lncRNA LOC102724169 plus cisplatin exhibit the synergistic anti-tumor effect in ovarian cancer with chronic stress

Abstract

Chronic stress has been proven to accelerate the development and progression of ovarian cancer, but the underlying molecular mechanisms have not been fully elucidated. In a combination survey of ovarian cancer with chronic stress (OCCS) mouse models and high-throughput sequencing, a key lncRNA named LOC102724169 on chromosome 6q27 has been identified, which functions as a dominant tumor suppressor in OCCS. Transcriptionally regulated by CCAAT enhancer binding protein (CEBP) beta (CEBPB), LOC102724169 shows low expression and correlates with poor progression-free survival (PFS) in OCCS patients. LOC102724169 is an instructive molecular inhibitor of malignancy of ovarian cancer cells, which is necessary to improve the curative effect of cisplatin therapy on ovarian cancer. This function stems from the inactivation of molecules in phosphatidylinositol 3-kinase (PI3K)/AKT signaling, repressing MYB expression and retaining the responsiveness of cancer cells to cisplatin. These findings provide a mechanistic understanding of the synergistic anti-tumor purpose of LOC102724169 as a bona fide tumor suppressor, enhancing the therapeutic effect of cisplatin. The new regulatory model of "lncRNA-MYB" provides new perspectives for LOC102724169 as a chronic stress-related molecule and also provides mechanistic insight into exploring the cancer-promoting mechanism of MYB in OCCS, which may be a promising therapeutic strategy for ovarian cancer.

Keywords: chronic stress; ciaplatin resistance; lncRNA LOC102724169; ovarian cancer; tumor suppressor.

Graphical abstract

Figure 1

Chronic stress-related lncRNAs identified through integrated analysis in ovarian cancer (A) Schematic diagram of stress nude mouse model. Blue open arrows indicate that mice were randomly divided into the stress group or the control group after adaptive growth for 1 week. Blue filled arrows indicate the stressed group, in which mice receive 2 h of immobilization daily. Green arrows indicate that SKOV3 cells were injected intraperitoneally in nude mice with/without periodic immobilization. Red arrows indicate mice were euthanized, and tumor tissues were collected for RNA sequencing. (B) Gross specimens of tumor tissue in stressed mice and control group. (C) Quantification of tumor nodules and tumor volume in stressed mice and the matched control group. (D) Heatmap shows part of differentially expressed mRNAs and lncRNAs in high-throughput sequencing of stressed mice and the matched control group. (E) Part of the KEGG pathway and their enriched genes of high-throughput sequencing in stressed mice and control group. (F) Correlation analysis of differentially expressed genes in sequencing results. (G) Several mRNAs and lncRNAs from high-throughput sequencing were validated in mice tumor tissue between the stress and control groups. (H) Cell viability of LOC102724169 (LOC) and HOTAIRM1 (HO) compared to the negative control (NC) detected by CCK-8. LOC102724169 reduced the value of IC₅₀ in both SKOV3 and HO8910 cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Statistical differences were calculated using a two-tailed Student’s t test. Data are represented as mean ± SEM.

Figure 2

Identification of lncRNA LOC102724169 transcriptionally regulated by CEBPB (A) Gene map of LOC102724169, a lncRNA on human chromosome 6q27. (B) RNA ISH assay suggested that LOC102724169 was mostly located in the nucleus of SKOV3 cells. Original magnification, ×200; scale bars, 5 μm). (C) JASPAR (http://jaspar.genereg.net/) was used to predict sites of putative CEBPB binding motif in the LOC102724169 promoter region. (D) Relative score and score of transcription factors predicted by JASPAR. (E) Relative expression of LOC102724169 in both SKOV3 and HO8910 cells transfected with CEBPB or control plasmid. (F) Dual-luciferase reporter assays examining the relative luciferase activities, which show a marked CEBPB binding activity in the BP3 site of LOC102724169. (G) ChIP assays of the enrichment of CEBPB on the LOC102724169 promoter relative to control IgG in both SKOV3 and HO8910 cells. (H) CEBPB expression levels measured in tumor tissues of stressed mice and non-stressed mice by immunohistochemistry analysis. Original magnification, ×200; scale bars, 5 μm). (I) Immunohistochemical score of CEBPB in stressed mice and matched control group. ∗∗p < 0.01. Statistical differences were calculated using a two-tailed Student’s t test. Data are represented as mean ± SEM. ISH, in situ hybridization; BP3, binding position 3.

