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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 52  |  Issue : 6  |  Page : 47-55
 

Transcatheter arterial infusion chemotherapy increases expression level of miR-142-5p in stage III colorectal cancer


1 Department of Interventional Oncology, Renji Hospital Shanghai Jiaotong University School of Medicine,160 Pujian Rd., Pudong, Shanghai 200127, China
2 State Key Laboratory of Genetic Engineering and Institute of Developmental Biology and Molecular Medicine School of Life Sciences, Fudan University, 220 Handan Rd., Shanghai 200433, China
3 Shanghai Institute of Medical Image Research, 180 Fenglin Rd., Shanghai 200032, China

Date of Web Publication24-Dec-2015

Correspondence Address:
B Zhai
Department of Interventional Oncology, Renji Hospital Shanghai Jiaotong University School of Medicine,160 Pujian Rd., Pudong, Shanghai 200127
China
X Wang
Shanghai Institute of Medical Image Research, 180 Fenglin Rd., Shanghai 200032
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.172513

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 » Abstract 

Objective: To investigate the expression level of miR-142-5p and its potential target gene endothelial PAS domain protein 1(EPAS1) in Stage III colorectal cancer during Transcatheter arterial infusion chemotherapy (TAI). Materials and Methods: Illumina high-throughput sequencing was used to obtain miRNA expression profiles of paired tumor and adjacent normal tissues from one patient received TAI 1 week before the operation and another patient directly underwent an operation. The expression levels of miR-142-5p was measured with both high-throughput sequencing and quantitative real time-polymerase chain reaction. Results: The expression levels of miR-142-5p, were significantly reduced in tumor tissues of stage III CRC, then significantly increased in tumor tissues receiving TAI and higher than tumor tissues without TAI. The apoptosis rate of HT-29 colon cancer cells was mildly increased after transfection with pre-miR-142. miR-142-5p could bind directly to the 3′untranslated region of endothelial PAS domain protein 1 and reduce its expression. Conclusions: miR-142-5p is a potential tumor suppressor in CRC and is upregulated in tumor tissues after TAI, suggesting its potential clinical values for testing the functionality of TAI and predicting the progress of CRC.


Keywords: Colorectal cancer, endothelial PAS domain protein 1, miR-142-5p, transcatheter arterial infusion chemotherapy


How to cite this article:
Shi D, Zhai B, Zheng Y, Ren R, Han M, Wang X. Transcatheter arterial infusion chemotherapy increases expression level of miR-142-5p in stage III colorectal cancer. Indian J Cancer 2015;52, Suppl S2:47-55

How to cite this URL:
Shi D, Zhai B, Zheng Y, Ren R, Han M, Wang X. Transcatheter arterial infusion chemotherapy increases expression level of miR-142-5p in stage III colorectal cancer. Indian J Cancer [serial online] 2015 [cited 2019 Aug 18];52, Suppl S2:47-55. Available from: http://www.indianjcancer.com/text.asp?2015/52/6/47/172513



 » Introduction Top


Colorectal cancer (CRC) is the third most common cancer in the United States and also the most common cancer of the digestive system.[1] Currently, the standard diagnostic method depends on the use of sensitive guaiac fecal occult blood test, fecal immunochemical test, computed tomography colonography, sigmoidoscopy, and colonoscopy.[2] CRC antigens such as a carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) are available as biomarkers for CRC diagnosis. However, these antigens fail to provide enough information for this disease because of their limited sensitivity and specificity for distinguishing CRC from other nonmalignant or malignant diseases.[3] In the treatment of CRC, the overall response rate and 3-year disease free survival rate were increased and liver metastasis was delayed due to various new drugs and treatment methods.[4] Moreover, preoperative transcatheter arterial infusion chemotherapy (TAI) combined with surgical resection was beneficial to patients with stage III CRC.[5] However, the molecular changes behind TAI are still unknown, and molecular markers are terribly lacking for testing the functionality of TAI in CRC.

