured in MG-63 and U2OS cells cotransfected with miR-101 mimics and EPHA7-WT (wild-type) or EPHA7-MUT (mutant). E and G, EPHA7 protein expression level was observed by western blot in MG-63 and U2OS cells with NC (negative control) mimics, miR-101 mimics, NC inhibitor, or miR-101 inhibitor transfection. Data are reported as means±SD. **P<0.01 (Student's t-test and ANOVA).

Figure 6 HCP5 regulated cell proliferation, apoptosis, migration, and invasion in osteosarcoma (OS) cells by controlling the expression of EPHA7 via targeting miR-101. A and B, Relative protein expression level of EPHA7 was determined by western blot in MG-63 and U2OS cells transfected with si-NC (negative control), si-HCP5#2, si-HCP5#2+miR-101 inhibitor, miR-101 inhibitor+si-NC, and miR-101 inhibitor+si-EPHA7. C and D, Cell proliferation was measured by MTT assay in MG-63 and U2OS cells containing diverse transfection. E, Cell apoptosis was examined using flow cytometry. F and G, Cell migration and invasion were detected in MG-63 and U2OS cells after transfection by transwell assay. Data are reported as means±SD. **P<0.01 (ANOVA).

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Down-regulation of HCP5 inhibits cell proliferation, migration, and invasion through regulating EPHA7 by competitively binding miR-101 in osteosarcoma

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[1] Correspondence: Min Xu: <ngchongzhiguril@163.com>