Colorectal carcinoma (CRC) is one of the most common cancers in the world, and identification of new CRC biomarkers will be helpful for the diagnosis and treatment of CRC. For isobaric tags for relative and absolute quantitation (iTRAQ) analysis, fresh CRC and adjacent, colonic adenoma, ulcerative colitis, Crohn's disease, and noncancerous colonic epithelial tissue were obtained from patients at the 2nd Xiangya Hospital of Central South University, China. The function of heterogeneous nuclear ribonucleoprotein M (HnRNP M) during the proliferation, invasion, and metastasis of CRC cells in vitro was evaluated. One hundred and twenty-six differentially expressed proteins were identified by iTRAQ analysis. The expression of HnRNP M exhibited progressive changes during the carcinogenic process and was validated by Western blot. The upregulation of HnRNP M correlated with cancer recurrence and regional lymph node metastasis. Furthermore, biological role exploration suggests that HnRNP M positively regulates cell cycle progression, promotes cell growth and invasion in vitro, and increases the colony-forming ability of LS174T cells. The present data demonstrate that the upregulation of HnRNP M is involved in human colorectal epithelial carcinogenesis and may serve as a carcinoma biomarker for CRC.
- heterogeneous nuclear ribonucleoprotein M
colorectal carcinoma (CRC) is one of the most common cancers in the world. Surgery is the first-line treatment for patients with early stage CRC (stage I or II) and has been reported to afford a positive prognosis (14). However, the early diagnosis rate of CRC is low, surgery is less effective in patients with more advanced CRC (stage III), and mortality is high. The majority of deaths from CRC are associated with liver metastasis (3), and, if left untreated, the median survival is only 6–12 mo (24). Therefore, early diagnosis, exact histological grading, and accurate prognostication of CRC are critical for guiding treatment and improving the prognosis of CRC. The identification of CRC biomarkers may be a convenient way to achieve these objectives. Much recent research has focused on the identification of new biomarkers: zinc finger protein 148, p42.3, and Yes-associated protein. These markers are expressed in patients with CRC and may serve as prognostic markers after surgery in patients with CRC (6, 17, 23). Additionally, vascular endothelial growth factor receptors 1,3,caveolin-1, CalCyclin (S100A6)-binding-protein (CacyBP), and Gankyrin (PSMD10) have been implicated in CRC aggressiveness and metastasis (1, 7, 9). However, at present, the mainstay for the diagnosis and histological grading of CRC is still the endoscopic examination and histological observation of biopsies, and tumor-node-metastasis staging is still the main method to predict the prognosis of CRC. Therefore, it is urgent to discover more effective biomarkers for CRC.
Recently, a new approach using isobaric tags for relative and absolute quantitation (iTRAQ) has been applied (18) to uncover new biomarkers. The multiplexing ability afforded by the iTRAQ reagents, which are available in four to eight different tags, when combined with two-dimensional (2D) LC-MS/MS analysis, is one of the more powerful quantitative proteomics methodologies available in the search for tumor biomarkers (16). This approach was demonstrated to be more sensitive than two-dimensional differential in-gel electrophoresis (2D-DIGE) and isotope-coded affinity tag (20) and was very suitable, especially for comparative studies in which more than two samples need to be evaluated in parallel (18, 22).
In this study, we aimed to elucidate the pathogenesis of CRC through proteomic analysis and to identify potential carcinoma biomarkers for CRC. We performed quantitative proteomic analysis using iTRAQ and identified the dysregulated expression of heterogeneous nuclear ribonucleoprotein M (HnRNP M) in CRC. Additionally, using the CRC cell line LS174T, we studied the carcinogenic properties of HnRNP M in vitro. Based on our results, we found that HnRNP M expression is positively correlated with the proliferation, invasion, and metastasis of CRC cells. As such, HnRNP M may serve as a new carcinoma biomarker for CRC.
MATERIALS AND METHODS
This study was approved and monitored by the Ethics Committee of the 2nd Xiangya Hospital of Central South University, China. All participants provided written informed consent. Fresh CRC and paracarcinoma (tissue 2 cm from the lesions), adenoma, ulcerative colitis (UC), Crohn's disease (CD), and noncancerous colonic epithelial tissue specimens and blood were obtained from patients at the 2nd Xiangya Hospital of Central South University, China, between 2010 and 2011. This included 27 cases of primary CRC and adjacent, 23 cases of colonic adenoma, 20 cases of UC, 20 cases of CD, and 29 cases of noncancerous colonic epithelial tissue from patients with non-CRC diseases, and were kept in −80°C for further analysis. This sampling was performed at the time of diagnosis or operation, and only after being verified by histopathology. The clinicopathological parameters of the 27 CRC tissue specimens used in the present study are shown in Table 1.