MicroRNAs (MiRNAs) are an abundant class of small noncoding RNAs of about 22 nucleotides in length that function as negative regulators of gene expression through their complementarities to specific messenger RNAs.[6] Recent studies have demonstrated that miRNAs contribute to the development of various cancers.[7] To reveal the miRNA profiles of CRC with TAI, we performed high-throughput sequencing for CRC tissues and adjacent normal tissues with and without TAI before operations. We identified 15 severely deregulated miRNAs, with 5 downregulated and 10 upregulated in tumor tissues without TAI, respectively, based on the obtained sequencing profiles. These 15 miRNAs showed different changes of their expression levels in tumor tissues with TAI. And we found that miR-142-5p was one of the most differentially expressed miRNAs when comparing tissues with and without TAI.

miR-142-5p is downregulated in various human malignancies, including, but not limited to, lung cancer,[8] human papillomavirus (HPV) positive squamous-cell carcinoma of the head and neck cell lines,[9] ovarian cancer,[10] and CRC.[11] These results strongly suggest that low expression level of miR-142-5p may contribute to pathogenesis and progression of human malignancies. After performing quantitative real time-polymerase chain reaction (qRT-PCR) experiments in 80 pairs of tumor and adjacent normal tissue samples (40 with and 40 without TAI, respectively), we found that miR-142-5p demonstrated lower expression levels in tumor tissues without TAI, and it was upregulated in tumor tissues with TAI when compared with tumor tissues without TAI.

To reveal the functional roles of miR-142-5p in CRC, we predicted target genes of miR-142-5p using several bioinformatic algorithms and databases and performed Gene Ontology (GO) and signaling pathway enrichment analysis for the predicted targets. Among predicted targets of miR-142-5p, endothelial PAS domain protein 1 (EPAS1), also named as hypoxia-inducible factor 2α (HIF-2α), is a recently discovered transcription factor protein and mainly exists in endothelial cells. EPAS1 is essential to hypoxia reaction.[12] Tumor is in chronic hypoxia state due to its biological behavior,[13],[14] which induces the expression of hypoxia associated factors in tumor tissues.[14] Then, this results in degradation of HIF-1α and retains EPAS1.[15] Increased activity of EPAS1 activates matrix metalloproteinasess in human and mouse osteoarthritic cartilage [16] and plasminogen activator inhibitor-1 in adenocarcinoma cells,[17] which are genes correlated with tumor cell migration and invasion,[18],[19] thus increases invasiveness of tumor cells.[15],[19],[20],[21] Existing researches also suggest that high levels of EPAS1 in the plasma are associated with a poor outcome in advanced CRC patients.[22] However, it is still unknown whether EPAS1 is regulated by some specific miRNAs.


 » Materials and Methods Top


Ethics statement

Our study was approved by the Bioethics Committee of Zhongshan Hospital, Fudan University (No. 2011-150); written informed consent and approval were given by the patients.

Patients' recruitment and treatment

Outpatients suspected of CRC were suggested to undergo abdomen and pelvis magnetic resonance imaging (MRI) scanning, flexible colonoscopy and serum biomarker examination, e.g., CEA and CA19-9. Patients who were considered as stage III CRC using MRI imaging were randomly assigned into two groups: Patients underwent surgical resection of CRC tumor directly and patients underwent TAI 1 week before surgical resection. Written informed consents were obtained from patients themselves at the time of patient recruitment. The selected patients' background and clinical-pathological features are summarized in [Table 1].
Table 1: Clinicopathologic characteristics of patients

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Clinical samples and cell culture

There were 40 patients in each of the two groups. The tumor and adjacent normal tissues were collected from these 80 CRC patients who had undergone surgical resection of CRC tumors at Zhongshan Hospital, Fudan University between 2010 and 2011. The tissue samples were put into liquid nitrogen immediately after being separated.

Human CRC cell line HT-29 was obtained from the Chinese Academy of Sciences Type Culture Collection. Cells were maintained in a McCoy's 5A medium supplemented with 2.2 g/L NaHCO3 and 10% fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% air at 37°C.

