The role of lncRNA MSC-AS1/miR-29b-3p axis-mediated CDK14 modulation in pancreatic cancer proliferation and Gemcitabine-induced apoptosis

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related death due to the failure of traditional therapies. In the present study, we attempted to construct a lncRNA-miRNA-mRNA network which may modulate PDAC cell proliferation and Gemcitabine-induced cell apoptosis starting from CDK14, a new member of the CDK family and an oncogene in many cancers. Based on TCGA data, a significant positive correlation was observed between lncRNA MSC-AS1 and CDK14. Moreover, MSC-AS1 expression was upregulated in PDAC tissues. Higher MSC-AS1 expression was correlated with poorer prognosis in patients with PDAC. MSC-AS1 knockdown in Panc-1 and BxPC-3 cells significantly inhibited the cell proliferation. Moreover, miR-29b-3p, which has been reported to act as a tumor suppressor, was predicted to bind to both MSC-AS1 and CDK14. Contrary to MSC-AS1, higher miR-29b-3p expression was correlated to better prognosis in patients with PDAC. In both PDAC cell lines, miR-29b-3p negatively regulated MSC-AS1 and CDK14. As confirmed using luciferase reporter gene and RIP assays, MSC-AS1 served as a ceRNA for miR-29b-3p to counteract miR-29b-mediated CDK14 repression. MSC-AS1 knockdown inhibited CDK14 protein levels and PDAC proliferation and enhanced gemcitabine-induced cell death and apoptosis while miR-29b-3p inhibition exerted an opposing effect; the effect of MSC-AS1 knockdown was partially attenuated by miR-29b-3p inhibition. Taken together, we demonstrated that MSC-AS1/miR-29b-3p axis modulates the cell proliferation and GEM-induced cell apoptosis in PDAC cell lines through CDK14. We provided a novel experimental basis for PDAC treatment from the perspective of lncRNA-miRNA-mRNA network.

Keywords: CDK14; apoptosis; lncRNA MSC-AS1; miR-29b-3p; pancreatic ductal adenocarcinoma (PDAC); proliferation.

Figure 1.

Selection of lncRNA MSC-AS1 and its correlation with the clinical parameters (a-b) Expression of CDK14 and MSC-AS1 in 45 paired PDAC and non-cancerous tissue specimens were examined using real-time PCR assays. (c) The correlation between CDK14 and MSC-AS1 was analyzed using Pearson’s analysis. (d) The overall survival of 45 cases patients with PDAC grouped by MSC-AS1 expression was analyzed using Kaplan-Meier method. The data are presented as mean ±SD of three independent experiments. *P < 0.05, **P < 0.01.

Figure 2.

MSC-AS1 knockdown inhibited PDAC cell proliferation (a) MSC-AS1 knockdown in Panc-1 and BxPC-3 cells was achieved by transfection of MSC-AS1 shRNA1 or shRNA2, as confirmed by real-time PCR. (b-c) Panc-1 and BxPC-3 cells were transfected with MSC-AS1 shRNA1 or shRNA2 and examined for cell proliferation using MTT assays. (d-e) Panc-1 and BxPC-3 cells were transfected with MSC-AS1 shRNA2 and examined the colony formation capacity. The data are presented as mean±SD of three independent experiments. *P < 0.05, **P < 0.01.

Figure 3.

Selection of miR-29b-3p and its correlation with the clinical parameters (a) A schematic diagram showing the selection of miRNAs related to CDK14 and MSC-AS1. (b) Expression of five selected miRNAs, miR-135a-5p, miR-29b-2-5p, miR-29b-3p, miR-148b-3p and miR-33a-5p in response to MSC-AS1 knockdown was examined in Panc-1 cells. (c) miR-29b expression in response to MSC-AS1 knockdown was examined in BxPC-3 cells. (d) Expression of miR-29b-3p in 45 paired PDAC and non-cancerous tissue specimens was examined. The data are presented as mean ± D of three independent experiments. *P < 0.05, **P < 0.01. (e) The overall survival of patients with PDAC recorded in TCGA database was analyzed using Kaplan-Meier method. (f) The overall survival of 45 cases patients with PDAC grouped by miR-29b-3p expression was analyzed using Kaplan-Meier method. (g-h) Correlation between miR-29b-3p and MSC-AS1, between miR-29b-3p and CDK14 was analyzed using Pearson’s analysis.

Figure 4.

MSC-AS1 serves as a ceRNA for miR-29b-3p to counteract miR-29b-mediated repression of CDK14 (a) miR-29b-3p expression was achieved by transfection of miR-29b-3p mimics or miR-29b-3p inhibitor into Panc-1 and BxPC-3 cells, as confirmed using real-time PCR. (b) Expression of MSC-AS1 in response to miR-29b-3p overexpression or miR-29b-3p inhibition was examined in Panc-1 and BxPC-3 cells. (c) Protein levels of CDK14 in response to miR-29b-3p overexpression or miR-29b-3p inhibition was examined in Panc-1 and BxPC-3 cells. (d) The interaction between miR-29b-3p and CDK14, between miR-29b-3p and MSC-AS1 was validated by luciferase reporter gene assays. (e) Association of miR-29b-3p and MSC-AS1 with AGO2 in Panc-1 and BxPC-3 cells. Detection of AGO2 using Immunoblotting assays. (f) RIP assay in Panc-1 and BxPC-3 cells transfected with vector-NC (negative control) or MSC-AS1 overexpressing vector followed by real-time PCR to detect MSC-AS1 and CDK14 associated with AGO2. The data are presented as mean ±SD of three independent experiments. *P < 0.05, **P < 0.01.

Figure 5.

MSC-AS1/miR-29b-3p axis modulates CDK14 and cell cycle-related proteins expression in PDAC cell. (a)Panc-1 and BxPC-3 cells were co-transfected with miR-29b-3p inhibitor and MSC-AS1 shRNA and then examined for protein levels of CDK14, c-Myc, cyclin D1, cyclin Y and cyclin D3 using westernblot, the protein density analysis were shown in (b-f). The data are presented as mean ±SD of three independent experiments. *P < 0.05, **P < 0.01, compared to control group; ##P < 0.01, compared to MSC-AS1 shRNA group.

Figure 6.

MSC-AS1/miR-29b-3p axis modulates PDAC cell proliferation through CDK14. Panc-1 and BxPC-3 cells were co-transfected with miR-29b-3p inhibitor and MSC-AS1 shRNA and then examined for cell proliferation (a-b) and colony formation capacity (c). The data are presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, compared to control group; ##P < 0.01, compared to MSC-AS1 shRNA group.

7.

MSC-AS1/miR-29b-3p axis modulates Gemcitabine-mediated PDAC cell death and apoptosis Panc-1 and BxPC-3 cells were co-transfected with miR-29b-3p inhibitor and MSC-AS1 shRNA in the presence or absence of 20 nM GEM treatment and examined for cell death (a) and apoptosis (b). The data are presented as mean ±SD of three independent experiments. *P < 0.05, **P < 0.01, compared to control group; ##P < 0.01, compared to GEM treatment group; &&P < 0.01, compared to MSC-AS1 shRNA + GEM group.