Figure 3

Chronic stress promoted tumor progression in EOC patients (A) Clinical sample validation of CEBPB expression in BOT and EOC. (B) Clinical sample validation of LOC102724169 expression in BOT and EOC. (C) Correlation analysis of CEBPB and LOC102724169 expression levels by a Pearson correlation coefficient (r = 0.4, p = 0.0061). (D) Validation of LOC102724169 expression between high-risk stress group and low-risk stress group. LOC102724169 showed low expression in the high-risk group. (E) Spearman rank regression was used to analyze the correlation between chronic stress and cisplatin resistance. (F) Kaplan-Meier analysis and a log-rank test were used to show progression-free survival curves of EOC patients with different expressions of LOC102724169. Data are represented as mean ± SEM. BOT, benign ovarian tumor; EOC, epithelial ovarian cancer.

Figure 4

LOC102724169 was necessary for reducing cell viability and inducing apoptosis of EOC cells (A) Relative expression of LOC102724169 was tested in EOC cell lines (A2780, SKOV3, HO8910, OVCAR5) and the normal ovarian cell line (IOSE80) using qRT-PCR assays. (B) Relative LOC102724169 expression was tested in SKOV3 and HO8910 cells transfected with LOC102724169 transcript or negative control using qRT-PCR assays. (C and D) IC₅₀ of SKOV3 and HO8910 cells transfected with LOC102724169 transcript was determined by CCK-8 assays using a wide range of cisplatin concentrations. (E and F) Caspase-3 activity assays indicated the expression of caspase-3 among different groups in both SKOV3 and HO8910 cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Data are represented as mean ± SEM.

Figure 5

LOC102724169 regulated the expression of MYB and affected the PI3K/AKT pathway in OCCS (A) Schematic overview of the workflow used to investigate the target gene of LOC102724169 in OCCS. (B) Relative expression of MYB in both SKOV3 and HO8910 cells transfected with LOC102724169 or control plasmid. (C) Relative expression of LOC102724169 in both SKOV3 and HO8910 cells transfected with MYB or control plasmid. (Da and Db) Knockdown of MYB decreased the IC₅₀ values of cisplatin in SKOV3 and HO8910 cells. (Dc and Dd) Overexpression of MYB in LOC-SKOV3/HO8910 cells increased the IC₅₀ values of cisplatin. (E) Validation of MYB expression between the high-risk stress group and low-risk stress group. (F) Correlation analysis of MYB and LOC102724169 expression levels by a Pearson correlation coefficient (r = −0.45, p = 0.0461). (Ga and Gb) Histograms represent the quantification of western blot bands in both SKOV3 and HO8910 cells. (Gc and Gd) Western blot analysis of MYB, caspase-3, and PI3K/AKT signaling-related molecule expression in different groups in both SKOV3 and HO8910 cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are represented as mean ± SEM.

Figure 6

LOC102724169 overexpression increased response rate to cisplatin (CDDP) in the OCCS mice model. (A) Schematic overview of different treatment regimens on stressed mice. Blue arrow indicates all mice kept for 2 h of immobilization daily during the whole experimental cycle. Green arrow indicates that SKOV3/ID8 cells were injected intraperitoneally in nude mice with periodic immobilization in nude mice/C57BL/6. Red arrow indicates after injection of EOC cells intraperitoneally, all stressed mice were randomly divided into four groups according to different treatments: (1) empty vector viruses (NC-virs/NC-SKOV3), (2) empty vector viruses plus CDDP (NC-virs/NC-SKOV3+CDDP), (3) overexpression of LOC102724169 viruses (LOC-virs/LOC-SKOV3), (4) overexpression of LOC102724169 viruses plus CDDP (LOC-virs/LOC-SKOV3+CDDP). (B) Macroscopic tumor tissue and metastases lesions of four groups are presented, respectively. Red circles mark tumor tissues; red arrows indicate liver metastasis. (C) The tumor tissues of those groups were presented. From top to bottom are NC-SKOV3, NC-SKOV3+CDDP, LOC-SKOV3, and LOC-SKOV3+CDDP. (D) Histogram showed different metastases lesions of four groups. (E) Quantification of tumor volume in four groups. (F) Quantification of tumor weight in four groups. (G) Expression of LOC102724169 was validated in mice tumor tissue in different groups. (Ha) Western blot analysis of MYB and other related molecule expressions in different groups were validated. (Hb) Histogram represents the quantification of bands. (I) NC-ID8/ LOC-ID8 cells were intraperitoneally injected into C57BL/6 mice. The tumor tissues of NC/LOC-ID8 and those treated with cisplatin groups are presented. (J) Histogram shows different metastasis lesions of four groups in C57BL/6 mice. (K) Quantification of tumor volume in four groups. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Statistical differences calculated using a two-tailed Student’s t test. Data are represented as mean ± SEM.