MicroRNAs expression profiles of colorectal cancer and adjacent normal tissues

Total RNA of the collected tissues were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The integrity of the RNA was checked by an ultraviolet spectrophotometry and 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA). Four RNA samples (2 pairs of tumor and adjacent normal tissues) of 2 patients, each from one of the two groups, were further performed small RNA deep sequencing using Illumina HiSeq2000 sequencer by following the corresponding protocols.

Analysis of sRNA sequencing libraries

The small RNA libraries were analyzed as mentioned previously.[23],[24] Briefly, the obtained reads with low-scored nucleotides were removed. There are more than 80 million qualified reads with 18–45 nucleotides, representing 1.76 million unique sequences, in the four samples. These unique sequences were exclusively aligned to pre-miRNAs, reference mRNA, ncRNAs, repeat elements, and lncRNAs with SOAP2[25] by allowing zero mismatches. The distributions of reads and unique sequences in these categories are shown in [Figure 1]a and [Figure 1]b, respectively. The frequencies of mature miRNAs were calculated with the reads perfectly aligned to mature human miRNAs in the miRBase and normalized to reads per ten million transcripts (RPTM).
Figure 1: The distributions of reads, top 30 abundant MicroRNAs and the log2 ratios of deregulated MicroRNAs in the sequenced tissues. (a) The distributions of obtained reads in the sequenced tissues. The reads were exclusive mapped to pre-MicroRNAs from miRBase (r19), refMRNAs from UCSC Genome Browser, classical noncoding RNAs (tRNAs, rRNAs, snRNAs, snoRNAs, etc., see Materials and Methods), long noncoding RNAs (lncRNAs) from GENCODE v7, and repeat elements (RepBase, v14) with SOAP2. (b) The distributions of unique sequences in the sequenced tissues. (c) The top 30 MicroRNAs with the largest numbers of normalized sequencing frequencies. (d) Log2 ratios of frequencies of the most deregulated MicroRNAs in tumor tissues over in adjacent normal tissues without and with transcatheter arterial infusion. The 30 MicroRNAs with the largest and 30 MicroRNAs with the smallest log2 ratios of tissues without transcatheter arterial infusion were drawn from bottom. Then, the log2 ratios of these MicroRNAs were calculated for tissues with transcatheter arterial infusion

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Analysis of MicroRNA expression using TaqMan quantitative real time-polymerase chain reaction

TaqMan probes and primers for miR-142-5p were provided by Roche Applied Science. Regarding the PCR condition, we followed the manufacturer's protocol. Stem-loop RT-PCR was used to quantify the expression levels of miRNAs. Expression of RNU6B was used as internal control. All the experiments were done in triplicate. Fold change was calculated according to the previous study.[26] The obtained relative expression levels (2-∅∅Ct) of different groups were compared using the Mann–Whitney U-test Wilcoxon test. Adjacent normal tissues without TAI were used as references for the miRNAs relative expression levels between all tissue samples.

Transfection of hsa-miR-142 precursor

For gain-of-function experiments, we cloned hsa-miR-142 precursor into pSUPER. As described by Balaguer et al.[27] HT-29 cells and HEK293T cells were transfected with PS_miR-142 or PS_null at a final concentration of 300 ng/L, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, Opti-MEM (Invitrogen) with 1 µg of the corresponding pSUPER.

Cell apoptosis assay

Cell apoptosis rate was determined by using an Annexin V assay (eBioscience, San Diego, CA) in combination with 7-AAD (eBioscience) according to the manufacturer's instructions. Cell apoptosis rate was evaluated by Fluorescence-Activating Cell Sorter (FACSCalibur, BD Biosciences, Bedford, MA). HT-29 colon cancer cells were plated in 6-well dishes, 48 h after transfected with PS_miR-142 or PS_null, cells were harvested for FACS analysis. All experiments were performed in triplicate.

