LncRNA LINC01305 silencing inhibits cell epithelial-mesenchymal transition in cervical cancer by inhibiting TNXB-mediated PI3K/Akt signalling pathway

Abstract

Cervical cancer (CC) remains one of the leading malignancies afflicting females worldwide, with its aetiology associated with long-term papillomavirus infection. Recent studies have shifted their focus and research attention to the relationship between long non-coding RNAs (lncRNAs) and CC therapeutic. Thus, the aim of the current study was to investigate the underlying mechanism of lncRNA LINC01305 on the cell invasion, migration and epithelial-mesenchymal transition (EMT) of CC cells via modulation of the PI3K/Akt signalling pathway by targeting tenascin-X B (TNXB). The expressions of LINC01305, TNXB, MMP2, MMP9, E-cadherin, vimentin, PI3K, Akt, p-PI3K, p-Akt and TNXB were detected in this study. After which, the cell invasion and migration abilities of the CC cells were determined respectively. Bioinformatics and the application of a dual luciferase reporter gene assay provided verification indicating that TNXB is the target gene of lncRNA LINC01305. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis methods revealed that the expressions of MMP2, MMP9, vimentin, PI3K, Akt, p-PI3K and p-Akt were decreased following the down-regulation of LncRNA LINC01305 or overexpression of TNXB. LncRNA LINC01305 silencing or TNXB overexpression was noted to decrease the migration and invasion of SiHa cells. Taken together, the key findings of the current study present evidence suggesting that lncRNA LINC01305 silencing suppresses EMT, invasion and migration via repressing the PI3K/Akt signalling pathway by means of targeting TNXB in CC cells, which ultimately provides novel insight and identification of potential therapeutic targets for CC.

Keywords: PI3K/Akt signalling pathway; TNXB; cervical cancer; epithelial-mesenchymal transition; invasion; long non-coding RNA LINC01305; migration.

Figure 1

TNXB is the target gene of lncRNA LINCRNA. A, The thermal image analysis of GSE63514. B, The prediction of the target gene of LINC01305 detected by bioinformatics website. C, The prediction of the biding site of LINC01305 and TNXB. D, The relative luciferase activity in each group; the experiments were analysed by independent sample t test. E, Results of binding site between LINC01305 and TNXB by RIP assay. *P < 0.05, compared with the co‐transfection group of TNXB‐wt and NC. lncRNA LINC01305, long non‐coding RNA LINC01305; TNXB, Tenascin‐X B; NC, negative control; RIP, RNA immunoprecipitation

Figure 2

LncRNA LINCRNA is located in nucleus of SiHa cells. A, Prediction of LINC01305 subcellular localization in different cells through lncatlas.crg analysis. B, Verification of LINC01305 subcellular localization in SiHa cells (×400) whereby the nucleus was represented by blue, green represented the fluorescence localization of LINC01305, and the ruler = 25 μm. lncRNA LINC01305, long non‐coding RNA LINC01305; DAPI, Diamidino‐phenylindole

Figure 3

SiHa, Hela and C‐33A cell lines exhibit up‐regulated LINC01305 but down‐regulated TNXB expressions. Data were expressed as mean ± SD (n = 3), and compared using one‐way ANOVA. *P < 0.05 compared with MS751 cells. #compared with Ca ski cells

Figure 4

Expressions of TNXB was up‐regulated due to LncRNA LINC01305 silencing. Expressions of genes in SiHa, Hela and C‐33A were expressed as mean ± SD (n = 3), and compared using one‐way ANOVA. *P < 0.05 compared with the blank group, RT‐qPCR, reverse transcription quantitative polymerase chain reaction. TNXB, Tenascin‐X B; NC, negative control

Figure 5

Protein expressions of MMP2, MMP9, vimentin, p‐PI3K and p‐Akt were reduced while E‐cadherin and TNXB protein expressions elevated owing to LncRNA LINC01305 silencing. A, The protein expressions of MMP2, MMP9, E‐cadherin, vimentin, PI3K, Akt, p‐PI3K, p‐Akt and TNXB in SiHa determined by western blot analysis, expressed as mean ± SD (n = 3), in which x‐axis represented different proteins in each group, and y‐axis represented relative protein expressions (the gray values of target bands to internal control bands). B, The electrophoresis bands of MMP2, MMP9, E‐cadherin, vimentin, PI3K, Akt, p‐PI3K, p‐Akt and TNXB proteins in SiHa cells. *P < 0.05 compared with the blank group. #P < 0.05, compared with siRNA‐TNXB group. MMP2, matrix metalloprotease 2; MMP9, matrix metalloprotease 9; p‐PI3K, phospho‐phosphatidylinositol 3‐kinase; p‐Akt, phospho‐protein kinase b; TNXB, Tenascin‐X B; NC, negative control

Figure 6

LncRNA LINC01305 silencing declined cell migration in SiHa cells. A, The images of scratch healing of SiHa cells in each group (100×), in which the ruler = 100 μm. B, The percentage of cell migration width of SiHa cells in each group expressed as mean ± SD (n = 3), in which x‐axis represented different groups, and y‐axis represented scratch healing percentage. *P < 0.05 compared with the blank group. #P < 0.05 compared with the siRNA‐TNXB group. TNXB, Tenascin‐X B; NC, negative control

Figure 7

LncRNA LINC01305 silencing reduces cell invasion in SiHa cells. A, The images of transwell invasion of SiHa cells in each group (200×), in which the ruler = 50 μm. B, The number of cell invasion of SiHa cells in each group expressed as mean ± SD (n = 3), in which x‐axis represented different groups, and y‐axis represented the number of cells that penetrating the Transwell chamber covered with Matrigel. *P < 0.05 compared with the blank group. #P < 0.05 compared with the siRNA‐TNXB group. TNXB, Tenascin‐X B; NC, negative control