Protein sample preparation and iTRAQ labeling.
Five fresh frozen tissues for each of the groups (normal noncancerous colonic epithelial tissue specimens, UC, CD, paracarcinoma, colorectal adenoma, and CRC) were obtained for proteomic analysis. Tissues were dissolved in lysis buffer (7 mol/l urea, 2 mol/l thiourea, 100 mmol/l DTT, 4% CHAPS, 0.5 mmol/l EDTA, 40 mmol/l Tris, 2% Nonidet P-40, 1% Triton X-100, 5 mmol/l phenylmethylsulfonyl fluoride, and 2% Phamarlyte) at 4°C for 1 h and then centrifuged at 12,000 rpm for 30 min. The supernatant was transferred to a fresh tube and stored at −80°C. The concentration of total proteins was determined using a 2D Quantification kit (Amersham Biosciences). To diminish the effect of sample biological variation on the results of a proteomics analysis, equal amounts of protein from five different samples were pooled to generate one common sample for each type of tissue (normal, UC, CD, paracarcinoma, adenoma, and CRC), in turn obtaining the four pooled protein samples used for iTRAQ labeling (21, 25). For each sample, a total of 100 μg of protein was precipitated by the addition of four volumes of cold acetone. The precipitated protein was then dissolved in solution buffer (Applied Biosystems) and denatured. Each sample was then digested with 20 μl of 0.25 μg/μl trypsin (Promega) solution at 37°C overnight and labeled with the iTRAQ tags according to the kit protocol (Applied Biosystems, Foster City, CA). The iTRAQ reagents were used to label the peptides from the normal, UC, CD, paracarcinoma, colorectal adenoma, and CRC groups. Next, the samples were mixed, desalted with Sep-Pak Vac C18 cartridges (Waters, Milford, MA), and dried in a vacuum concentrator. The labeled samples were pooled before further analysis.
The iTRAQ-labeled, mixed peptides were fractionated by strong cation exchange (SCX) chromatography on a 20AD high-performance liquid chromatography (HPLC) system (Shimadzu; Kyoto, Japan) using a polysulfoethyl column (2.1×100 mm, 5 μm, 200 Å; The Nest Group, Southborough, MA). The peptide mixture was reconstituted in buffer A [10 mM KH2PO4 in 25% acetonitrile (ACN; Fisher Scientific, Fair Lawn, NJ), pH 2.6] and loaded on the column. Buffer A was identical in composition to the loading buffer; buffer B was made by adding 350 mM KCl to buffer A. The peptides were separated at a flow rate of 200 μl/min for 60 min with a gradient of 0–80% buffer B. The absorbance at 214 and 280 nm was monitored; a total of 25 SCX fractions were collected along the gradient. The fractions were vacuum dried and then resuspended in 50 μl HPLC buffer A [5% ACN, 0.1% formic acid (TEDIA)], loaded across the ZORBAX 300SB-C18 reversed-phase column (5 μm, 300 Å, 0.1×150 mm; Microm BioResources, Auburn, CA), and analyzed on a QSTAR XL System (Applied Biosystem) coupled with a 20AD HPLC system (Shimadzu). The flow rate of elution was 0.3 μl/min, with a gradient 5–35% HPLC buffer B (95% ACN, 0.1% formic acid) for 90 min. Survey scans were acquired from mass-to-charge ration (m/z) 400–1,800 with up to four precursors selected for MS/MS from m/z 100–2,000. Each SCX fraction was analyzed in triplicate.
Data analysis and bioinformatics.