Dual-luciferase assay for miR-142-5p target validation

A 51 nucleotide sequence around the miR-142-5p complementary site in 3′UTR of EPAS1 were inserted between the NheI-XhoI restriction sites in the 3′UTR of the luc2 gene in pmirGLO vector (E1330, Promega, Madison, WI). Another sequence with mutated nucleotides at position 2-6 of miR-142-5p complementary site in the 3′UTR of EPAS1 was used as a control. HEK293T cells were transfected with 250 ng of vector, 5 μg of miRNAs, and 2 μl of Lipofectamine 2000 (Invitrogen) in a 100 μl of Opti-MEM. The activities of firefly and Renilla luciferases in cell lysates were determined with a dual-luciferase assay system (Promega). Normalized data were calculated as the quotient of firefly/Renilla luciferase activities.

Statistical analysis

Data are expressed as the mean ± standard deviation from at least three separate experiments. Differences between groups were analyzed using Student's t-test. A value of P < 0.05 was considered statistically significant.


 » Results Top


MicroRNAs transcriptomes of colorectal cancer tissues

For comprehensive screening of miRNAs that were differentially expressed in tumor and adjacent normal tissues with and without TAI, we performed high-throughput sequencing for two pairs of tumor and adjacent normal tissues with each pair from one of the two groups with and without TAI, respectively, using Illumina HiSeq 2000 sequencer. As shown in [Figure 1]a, the reads mapped to miRNAs constitute the largest parts among different categories in all tissues and the parts of unique sequences mapped to miRNAs are much smaller than those of reads [Figure 1]b. Next, after aligning the obtained sequences to mature miRNAs, we detected 395 miRNAs or miRNA* (the Complementary strand of a mature miRNA) with at least 100 reads in the four sequenced samples collectively. As shown in [Figure 1]c, miR-143-3p, miR-10a-5p, miR-192-5p, miR-26a-5p, and miR-21-5p are the top five miRNAs that have the highest abundances for the four sequenced tissues.

To identify deregulated miRNAs in tumor tissues, we calculated the log2 fold changes for 131 miRNAs with the normalized frequency of at least 1000 RPTM in at least one of the four libraries. We totally identified 15 severely deregulated miRNAs in tumor tissues without TAI, with a log2 ratio of normalized frequency >1 or <−1 [Figure 1]d.

Target analysis of miR-142-5p

As shown in [Figure 1]d, two miRNAs, miR-451a, and miR-142-5p, severely downregulated in tumor tissue without TAI, were upregulated in tumor tissue with TAI. Here, we focused on miR-142-5p and tried to reveal its function in CRC by identifying its targets. We used four miRNA target prediction algorithms, i.e., TargetScan (Lewis et al., 2005) (release 5.2), PITA,[28],[29] and StarBase,[30] to predict targets of miR-142-5p. The targets predicted by at least two algorithm and the experimentally verified targets in miR2Disease,[31] miRecords,[32] and TarBase [33] were used to perform further GO and pathway enrichment analysis.

The significantly enriched GO terms and pathways are shown in [Table 2] and [Table 3], respectively. As shown in [Table 2], the targets of miR-142-5p involve in a lot of functions and pathways. As one of the targets of miR-142-5p, EPAS1 appears in several significant GO terms, including protein binding (GO: 0005515), DNA binding (GO: 0003677), and DNA-dependent transcription (GO: 0006351) [Table 2].
Table 2: The significantly enriched GO terms of miR-142-5p targets

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Table 3: The enriched signaling pathways of miR-142-5p targets

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Our results also suggest that miR-142-5p plays a role in many signaling pathways, such as transforming growth factor-beta signaling (P = 2.3 × 10−4, Hypergeometric test) and EGFR1 signaling (P = 6.8 × 10−3, Hypergeometric test), as shown in [Table 3]. Targets of miR-142-5p are significantly enriched in many cancer pathways [Table 3], including CRC (P = 0.014, Hypergeometric test), pancreatic cancer (P = 0.014, Hypergeometric test), renal cell carcinoma (P = 0.028, Hypergeometric test), and chronic myeloid leukemia (P = 0.033, Hypergeometric test), suggesting miR-142-5p might be involved in multiple cancers.

