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. 2024 Jan 27;15(6):1718-1730.
doi: 10.7150/jca.91184. eCollection 2024.

LncRNA PRKCA-AS1 promotes LUAD progression and function as a ceRNA to regulate S100A16 by sponging miR-508-5p

Chaohui Wu  1 , Jiansheng Yang  1 , Xianbin Lin  1 , Jingyang Wu  1 , Chuangcai Yang  1 , Shuchen Chen  2
Affiliations

LncRNA PRKCA-AS1 promotes LUAD progression and function as a ceRNA to regulate S100A16 by sponging miR-508-5p

Chaohui Wu et al. J Cancer. .

Abstract

Objective: This study aimed to elucidate the underlying mechanism of LncRNA PRKCA-AS1 in lung adenocarcinoma (LUAD). Methods: The expression of LncRNA PRKCA-AS1, miR-508-5p and S100A16, in LUAD tissues or cell lines (NCI-H520 and H1299) was analyzed with qRT-PCR. The clinical diagnostic value of LncRNA PRKCAAS1, miR-508-5p and S100A16 in LUAD were analyzed by receptor operating characteristic (ROC) curve. Then we knockdown or overexpression of PRKCAAS1 in NCI-H520 and H1299 cells, and the cell function test was applied to detect the activity and metastasis level of cells in different transfection groups. Then Pearson correlation analysis was used for the correlation between miR-508-5p and PRKCA-AS1. The dual-luciferase reporter experiment and CHIRP analysis was conducted to verify the target binding relationship of PRKCA-AS1, miR-508-5p or S100A16. FISH assay analyzed the colocalization of PRKCA-AS1 and miR-508-5p in NCI-H520 and H1299 cells. Rescue experiment and tumorigenesis experiment in nude mice further explore the regulatory mechanisms of LncRNA PRKCA-AS1, miR-508-5p and S100A16 on LUAD progression in vitro and in vivo. Results: From the results, PRKCA-AS1 and S100A16 were up-regulated in LUAD tissues, while miR-508-5p was downregulated compared with the adjacent tissues. And gain-of-function revealed that PRKCA-AS1 knock-down apparently suppressed the cell proliferation and metastasis, whereas miR-508-5p inhibitors or S100A16 overexpression showed a opposite effect. In addition, there is evidence that PRKCA-AS1, miR-508-5p and S100A16 have a targeted regulatory relationship. Moreover, rescue experiment and tumorigenesis experiment in nude mice further confirmed that LncRNA PRKCA-AS1 regulates S100A16 through sponging miR-508-5p to regulate LUAD progression in vitro and in vivo. Conclusion: These results demonstrated that LncRNA PRKCA-AS1 might regulate LUAD by acting as a ceRNA via sponging miR-508-5p and regulating S100A16 expression, indicating that manipulation of PRKCA-AS1 might be a potential therapeutic strategy in LUAD.

