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Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, with the mortality increasing steadily over the last decade. Myosin VI (MYO6) expression is found to be elevated in some types of human carcinoma cell types, suggesting that it may be a sensitive biomarker for the diagnosis and follow-up. In this study, we first used the Oncomine database to explore the expression of MYO6 in CRC tissues, and then constructed a plasmid of RNA interference targeting MYO6 gene. After transfection of lentivirus targeting MYO6 into SW1116 cells, cell viability and proliferation were measured with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay. Cell cycle distribution was assayed by flow cytometry and apoptosis was evaluated by Annexin V. MYO6 expression was detected by quantitative real-time polymerase chain reaction and western blot analysis. It was found that MYO6 mRNA was upregulated in CRC tissues using data mining of public Oncomine microarray datasets. Depletion of MYO6 significantly inhibited cell proliferation and colony formation. In addition, knockdown of MYO6 slightly arrested cell cycle in G0/G1 phase, but remarkably increased the proportion of the sub-G1 phase of cell with the increase of apoptotic cells. These results suggest that MYO6 may promote cell growth and may be used as a potential target for anticancer therapy of CRC.
Despite a remarkable decline in mortality over the past decade, colorectal cancer (CRC) remains one of the most common cancers worldwide . It is the third most common cancer and the third leading cause of cancer-related death in men and women, involving 136,830 diagnosed patients and 50,310 deaths in the USA in 2014 . The incidence and mortality of CRC in China are lower than those in western countries, though they have been gradually increasing in recent years [3,4]. Even after excision, the 2-year recurrence rate is as high as 75%, and the 5-year survival rate is only 26.8% [5,6]. Therefore, developing new therapeutic drugs for CRC remains a challenge. Colorectal carcinogenesis involves multistep molecular processes with sequential activation of oncogenes and inactivation of tumor suppressor genes. More and more oncogenes and tumor suppressor genes have been identified during the course of CRC in recent years.
Most of myosins in the myosin superfamily are defined as actin-based motor molecules that mainly participate in cellular processes based on actin and move on the actin filaments driven by adenosine triphosphate (ATP) hydrolysis [7,8]. Unconventional myosins have been implicated in F-actin-mediated cellular functions such as cell motility, vesicular trafficking, as well as intracellular transport of macromolecules. And members of myosins also play an important role in signal transduction [9–11]. Myosin VI (MYO6) is a unique member of the unconventional myosin family, and a reverse-direction motor protein that moves toward the minus-end of actin filaments . The MYO6 protein has a motor domain containing an ATP-actin-binding site and a globular tail that interacts with other proteins. An interesting link between MYO6 and human cancers has been described in previous studies. Yoshida et al.  found that there was a positive correlation between aggressive ovarian cancer and MYO6 overexpression. Some recent studies demonstrated that MYO6 acted as a novel oncogene in human prostate cancer [14–17]. It was found that knockdown of MYO6 gene by siRNA inhibited prostate tumor growth and metastasis by decreasing the secretion of prostate specific antigen and the vascular endothelial growth factor. Similar results were also obtained in human lung cancer cells .
However, no study on MYO6 expression in CRC has been reported so far. In this study, we first observed the overexpression of MYO6 in CRC by the data mining from the public Oncomine microarray database. To investigate the role of MYO6 in CRC cells, we silenced MYO6 gene in CRC cell line SW1116 using lentivirus-mediated gene delivery method, and analyzed the biological function after the knockdown of MYO6 in vitro. Our results suggest that inhibition of MYO6 by siRNA might be a potential therapeutic strategy for the treatment of CRC.
To determine the expression level of MYO6 in CRC, five datasets (Sabates-Bellver Colon, Skrzypczak Colorectal, Hong Colorectal, Kaiser Colon, and TCGA Colorectal) in Oncomine database (https://www.oncomine.org) were selected . The expression difference of MYO6 gene was compared between the CRC tissues and normal tissues. The log-transformed and normalized expression values of MYO6 were extracted, analyzed, and read from the bar chart.
Human colon cancer SW1116 cells (Cat No. TCHu 174) and human embryonic kidney 293T cells (Cat No. GNHu17) were obtained from the Cell Bank of Chinese Academy of Science (Shanghai, China). SW1116 and 293T cells were maintained in Dulbecco's modified Eagle's medium (Hyclone, Logan, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Cromwell, USA), respectively. Both cell lines were cultured at 37°C in a 5% CO2 humidified incubator.
