Altered p53 regulation of miR-148b and p55PIK contributes to tumor progression in colorectal cancer

Abstract

MicroRNAs are a class of small non-coding RNAs that regulate the expressions of many genes. Previously, we found that the expression of p55PIK, an isoform of phosphatidylinosotol 3-kinase that has important roles in the regulation of cell cycle, is increased significantly in several types of cancer and contributes to the tumor growth. However, the mechanism for this increased p55PIK expression is not well understood. In this study, we show that miR-148b binds specifically to the 3′-untranslated region of p55PIK and significantly suppresses p55PIK expression. MiR-148b overexpression abolished p55PIK stimulation of cell proliferation and cell cycle progression in colorectal cancer (CRC) cell lines and decreased tumor growth in vivo. Furthermore, we demonstrated that p53 directly activates the transcription of miR-148b by binding to its promoter. In CRC cell lines and tissues, p53 expression was associated with miR-148b expression, and both were negatively associated with p55PIK expression. Our study shows that the p53/miR-148b/p55PIK axis has an important role in cell proliferation and tumor growth, and may represent a novel therapeutic target for treating cancers containing p53 mutations or losses.

Introduction

MicroRNAs (mRNAs) are a class of small non-coding RNAs, which bind to partially complementary sequences in the 3′-untranslated region (UTR) of specific target mRNAs, resulting in either mRNA degradation or translation inhibition.1,2 Growing evidence suggests that mRNAs have an important role in various biological processes, including cell proliferation, development and differentiation.3,4 Furthermore, abnormal expression of mRNAs have been observed in various types of cancer and may be involved in modulating cancer cell behaviors.2,3,5

Microarray studies have identified a number of mRNAs that are dysregulated in colorectal cancer (CRC).6,7 Recently, miR-148b has been shown to be deregulated in some cancers such as ovarian cancer and lung cancer,8,9 whereas it is downregulated in pancreatic cancer, gastric cancer and CRC.10, 11, 12 However, the downregulation mechanism of mir-148b and the role of miR-148b in CRC is still unknown.

P55PIK (p55γ) is a regulatory subunit of phosphatidylinosotol 3-kinase, which is encoded by PIK3R3.13 Several groups have shown that it is overexpressed in gastric and ovarian cancers.14,15 Previously, we showed that p55PIK was overexpressed in CRC, and its overexpression accelerated cell cycle progression and promoted cell proliferation by interacting with cell cycle regulators, such as retinoblastoma protein or proliferation cell nuclear antigen.16, 17, 18 Blocking p55PIK signaling with a peptide inhibitor markedly reduced the size of CRC tumors in nude mice suggesting that it had a critical role in tumor growth. p55PIK also stimulated angiogenesis in CRC cell by activating the nuclear factor-kappa B pathway.19,20 Although p55PIK overexpression may be important in tumorigenesis, the mechanism for its overexpression in CRC is not fully understood.

P53 (which is encoded by TP53 gene) is one of the most commonly mutated tumor suppressors, and alters the expression of a large set of target genes leading to cell cycle arrest, apoptosis, increased DNA repair and/or inhibition of angiogenesis.21 More recently, there are reports that p53 may regulate mRNAs by binding to target mRNAs promoters or by regulating Dicer expression.22,23 P53 directly regulated the expression of the miR-34 genes, miR-34a and miR-34b/c, leading to a cell cycle arrest.24,25 Moreover, p53 directly induced the expression of the miR-200 subfamilies, miR-200c/141 and miR-200a/200b/429, which were previously shown to antagonize epithelial-mesenchymal transition by targeting the epithelial-mesenchymal transition-inducing transcription factors ZEB1 and ZEB2.26,27

In this manuscript, we show that p53, miR-148b and p55PIK form a tightly-linked transcription system that can become dysregulated in CRC. First, miR-148b is a direct transcriptional target of p53. Next, upregulation of miR-148b by p53 suppresses the expression of p55PIK protein by mir-148b binding to the 3'-UTR of p55PIK. This coordinated regulation of p55PIK by miR-148 and p53 leads to cell cycle arrest and inhibition of cell proliferation. In contrast, dysregulation of the p53/miR-148b/p55PIK axis has an important role in tumor progression in vivo. In p53 loss or mutated CRC cells and tissues, miR-148b is downregulated, leading to increased expression of p55PIK and tumor progression. Taken together, our findings suggest that the p53/miR-148b/p55PIK axis may be a novel therapeutic target in p53-mutated CRC.