Validation of the expression levels of miR-142-5p

We used TaqMan qRT-PCR to assess the expression of miR-142-5p in 80 pairs of tumor and adjacent normal tissues, 40 pairs from each of the two groups, respectively. We found that miR-142-5p is significantly downregulated more than 2 folds (P < 10−8, Mann–Whitney U-test Wilcoxon test) in tumor tissues without TAI [Figure 2]b, which is consistent with the sequencing results [Figure 2]a. But its expression is upregulated about 2 folds in tumor tissues that received TAI when compared with tumor tissues without TAI (P< 10−9, Mann–Whitney U-test Wilcoxon test, [Figure 2]b).
Figure 2: The expression levels of miR-142-5p and endothelial PAS domain protein 1. (a) The normalized frequencies (Reads Per 10 Million transcripts) of miR-142-5p in the sequenced tissues. (b) The expression levels of miR-142-5p using quantitative real-time polymerase chain reaction (n=40). P-values were calculated using Mann–Whitney U-test Wilcoxon test. (c) The expression levels of endothelial PAS domain protein 1 using quantitative real-time polymerase chain reaction (n=40). The values were standardized to β2MG. (d) The scatter plot of the expression levels of miR-142-5p and endothelial PAS domain protein 1 in samples without transcatheter arterial infusion. (e) The scatter plot of the expression levels of miR-142-5p and endothelial PAS domain protein 1 in samples with transcatheter arterial infusion. (f) The scatter plot of the expression levels of miR-142-5p and endothelial PAS domain protein 1 in refined samples whose ratios of expression changes ∅EEP AS1/∅EmiR-142-5p is negative, where ∅E is the expression level of endothelial PAS domain protein 1/miR-142-5p in tumor tissue minus in adjacent normal tissues. Sample size n is 35 and 31 for the group without transcatheter arterial infusion and with transcatheter arterial infusion, respectively. In part d, e, and f, the values of a pair of tumor tissue and adjacent normal tissue were linked by a blue line. The values and error bars in part b and c were arithmetic mean values and standard deviations, respectively

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Validation of the expression levels of endothelial PAS domain protein 1

The expression levels of EPAS1 were quantified using qRT-PCR for all 80 pairs of samples, 40 for each of the two groups, respectively. Our results indicate that the expression level of EPAS1 is marginally higher in tumor tissues without TAI (P = 0.08, Mann–Whitney U-test Wilcoxon test, [Figure 2]c) and is reduced in tumor tissues with TAI (P < 0.05, Mann–Whitney U-test Wilcoxon test, [Figure 2]c). In adjacent normal tissues with TAI, EPAS1 shows an increased expression level with a large variance, suggesting inconsistent changes. Thus, we plotted the relative expression levels of miR-142-5p and EPAS1 in [Figure 2]d and [[Figure 2]e for 40 samples without TAI and with TAI, respectively. There is a small negative correlation coefficient between expression levels of miR-142-5p and EPAS1 (P > 0.05, Student's t-test, for both groups). Some paired samples, 5 and 9 samples in the group without and with TAI, respectively, show positive correlations between the expression levels of miR-142-5p and EPAS1. After removing these 14 samples, we obtained a correlation coefficient value of − 0.16 (P = 0.06, Student's t-test) for the remaining 66 samples as shown in [Figure 2]f. We also found that the expression level of EPAS1 does not show a significant change (P = 0.38, Mann–Whitney U-test Wilcoxon test) in tumor tissues between the removed 5 samples without TAI and 9 samples with TAI, although miR-142-5p has a significantly higher expression level in the 9 tumor samples with TAI (P < 0.01, Mann–Whitney U-test Wilcoxon test) than in the 5 tumor samples without TAI. These results suggest that miR-142-5p represses EPAS1 in most, but not all, samples.