The short hairpin RNA (shRNA) sequences specifically targeting the nucleotides of human MYO6 gene (NM_004999) were designed through Sigma-Aldrich web-based tool (http://www.sigmaaldrich.com/). BLAST homology search was used to verify the 100% similarity of oligonucleotides and the effective cDNA sequences of MYO6 genes from various species. The non-silencing shRNA was used as a control that has no homology with any gene in human as determined by screening with NCBI RefSeq. The shRNA sequences were as follows—MYO6 shRNA (S1): 5′-GTGAATCCAGAGATAAGTTTACTCGAGTAAACTTATCTCTGGATTCACTTTTT-3′, MYO6 shRNA (S2): 5′-CCAGATTTAACCATTCCATAACTCGAGTTATGGAATGGTTAAATCTGGTTTTTT-3′ and the negative control shRNA was 5′-GCGGAGGGTTTGAAAGAATATCTCGAGATATTCTTTCAAACCCTCCGCTTTTTT-3′. The double-stranded DNA fragments were formed in the annealing reaction system. The pFH-L vector (Shanghai Hollybio, Shanghai, China) was linearized by EcoRI and BamHI restriction enzyme digestion. Pure linearized vector fragments and double-stranded DNA fragments were collected and combined together during a 16 h reaction. Each DNA was used to transform the Escherichia coli strain DH5α and purified with a plasmid purification kit (Qiagen, Valencia, USA). The ligation product was confirmed by polymerase chain reaction (PCR) and sequencing. The generated plasmids were named as pFH-Lv-shMYO6 (S1), pFH-Lv-shMYO6 (S2), or Lv-shCon. Recombinant lentiviral vectors and packaging pHelper plasmids (pVSVG-I and pCMVΔR8.92) (Shanghai Hollybio) were cotransfected into 293T cells. Supernatants containing lentivirus expressing shMYO6(S1), shMYO6(S2), or control shRNA (shCon) were harvested 48 h after transfection. Then, the lentiviruses were puriﬁed via ultracentrifugation, and the viral titer was determined by counting the green fluorescent protein (GFP)-positive cells. Viral titer was determined by the method of end point dilution through counting the numbers of infected GFP-positive cells at ×100 magnification under a fluorescence microscope (Olympus, Tokyo, Japan) 3 days after infection to 293T cells. Titer (IU/ml) = the numbers of green fluorescent cells × dilution factor/volume of virus solution . For lentivirus infection, SW1116 cells (40,000 cells/well) were seeded in six-well plates and transduced with shMYO6(S1), shMYO6(S2), or shCon at a multiplicity of infection of 25. Infection efficiency was determined through counting the GFP-positive cells under a fluorescence microscope 72 h after infection, and the knockdown efficiency of MYO6 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis.
After 5 days of lentivirus infection, total cellular RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, USA). RNA quantity and quality were determined by spectrophotometry and agarose gel electrophoresis, respectively. Extracted total RNA samples (1 μg) was reverse transcribed into cDNA with oligod(T) primers according to the manufacturer's protocol for reverse transcription PCR (Promega, Fitchburg, USA). qRT-PCR was carried out by using the GXD kit iqSYBR Green (BioRad, Hercules, USA) according to the manufacturer's instructions with the CFX96 Touch™ Real-Time PCR Detection System (BioRad). Primer sets used were as follows: for β-actin, 5′-GTGGACATCCGCAAAGAC-3′ (forward), 5′-AAAGGGTGTAACGCAACTA-3′ (reverse) and for MYO6, 5′-AATCACTGGCTCACATGCAG-3′ (forward), 5′-AATGCGAGGTTTGTGTCTCC-3′ (reverse). The cycling conditions were as follows: initial denaturation 95°C for 60 s, 95°C for 5 s, 60°C for 20 s, 40 cycles. Data analysis was performed using the method.