Results

miR-148b blocks cell cycle progression and inhibits cell proliferation in CRC cell

miR-148b expression was detected by qRT–PCR in each several CRC lines (data not show). The expression levels of miR-148b were low in LoVo and SW48 cells and high in HCT116 and HT29 cells. We then transfected miR-148b mimic (miR-148b) or miR-148b-negative control (miR-NC) in LoVo cells. After transfection, cells were counted daily for the next 4 days. MiR-148b significantly decreased cell proliferation by 43% after 4 days when compared with miR-NC (Figure 1a). Conversely, when HCT116 cells were transfected with inhibitor of miR-148b (Inh-148b) or miR-148b inhibitor-negative control (Inh-NC), Inh-148b increased cell proliferation by 50% after 4 days when compared with Inh-NC (Figure 1b). Moreover, the results demonstrated that S-phase cells were significantly decreased in LoVo cells following transfection with miR-148b after 48 h (Figure 1c). In contrast, the S-phase cells were increased in HCT116 cells following transfection with Inh-148b after 48 h (Figure 1d). Similar data with above can be found in SW48 and HT29 cells using miR-148b and Inh-148b, respectively (data not show). In addition, bromodeoxyuridine (BrdU) incorporation was measured to detect the DNA synthesis in CRC lines transfected with miR-148b or Inh-148b. MiR-148b inhibited DNA synthesis in LoVo cells (Figure 1e), whereas Inh-148b promotes DNA synthesis in HCT116 cells (Figure 1f).

Figure 1
figure1

miR-148b blocks cell cycle progress and inhibits cell proliferation in CRC cell. (a) (Left) miR-148b expression levels in LoVo cells after transfection with miR-148b mimics (miR-148b) or the negative control (miR-NC); (right) LoVo cell number was counted 0–4 days after transfection with miR-148b mimics (miR-148b) or the negative control (miR-NC); (b) (left) miR-148b expression levels in HCT116 cells after transfection with miR-148b inhibitor (Inh-148b) or the negative control (Inh-NC); (right) HTC116 cell number was counted 0–4 days after transfection with miR-148b inhibitor (Inh-148b) or the negative control (Inh-NC); (c) LoVo cells were transfected with miR-148b or miR-NC, after 48 h, cell cycle distribution was measured by propodium iodide staining and flow cytometry; (d) HCT116 cells were transfected with Inh-148b or Inh-NC, after 48 h, cell cycle distribution was measured by propodium iodide staining and flow cytometry; (e) LoVo BrdU-positive cells were counted by BrdU incorporation assay on 4 days after transfected with miR-148b mimics (miR-148b) or the negative control (miR-NC); (f) HCT116 BrdU-positive cells was counted by BrdU incorporation assay on 4 days after transfection with miR-148b inhibitor (Inh-148b) or the negative control (Inh-NC). The results above were reproducible in three independent experiments.

