MIR600HG suppresses metastasis and enhances oxaliplatin chemosensitivity by targeting ALDH1A3 in colorectal cancer

Abstract

Background: Metastasis and chemoresistance indicate a poor prognosis in colorectal cancer (CRC) patients. However, the mechanisms that lead to the development of chemoresistance and metastasis in CRC remain unclear.

Materials and methods: We combined clinical and experimental studies to determine the role of MIR600HG in CRC metastasis and chemoresistance. The statistical analysis was performed using GraphPad Prism software, version 8.0.

Results: We detected down-regulated expression of long non-coding RNA (lncRNA) MIR600HG in CRC specimens and cell lines compared with normal controls, and the expression level of MIR600HG was inversely correlated with the overall survival of CRC patients. The inhibition of MIR600HG stimulated CRC cell metastasis and chemoresistance. In addition, our data showed that the inhibition of MIR600HG stimulated CRC stemness, while the overexpression of MIR600HG suppressed stemness. Importantly, our animal experiments showed that MIR600HG inhibited tumour formation and that the combination of MIR600HG inhibition and oxaliplatin (Oxa) treatment significantly inhibited tumour growth compared with that with either intervention alone. Furthermore, we demonstrated that MIR600HG exerts its anticancer role by targeting ALDH1A3 in CRC.

Conclusions: Our data suggest that MIR600HG functions as a tumour suppressor and that the overexpression of MIR600HG inhibits tumour invasion and enhances chemosensitivity, providing a new strategy for CRC treatment.

Keywords: ALDH1A3; Chemosensitivity; MIR600HG; Metastasis; colorectal cancer.

Figure 1. Decreased expression of MIR600HG correlates with poor survival of CRC patients

(A) MIR600HG expression was significantly down-regulated in CRC patients analysis from TCGA database. (B) MIR22HG expression was significantly down-regulated in CRC specimens. The expression levels of MIR600HG were measured using qRT-PCR in normal tissue (n=50) and tumour tissues (n=50). (C) Survival rates for CRC patients with low (n=20) and high (n=20) MIR600HG expression. (D) CRC cell lines showed lower expression level of MIR600HG compared with human normal colon epithelial (HcoEpiC) cells. **, P<0.01; ***, P<0.001.

Figure 2. MIR600HG negatively regulates CRC cells metastasis and chemosensitivity

(A) Caco2 cells were transfected with negative control oligonucleotide, MIR600HG mimics or MIR600HG inhibitor. After 72 h of transfection, isolated mRNAs were subjected to qRT-PCR. (B) MIR600HG enhanced the sensitivity of Caco2 cells to Oxaliplatin treatment. Caco2 cells were transfected with the MIR600HG and treated with or without 0.2 µM Oxaliplatin for 48 h and then subjected to a CCK-8 assay. (C) Knockdown MIR600HG expression inhibited the sensitivity of Caco2 cells to Oxaliplatin treatment. Caco2 cells were transfected with the siMIR600HG and treated with or without 0.2 µM Oxaliplatin for 48 h and then subjected to CCK-8 assay. (D) The MIR600HG overexpression enhanced Oxaliplatin-induced apoptosis in Caco2 cells. MIR600HG overexpression Caco2 cells were treated with DMSO or 0.2 μM Oxaliplatin for 24 h and analysed with flow cytometry. (E) MIR600HG stimulates Oxaliplatin-induced expression of cleaved- and total-PARP in CRC cells. Caco2 cells were transfected with the MIR600HG or treated with 0.2 µM Oxaliplatin for 48 h, and then subjected to Western blotting. (F) MIR600HG negatively regulates Caco2 cell metastasis. After 24 h of transfection, cells were subjected to an invasion assay. (G) MIR600HG inhibited EMT in CRC cells. Caco2 cells were transfected with negative control oligonucleotides, MIR600HG mimics or MIR600HG inhibitor. After 72 h of transfection, cells were subjected to Western blot analysis for the detection of the expression of the indicated proteins. NC, negative control oligonucleotides; mimics, MIR600HG mimics; inhibitor, MIR600HG inhibitor; Oxa, Oxaliplatin; *, P<0.05; **, P<0.01.