After 7 days of lentivirus infection, cells were harvested and lysed in ice-cold lysis buffer [50 mM Tris, 2% sodium dodecyl sulphate (SDS), 5% glycerinum, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, pH 6.8]. Total protein concentrations of the lysate were determined using BCA Protein Assay Kit (Pierce Biotechnology, Rockford, USA). A total of 30 μg protein in each lane was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis gel and transferred to polyvinylidene fluoride membrane (BioRad), and incubated with rabbit anti-MYO6 (Sigma, St Louis, USA) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 40,000; Proteintech, Rosemont, USA) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1 : 5000; Santa Cruz Biotechnology, Santa Cruz, USA). Signals were detected using the ECL-PLUS Kit (Amersham, Piscataway, USA) according to the manufacturer's protocol. GAPDH was used as the internal standard.
The effect of MYO6 on cell viability was analyzed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on growth curves of SW1116 cells in vitro. Briefly, cells were seeded in 96-well plates at a concentration of 2500 cells/well in 200 μl/well after 3 days of lentivirus infection. Cells were then further cultured in this manner for 1–5 days. Four hours before the termination of culture, MTT (5 mg/ml; Sigma) was added at a volume of 20 μl/well. Afterward, the acidic isopropanol (0.01 M HCl, 10% SDS and 5% isopropanol) was added at a volume of 100 μl/well and incubated overnight at 37°C. The absorbance at 595 nm of each well was determined using the Epoch Microplate Spectrophotometer (BioTek, Shoreline, USA).
After 3 days of lentivirus infection, SW1116 cells (500 cells/well) were seeded in 6-well plates and incubated for 10 days to form normal colonies. The media were replaced every 3 days. Then the cells were fixed with paraformaldehyde for 30 min at room temperature. The fixed cells were washed twice with phosphate buffered saline (PBS), stained with 1% crystal violet (Beyotime, Shanghai, China) for 10 min, washed with ddH2O and air dried. The total number of colonies that contain >50 cells was counted under a light microscope. Image analysis was conducted using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, USA).
After 4 days of lentivirus infection, SW1116 cells were seeded in 6-cm dishes at a density of 80,000 cells/dish and cultured for 40 h. Cells were then released by digestion with trypsin and harvested. After centrifugation, the cell pellet was washed twice with precooled PBS and fixed with precooled 70% ethanol overnight at 4°C. Then cells were washed twice with precooled PBS and resuspended in PBS containing 10% FBS, filtered through a 400-mesh sieve, and stained with propidium iodide solution (PI, 50 μg/ml, 100 μg/ml RNase in PBS) at 37°C in the dark for 30 min. Finally, cells were analyzed for the cell cycle phase by FACScan (Beckman Coulter, Brea, USA). The percentages of cells at various phases of the cell cycle were analyzed using the ModFit (Verity Software House, Topsham, USA) software.
To determine the apoptosis of SW1116 cells, cells were subject to two different treatments [shCon and shMYO6(S2)]. Infected by lentivirus for 72h, thereafter, the cells were resuspended and reseeded in 6-cm dishes at a density of 80,000 cells/dish. After 24 h, the cells were collected, washed, and subjected to Annexin V-APC/7-AAD double staining according to the manufacture's instruction (KeyGEN Biotech, Nanjing, China). Flow cytometry analysis was performed on Cell Quest FACS system (BD Biosciences, San Jose, USA) to assess the apoptotic and viable cell fractions.
The results were expressed as the mean ± SD. Differences between the two groups were assessed using a two-tailed t-test. The P< 0.05 was defined as a significant difference. Statistical analysis was performed with GraphPad Prism 6 (GraphPad Software, Inc., San Diego, USA).
By using the Oncomine database mining, we examined and analyzed the expression level of MYO6 in CRC tissues. As shown in Fig. 1, all of the five datasets showed that MYO6 expression is significantly elevated in CRC compared with the normal tissues. Sabates-Bellver Colon dataset  showed MYO6 expression in colon adenoma (n = 25), and rectal adenoma (n = 7) was higher than that in the normal tissues (P = 8.52 × 10−8 and P = 3.62 × 10−4, respectively) (Fig. 1A). It was also found that MYO6 expression was significantly increased in Skrzypczak Colorectal dataset (n = 45, P = 2.80 × 10−6) (Fig. 1B) . Another independent Hong Colorectal dataset  revealed that MYO6 was upregulated in colorectal carcinoma (n = 70, P = 0.004) compared with normal colon tissues (Fig. 1C).
