LINC00473 promotes the Taxol resistance via miR-15a in colorectal cancer

Abstract

Dysregulation of long non-coding RNAs (LncRNAs) participated into the initiation and progression of different diseases via direct regulation of proteins or indirect regulation of microRNA (miRNA)-target genes. LINC00473 is a novel carcinoma-related LncRNA and up-regulated in many cancers for tumor growth and metastasis, but its role in chemotherapy resistance is unclear. We here investigated the function of LINC00473 in colorectal cancer (CRC) in vitro and in vivo The CRC tissues (n=20) and relative normal tissues were collected and found that LINC00473 was overexpressed in CRC tissues when compared with which in normal tissues. Highly expressed LINC00473 predicted large tumor size, high TNM stage of CRC patients. Interestingly, the tumor suppressor miR-15a was down-regulated and negatively correlated with LINC00473 levels in CRC. LINC00473 harbored the binding sites for miR-15a and reduced its availability in CRC cell line HCT116. Knockdown of LINC00473 elevated the expression of miR-15a. Moreover, in the Taxol-resistant HCT116, the LINC00473 level was further increased than that in HCT116. Knockdown of LINC00473 restored the Taxol-induced cytotoxicity, inhibited the cell vitality, colony formation and induced apoptosis, impaired the ability of migration or invasion, but these effects could be abrogated by the inhibition of miR-15a. Mechanistically, the BCL-2-related anti-apoptosis pathway was activated and the multidrug-resistant (MDR) genes LRP, MDR1 were up-regulated by LINC00473. Furthermore, inhibition of LINC00473 in vivo could overcome the Taxol resistance of CRC cells, could recover the expression of tumor suppressor miR-15a and chemotherapy-induced tumor regression, indicating that LINC00473 functioned as oncogene in CRC via miR-15a.

Keywords: LINC00473; apoptosis; chemotherapy; colorectal cancer; miR-15a.

Figure 1. LINC00473 is overexpressed in CRC patients

(A) The expression of LINC00473 was analyzed in CRC tissues and relatively normal tissues (n=20) were by Q-PCR. (B–E) Correlations between the expression of LINC00473 and clinicopathological characteristics (tumor size, TNM stage, differentiation and LNM status) of CRC patients were analyzed. *P<0.05, **P<0.01, ***P<0.001; data represent the means ± s.d.

Figure 2. LINC00473 interacts with miR-15a and inhibits its expression in CRC

(A) The predicted miR-15a-binding sites for LINC00473. (B) The expression of miR-15a was analyzed in CRC tissues and relatively normal tissues were by Q-PCR. (C) The correlation between miR-15a and LINC00473 in CRC tissues was determined. (D) The expressions of miR-15a and LINC00473 were confirmed in normal human colon epithelial cell line FHC and four CRC cell lines HCT116, SW620 and LoVo. (E) Knockdown of LINC00473 could increase the miR-15a levels in HCT116 cell line. (F) The HCT116 cell line was transfected with miR-15a mimics, psiCHECK2-LINC00473-wild or mut and negative control, and then the luciferase activity was measured. **P<0.01, ***P<0.001, data represent the means ± s.d.

Figure 3. LINC00473 promotes Taxol resistance via miR-15a in CRC cells

(A) The IC50 of Taxl in HCT116/Taxol and HCT116 was determined by CCK-8. (B) The expressions of miR-15a and LINC00473 in HCT116/Taxol and HCT116 cells were determined by Q-PCR. (C) The HCT116/Taxol cells were transfected of siRNA of LINC00473 with or without miR-15a inhibitors, then the cells were treated with 0.5 μg/ml Taxol and the cell vitality was estimated by CCK-8. Besides, (D) the proliferation, (E) apoptosis and (F) the ability of migration and invasion were determined by colony formation assay, flow cytometry and transwell assay. *P<0.05, **P<0.01, ***P<0.001, data represent the means ± s.d.

Figure 4. The apoptosis pathway and MDR genes were regulated by LINC00473

(A) The expressions of apoptosis-related Bcl-2, caspase-3, BAX and MDR-related LRP and MDR1 were determined by WB. (B) The grayscale value of each protein was analyzed. *P<0.05, **P<0.01, data represent the means ± s.d.

Figure 5. Knockdown of LINC00473 promotes the tumor regression of CRC in vivo

HCT116/Taxol cells with/without LINC00473 knockdown were subcutaneously injected in rear flank of nude mice (six per group) and then the mice were treated with 6 mg/kg Taxol (i.p). (A) The mean tumor size (mm³) and (B) tumor weight (g) was analyzed. (C) The expression of miR-15a in tumor tissues was estimated by Q-PCR. (D) The expression of Ki-67 was determined by immunohistochemistry and (E) the expressions of apoptosis-related Bcl-2, caspase-3, BAX and MDR-related LRP and MDR1 in tumor tissues were determined by WB. **P<0.01, data represent the means ± s.d.