MiR-148b inhibits cell proliferation in vivo

To further evaluate the function of miR-148b on cell proliferation, we established a stable cell line that had high or low expression of miR-148b by transfecting with vectors that expressed pre miR-148b sequence or anti-pre miR-148b sequence in LoVo cells (obtained from Genecopier Corp., Guangzhou, China). These cells then were cultured, collected, washed and resuspended in culture medium (about 2 × 107/ml) and injected into subcutaneous of athymic nude mice (100 μl/tumor). Tumors were measured every two days after they became visible to the naked eye. All mice were killed after 4 weeks, and tumors were collected and weighed. The tumor volume was calculated by the formula: volume=length × width2/2 and tumor growth curve was drawn as shown (Figure 2a). After 4 weeks, the tumor volume of mice injected with the empty vector was 204.64±65.67 mm3, whereas the tumor volumes of mice injected with miR-148b or anti-148b were 94.82±43.6 mm3 and 414.42±97.6 mm3, respectively. The tumor weight of mice injected with empty vector was 0.157±0.093 g, whereas the tumor weights of mice injected with miR-148b or anti-148b were 0.068±0.052g and 0.543±0.156g, respectively (Figure 2b). qRT–PCR demonstrated that miR-148b expression levels were increased in miR-148b transfected tumors compared with control tumors, whereas miR-148b expression levels were markedly decreased in anti-148b transfected tumors compared with control tumors (Figure 2c). MiRNA exerts its function by binding to the 3′-UTR of target genes through partial sequence homology.1 The sequence analysis using information available online (http://www.targetscan.org/) identified a putative sequence for miR-148b in the 3′-UTR of p55PIK, suggesting p55PIK a potential target of miR-148b. We then detected the expression of p55PIK in each tumor by western blot and immunohistochemistry. Our results show that the p55PIK expression was negatively correlated with miR-148b expression in each tumor (Figures 2d and e).

Figure 2
figure2

miR-148b inhibits cell proliferation in vivo. LoVo cells were cultured, collected, washed and resuspended in culture medium (about 2 × 107/ml) and injected into subcutaneous of athymic nude mice (100 μl/tumor). (a) The tumor growth curves of four groups during 3 weeks, the tumor volumes and the general health of mice are monitored before they are killed. The length, width and depth of tumors are measured every 3 days to determine tumor volume (mean±s.d., n=6); (b) after 3 weeks, all the mice were killed and the tumors were collected. The mean tumor weight of each group was calculated. (c) The expression of miR-148b was detected by qRT–PCR in each group; (d) the expression of p55PIK was detected by western blot in each group; (e) the expression of p55PIK was detected by immunohistochemistry in each group.

P55PIK is a potential target of miR-148b in CRC cell line

To confirm whether miR-148b targeted to p55PIK, we cloned 3′-UTR sequences that contain the predicted target site (wild type) or mutated sequences (mutant type) of miR-148b into the pGL3 control vector, respectively (Figure 3a). Our data showed that co-transfection with miR-148b mimics significantly decreased the firefly luciferase activity of the reporter with wild-type 3′-UTR of p55PIK, but had no effect on the mutant reporter (Figure 3b). Conversely, co-transfection of miR-148b inhibitor (Inh-148b) notably increased the firefly luciferase activity of reporter with wild-type 3′-UTR of p55PIK, but no that of the mutant reporter (Figure 3c). We then examined the effect of miR-148b on endogenous expression of p55PIK by western blot and qRT–PCR. Transfection of miR-148b mimics in LoVo cells led to a decrease of p55PIK protein expression, but had no effect on mRNA expression (Figure 3d). In contrast, transfection of miR-148b inhibitor in LoVo cells significantly upregulated p55PIK protein expression (Figure 3e). These results demonstrated that miR-148b directly targets the 3′-UTR of p55PIK and regulates the protein expression of p55PIK.

Figure 3
figure3

P55PIK is a potential target of miR-148b in CRC cell line. (a) Schematic representation of the 3′-UTR of p55PIK with the predicted target site for miR-148b. The mutant site of p55PIK 3′-UTR was indicated (dotted line); (b) reporter constructs containing either wild-type p55PIK 3′-UTR or p55PIK 3′-UTR with mutation at the predicted miR-148b target sequence were co-transfected into LoVo cells, along with miR-148b mimics (miR-148b), miR-con and relative luciferase activity was assayed. (c) Reporter constructs containing either wild-type p55PIK 3′-UTR or p55PIK 3′-UTR with mutation at the predicted miR-148b target sequence were co-transfected into LoVo cells, along with miR-148b inhibitor (Inh-148b) or Inh-con and relative luciferase activity was assayed. (d) Western blot and qRT–PCR (mean±s.d.; n=3) detect the expression of p55PIK after LoVo cells were transfected with the miR-148b or miR-con; (e) western blot and qRT–PCR (mean±s.d.; n=3) detect the expression of p55PIK after LoVo cells were transfected with the Inh-148b or Inh-con.