Similarly, TCGA Colorectal dataset (http://tcga-data.nci.nih.gov/tcga/) showed that the expression level of MYO6 was also significantly higher in most CRC types than that in normal tissues (Fig. 1D), including cecum adenocarcinoma (n = 22, P = 1.35 × 10−5), colon adenocarcinoma (n = 101, P = 1.45 × 10−6), colon mucinous adenocarcinoma (n = 22, P = 1.41 × 10−5), rectal adenocarcinoma (n = 60, P = 9.84 × 10−10), and rectal mucinous adenocarcinoma (n = 6, P = 0.004), except rectosigmoid adenocarcinoma (n = 3) and rectosigmoid mucinous adenocarcinoma (n = 1) based on small sample numbers. Moreover, the result of Kaiser Colon dataset was similar to the above-mentioned findings, revealing that MYO6 was significantly upregulated in rectal mucinous adenocarcinoma (n = 4) compared with normal colon tissues and slightly increased in other CRC types, including cecum adenocarcinoma (n = 17), colon adenocarcinoma (n = 41), colon mucinous adenocarcinoma (n = 13), colon signet ring cell adenocarcinoma (n = 2), colon small cell carcinoma (n = 2), rectal adenocarcinoma (n = 8), rectal mucinous adenocarcinoma (n = 4), rectosigmoid adenocarcinoma (n = 10), and rectosigmoid mucinous adenocarcinoma (n = 2), except rectal signet ring cell adenocarcinoma (n = 1) (Fig. 1E) . These data suggest that MYO6 plays a potential role in colon carcinogenesis.
For functional study of MYO6 in CRC cells, the MYO6 gene was knocked down in SW1116 cells using a lentivirus-delivered shRNA specifically targeting human MYO6 [shMYO6 (S1)/(S2)]. The infection efficacy was verified by the ratio of GFP-positive cells over 80% (Fig. 2A). To examine the knockdown efficacy, mRNA and protein in the SW1116 cells from different treatment groups were extracted for qRT-PCR and western blot analysis. As shown in Fig. 2B, MYO6 mRNA levels in groups transferred with shMYO6(S1) and shMYO6(S2) were significantly lower than those in shCon group. The knockdown efficacy of MYO6 by shMYO6(S1) and shMYO6(S2) was 72% and 79.4%, respectively, suggesting that shRNA knockdown was specific and the loss-of-function outcome was not possibly caused by off-target effect. Western blot analysis also showed that MYO6 protein was reduced following MYO6 knockdown (Fig. 2C). These results suggested that MYO6 shRNA could significantly downregulate MYO6 expression in SW1116 cells.
MTT test showed that the OD value that represented the cell proliferation rate was increased from 1 to 3.2 in shCon group within 5 days after lentivirus transduction, while cell proliferation in shMYO6(S1) or shMYO6(S2) group was relatively slow, increasing from 1 to 2.6 in shMYO6(S1) group and from 1 to 1.2 in shMYO6(S2) group (P< 0.001; Fig. 3A) respectively, indicating that the proliferation of SW1116 cells was significantly impeded following MYO6 knockdown.
In addition, the colony formation capacity of SW1116 cells was examined after MYO6 knockdown. Representative photographs of the number of cells per colony and the number of colonies in per well were shown in Fig. 3B. Both cell and colony numbers were much smaller in shMYO6(S1) and shMYO6(S2) groups than those in shCon group. There were ~109 ± 4 colonies in shCon group vs. 78 ± 2 colonies in shMYO6(S1) group, indicating that the colony number was reduced by nearly 30% (P< 0.005; Fig. 3C). The colony number (22 ± 3) was reduced by ~80% in shMYO6(S2) group, compared with that in the shCon group (P< 0.001; Fig. 3C).
To investigate whether cell cycle arrest contributed to cell growth inhibition, the cell cycle distribution in SW1116 cells was analyzed using fluorescence-activated cell sorting (FACS) (Fig. 4A). The results showed that knockdown of MYO6 increased the percentage of G0/G1-phase cells in shMYO6(S2) group (54.50% ± 0.30% vs. 47.95% ± 0.13%; P< 0.001) and decreased the percentage in G2/M-phase cells (22.17% ± 0.52% vs. 28.59% ± 0.20%; P< 0.001) in comparison with the shCon group (Fig. 4B). Although the difference in G0/G1 and G2/M phases was small between shMYO6(S2) and shCon groups, it was worth noting that the shMYO6(S2) group exhibited a higher proportion of sub-G1 phase than the shCon group (3.20% ± 0.08% vs. 1.30% ± 0.12%; P< 0.001), suggesting that MYO6 silencing induced cell apoptosis.