For peptide identification and quantification, the MS data were compared against the human database of UniProtKB/Swiss-Prot (version 3.52, November 2008) from the EBI website (http://www.ebi.ac.uk./IPI/IPIhelp.html) using the ProteinPilot software (version 3.0, revision 114732; Applied Biosystem). The parameters applied were as follows: trypsin was used as the enzyme; methyl methanethiosulfonate of cysteines residues were fixed modifications; the Paragon Algorithm (Applied Biosystem) was used, which was followed by the Pro Group Algorithm (Applied Biosystem). Other parameters, such as parent ion accuracy, fragment ion mass accuracy, tryptic cleavage specificity, and allowance for number of missed cleavages, were provided and processed by the ProteinPilot software. The protein confidence threshold cutoff was 1.3 (unused ProtScore) with at least one peptide with 95% confidence. The average iTRAQ ratios from the triplicate experiments were calculated for each protein. The false discovery rate (FDR) for protein detection was calculated as FDR = (2 × reverse)/(forward + reverse).
Western blot analysis.
The protein was electrophoretically separated on a SDS-PAGE that contained a 10% separation gel and a 5% spacer gel. A total of 50 μg of each sample was loaded in each well. After electrophoresis, the proteins were electrotransferred to polyvinylidene difluoride membranes and blocked in 5% skimmed milk/TBS with Tween 20 for 2 h. It was then incubated overnight at 4°C with rabbit anti-HnRNP M (1:200) (ab116526; Abcam) or rabbit anti-β-actin (1:2,000) (ab156302; Abcam). After three washes with PBS, the membranes were incubated with goat anti-rabbit IgG secondary antibody (1:2,000) (ab97200; Abcam) at room temperature for 1 h. They were then visualized by diaminobenzidine staining. The images were obtained using the Alpha multifunctional gel imaging system and analyzed using the Alpha View software. The relative expression of the protein of interest was calculated based on the densitometric value ratio of the band corresponding to the protein of interest to that of β-actin. Different levels of HnRNP M are defined as low <0.5, moderate 0.5–1.5, and high >1.5.
The measurement of carcino-embryonic antigen expression in colorectal epithelial carcinogenic process.
Serum samples were isolated from whole blood for each group (normal, colorectal adenoma, and CRC) by centrifugation at 3,000 g for 10 min. Serum carcino-embryonic antigen (CEA) was measured with a one-step, solid-phase enzyme immunoassay (EIA) commercial kit (Cobas Core CEA EIA; Roche, Basel, Switzerland). The assay was carried out according to the manufacturers' instructions. Different levels of CEA measured by EIA are defined as low <5 μg/l, moderate 5–25 μg/l, and high >25 μg/l.
Construction of recombinant pSUPER plasmid expressing HnRNP M-short-hairpin RNA and enhanced green fluorescent protein-short-hairpin RNA.
Plasmid expressing short-hairpin RNA (shRNA) targeting human HnRNP M (pSUPER/siHnRNPM) and enhanced green fluorescent protein (eGFP) (pSUPER/sieGFP) were constructed. The target sequences for eGFP (GenBank accession no. U55763) were chemically synthesized (Takara) using the following complementary oligonucleotides: sieGFPa: 5′-gatccccGCTGACCCTGAAGTTCATCttcaagagaGATGAACTTCAGG-GTCAGCtttttggaaa-3′, sieGFPs: 5′-agcttttccaaaaaGCTGACCCTGAAGTTCATCtctcttgaaGATGA-ACTTCAGGGTCAGCggg-3′. The target sequences for HnRNP M (GenBank accession no. BC019580) were chemically synthesized (Takara) using the following complementary oligonucleotides: siHnRNPMa: 5′-gatccccCTATCAAATAGCAGCTGGCCAttcaagagaTGGCCAGC-TGCTATTTGATAGtttttggaaa-3′, siHnRNPMs: 5′-agcttttccaaaaaCTATCAAATAGCAGCTGGCCAt-ctcttgaaTGGCCAGCTGCTATTTGATAGggg-3′.
Cell culture and stable transfection.
For stable transfection for the CRC cell line, LS174T cells were seeded at 5 × 105 cells/well in six-well tissue culture plates and transfected with pSUPER/siHnRNPM or pSUPER/sieGFP using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer's instructions. They were then validated by Western blot assay.
Cell cycle analysis by flow cytometry.