miR-148b suppresses cell proliferation in colon cancer cells by targeting p55PIK

Our previous studies showed that p55PIK is upregulated in human CRC samples and its overexpression accelerates cell cycle progress and promotes cell proliferation by regulation of cell cycle regulators.16 To further evaluate whether the effects of miR-148b on cell cycle regulation and cell proliferation is mediated by p55PIK, we knocked down p55PIK expression by RNA interference (si-p55PIK) in LoVo cells. Transfection of si-p55PIK significantly blocked cell cycle progression at G0/G1 phase and inhibited DNA synthesis in LoVo cells (Figure 4a). In contrast, overexpression of p55PIK by transfection of a pCDNA3.1 vector carrying p55PIK sequences (p55PIK) in LoVo cells accelerated S- phase progression and increased DNA synthesis (Figure 4b). Next, we employed a 'rescue' experiment by co-transfecting LoVo cell with mimics of miR-148b and p55PIK. Our data show that transfection of miR-148b induced p55PIK protein level downregulation, cell cycle blockade and decreased DNA synthesis; however, these effects could be rescued when these cells were transfected with p55PIK expression vector (Figures 4c and d). Furthermore, we co-transfected LoVo cells with miR-148b inhibitor in the absence or presence of si-p55PIK. Transfection of LoVo cells with Inh-148b increased p55PIK protein level that was abrogated by si-p55PIK (Figure 4e). Inh-148b promoted cell cycle progression and DNA synthesis; however, these effects were abrogated when cells were co-transfected with Inh-148b and si-p55PIK (Figure 4f).

Figure 4
figure4

miR-148b suppresses cell proliferation in colon cancer cell by targeting p55PIK. (a) LoVo cells were transfected with p55PIK siRNA (si-p55PIK) or siRNA-negative control (si-NC), DNA synthesis was measured by BrdU incorporation; (b) LoVo cells were transfected with pCDNA3.1-p55PIK (p55PIK) or pCDNA3.1 control vector (pCDNA3.1), DNA synthesis was measured by BrdU incorporation; (c) LoVo cells were transfected with miR-148b or co-transfected with miR-148b and p55PIK, the p55PIK expression was detected in each group by western blot; (d) LoVo cells were transfected with miR-148b or co-transfected with miR-148b and p55PIK, the DNA synthesis was measured by BrdU incorporation in each group (mean±s.d.; n=3); (e) HCT116 cells were transfected with Inh-148b or co-transfected with Inh-148b and si-p55PIK, the p55PIK expression was detected in each group by western blot; (f) HCT116 cells were transfected with Inh-148b or co-transfected with Inh-148b and si-p55PIK, DNA synthesis was measured by BrdU incorporation in each group (mean±s.d.; n=3).

TP53 binds to the promoter of miR-148b and transcrptionally upregulates miR-148b

Analysis of the miR-148b promoter region (about 2 kb upstream of the miR-148b stem loop) using the CONSITE program predicted six p53-binding sites (CATG) that might regulate miR-148b. We next performed chromatin immunoprecipitation (ChIP) assay in LoVo cells and showed that endogenous p53 could bind to the second and third CATG region (Figure 5a). We ,thus, constructed reporter vectors containing the miR-148b promoter and a promoter in which we mutated the p53-binding domain (Figure 5b). MiR-148b expression was upregulated in p53-elevated cells and downregulated in p53 downregulated cells (Figure 5c). Overexpression of p53 significantly increased luciferase activity driven by the second and third CTAG of the miR-148b promoter. Knockdown of p53 significantly decreased miR-148b promoter activity (Figure 5d). In contrast, overexpression and knockdown of p53 had no effects on the luciferase activities of the miR-148b promoter containing the mutated second or/and third CTAG (Figure 5e). Moreover, we used p53-knockout cells (HCT116p53/) to determine whether miR-148b is a direct target of p53. Data showed, in HCT116p53/ cells, mir-148b was downregulated and p55PIK was overexpressed, in comparison with the HCT116 cells with wild-type p53. When p53 was overexpressed in HCT116p53/ cells, the expression of mir-148b was upregulated and the expression level of p55PIK was downregulated (Figure 5f). Taken togather, our results demonstrate that p53 upregulates miR-148b expression through direct binding to the miR-148b promoter.

