miR-150-5p represses TP53 tumor suppressor gene to promote proliferation of colon adenocarcinoma

MicroRNAs (miRNAs) play a critical role in regulation of numerous biological processes and pathogenesis of a variety of diseases. In addition, miRNAs contribute to carcinogenesis by acting as oncogenic or tumor suppressive. Circulating miRNAs including miR-150-5p are associated with colorectal cancer progression, and the putative targets of miR-150-5p include tumor suppressor gene, TP53. Here we sought to investigate the role of miR-150-5p-TP53 signaling pathway in proliferation of colon cancer and to determine expression levels of miR-miR-150-5p and TP53 in colon adenocarcinoma and adjacent non-cancerous tissue samples, or in human colon adenocarcinoma cell lines. MTT assay was used to determine proliferation and apoptosis in cell lines. Furthermore, we used Western blot to determine levels of cell cycle regulators with anti-miR-150-5p or apoptosis with overexpression of TP53. Our results show that expression levels of miR-150-5p were significantly elevated in clinical specimens from cancer patients. We further showed that inhibition of miR-150-5p increased TP53, and in turn, suppression of proliferation of colon adenocarcinoma. Moreover, inhibition of miR-150-5p or overexpression of TP53 caused cell arrest or apoptosis in colon adenocarcinoma. Our results support that miR-150-5p-TP53 pathway plays an important role in regulation of proliferation, cell arrest, and apoptosis in colon cancer, and could be an attractive target for therapy.

activated, and such an activation of p53 can either transactivate or repress downstream target genes that in turn regulate cell cycle arrest, apoptosis, DNA repair, and angiogenesis and even metastasis 17 .
Although multiple studies have revealed the potential tumor-suppressive effect of TP53 in human tumor, the molecular function of TP53 in CRC has remained further defined. Expression of miR-150-5p has been linked with TP53-downstream target genes 15 . Interestingly, a preliminary bioinformatics analysis indicates that TP53 is a potential target of miR-150-5p 18 . In the present study, we sought to investigate the role of miR-150-5p-TP53 signaling in CRC, and we demonstrate that such a signaling plays a critical role in proliferation and progression of CRC.

Materialas and Methods
Subjects. We collected specimens from a total of 10 CRC patients after surgical resection. The study and all methods were carried out in accordance with relevant guidelines and regulations. all experimental protocols were approved by the Ethic Committee of China-Japan Friendship Hospital. The general information for patients is provided in Table 1. Informed consent was obtained from all subjects.
Cell culture. Normal human colon epithelial cell line CCD 841 CoN (ATCC, USA), and human colon carcinoma cell lines HT29, T84, and LS174 (ATCC, USA) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, as recently described 19 . Cells were cultured in a humidified incubator at 37 °C and 5% CO 2 .
Western blot. Whole cell lysates were suspended in 1 × SDS loading buffer, boiled at 95 °C for 5 min, and centrifuged. Supernatants were separated on SDS-10% PAGE and transferred onto PVDF membranes (Bio-Rad, USA). Membranes were blocked in 5% milk in PBST (10 mM phosphate buffer, pH 7.2; 150 mM NaCl; and 0.1% Tween-20) for 60 min, washed three times, and incubated with appropriate antibody at 4 °C overnight. Membranes were washed three times with PBST, incubated with horseradish peroxidase-conjugated secondary antibody at 1:5,000, and developed by Immun-Star HRP Substrate (Bio-Rad, CA, USA). Blots were probed with β-actin antibody (Sigma-Aldrich, USA) as the loading control.
Dual Luciferase Assay. 3′-UTR segment of TP53 gene corresponding to predicted target site was amplified by PCR from human genomic DNA using primers that included a XbaI and EcoRI tails on the 5′ and 3′ strands, respectively, as previously described 20 . PCR products were restriction digested with both XbaI and EcoRI DNA restriction endonucleases, gel purified, and ligated into pGL3 vector (Promega, USA). HT29 cells were transfected with the firefly luciferase UTR-report vector, control Renilla luciferase pRL-TK vector (Promega, USA) with Lipofectamine 2000 reagent, according to the manufacturer's protocol (Invitrogen, USA). Twenty-four hours after transfection, cells were lysed with a 1x passive lysis buffer and the activity of both Renilla and firefly luciferases were assayed using the dual-luciferase reporter assay system (Promega, USA), according to the manufacturer's instructions.