Figure 3. Analysis of MIR600HG downstream regulatory pathway

We constructed MIR600HG overexpressing Caco2 cell lines for RNA sequencing with normal controls. (A) PCA analysis can find that the two groups of samples are almost completely different. (B) The heat map also shows the sample. There is a large difference between them. (C) The volcanic map was performed on the two groups of samples (tools, R language limma package). The volcano map showed that compared with the miR600HG low expression group, the miR600HG high expression group had a large number of proteins with low expression. (D) KEGG Enrichment analysis, taking the first 30 signal pathways as a map, suggesting that MIR600HG may affect the process of colon cancer by affecting the pluripotency of colon cancer cells.

Figure 4. MIR600HG targets ALDH1A3

(A) The MIR600HG seed sequence is complementary to the 3′ UTR of ALDH1A3. (B,C) MIR600HG inhibited ALDH1A3 mRNA and protein expression. After 72 h transfection of MIR600HG and siMIR600HG, using qRT-PCR and Western blot measurement. (D) Activity of the luciferase gene linked to the 3′ UTR of ALDH1A3. The luciferase reporter plasmids of wild-type (WT) or mutated 3′ UTR sequences of ALDH1A3 (MT) were transfected into HEK-293 cells with or without the MIR600HG. (E) The expression levels of ALDH1A3 and MIR600HG showed a negative correlation in CRC patients. Tumour samples were obtained from ten patients with CRC, and the expression of ALDH1A3 and MIR600HG were measured by RT-qPCR. NC, negative control inhibitor, MIR600HG inhibitor; ns, no significance; **, P<0.01.

Figure 5. MIR600HG negatively regulates CRC stemness

(A) The inhibition of MIR600HG stimulated osteosphere formation in Caco2 cells, whereas the overexpression of MIR600HG inhibited osteosphere formation. (B) MIR600HG negatively regulated CSC marker protein expression. Caco2 cells were transfected with the MIR600HG and siMIR600HG. After 72 h of transfection, cells were subjected to Western blot analysis. NC, negative control oligonucleotides; inhibitor, MIR600HG inhibitor; *, P<0.05; **, P<0.01.

Figure 6. ALDH1A3 is a functional target of MIR600HG that modulates CRC metastasis and chemosensitivity of Oxaliplatin

(A) The cell viability analysis results show that the overexpression of ALDH1A3 restored the cells viability inhibited by MIR600HG and decreased chemosensitivity of Oxaliplatin. (B) Silencing of ALDH1A3 suppressed MIR600HG inhibition-induced cells viability and increased chemosensitivity of Oxaliplatin. (C) The apoptosis analysis ALDH1A3 overexpression attenuated the MIR600HG induced cell apoptosis, decreased chemosensitivity of Oxaliplatin. (D) Transwell experiments showed that the overexpression of ALDH1A3 attenuated the MIR600HG-induced metastasis effect. NC, negative control oligonucleotides; inhibitor, MIR600HG inhibitor; ALDH1A3: ALDH1A3 plasmid; siALDH1A3, siRNA of ALDH1A3; *, P<0.05; **, P<0.01.

Figure 7. MIR600HG overexpression in combination with Oxaliplatin inhibits tumour relapse

(A,B) Tumour volume and weight in xenografts treated with Oxaliplatin, MIR600HG or both at days 29, 32 and 36. Caco2 cells and injected s.c. into nude mice (n=9/group, 1 × 10⁴ cells/mouse). (C) The level of CD133 mRNA derived from xenograft model tumour analysis by RT-PCR. (D) Ki-67 immunohistochemistry assay show that combination of MIR600HG inhibition and Oxaliplatin treatment more significantly inhibits cell proliferation in xenograft tumour. (E) A schematic model of MIR600HG target ALDH1A3 inhibited CRC metastasis and chemosensitivity. NC, negative control oligonucleotides; inhibitor, MIR600HG inhibitor; Oxa, Oxaliplatin. *, P<0.05; **, P<0.01.