Figure 5
figure5

TP53 binds to the promoter of miR-148b and transcrptionally upregulates miR-148b. (a) ChIP assay was performed in LoVo cells and indicated that endogenous p53 could bind to the second and third CATG region of miR-148b promoter. (b) Location and sequence of predicted p53-binding sites in the promoter of miR-130b gene. Mutated residues (red) are indicated at the bottom; (c) (left) LoVo cells were transfected with pCDNA3.1-p53 (p53) or pCDNA3.1 control vector (pCDNA3.1), miR-148b expression was detected by qRT–PCR (mean±s.d.; n=3); (right) HCT116 cells were transfected with p53 siRNA (si-p53) or siRNA-negative control (si-NC), miR-148b expression was detected by qRT–PCR (mean±s.d.; n=3); (d) (left) LoVo cells were transfected with pCDNA3.1-p53 (p53) or pCDNA3.1 control vector (pCDNA3.1) as well as miR-148b promoter, relative luciferase activity was assayed. (Right) HCT116 cells were transfected with p53 siRNA (si-p53) or si-NC as well as miR-148b promoter, relative luciferase activity was assayed. (e) (Left) LoVo cells were transfected with pCDNA3.1-p53 (p53) or pCDNA3.1 control vector (pCDNA3.1) as well as miR-148b promoterMUT, miR-148b promoterMUT relative luciferase activity was assayed. (Right) HCT116 cells were transfected with p53 siRNA (si-p53) or si-NC as well as miR-148b promoterMUT, relative luciferase activity was assayed. (f) (left) The expression level of p53, p55PIK and mir-148b were detected in HCT116 wild type and HCT116p53/ cells. (Right) HCT116p53−/− cells, transfected with pCDNA3.1-p53 (p53) or pCDNA3.1 control vector (pCDNA3.1), the expression level p55PIK and mir-148b were detected.

TP53 downregulates p55PIK expression level through the upregulation of miR-148b

We then demonstrated whether TP53-mediated miR-148b upregulation contributes to p55PIK protein suppression. As shown in Figures 6a and b, the expression level of p55PIK protein in LoVo cells and SW48 cells was decreased when TP53 was overexpressed. In contrast, knockdown of TP53 increased p55PIK expression. Of note, the expression levels of p55PIK mRNA were only slightly decreased in the TP53-overexpressing cells and increased in the TP53 knockdown cells (Figure 6c), suggesting that TP53/miR-148b mainly regulated p55PIK expression at the post-transcriptional level.

Figure 6
figure6

TP53 downregulates p55PIK expression level through the upregulation of miR-148b. (a) p55PIK and p53 expression levels in LoVo cells transfected with pCDNA3.1-p53 (p53) or pCDNA3.1 control vector (pCDNA3.1), glyceraldehyde 3-phosphate dehydrogenase expression is also analyzed with an appropriate antibody to show the equal loading of proteins in every well; (b) p55PIK and p53 expression levels in HCT116 cells transfected with p53 siRNA (si-p53) or siRNA-negative control (si-NC), glyceraldehyde 3-phosphate dehydrogenase expression is also analyzed with an appropriate antibody to show the equal loading of proteins in every well; (c) p55PIK mRNA level in cells transfected with each plasmids as shown. (d) p55PIK and p53 expression levels in LoVo cells transfected with pCDNA3.1-p53 (p53) or pCDNA3.1 control vector (pCDNA3.1), along with miR-148b inhibitor (Inh-148b) or Inh-con, glyceraldehyde 3-phosphate dehydrogenase expression is also analyzed with an appropriate antibody to show the equal loading of proteins in every well; (e) p55PIK and p53 expression levels in HCT116 cells transfected with p53 siRNA (si-p53) or si-NC, along with miR-148b mimics (miR-148b) or the negative control (miR-NC), glyceraldehyde 3-phosphate dehydrogenase expression is also analyzed with an appropriate antibody to show the equal loading of proteins in every well.