For cell cycle analysis, 1 × 106 saline-treated LS174T cells (NS group) or pSUPER/siHnRNPM (HnRNPM-RNAi group)-, or pSUPER/sieGFP (control group)-transfected LS174T cells were harvested and fixed with 70% cold ethanol. After incubation at 4°C overnight, the cells were resuspended in FACS buffer containing RNase A (0.2 μg/ml) and incubated at 37°C for 30 min. They were then incubated at 4°C in the dark for 30 min in FACS buffer containing propidium iodide (20 μg/ml; Sigma-Aldrich). The stained cells were analyzed on a FACScan flow cytometer (Becton-Dickinson) with excitation at 488 nm and emission at 675 nm using long-pass (FL4, mitoxantrone) filters. The data were analyzed by the ModFIT/LT software (Moflo XDP flow cytometer; Beckman Coulter). The proliferation index (PI) was calculated using the formula: PI = (S + G2/M)/(G0/G1+ S + G2/M).
Analysis of cell growth in vitro.
For the MTT assay, aliquots of LS174T cell suspension, containing 2,000 cells in 200 μl of medium, were transferred to 96-well tissue culture plates and grown for 4 days. At 24, 48, 72, and 96 h, 20 μl of MTT (5 mg/ml; Sigma-Aldrich) were added to wells; the medium was removed after 4 h of incubation. Dimethyl sulfoxide (150 μl; Merck) was added to each well for 10 min at room temperature. The absorbance of each well was read with a Bio-Tek Instruments EL310 Microplate Autoreader at 490 nm (MK3 microplate reader THERMO). The optical density (OD) value of each well was measured at 570 nm wavelength. The percentage of cell growth was calculated by comparing the 570-nm absorbance readings with that obtained on the 1st day. Each experiment was performed at least three times and in triplicate.
In vitro cell invasion assay.
The invasiveness of saline-treated LS174T cells or pSUPER/siHnRNPM- or pSUPER/sieGFP-transfected LS174T cells was evaluated in 24-well Transwell chambers (Costar, Cambridge, MA), according to the manufacturer's instructions. The upper and lower culture compartments of each well were briefly separated by polycarbonate membranes (8 μm pore size). The membranes were precoated with 100 μg/cm2 of collagen matrix (Matrigel; Collaborative Biomedical Products, Bedford, MA), which was reconstituted by adding 0.3 ml of serum-free medium to the well for 2 h. To assess the ability of the cells to penetrate the precoated polycarbonate membrane, 3×105 cells in 0.3 ml of DMEM containing 1% FBS were placed in the upper compartment of wells, and 0.5 ml of DMEM containing 10% FBS was placed in the lower compartment. The Transwell chambers were incubated for 48 h at 37°C in a humidified 5% CO2 atmosphere. The cells that had invaded and that were attached underneath the chamber membrane were stained with a Diff-Quik stain kit (Dade Behring, Newark, DE) and were counted in six random fields with an inverted microscope at ×200 magnification. Invasive ability was defined as the average number of cells that penetrated the matrix-coated membrane per field. Three independent experiments were performed in triplicate. Five hundred microliters of 10% acetic acid were then used to dissolve the dye on the membrane. This was transferred to 96-well plates (150 μl/well) and used to measure the OD values of each well at 570 nm wavelength.
Clonogenic survival assay.
Saline-treated LS174T cells or pSUPER/siHnRNPM- or pSUPER/sieGFP-transfected LS174T cells were trypsinized, and aliquots of cell suspension containing 200 cells in 2 ml of medium were plated in six-well culture plates and cultured for 12 days. The cultured cells were fixed with 4% paraformaldehyde for 15 min and stained with Giemsa dissolved in Hanks' buffer in the dark for 10–30 min. The number of surviving colonies was counted. A colony was defined as >50 cells according to visible cloning. At least five fields were assessed using a ×10 objective lens, and the number of cells per field (>5 clones) was counted. The survival fraction was calculated as the numbers of colonies divided by the number of cells seeded times plating efficiency. Plating efficiencies were calculated as colonies per 105 cells. Cell survival was calculated as follows: (colony numbers of irradiated cells/plating cell numbers)/(colony numbers of sham nonirradiated cells/plating cell numbers) × 100%. Three independent experiments were performed.
All observations were confirmed by at least three independent experiments. t-Test, Spearman, Mann-Whitney U-test, ANOVA test, and least-significant difference (LSD) t-tests were used in this study. These analyses were performed using the Statistical Package for Social Science software (SPSS for Windows, version 13.0). A value of P < 0.05 or P < 0.01 was considered to be statistically significant.
Identification of HnRNP M, a differentially expressed protein in CRC, by iTRAQ.