Statistical analysis.
All results were expressed as mean ± standard deviation. We used Student's t-test or ANONA (One-way) to compare two and three groups, respectively. P < 0.05 was considered significantly different.
Results miR-150-5p directly targets TP53 in CRC. TP53 is a tumor suppressor gene as well as putative target of miR-150-5p 18 . To investigate whether miR-150-5p targets TP53 in CRC, we first performed bioinformatic analysis, using TargetScan and found that there are multiple miR-150-5p target sites in TP53 mRNA (Fig. 1A).
To validate a direct interaction between miR-150-5p and TP53, we cloned a miR-150-5p target site at 3′-UTR of TP53 mRNA upstream of firefly luciferase (FL) reporter gene, so that it could be regulated by miR-150-5p. In addition, a mutated version of this target site was used as the negative control. Colon adenocarcinoma HT29 cells were transfected with either the control FL plasmid or FL plasmid with miR-150-5p target site or the FL plasmid with mutation of miR-150-5p target site. Also, Renilla luciferase reporter plasmid was co-transfected as an internal reference. As shown in Fig. 1B, a significant decrease in FL activity was observed in cells transfected with FL reporter with wild type of miR-150-5p target site. In contrast, no repression of FL activity was obtained in cells transfected with miR-150-5p-mutation FL reporter plasmid (Fig. 2B). Further, we confirmed a direct targeting of TP53 mRNA by miR-150-5p in HT29 cells. We found that TP53 increased at both protein and mRNA levels when cells were treated with miR-150-5p specific inhibitors (Fig. 1C). Taken together, these results indicate that TP53 is a direct target of miR-150-5p in CRC cells.
Coupled increase of miR-150-5p and decrease of TP53 in CRC. Next, we sought to determine miR-150-5p and TP53 levels in in tissue specimens derived from CRC patients. Total RNAs were extracted from 10 colon cancer and adjacent non-cancerous tissue samples for the assessment of miR-150-5p and TP53 mRNA levels by quantitative RT-PCR. TP53 protein levels were assessed by Western blots as well. In the meantime, the same tissues were subjected to purification of colon cancer cells followed by immunofluorescence staining (IF). We found that miR-150-5p was significantly up-regulated in CRC cancer tissues ( Fig. 2A). In contrast, TP53 mRNA and protein levels decreased by an average of >45% (Fig. 2B,C), compared to the non-cancerous adjacent colon mucosa. In addition, weak cytoplasmic staining for TP53 was observed in the cancerous tissues, in contrast to strongly positive staining of COX-2, another biomarker for CRC 22 (Fig. 2D). www.nature.com/scientificreports www.nature.com/scientificreports/ Next, we used RT-PCR to determine levels of miR-150-5p and TP53 in different colon adenocarcinoma cell lines and similar results were obtained in all three colon adenocarcinoma cell lines as found in the primary tissues (Fig. 2E). Collectively, these results suggest that miR-150-5p and TP53 are negatively correlated in CRC, suggesting a possible regulation of TP53 by miR-150-5p. miR-150-5p stimulates cell proliferation in vitro. HT29 cells were transfected with non-specific miRNA mimics, anti-miR-150-5p, or anti-miR-150-5p combined with TP53 siRNAs, as indicated, and MTT assays were performed to determine cell proliferation. As shown here, miR-150-5p mimics promoted, but miR-150-5p inhibitors inhibited cancer cell growth (Fig. 3A). Consistent with this, depletion of TP53 also promoted cancer growth, and abolished anti-miR-150-5p mediated inhibition of cell proliferation (Fig. 3B,C). These results further confirm that miR-150-5p directly inhibits TP53 to exert oncogene functions and promote CRC cancer cell proliferation.

Inhibition of miR-150-5p affects the expression of key cell cycle factors. TP53 has been found
to induce cell cycle arrest in G2/M phase in clear cell renal cell carcinoma (ccCRC) 23 . Here we tested the role of miR-150-5p-TP53 signaling in regulation of cell cycle in CRC. The expression of cell cycle regulators associated with G2/M phase transformation was investigated in HT29 cells treated with either anti-miR-150-5p or the control non-specific miRNA mimics. As shown in Fig. 4, protein levels of cyclinB1 and CDK1 were significantly decreased in cells transfected with anti-miR-150-5p, in contrast to upregulation of TP53. These results support that miR-150-5p-TP53 signaling pathway modulates cell cycle progression via expression of cell cycle regulators.