Keywords: LncRNA PRKCA-AS1; S100A16; ceRNA; lung adenocarcinoma; miR-508-5p.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
LncRNA PRKCA-AS1 is highly expressed in LUAD. (A) The expression of PRKCA-AS1 in LUAD and adjacent tissues was detected with qRT-PCR. (B) Information collected about 60 patients. (C) ROC curve analysis of clinical diagnostic significance of LncRNA PRKCAAS1. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 2
Figure 2
Differential expression of PRKCAAS1 affected LUAD Cell Proliferation and metastasis. (A-B) The transfection efficiency of PRKCAAS1 was confirmed by qRT-PCR. (C) CCK-8 assay was used to detect the vitality of NCI-H520 and H1299 cell. (D) Colony formation experiments was used for proliferation ability of NCI-H520 and H1299 cell. (E-F) The transwell assay analyzed the cell migration and invasion abilities in NCI-H520 and H1299 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 2
Figure 2
Differential expression of PRKCAAS1 affected LUAD Cell Proliferation and metastasis. (A-B) The transfection efficiency of PRKCAAS1 was confirmed by qRT-PCR. (C) CCK-8 assay was used to detect the vitality of NCI-H520 and H1299 cell. (D) Colony formation experiments was used for proliferation ability of NCI-H520 and H1299 cell. (E-F) The transwell assay analyzed the cell migration and invasion abilities in NCI-H520 and H1299 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 3
Figure 3
MiR-508-5p was down-regulated in LUAD and negatively regulated by LncRNA PRKCA-AS1. (A) The expression of miR-508-5p in LUAD and adjacent tissues was detected with qRT-PCR. (B) ROC curve analysis of clinical diagnostic significance of miR-508-5p. (C) Pearson Correlation analysis was used for the relationship of miR-508-5p and PRKCA-AS1. (D) The targeting relationship of PRKCA-AS1 and miR-508-5p was verified with CHIRP assay. (E) The location of PRKCA-AS1 and miR-508-5p was determined by FISH analysis. (F) The expression of miR-508-5p in different transfection groups was detected with qRT-PCR. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 4
Figure 4
LncRNA PRKCA-AS1 regulated S100A16 and AKT pathway by sponging miR-508-5p. (A) Online websites predict the binding sites of miR-508-5p and S100A16. (B) Dual-luciferase reporter assay verified the binding relationship between the two. (C) The expression of S100A16 in LUAD and adjacent tissues was detected with qRT-PCR. (D) ROC curve analysis of clinical diagnostic significance of S100A16. (E) The mRNA level of S100A16 in different transfection cells was analyzed by qRT-PCR. (F) Western blot was used to detect the protein expression of S100A16, AKT, p-AKT, E-cadherin, N-cadherin, and Vimentin. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 4
Figure 4
LncRNA PRKCA-AS1 regulated S100A16 and AKT pathway by sponging miR-508-5p. (A) Online websites predict the binding sites of miR-508-5p and S100A16. (B) Dual-luciferase reporter assay verified the binding relationship between the two. (C) The expression of S100A16 in LUAD and adjacent tissues was detected with qRT-PCR. (D) ROC curve analysis of clinical diagnostic significance of S100A16. (E) The mRNA level of S100A16 in different transfection cells was analyzed by qRT-PCR. (F) Western blot was used to detect the protein expression of S100A16, AKT, p-AKT, E-cadherin, N-cadherin, and Vimentin. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 5
Figure 5
LncRNA PRKCA-AS1 regulates LUAD progression by modulating S100A16/AKT via sponging miR-508-5p in vitro. (A) The mRNA expression of PRKCA-AS1, miR-508-5p and S100A16 were assessed by qRT-PCR. (B) CCK-8 assay was used to detect the vitality of NCI-H520 and H1299 cell in different transfected groups. (C) Colony formation experiments was used for proliferation ability of cells in different transfected groups. (D-E) The transwell assay analyzed the cell migration and invasion abilities in cells in different transfected groups. (F) Western blot was used to detect the protein expression of S100A16, AKT, p-AKT, E-cadherin, N-cadherin, and Vimentin. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 5
Figure 5
LncRNA PRKCA-AS1 regulates LUAD progression by modulating S100A16/AKT via sponging miR-508-5p in vitro. (A) The mRNA expression of PRKCA-AS1, miR-508-5p and S100A16 were assessed by qRT-PCR. (B) CCK-8 assay was used to detect the vitality of NCI-H520 and H1299 cell in different transfected groups. (C) Colony formation experiments was used for proliferation ability of cells in different transfected groups. (D-E) The transwell assay analyzed the cell migration and invasion abilities in cells in different transfected groups. (F) Western blot was used to detect the protein expression of S100A16, AKT, p-AKT, E-cadherin, N-cadherin, and Vimentin. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 5
Figure 5
LncRNA PRKCA-AS1 regulates LUAD progression by modulating S100A16/AKT via sponging miR-508-5p in vitro. (A) The mRNA expression of PRKCA-AS1, miR-508-5p and S100A16 were assessed by qRT-PCR. (B) CCK-8 assay was used to detect the vitality of NCI-H520 and H1299 cell in different transfected groups. (C) Colony formation experiments was used for proliferation ability of cells in different transfected groups. (D-E) The transwell assay analyzed the cell migration and invasion abilities in cells in different transfected groups. (F) Western blot was used to detect the protein expression of S100A16, AKT, p-AKT, E-cadherin, N-cadherin, and Vimentin. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 6
Figure 6
LncRNA PRKCA-AS1 regulates LUAD progression by modulating S100A16/AKT via sponging miR-508-5p in vivo. (A) The tumor volume within 30 days after inoculation in mice was analyzed. (B) HE staining experiment was used to observe tumor tissue in mice. (C) The mRNA expression of PRKCA-AS1, miR-508-5p and S100A16 in tumor tissues were assessed by qRT-PCR. (D) Western blot was used to detect the protein expression of S100A16, AKT, p-AKT, E-cadherin, N-cadherin, and Vimentin. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 6
Figure 6
LncRNA PRKCA-AS1 regulates LUAD progression by modulating S100A16/AKT via sponging miR-508-5p in vivo. (A) The tumor volume within 30 days after inoculation in mice was analyzed. (B) HE staining experiment was used to observe tumor tissue in mice. (C) The mRNA expression of PRKCA-AS1, miR-508-5p and S100A16 in tumor tissues were assessed by qRT-PCR. (D) Western blot was used to detect the protein expression of S100A16, AKT, p-AKT, E-cadherin, N-cadherin, and Vimentin. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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