Long non-coding RNA POU6F2-AS2 promotes cell proliferation and drug resistance in colon cancer by regulating miR-377/BRD4

Abstract

The aim of this study was to explore the molecular mechanism of lncRNA POU6F2-AS2 in proliferation and drug resistance of colon cancer. Total paired 70 colon cancer and adjacent normal tissues were collected from colon cancer patients. Colon cancer and normal colonic epithelial cells were purchased. POU6F2-AS2 was up- or down-expressed by vectors. LC50 of all cell lines before and after transfection with these plasmids was detected. qRT-PCR was used to detect the expression of POU6F2-AS2, miR-377 and BRD4 before or after transfection. In situ hybridization was also undertaken to detect the level of POU6F2-AS2. Different concentrations of 5-Fu (0, 1, 2.5, 5, 10, 20, 40 and 80 μg/mL) were used for 5-FU insensitivity assay. CCK-8 and crystal violet staining assay were used for detecting cell proliferation, and flow cytometry was used for identifying cell cycle distribution and apoptosis. In order to detect the fragmented DNA in apoptotic cells, TUNEL assay was used. RNA pull-down assay and luciferase reporter assay were used to verify the binding site. Rescue assay confirmed the subtractive effect of miR-377 inhibitors. POU6F2-AS2 was highly expressed in colon cancer, which was associated with clinical pathology. Up-regulated POU6F2-AS2 promoted cell proliferation and cell cycle of colon cancer cells. Overexpression of POU6F2-AS2 inhibited the expression of miR-377 and then up-regulated the expression of BRD4. Up-regulated BRD4 ultimately promoted cell proliferation and cell survival Down-regulated POU6F2-AS2 showed enhanced sensitivity of 5-FU. POU6F2-AS2 promoted cell proliferation and drug resistance in colon cancer by regulating miR-377/BRD4 gene.

Keywords: LncRNA POU6F2-AS2; cell proliferation; colon cancer; drug resistance; miR-377/BRD4.

Figure 1

POU6F2‐AS2 expression level and related survival curve. A, POU6F2‐AS2 expression level in colon cancer tissue and adjacent normal tissues were detected by RT‐PCR, ^*** P < .001. B, In situ hybridization for POU6F2‐AS2 in colon cancer tissue and adjacent normal tissues. C, POU6F2‐AS2 expression level in colon cancer cell lines (HT‐29, HCT‐116, SW620 and OUMS23) and non‐cancerous colon mucosal epithelial cell lines (NCM460) were detected by RT‐PCR. ^** P < .01 and ^*** P < .001 vs NCM460. D, survival curve of colon cancer patients with low and high POU6F2‐AS2 expression level by Kaplan‐Meier survival analysis. Mean ± standard deviation was used to present the data

Figure 2

Overexpression of POU6F2‐AS2 promoted cell proliferation and cell cycle of colon cancer cells. A, The expression of POU6F2‐AS2 in HT‐29 and SW620 cell lines after transfected by pBabe‐puro‐POU6F2‐AS2 plasmid. B, The proliferation of HT‐29 and SW620 cell lines after transfected by pBabe‐puro‐POU6F2‐AS2 plasmid. C, Cell cycle of HT‐29 and SW620 cell lines after transfected by pBabe‐puro‐POU6F2‐AS2 plasmid. D, Clone number of HT‐29 and SW620 cell lines after transfected by pBabe‐puro‐POU6F2‐AS2 plasmid. E, The apoptosis of HT‐29 and SW620 cell lines after transfected by pBabe‐puro‐POU6F2‐AS2 plasmid. Mean ± standard deviation was used to present the data. ^*** P < .001

Figure 3

Down‐regulation of POU6F2‐AS2 inhibited cell proliferation and induced cell cycle arrest of colon cancer cells. A, The expression of POU6F2‐AS2 in HT‐29 and SW620 cell lines after transfected by pLKO.1‐POU6F2‐AS2 plasmid; B, the proliferation of HT‐29 and SW620 cell lines after transfected by pLKO.1‐POU6F2‐AS2 plasmid; C, cell cycle of HT‐29 and SW620 cell lines after transfected by pLKO.1‐POU6F2‐AS2 plasmid; D, clone number of HT‐29 and SW620 cell lines after transfected by pLKO.1‐POU6F2‐AS2 plasmid; E, the apoptosis of HT‐29 and SW620 cell lines after transfected by pLKO.1‐POU6F2‐AS2 plasmid. Mean ± standard deviation was used to present the data. ^*** P < .001

Figure 4

Down‐expression of POU6F2‐AS2 leads to 5‐FU insensitivity. A, LC50 assay. B, Cell viability by CCK8 assay after transfected by pBabe‐puro‐POU6F2‐AS2 plasmid or pLKO.1‐POU6F2‐AS2 plasmid. C, Tumour volume after transfection and drug treatment in four groups (pLKO.1 + Vehicle group, pLKO.1 + 5‐FU group, pLKO.1‐POU6F2‐AS2 + Vehicle group and pLKO.1‐POU6F2‐AS2 + 5‐FU group). D, The proportion of TUNEL‐positive cells in four groups. E, The proportion of Ki67‐positive cells in four groups. F, The protein levels of cancer resistance‐related gene (P‐gp, MRP2 and BRCA2). Mean ± standard deviation was used to present the data. ^** P < .01 and ^*** P < .001

Figure 5

POU6F2‐AS2 combined with miR‐377. A, The binding site of POU6F2‐AS2 and miR‐377 predicated by catRAPID analysis. B, Pull‐down assay for the enrichment of miR‐377 in HT‐29 cell lines. C, miR‐377 expression level in HT‐29 after pBabe‐puro‐POU6F2‐AS2 plasmid transfected. D, miR‐377 expression level in SW620 after pBabe‐puro‐POU6F2‐AS2 plasmid transfected. E, miR‐377 expression level in HT‐29 after pLKO.1‐POU6F2‐AS2 plasmid transfected. F, miR‐377 expression level in SW620 after pLKO.1‐POU6F2‐AS2 plasmid transfected. Mean ± standard deviation was used to present the data. ^*** P < .001

Figure 6

The fluorescence activity and the correlation among miR‐377, BRD4 and POU6F2‐AS2. A, The fluorescence activity after transfected by miR‐377 mimics; B, The fluorescence activity after transfected by pLKO.1‐POU6F2‐AS2 plasmid; C, D and E, The expression of POU6F2‐AS2 was negatively correlated with miR‐377, the expression of miR‐377 was negatively correlated with BRD4, and the expression of POU6F2‐AS2 was positively correlated with BRD4. Pearson correlation analysis was calculated the relationship between lncRNA and miRNA. F, G, The expression of BRD4 in colon cancer cells was examined by Western blot assay. ^*** P < .001

Figure 7

Rescue experiments. A, Clone number of HT‐29 and SW620 after transfected by pLKO.1‐POU6F2‐AS2 plasmid or miR‐377 inhibitors. B, Cell proliferation after transfected by pLKO.1‐POU6F2‐AS2 plasmid or miR‐377 inhibitors C, The proportion of apoptosis was detected after transfected by pLKO.1‐POU6F2‐AS2 plasmid or miR‐377 inhibitors. D, Western blot detected the expression levels of PARP and C‐PARP after transfected by pLKO.1‐POU6F2‐AS2 plasmid or miR‐377 inhibitors in 5‐FU treated cells. E, LC50 assay. Mean ± standard deviation was used to present the data. ^*** P < .001