A total of 116 paired CRC tissues and matched normal mucosae were collected from patients with CRC who underwent tissue biopsy at the Department of Gastroenterology, Shanghai General Hospital of Nanjing Medical University between September 2010 and March 2016. Patients comprised 60 men and 56 women (mean age, 64 years; age range, 32–89 years). All specimen were histopathologically confirmed as CRC. No patients had received local or other related anticancer therapies prior to tumor biopsy. The tumor grade and clinical stages were classified according to the guidelines of the American Joint Committee on Cancer (AJCC) (16). The present study was approved by the Ethics Committee of Shanghai General Hospital of Nanjing Medical University, and written informed consent was obtained from all patients prior to enrollment in the present study.

Total RNA was isolated from fresh CRC and adjacent normal mucosae tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First strand cDNA was synthesized from 2 µg RNA using a Prime-Script PCR reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. By using the LightCycler^®480 system (Roche Applied Science), the SYBR Premix Ex-Taq II kit (Takara Bio, Inc.) was applied to perform qPCR. The cycling conditions were as follows: Initial denaturation (2 min at 95°C) followed by 40 cycles of denaturation (10 sec at 95°C), annealing (30 sec at 59°C), elongation (30 sec at 72°C) and a final extension (30 sec at 72°C). The amplified samples were then maintained at 4°C. The primer sequences used for MIR100HG detection were as follows: MIR100HG forward, 5′-CCCAGTGCAAGGACAAAGA-3′ and reverse, 5′-GCAGAGGAGGTGTCTTCAGG-3′; and GAPDH forward, 5′-GGGAAATTCAACGGCACAGT-3′ and reverse, 5′-AGATGGTGATGGGCTTCCC-3′. The relative expression levels of MIR100HG were normalized to the endogenous control GAPDH and expressed as 2^−ΔΔCq (17).

The human CRC LoVo, DLD-1, RKO, SW48, HCT8 and HCT116 cell lines, and the normal intestinal mucous epithelium FHC cell line were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences. All cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin and placed at 37°C in a humidified incubator containing 5% CO₂.

The CRC cell line LOVo overexpressing MIR100HG was established by transfection with lentiviral vectors encoding human MIR100HG (PCDH-MIR100HG). In addition, MIR100HG-knockdown CRC cell line HCT116 were constructed by transfection with MIR100HG short hairpin RNA (shRNA). The sequences used for overexpression and knockdown were previously described (18).

By using the two transfected CRC cell lines LoVo and HCT116 and their control groups, transwell assays were used to assess tumor cell migration and invasion. A Transwell 24-well Boyden chamber with a polycarbonate membrane (pore size, 8.0 µm; Corning Inc.) was used for cell migration (without Matrigel^® coating; BD Biosciences) and invasion (with Matrigel^® coating, prepared on ice) assays, according to the manufacturer's protocol. Briefly, 5×10⁵ cells/ml were seeded in 200 µl serum-free DMEM medium in the upper chamber, whereas DMEM medium supplemented with 500 µl 10% FBS was added to the lower chamber. Following incubation for 24 h, cells were fixed with 4% polyoxymethylene and stained with 0.1% crystal violet at room temperature. Cells that had moved to the lower side of the membrane were counted in 10 visual fields and the average value was calculated. Cells images were captured using a light microscope (Olympus Corporation; magnification, ×200). Each experiment was performed independently three times.

Nude mice were purchased from Shanghai Research Center for Model Organisms, Inc. Mice had an average weight of 20 g. Mice were placed in plastic cages with airtight air filter at the temperature of 18–22°C, humidity of 40–60%, under a light/dark cycle of 10/14 h each day, and had free access to food and water. For the in vivo cell metastatic assay, following anesthesia with ether inhalation, MIR100HG-overexpressing CRC cells LoVo, sh-MIR100HG knockdown CRC cells HCT116 and their control groups were injected (200 µl at the density of 1×10⁶/ml in PBS) into the tail vein of nude mice (4 weeks old male BALB/C nude mice; n=3 for each group). After 4 weeks, mice were euthanized by cervical dislocation, and livers were collected and immediately fixed with 4% formaldehyde at room temperature for 24–48 h. After paraffin embedding, liver tissue was cut into 4–7 µm-thick sections. After hematoxylin-eosin staining at room temperature for 3–5 min, the tumor colonies formed in the livers were detected by light microscopy and quantified using GraphPad Prim 7 (GraphPad Software, Inc.). All experimental procedures and animal studies involving mice were in accordance with the Shanghai General Hospital of Nanjing Medical University Animal Care and Use guidelines. Ethical approval for the animal study was obtained from the Ethics Committee of Shanghai General Hospital of Nanjing Medical University.