A distinct sub-G1 peak was observed in MYO6 knockdown cells. To further confirm the effect of MYO6 on cell apoptosis, Annexin V-APC/7-AAD double labeling of SW1116 cells was conducted to assess whether MYO6 knockdown cells really underwent apoptosis (Fig. 5A). As shown in Fig. 5B, the percentage of early apoptotic cells was increased from 5.7% to 19.6% in shMYO6(S2) group. Likewise, compared with control group shCon, the percentage of late apoptotic cells in SW1116 cells treated with shMYO6(S2) was increased from 2.1% to 24.0%. The above results suggested that the inhibition of MYO6 gene induced a strong proapoptotic effect in human CRC SW1116 cells.
Oncomine is the biggest cancer microarray database currently available, as well as a web-based data mining platform to dig the cancer gene information . In the present study, we used data mining of five independent microarray datasets (Sabates-Bellver Colon, Skrzypczak Colorectal, Hong Colorectal, Kaiser Colon, and TCGA Colorectal) in the Oncomine database and proved the overexpression of MYO6 in CRC. The similar correlation has also been reported between MYO6 expression and other cancers such as prostate cancer [25,26], lung adenocarcinoma , hepatocellular carcinoma , and centroblastic lymphoma . In addition, Yoshida et al.  found that elevation of MYO6 level was positively correlated with the aggressiveness of ovarian cancer both in vitro and in vivo, implying that MYO6 plays a potential role in the carcinogenesis or malignant transformation of tumors, and is a valuable biomarker for ovarian cancer.
MYO6 plays an important role in cellular transport and anchoring, and also participates in different cellular processes such as cell endocytosis, migration, cell division, and cytokinesis. At present, most of the research results showed that high MYO6 expression could accelerate tumor cell migration. In prostate cancer cell line LNCaP, knockdown of MYO6 impaired cell migration and anchorage independent growth ability by regulating the expression of tumor suppressor genes TXNIP (thioredoxin-interacting protein 1), but did not affect the proliferation rate of cells in the culture medium . Consistent with this, Yoshida et al.  found that silencing the MYO6 expression could hinder the initial migration process in ovarian cancer. Likewise, MYO6 expression was positively correlated with the expression of epithelial-mesenchymal transition markers E-cadherin and β-catenin in human renal cell carcinoma and border cells that behave like cancer cells [30,31]. However, there are few reports showing the potential biological functions of MYO6 in tumor cell proliferation especially in CRC.
Detection of cell proliferation and apoptosis is the most common way to evaluate and measure the tumor responses to oncogenes or tumor suppressor genes. Herein, we found that knockdown of MYO6 by small interfering RNA inhibited the growth and proliferation of CRC SW1116 cells, and also arrested cell cycle progression to some extent. Meanwhile, knockdown of MYO6 significantly increased the proportion of sub-G1 cells and led to cell apoptosis in SW1116 cells. Sub-G1 phase cells are usually considered to be the result of apoptotic DNA fragmentation because DNA is resolved by endonucleases in cells during apoptosis . Generally speaking, the DNA content in apoptotic cells is less than normal diploid cells in the nucleus, leading to a sub-G1 peak, which can be used to confirm the relative number of apoptotic cells in DNA histogram of FACS [33,34]. Consistent with these findings, Jung et al.  demonstrated that silencing of MYO6 could attenuate activation of p53 and impair the integrity of Golgi complex, thus enhancing the susceptibility of MYO6-deficient cells to DNA damage-induced apoptosis.
Collectively, this is the first study to report that MYO6 is a novel regulator of proliferation and apoptosis in CRC. Overexpression of MYO6 mediates tumor cell survival through enhancing cell growth and inhibiting apoptosis. Although the precise mechanism underlying the effect of MYO6 on CRC cell proliferation and apoptosis needs to be further defined, this study provides a basic conception for recognizing novel target molecules that may control the development and progression of human CRC. Further research is required to provide more convincing evidence to support the possibility of using MYO6 as a new and potential therapeutic target against CRC.
This work was supported by the grants from the National Natural Science Foundation of China (No. 81372311) and Shanghai Health and Family Planning Commission Research Project (No. 20134024).