miR-142 transfection induced cell apoptosis

Having discovered that miR-142-5p is downregulated in CRC and upregulated in tumor tissues received TAI, we next performed functional studies to determine whether miR-142-5p had in vitro tumor-suppressive features following transfection of miR-142-5p precursor into CRC cell lines. We performed Annexin V/7-AAD cell apoptosis assays after transfection of either miR-142-5p precursor or a null vector into human colon cancer cell line HT-29. As shown in one of the FACS experiments for the cell lines transfected with PS_null (control) and PS_miR-142 vector, a higher percentage of cells demonstrated apoptosis signals, lower-right corners of [Figure 3]a and [Figure 3]b, respectively, in cells transfected with PS miR-142 vector.
Figure 3: Cell apoptosis rate of HT-29 cell lines transfected with null and miR142-5p plasmid. (a) Fluorescence-activated cell sorting image of an HT-29 cell line sample transfected null plasmid. (b) Fluorescence-activated cell sorting image of an HT-29 cell line sample transfected with miR-142 plasmid. (c) The cell apoptosis rates for the HT-29 cell lines transfected with null and plasmid. P value was calculated with Student's t-test. The values and error bars in part C were arithmetic mean values and standard deviations, respectively

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Overexpression of miR-142-5p significantly induced cell apoptosis (P< 10−3, Student's t-test, [Figure 3]c), suggesting the specificity of miR-142-5p as a tumor suppressor in CRC.

miR-142-5p directly targets endothelial PAS domain protein 1

We next explored the functional interaction between miR-142-5p and EPAS1. Luciferase reporter plasmids harboring wild-type and mutated miR-142-5p binding site at the EPAS1 3′UTR region was constructed [Figure 4]a and [Figure 4]b. The transient transfection of HEK293T cells with the wide-type reporter plasmid and the miR-142-5p precursor led to a significant decrease (P < 10−9, Student's t-test) of luciferase activity in comparison with the control precursor [Figure 4]c. However, luciferase activity was unaffected in cells transfected with the null plasmid and plasmid with the mutated miR-142-5p complementary site (P > 0.05, Student's t-test, [Figure 4]c). Moreover, the mutation in the miR-142-5p complementary site of EPAS1 3′UTR region also resulted in a significant restoration of the luciferase expression (P< 10−5, Student's t-test, [Figure 4]c), which verified that miR-142-5p repressed EPAS1 through the predicted complementary site in [Figure 4]a.
Figure 4: Endothelial PAS domain protein 1 is a direct target of miR-142-5p. (a) Human wide type (upper) and mutated (lower) miR-142-5p complementary site of endothelial PAS domain protein 1. (b) The plasmids used. (c) The luciferase expression levels of NONE (untreated cells), PS_null+PGLO_null, PS¬_miR-142+PGLO_null, PS_null+PGLO_endothelial PAS domain protein 1, PS_miR-142+PGLO_endothelial PAS domain protein 1, and PS_miR-142+PGLO_endothelial PAS domain protein 1_mut, from left to right, respectively. P value was calculated with Student's t-test. The values and error bars in part C were arithmetic mean values and standard deviations, respectively

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 » Discussion Top


We obtained the miRNA profiles of CRC tissues with and without TAI using high-throughput sequencing. Our results show that some typical tumor-related miRNAs are deregulated in CRC tissues. Two miRNAs, miR-451, and miR-142-5p have demonstrated increased expression levels in tumor tissues with TAI compared with that without TAI. Godlewski et al. found that miR-451 is downregulated in migrating glioma cells.[34] miR-451 could repress CAB39 and reduce migration and enhance proliferation of glioma cells.[34] Another study also found that miR-451 targets Ras-related protein 14 (RAB14) and functions as a tumor suppressor and downregulated in human nonsmall cell lung cancer.[35] These findings suggest that the reduced metastasis levels of CRC after TAI [5] could, at least partially, be resulted from the increased miR-451 expression levels in tumor tissues received TAI.