TP53 triggers cell apoptosis in CRC cells. Evasion of apoptosis is a critical event during malignant trans-
formation, and TP53 has been found to induce apoptosis in various cancer cells 24 . Hence, we examined the effects of TP53 on induction of CRC cell apoptosis. As shown in Fig. 5, the expression levels of cleaved caspase-3 and caspase-9, which are molecular hallmarks for cell apoptosis, were significantly increased in HT29 cells transfected with pc-DNA3-TP53. Collectively, these results suggest that TP53 overexpression exerts significant effects on the proliferation of CRC cells.

Discussion
The molecular mechanisms underlying CRC initiation and progression remain largely unknown, and, therefore, a comprehensive detailing of all such mechanisms is critical. This alone will facilitate discovery of novel biomarkers for early diagnosis and therapy, thus improving the outcome of CRC patients. Over the past decade, miRNAs have emerged as a new and promising class of gene regulators involved in cancer progression 25,26 . Here, we show, for the first time, that miR-150-5p plays a critical role in colon tumorigenesis by inhibiting the tumor suppressor gene TP53 to promote cancer proliferation. Our results suggest that miR-150-5p can be used for early stage diagnosis and a therapeutic target for therapy of CRC. These collective findings show an important and new www.nature.com/scientificreports www.nature.com/scientificreports/ role of miR-150-5p-TP53 signaling pathway in carcinogenesis of CRC, and provide a molecular detaining for the correlation between elevation of miR-150-5p and cell proliferation in CRC. Therefore, our study offers a concrete base for further understanding of the complex biology involved in CRC cancer progression.
miRNAs are well-known to play a role in cancer progression. As demonstrated previously by several groups, the same miRNA might play a distinct role in different types of cancer. For example, miR-146a acts as oncogenic as well as tumor suppressive in distinct cell types 27,28 . Consistent with this observation, patients with higher miR-150-5p levels usually have a worse survival in melanoma and inhibition of miR-150-5p enhanced cell apoptosis via upregulation of PDCD4-mediated activation of caspase-8 and p21 29 . However, another study has shown that miR-150  www.nature.com/scientificreports www.nature.com/scientificreports/ might serve as a potential therapeutic sensitizer through inhibition of the AKT pathway in NK/T cell lymphoma treatment 30 . Therefore, more studies are needed to elucidate the role of miR-150-5p in other types of cancer.
Our results are consistent with the earlier published literature that TP53 plays a protective role in blocking a decisive step in oncogenesis. Activation of TP53 can induce both the mitochondrial and the death-receptor-induced apoptotic pathways 31 . Activation of TP53 leads to expression of pro-apoptotic Bcl-2 (B-cell lymphoma-2) family proteins, mainly Bax, Noxa and PUMA, but inhibits the pro-survival Bcl-2, and in turn, permeabilization of outer mitochondrial membrane. Then cytochrome c releases from the mitochondria binds to APAF-1, and induces the activation of the initiator caspase-9, eventually resulting in the activation of executioner caspase-3, -6 and -7. On the other hand, activated TP53 increases some death receptors and form death-inducing signaling complex with caspase-8, to induce apoptosis. The progression of cell cycle is tightly controlled by cyclins and cyclin-dependent kinases (CDK). p21 is one member of CDK inhibitor family, which stimulates cell cycle transition from G1 to S phase, as well as one of the major mediator of p53-induced growth arrest. In response to DNA damage, p53 induces not only cell cycle G1 phase arrest, but also G2/M checkpoint arrest 32 .
In conclusion, our results support a role of miR-150-5p-TP53 signaling pathway in the proliferation, cell cycle progress, cell apoptosis and invasion/migration of CRC cells. Furthermore, miR-150-5p acts an oncogene and its target TP53 acts as a tumor suppressor in CRC pathogenesis. Due to current lack of appropriate biomarkers and therapeutic targets in CRC, these results suggest that miR-150-5p-TP53 signaling may provide novel insights into the improvement of clinical therapeutic strategies for CRC patients.

Data Availability
All the data is available within this manuscript.