Each experiment was performed independently three times. Data were expressed as the means ± standard deviation and were analyzed using SPSS v22.0 software (IBM Corp.). Student's t-test or one-way analysis of variance followed by Student-Newman-Keuls post hoc test were used to analyze the differences in MIR100HG expression among the continuous variables. A χ² test or Fisher's exact test was appropriately used to determine the statistical significance between MIR100HG expression and the patients' clinicopathological characteristics. Kaplan-Meier analysis and the log-rank test were used for the comparison of patients' survival curves. The hazard ratio with 95% confidence interval in the Cox proportional hazards model was calculated to measure the hazard risk of individual factors for disease-free survival (DFS) and overall survival (OS). All graphs were plotted using GraphPad Prism v5.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

A total of 40 paired CRC and normal specimen were randomly selected from the 116 patients to detect MIR100HG expression by RT-qPCR. The results demonstrated that MIR100HG expression was significantly increased in CRC tissues compared with adjacent normal colon mucosae (Fig. 1A). Furthermore, MIR100HG expression was examined in CRC tissues at different stages. The results revealed that MIR100HG expression was significantly higher in advanced-stage (III+IV) CRC tissues compared with low stage (I+II) CRC tissues (Fig. 1B). These results demonstrated that MIR100HG was significantly elevated in CRC and may be associated with an aggressive phenotype in patients with CRC.

Subsequently, the association between MIR100HG expression and TNM stage in the 116 patients with CRC was investigated (19). MIR100HG expression was first evaluated in the 116 paired samples by RT-qPCR. A mean value of 5.55 was calculated. CRC samples with an expression level of MIR100HG ≥5.55 and <5.55 were classified in the high and low expression groups, respectively. Briefly, 75.0% (87/116) of specimens exhibited low MIR100HG expression in normal colorectal mucosa tissues. Furthermore, 25.0% (29/116) of normal colorectal mucosa tissues presented with high MIR100HG expression, whereas 68.1% (79/116) of CRC samples presented with high MIR100HG expression (Table I). In addition, high MIR100HG expression was positively associated with T stage, lymph node metastasis, distant metastasis, AJCC stage and histological differentiation in CRC samples (Table II). These results further indicated that MIR100HG overexpression may serve a role in CRC metastasis and in the prognosis of patients with CRC.

The prognostic value of MIR100HG expression in CRC was evaluated by assessing the DFS and OS of the 116 patients with CRC using Kaplan-Meier analysis and log-rank test. The results demonstrated that the DFS and OS of patients with CRC and high MIR100HG expression were shorter compared with those of patients with CRC and low MIR100HG expression (Fig. 1C and D). These results suggested that high MIR100HG expression may predict a poor prognosis and survival. Furthermore, the results from univariate and multivariate Cox regression analyses demonstrated that MIR100HG expression, AJCC stage, T classification, N classification, M classification and tumor differentiation may be considered as independent prognostic factors for DFS and OS (Tables III and IV). These findings suggested that high MIR100HG expression in CRC tissues may be considered as a prognostic factor for patients with CRC.

To further confirm the clinical significance of MIR100HG expression in CRC, the association between MIR100HG expression and the clinicopathological characteristics of patients with CRC was assessed (Table II). The results demonstrated that high MIR100HG expression was significantly associated with AJCC stage (P=0.024), pathological T stage (P=0.048), N stage (P=0.020), M stage (P=0.023) and tumor differentiation (P=0.035). However, no association was identified between high MIR100HG expression and other clinical parameters, including age, sex, tumor size, tumor location and vascular invasion status (P>0.05; Table II). These findings suggested that high MIR100HG expression may be associated with CRC migration and invasion.

To further investigate the role of MIR100HG in CRC cells, MIR100HG expression in various CRC cell lines (LoVo, DLD-1, RKO, SW48, HCT8 and HCT116) and the normal intestinal mucous epithelium FHC cell line was assessed by RT-qPCR. The results demonstrated that CRC cell lines exhibited higher MIR100HG expression compared with the FHC cell line (Fig. 2A). Subsequently, the HCT116 cell line, which exhibited the highest expression levels of MIR100HG, was selected to establish a cell line with knockdown of MIR100HG. Conversely, the LoVo cell line, which displayed the lowest MIR100HG expression level, was selected to establish an overexpression model for MIR100HG. The results of RT-qPCR analysis demonstrated that the transfections were successful (Fig. 2A).

In vitro Transwell assays were used to assess the function of MIR100HG in CRC progression. MIR100HG overexpression significantly enhanced the migration and invasion of LoVo cells (P<0.001; Fig. 2B), whereas MIR100HG-knockdown significantly inhibited the migration and invasion of HCT116 cells (P<0.001; Fig. 2C). These results suggested that MIR100HG overexpression may promote the aggressive phenotype of CRC cells in vitro.

Subsequently, by injecting CRC cells LoVo and HCT116 into tail veins of nude mice, the impact of MIR100HG on the migratory and invasive capacities of CRC cells was investigated. After 4 weeks, the results demonstrated that higher and lower numbers of liver metastatic colonies were formed in the MIR100HG-overexpression and knockdown groups, respectively, compared with their corresponding controls (P<0.001; Fig. 2D). MIR100HG overexpression may therefore enhance the migratory and invasive capacities of CRC cells in vitro and in vivo.