A total of 1,587 nonredundant proteins were repeatedly identified by triplicate iTRAQ labeling and 2D LC-MS/MS analyses, 87.1% of which were identified with two peptide matches. The FDR for protein identification was based on searching against a reversed database and was 0.0093, 0.0156, and 0.0141 in the triplicate experiments. To identify proteins that are differentially expressed during the colorectal carcinogenic process, protein expression profiles were compared between the different stages of this process with regard to normal, UC, CD, paracarcinoma, adenoma, and CRC groups. The proteins that met all of the following criteria were confidently considered to be differentially expressed: 1) proteins were repeatedly identified by the triplicate experiments; 2) proteins were identified based on ≥2 peptides; and 3) proteins showed an averaged ratio-fold change 1.3 or ≤0.7 with regard to comparing the normal group with the other groups (≥1.3 were overexpressed, ≤0.7 was underexpressed; LSD t-test, P < 0.05). As a result, 126 proteins were found to be differentially expressed between all the different groups. The names of these 126 proteins and the stages at which their expression have been significantly changed are shown in Table 2. Among these differentially expressed proteins, HnRNP M showed a progressive change during the carcinogenic process. CEA, the oldest and most widely used clinical tumor marker in CRC, actively prevents apoptosis during the metastatic process. The HnRNP M4 protein, which belongs to the HnRNP M subfamily (14), mediates the prometastatic properties of CEA in colon cancer cells. We were interested in whether the HnRNP M can also be a tumor marker for CRC and hoped to discover its function in the carcinogenic process. The relative quantification of proteins using iTRAQ was performed on MS/MS scans and was given by the ratio of the peak areas (Fig. 1, A, B, and C). The relative changes in the expression levels of HnRNP M in normal control, UC, CD, paracarcinoma, adenoma, and CRC were shown in Fig. 1D. The change index showed that HnRNP M was more highly expressed in adenoma and CRC than in normal, UC, CD, and paracarcinoma tissues. Additionally, it was more highly expressed in CRC than in adenoma (P < 0.05), and there was no significant difference among normal, UC, and CD (P > 0.05). To verify the function of HnRNP M in the carcinogenic process, we used Western blot to quantify HnRNP M in the normal, paracarcinoma, adenoma, and CRC tissues. As shown in Fig. 1E, HnRNP M expression increased along with the evolution of colorectal epithelial carcinogenesis, which was consistent with the findings of the iTRAQ.
Correlation of HnRNP M expression in CRC with clinicopathological factors and the value of HnRNP M as biomarker for colorectal cancer.
The expression levels of HnRNP M and CEA were detected by Western blotting and EIA in an independent set of archival tissue and serum specimens, respectively, for normal control, adenoma, and CRC groups. Table 1 shows the correlation of several clinicopathological factors with HnRNP M expression in 27 cases of CRC. HnRNP M expression levels positively correlated with the regional lymph node metastasis and recurrence (P < 0.05) but had no significant correlation with other clinicopathological factors (P > 0.05). As shown in Table 3, HnRNP M and CEA expression progressively increased with the evolution of colorectal epithelial carcinogenesis (P < 0.05). The characteristics for patients with adenoma are shown in Table 4. There was no significant correlation between the expression level of HnRNP M with the clinicopathological factors of adenoma (P > 0.05; Table 4).
The specific inhibition of HnRNP M gene expression reduces the growth of LS174T cells in vitro.
To determine whether or not biochemical changes caused by HnRNP M suppression had functional consequences, we evaluated the impact of stable transfected HnRNP M-shRNA and eGFP-shRNA on cell cycle progression. Successful silencing of HnRNP M was confirmed by Western blot (Fig. 2A). FACS analysis of LS174T cells revealed a significant increase in the percentage of cells in the G1 phase with a corresponding decrease in the percentage of cells in the G2 and S phases in the HnRNPM-RNAi group compared with the control group (P < 0.05, Fig. 2B). Taken together, our data suggest that HnRNP M positively regulates cell cycle progression in the CRC cell line LS174T.
The cell proliferation index of the HnRNPM-RNAi group was lower than that of the control group, as shown in Fig. 2C. Next, the effects of the stable inhibition of HnRNP M expression on CRC cell growth were evaluated by MTT assay. As shown in Fig. 2D, the cell viability at 24, 48, 72, and 96 h after HnRNP M-shRNA transfection was significantly reduced compared with that of the other two groups (P < 0.05). This difference was particularly obvious in the 72- and 96-h groups. As such, we report that the knockdown of HnRNP M inhibits the growth of LS174T cells. The data represent the average of three experiments, and the error bars reflect the SD from this average.