To further evaluate a direct role of TP53 on the regulation of p55PIK expression, we employed a 'rescue' experiment by co-transfecting the LoVo cells with a pcDNA3.1 vector carrying TP53-expressing cassette and miR-148b inhibitor. Western blotting demonstrated that transfection of TP53 significantly decreased the expression of p55PIK, and it was reversed by co-transfection of TP53 and miR-148b inhibitor (Figure 6d). In addition, transfection of TP53 siRNA (si-TP53) increased the expression of p55PIK, and it reversed by co-transfection of si-TP53 and miR-148b mimics (Figure 6e).

Relationship between TP53, miR-148b and p55PIK in colon cancer cell line and clinical samples

We next examined whether the p53/miR-148b/p55PIK axis, identified by our study, is clinically relevant. In 35 CRC specimens, we found that miR-148b expression inversely correlated with p55PIK protein level expression (Figure 7a) (P<0.01). We also observed a statistically inverse correlation between the expression of p53 and p55PIK, whereas there was a positive correlation between the expression of p53 and miR-148b in six CRC cell lines (Figure 7b). Moreover, immunohistomical analysis of 10 freshly collected colorectal tumors revealed that miR-148b expression inversely correlated with p55PIK and p53 expression and p55PIK expression level inversely correlated with p53 expression in majority of clinical cancer samples (eight of ten). It was interesting to notice that in two tumor samples (#1 and #4, * labeled) harboring a mutant p53, there was a low expression of p55PIK protein (Figure 7c). These clinical data further strengthen the observation that p53 upregulates miR-148b expression and downregulates p55PIK protein in CRC.

Figure 7
figure7

Relationship between TP53, miR-148b and p55PIK in colon cancer cell line and clinical samples. (a) P55PIK and miR-148b expressions in CRCs. Box plots show the fold changes in mRNA expression level for comparisons of tumor versus normal. (b) P55PIK, miR-148b and p53 expression levels in different colon cancer cell lines, mir-148b expression relative fold change was compared with U6, *showed bringing p53 loss function mutations. (c) P55PIK, miR-148b and p53 expression levels in normal tissues (N) and tumor tissues (T). Ten patients are analyzed in western blot; miR-148b expression level was compared by tumor versus normal, *showed bringing p53 loss function mutations.

Discussion

In this study, we examined the role of miR-148b in CRC. We found that overexpression of miR-148b inhibited cell proliferation and blocked cell cycle at G0/G1 phase in CRC cells. In contrast, decreased miR-148b expression promoted cell growth and cell cycle progression. We examined the potential target genes of miR-148b, and found that one of it was p55PIK, a protein that we previously showed is overexpressed in many cancers as well as induces cell proliferation and tumor growth. We showed that miR-148b regulated the expression of p55PIK protein by targeting the 3′-UTR of p55PIK mRNA, but had little or no effect on p55PIK mRNA level suggesting that its effects were likely post-transcriptional. In addition, we showed that overexpression of miR-148b decreased tumor growth, whereas knockdown increased tumor growth in vivo. The expression of miR-148b negatively correlated with the expression p55PIK in these tumors suggesting that miR-148b may be mediating its effects through its negative regulation of p55PIK expression. In this connection, 'rescue' experiments using miR-148b mimic and p55PIK overexpression in CRC cells demonstrate that miR-148b’s effects on cell cycle progression and cell proliferation were mediated by p55PIK.

We previously showed that the p55PIK regulatory subunit is important for cell proliferation, cell differentiation, tumor angiogenesis and tumor growth.16, 17, 18, 19, 20 The unique N-terminal domain of p55PIK specifically binds to important cell cycle regulators such as retinoblastoma protein;18 In addition, a peptide inhibitor that blocked p55PIK interaction with retinoblastoma protein inhibited cell cycle progression, tumor angiogenesis and induced cell differentiation19,20 and demonstrated the importance of p55PIK in these functions. Our data showing miR-148b-negative regulation of p55PIK protein expression and its effects on cell proliferation and tumor growth are consistent with all these previous findings on the important role of p55PIK in cancer. miR-148b previously was reported to have an important role in tumor suppression in pancreatic cancer by targeting AMPKα1 to inhibit cell proliferation and invasion, induce cell apoptosis and enhance chemosensitivity.28 However, we found that miR-148b had no significant effect on apoptosis in CRC cells, but was able to inhibit cell invasion (data not shown).