Monzo et al. found that miR-142-5p is downregulated in stage II CRC.[36] But the underlying mechanism of miR-142-5p in CRC is unclear until now. In this study, we found that low expression level of miR-142-5p, as well as miR-451, in CRC tissues is upregulated after receiving TAI, suggesting miR-142-5p could be used as a diagnostic biomarker for CRC patients treated with TAI. We verified that EPAS1 is a direct target of miR-142-5p.

Accurate prediction of the functionality of TAI might be of great clinical value. Although both miR-142-5p and miR-451 are responsive to TAI, the higher expression level of miR-142-5p suggests that it is a more suitable candidate for examining whether the TAI has realized the expected antimetastasis goal in practice. Kovalchuk et al. also noticed that miR-451 could not be detected by microarray in breast adenocarcinoma cells due to low expression level.[37]

Some miRNAs, such as miR-21 and miR-10a/b-5p, demonstrated increased expression levels in tumor tissues without TAI. miR-21 has been reported to be upregulated in CRC tissues,[38],[39] plasma of CRC patients,[40] and other cancers.[41] Some studies have reported the reduced expression of miR-10b in CRC,[39] which is inconsistent with our sequencing results. miR-10b promotes tumor invasion and metastasis in breast cancer by targeting HOXD10.[42] miR-10a plays a prometastatic role in pancreatic cancer cells [43] but shows inconsistent expression changes in different CRC cell lines.[44] miR-10a also targets a tumor suppressor HOXA1.[45] These evidences suggest that miR-10 family have complex roles in different cancer.[42]

Our data indicate that miR-142-5p may act as tumor suppressor in the development of colon carcinogenesis, supported by the fact that it can induces colon cancer cell line HT-29 apoptosis to some extent (0.53% vs. 3.17%, P < 0.001, Student's t-test). This was consistent with existing researches of miR-142-5p on lung cancer,[8] human embryonic colon,[36] and HPV-positive squamous cell carcinoma of the head and neck.[9] In addition, pancreatic cancer patients with high miR-142-5p expression had significantly longer survival times in the gemcitabine-treated group.[46] However, its expression was high in some CRC cell lines,[38] germinoma,[47] and brain tumors.[48] These studies indicate that miR-142-5p may differentially express according to tissue types, and its expression may be reversed as results of some treatments. The pathway enrichment analysis of miR-142-5p targets also suggests that miR-142-5p may be involved in several other cancers. Although further studies are needed, our results suggest that miR-142-5p may act as a tumor suppressor in CRC, and it is also regulated by TAI.

Because many human cancers associate with tumor angiogenesis, it is plausible to speculate that EPAS1, which is often upregulated in cancer tissue according to tumor biological characteristics, may play a role in colon tumorigenesis. Overexpression of EPAS1 has been documented in renal cell carcinoma,[49] and it may also promote renal cell carcinoma tumorigenesis. We found that EPAS1 was slightly upregulated in CRC tumor tissues, and its expression level was downregulated in CRC tumor tissues with TAI when compared with tumor tissues without TAI. We verified that miR-142-5p interacted with EPAS1 using luciferase experiments. We witnessed a modest negative correlation coefficient of −0.16 (P = 0.06, Student's t-test) between the expression levels of miR-142-5p and EPAS1 in 66 out of 80 selected patients. These suggest that miR-142-5p represses EPAS1 in most of our selected patients.

Taken together, this study reports that miR-142-5p is significantly downregulated in stage III CRC, and potentially acts as a tumor suppressor of this disease and its expression levels can be modulated by TAI. Overexpression of miR-142-5p can mildly increase apoptosis rate of colon cancer cells in vitro, potentially by repressing tumor growth through its interaction with EPAS1. These findings raise the possibility that in the future, miR-142-5p could be employed as a therapeutic target and diagnostic marker of CRC, especially for diagnosing the functionality of TAI.

Acknowledgments

The research was supported in part by National Basic Research Program of China (No. 2010CB945500) to YZ. We sincerely thank Prof. Jianmin Xu and colleagues for providing clinical tissue samples and their collaborations in clinical work. We also thank Dr. Chao Peng and Dr. Xinrong Du for their helpful discussions in experiments.

 
 » References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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