The effects of the specific inhibition of HnRNP M gene expression on the invasiveness and clonogenic survival of LS174T cells in vitro.
Because HnRNP M may play an important role in regulating metastatic potential, we examined the invasiveness of LS174T cells in vitro, and Transwell assay was performed. Direct enumeration of the three independent Transwell invasion trials showed that the invasive cell numbers of the control group, normal chow group, and HnRNP M-RNAi group were 51.3 ± 1.2, 50.3 ± 1.5, and 44.7 ± 2.1, respectively (Fig. 3, A and B). The indirect counting result of the three experiments showed that the invasion abilities of the three groups were 0.491 ± 0.017, 0.486 ± 0.012, and 0.405 ± 0.007, respectively (Fig. 3C). In both the direct and indirect assay, the invasion ability of the HnRNPM-RNAi group was lower than that of the control group. Given our observation that HnRNP M can promote the invasiveness of LS174T in vitro, HnRNP M may promote the invasiveness of cancers in vivo.
Because the survival ability for invasive cells is also important for metastasis of CRC, we examined the effect of HnRNP M expression in LS174T cells by performing a clonogenic survival assay. As shown in Fig. 3, D and E, the number of colonies formed in the normal chow group was not significantly different from that of the control group (P > 0.05), whereas the number of colonies formed in the HnRNPM-RNAi group was significantly less than that of the other two groups (P < 0.05). These results show that the expression of HnRNP M can promote colony-forming ability in vitro.
CRC is one of the leading causes of cancer lethality. Identification of new CRC biomarkers will be helpful for the diagnosis and treatment of CRC and may provide new insights into its pathogenesis. In this study, we found a total of 1,587 nonredundant proteins repeatedly identified by triplicate iTRAQ labeling and 2D LC-MS/MS analyses and the expression of 126 proteins that changed significantly with regard to the different stages of CRC. Among these differentially expressed proteins, HnRNP M showed a progressive change in expression during the carcinogenic process (P < 0.05), and there was no significant difference among normal, UC, and CD (P > 0.05). Additionally, tumors with HnRNP M upregulation tended to have more frequent recurrence and regional lymph nodes. HnRNP M and CEA expression progressively both increased with evolution of colorectal epithelial carcinogenesis. The serum CEA test is recommended by the American Society of Clinical Oncology and the European Group on Tumor Markers as a prognostic biomarker for CRC following curative resection. However, the effectiveness of CEA as a preoperative and postoperative marker for CRC remains to be evaluated. The HnRNP M4 protein belongs to the HnRNP M subfamily; the CEA receptor, which binds Tyr-Pro-Glu-Leu-Pro-Lys in CEA (15), is involved in the antiapoptotic function of CEA and mediates the prometastatic properties of CEA in colon cancer cells. Therefore, we speculate whether HnRNP M can also be a marker for CRC, and our data provide evidence that HnRNP M is highly expressed in CRC and may be a biomarker for the detection of CRC, enabling the identification of preneoplastic adenoma. The use of HnRNP M may increase the sensitivity of detecting CRC.
HnRNP M is an abundant component of human HnRNP complexes that can influence pre-mRNA splicing by regulating its own pre-mRNA splicing (10) or the alternative splicing of fibroblast growth factor receptor 2 (11). The gene that encodes HnRNP M is located on human chromosome 19 (8). Recent studies have shown HnRNP M to be directly involved in the spliceosome machinery through its interaction with the CDC5L/PLRG1 spliceosomal subcomplex, where it can influence both constitutive and alternative splicing. This provides new insights into the mechanism by which HnRNP M can modulate both the 50′ and 30′ splice site choice (13).
To discover the function of HnRNP M in CRC, we studied HnRNP M's biological significance by inhibiting its expression and found that HnRNP positively regulates cell cycle progression in CRC cells. As such, we hypothesize that HnRNP M may promote DNA replication by decreasing the percentage of cells in the G1 phase and promoting the growth of CRC cells. This further supports HnRNP M as a biomarker of CRC. However, the mechanisms underlying the functional effects of HnRNP M remain unknown. Previous research has shown that several processes are involved in alternative RNA processing: splicing, polyadenylation, and nucleocytoplasmic transport of mRNA, which potentially begins with transcript-specific packaging by proteins of the HnRNP complex. Recent reports have also indicated that HnRNPs play an important role in both transcript-specific packaging and alternative splicing of pre-mRNAs (5). In the G1 phase, cells prepare precursor substances for the synthesis of DNA, so we supposed that HnRNP M may bind to pre-mRNA to accelerate the production of these precursor substances, decrease the number of cells in the G1 phase, and promote the growth of CRC cell.