P53 is one of the most commonly mutated tumor suppressors, regulating multiple cellular processes coordinately to maintain genome integrity in cells.21 Functional loss of p53 in CRC has been proposed as a late event in the transition from adenoma to carcinoma, and occurs with higher frequencies in distal colon and rectal tumors than proximal tumors.29 P53 loss of function also is associated with changes in DNA methylation and microsatellite instability.21 As a transcription factor and tumor suppressor, p53 drives the expression of many genes and mRNAs.22,23 In this study, we demonstrated that p53 upregulated miR-148b expression by directly binding to the miR-148b promoter. In addition, the miR-148b expression level was associated with p53 expression level in patient CRC samples and CRC cell lines. Thus, our study strongly suggests that p53 mutation or loss may be a major cause for dysregulated miR-148b expression observed in some cancers.

Although p55PIK is upregulated in many cancers, the mechanism for p55PIK overexpression is not known. In this study, we showed that p55PIK expression is directly-linked to p53 expression through the latter’s regulation of miR-148b expression. Hence, decreased p53 expression and/or loss of function may be a major cause for p55PIK overexpression. Moreover, we showed that the p53/miR-148b/p55PIK axis has an important role in cell proliferation, DNA synthesis and tumor progression. We also found that miR-148b is a new target of p53 that directly binds to the miR-148b promotor region. As overexpression of miR-148b can inhibit cell proliferation and block cell cycle progression by targeting p55PIK, our study suggests that p53/miR-148b/p55PIK axis is new and potentially effective target for the treatment of CRCs harboring p53 mutations.

Materials and methods

Ethics statement

All research involving human participants had been approved by the Huazhong University of Science and Technology Ethics committee, and we had obtained informed consent that was written from all participants involved in this study. All animal work had been conducted according to Huazhong University of Science and Technology animal study guidelines.

Cell culture and antibodies

All CRC cell lines (SW48, LoVo, SW480, SW620, KM12, Caco2, HT29 and HCT116) and HEK293a cell line were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). All cell were cultured at 37 °C, 5% CO2 in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclon, Thermo Scientific, Rockford, IL, USA). The primary antibodies of glyceraldehyde 3-phosphate dehydrogenase, p55PIK p53 and BrdU were purchased from Santa Cruz Company (Santa Cruz, CA, USA) and the fluorescent sencondary antibodies were bought from BD Bioscience (BD Bioscience, San Jose, CA, USA).

BrdU incorporation and cell cycle analysis

BruU incorporation was done as described previously.24 Briefly, cells were cultured in a six-well plate overnight and treated with different processing for indicated time, following 30-min incubation with BrdU at 10 μg/ml. after aspirating the medium, the cell were immediately fixed at −20 °C for at least 8 h. After immunostaining against BrdUrd, DNA synthesis was determined by counting the percentage of BrdU+ cells in total cells. The experiments were repeated independently at least three times and the results were statistically analyzed.

For the flow cytometry analysis, cells were detached by trypsinization, washed three times with cold phosphate-buffered saline and resuspended in 80% ethanol for at least 8 h at −20 °C. After fixation, cells were treated with staining solution (3.4 mM Tris-Cl (pH 7.4), propodium iodide, 0.1% triton X-100 buffer and 100 μg/ml RNase A). Finally, the stained cells were analyzed using FACSCalibur from BD Biosciences (Franklin Lakes, NJ, USA). The experiments were repeated independently at least three times and the results were statistically analyzed.

Luciferase activity assay

Luciferase activity assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. HEK293A cell of about 80% confluence were seeded in 24-well plates. For p55PIK 3′-UTR luciferase reporter assay, 100 ng wild type or mutant luciferase reporter constructs were co-transfected into HEK293A cells in a 24-well plate with 100 nM miR-148b or 100 nM miR-NC by using lipofectamine 2000. Luciferase activity assay was performed 48 h after transfection using the Dual-Luciferase Assay System. Firefly luciferase activity was normalized to the corresponding Renilla luciferase activity. All experiments were performed three times.