Metastasis is the most insidious and life-threatening aspect of cancer. Regional metastasis is one of the major reasons for poor outcomes in patients with CRC; the identification of metastasis-associated proteins and the clarification of the mechanisms related to metastasis may provide new and specific targets for anticancer therapy. In our research, we used the Transwell assay to analyze the invasiveness of LS174T cells in vitro and found that HnRNP M could promote the invasion of LS174T. Also, the clonogenic survival assay demonstrated that the expression of HnRNP M can promote the colony-forming ability of cells. These data suggest that HnRNP M might enhance metastatic potential and promote the invasion of cancers. However, the mechanism by why this occurs is still unknown; further research on this subject is needed. The contribution of HnRNP M to CRC metastasis suggests that HnRNP M may be useful for the early diagnosis of CRC before metastasis, as well as a new target for anticancer therapy. The inhibition of the expression of HnRNP M could potentially reduce tumor growth and CRC metastasis and increase the life expectancy of CRC patients. The host's microenvironment is a major factor in determining the ability of tumor cells to grow in foreign tissue; it is possible that HnRNP M may also modulate the tumor's microenvironment. It has been reported that CEA can alter macrophage activity, both at the primary tumor site and also at secondary sites such as in the liver and lung (Kupffer cells and alveolar macrophages). This is significant because macrophages play a particularly prominent role in invasion by promoting cancer cell motility and angiogenesis (4, 12). CEA, the oldest and most widely used clinical tumor marker in CRC, actively prevents apoptosis during the metastatic process when cells detach from the extracellular matrix (19). This may also be a survival mechanism during metastasis. The mechanism by which CEA inhibits anoikis occurs via the binding of CEA to the death receptor, DR-5, thus reducing caspase 8 activity. HnRNP M4, a member of the HnRNP M family, is regarded to be a surface receptor for CEA (2) and may be involved in the antiapoptotic and prometastatic properties of CEA in colon cancer cells. However, more evidence for this is required.
According to our knowledge, this study is the first to identify the differential expression of HnRNP M in CRC and normal tissue and demonstrate its role in the proliferation, invasion, and metastasis of CRC cells. This may provide a better understanding regarding the carcinogenic process and the metastasis of CRC. Additionally, it may help in tailoring the use of chemotherapy to each patient, ultimately improving patients' outcome. The number of specimens analyzed was relatively small, which may limit the validity of these results. Additionally, the specific mechanisms underlying how HnRNP M modulates the proliferation, invasion, and metastasis of CRC cells are not well understood. The next step in our research is to analyze larger patient cohorts and run blinded samples to confirm the usefulness of HnRNP M in CRC diagnosis. Further studies on its mechanism will also be carried out.
In conclusion, our study determined that the expression of HnRNP M was higher in CRC than in normal, paracarcinoma, and adenoma groups. HnRNP M expression was closely correlated with proliferation, invasion, and metastasis of CRC cells and may serve as a new carcinoma biomarker for CRC. Further studies will be required to determine how HnRNP M may be applied in a clinical setting and increase the life expectancy of CRC patients.
This work was supported by National Natural Science Foundation of China (no.81172299), Research Program from Science and Technology Department of Hunan Province, China (S2012F1023).
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: S.C., J.Z., L.D., C.L., D.L., C.O., F.L., and X.L. conception and design of research; S.C., J.Z., L.D., and X.L. analyzed data; S.C. and J.Z. drafted manuscript; S.C., C.O., and X.L. edited and revised manuscript; J.Z., L.D., Y.Z., and C.L. performed experiments; L.D., Y.Z., and X.L. interpreted results of experiments; D.L., F.L., and X.L. approved final version of manuscript; X.L. prepared figures.
We acknowledge the Department of Chemistry and the Institute of Biomedical Science, Fudan University, People's Republic of China, for technical support.
- Copyright © 2014 the American Physiological Society