Western blot analysis

Western blot was performed as described recently.19 Briefly, cells were collected and total cell lysate were denatured and resolved on SDS–polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes. After blocking in 5% skim milk, membranes were probed with primary antibodies followed by horseradish peroxidase-linked secondary antibodies. The membrane was visualized using electrochemiluminescence: ECL (Pierce Biotechnology, Rockford, IL, USA) and exposed by the chemiluminescence instrument.

mRNA isolation and real-time PCR assay for miR-148b and p55PIK

Total mRNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription was performed using an RT–PCR kit (Fermentas, Beijing, China). Real-time experiments were conducted on an iQ5 Multi-color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using SYBR Green Real-time PCR Master Mix (TOYOBO, Shanghai, China). The PCRs consisted of 1 min at 95 °C followed by 40 cycles of denaturation for 15 s at 95 °C, annealing for 30 s at 60 °C and a primer extension for 40 s at 68 °C.

MicroRNA and RNA interference

MiR-148b mimics (miR-148b), miR-148b-negative control (miR-NC), miR-148b inhibitor (Inh-148b), miR-148b inhibitor-negative control (Inh-NC), siRNA duplexes targeting human TP53 gene (si-TP53) and p55IK gene (si-p55IK) were synthesized and purified by RiboBio (Ribobio Co., Guangzhou, China). SiRNA duplexes with non-specific sequences were used as siRNA-negative control. RNA oligonucleotides were transfected by using Lipofectamine 2000 (Invitrogen) and medium was replaced 6 h after transfection. A final concentration of 100 nM miR-148b, 100 nM Inh-148b, 100 nM si-p55IK and 100 nM si-TP53 was used, and the expression levels of miR-148b and mRNA were quantified 48 h after transfection.

Animal study

Cells were cultured, collected, washed and resuspended in culture medium (about 2 × 107/ml) and then injected into subcutaneous of athymic nude mice (100 μl/tumor). Tumors were measured every other two days after they were visible to the naked eye. Finally, all mice were killed and all tumors were collected and the tumor weight was measured. The tumor volume was calculated by the formula: volume=length × width2/2 and tumor growth curve was drawn.

ChIP assay

The ChIP assay kit (Beyotime Institute of Biotechnology, Jiangsu, China) was purchased from company and the experiment was performed by following the protocol. To examine the changes in p53-binding activity on the miR-148b promoter, ChIP assays were carried out using anti-p53 antibody (Cell Signaling Technology, Danvers, MA. USA, Mouse mAb #2524) as the handbook described (Millipore, Merck KGaA, Darmstadt, Germany, #17–371). In briefly, cells were cross-linked with 1% formaldehyde for 10 min at 37 °C, collected in SDS lysis buffer, and DNA was shredded to fragments of 200–1000 bp by sonication. Antibodies against p53 or control were added to each aliquot of pre-cleared chromatin and incubated overnight. Protein G-agarose beads were added and incubated for 2 h at 4 °C. After reversing the cross-links, DNA was isolated and used for PCR reactions. Primers used for ChIP assay: forward 5′-IndexTermTTGAGGCGGTCTCACTCTTT-3′ and reverse 5′-IndexTermAGACCAACCTGGGCAACAC-3′ and control primers for glyceraldehyde 3-phosphate dehydrogenase.

Statistical analysis

All statistical analyses were carried out using the SPSS 10.0 (SPSS Japan Inc., Tokyo, Japan) statistical software package. Means±s.d. were calculated and two-tailed Student’s t-test was performed using the data analysis tools provided by the software. P<0.05 in all cases was considered statistically significant.

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Acknowledgements

This study was supported by National Natural Science Foundation (No. 81300411, No. 81372662, No. 81072431, No.81272278, No.31000612), '973' Program (No. 2009CB521802) and Program for Changjiang Scholars and Innovative Research Team in University (No.PCSIRT1131).

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Correspondence to J Gong or J Hu.

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Wang, G., Cao, X., Lai, S. et al. Altered p53 regulation of miR-148b and p55PIK contributes to tumor progression in colorectal cancer. Oncogene 34, 912–921 (2015). https://doi.org/10.1038/onc.2014.30

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