shRNA‑induced knockdown of the SPERT gene inhibits proliferation and promotes apoptosis of human colorectal cancer RKO cells

  • Authors:
    • Long‑Zhi Zheng
    • Si‑Zeng Chen
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  • Published online on: May 21, 2018     https://doi.org/10.3892/or.2018.6455
  • Pages: 813-822
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Abstract

Colorectal cancer is the third most common type of cancer and the fourth leading cause of cancer‑related deaths worldwide. Although several genes have been identified to contribute to the pathogenesis of colorectal cancer, there are still many genes with unidentified functions in colorectal cancer. This study aimed to investigate the effect of shRNA‑induced knockdown of the SPERT gene on the proliferation and apoptosis of human colorectal cancer RKO cells. SPERT was screened based on the TCGA dataset, and SPERT expression, cell growth, proliferation and apoptosis were detected in shSPERT‑ and shCtrl‑transfected RKO cells. In addition, the SPERT‑related biological pathways were detected using a PathScan® Signaling Antibody Array Kit. We detected lower SPERT expression in shSPERT‑transfected RKO cells than in shCtrl‑transfected cells at both the translational and transcriptional levels (P<0.05), and an MTT assay revealed a clear‑cut decrease in the proliferation of shSPERT‑transfected RKO cells relative to shCtrl‑transfected RKO cells (P<0.01). A Caspase‑Glo® 3/7 assay detected an increase in the caspase‑3/7 activity and the number of apoptotic cells in the shSPERT‑transfected RKO cells than in the shCtrl‑transfected cells (P<0.01), and flow cytometry detected a higher apoptotic rate in the shSPERT‑transfected RKO cells than in the shCtrl‑transfected cells (20.65±0.26 vs. 5.93±0.06%, respectively, P<0.01). Elevated levels of phosphorylated p44/42 MAPK (ERK1/2), Akt, Bad, HSP27, p38 MARK and Chk2, and elevated PARP and caspase‑3 expression levels were detected in shSPERT‑transfected RKO cells compared with the shCtrl‑transfected cells (P<0.05). The results of the current study demonstrated that knockdown of SPERT suppresses colorectal cancer cell growth and promotes apoptosis. SPERT may serve as an oncogene and may be a potential target for the treatment of colorectal cancer.

Introduction

Colorectal cancer (CRC) is the third most common type of cancer and the fourth leading cause of cancer-related deaths worldwide (1). Globally, ~1.2 million patients are newly diagnosed with CRC each year, and >600,000 succumb to this malignancy (2). At the time of their diagnosis, ~25% of patients with CRC already have distal liver and lung metastases, which often means that they have missed the opportunity for radical surgery. In addition, 35–55% of patients with CRC with disease progression may develop distal liver and lung metastases, resulting in a marked decline in survival (35). It is estimated that stage I patients with CRC have a 5-year survival rate of 80–90%, while patients with advanced disease have a 5-year survival rate of <10% (6). Early diagnosis and early treatment are therefore of great significance in improving the survival and prognosis of patients with CRC.

During the past several decades, there has been a stable but slow improvement in the prognosis of CRC (1). However, this could be greatly improved by precision medicine, which facilitates the improvement of disease diagnosis and the development of novel treatments, and helps select the optimal treatment strategy for CRC based on gene mutation detection (7). Based on analyses of cohorts comprised of twins from Sweden, Denmark and Finland, heritable factors are thought to contribute ~35% to CRC (8). To date, a number of genes (including APC, p53 and KRAS) have been identified to be involved in the pathogenesis of CRC; however, there is still a large number of unidentified genes associated with the pathogenesis of CRC (9,10). Although several new hereditary CRC susceptibility genes have been identified, including DNA repair genes, DNA replication genes and genes related to the maintenance of genome stability (11), there is still no compelling evidence to support candidate genes for routine genetic diagnosis (12). Since the development of most CRC cases cannot be explained by known genes, the identification of CRC susceptibility genes is a high priority and is urgently needed.

As a comprehensive and coordinated effort to accelerate our understanding of the molecular basis of cancer, The Cancer Genome Atlas (TCGA) has generated comprehensive, multi-dimensional maps of the key genomic changes in multiple cancer types (13). In the present study, we screened a gene that has been shown to be differentially expressed in the development and progression of CRC, Spermatid-associated (SPERT), based on the TCGA dataset. Subsequently, we examined the effect of SPERT knockdown on the proliferation and apoptosis of the human CRC cell line RKO, and explored the mechanisms underlying these effects.

Materials and methods

Screening of target genes and evaluation of correlations between gene expression and clinicopathological characteristics

The TCGA dataset contains colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) data. When we analyzed the difference between cancer and peri-cancer expression for candidate gene screening, we selected paired samples from RNA-sequencing (RNA-Seq) and RNA-Seq Version 2 (RNA-SeqV2) to analyze the data (50 pairs). There were 41 pairs of colon cancer samples with pathological information and 9 pairs of rectal cancer samples with pathological information. For each gene symbol, the transcript with the highest expression was used for analysis (original reads >50), and transcripts were normalized with the Trimmed Mean of M-values (TMM) method (14). The following criteria were used for the screening of candidate genes: i) genes that have already been reported to be involved in CRC were excluded; ii) multi-transmembrane protein genes were excluded, because multiple transmembrane proteins generally have large molecular weights that are not easy to perform gene manipulation (e.g., knocked down or overexpressed), and most transmembrane proteins play the role of signal transmission and transduction; iii) genes with undefined annotations (such as open reading frames) were excluded; iv) genes with >100 publications in PubMed were excluded intended to ensure the originality and innovation of the genes we have screened. In order to render the analysis data more convincing, the range of analysis was extended to 624 RNA-Seq samples of colorectal cancer from the TCGA dataset after obtaining the differentially expressed SPERT gene in pairs of samples, to further analyze the difference of SPERT gene expression in different pathological stages of colorectal cancer.

Construction of shRNA lentiviral vectors

According to the RNA interference (RNAi) sequence, multiple RNAi target sequences (19–21 nucleotides in length) were designed, using the SPERT gene as a template. Following assessment by the design software, the sequence 5′-ACAAGATCCTACAGGTCTT-3′ was selected as an RNAi target (shSPERT): Forward primer, 5′-TACTTCTCCCCATCCGCCTCC-3′ and reverse primer, 5′-GCGACGTGGTCCTTCTTCACC-3′. The scramble sequence 5′-TTCTCCGAACGTGTCACGT-3′ served as an RNAi negative control (shCtrl): Forward primer, 5′-CCATGATTCCTTCATATTTGC-3′ and reverse primer, 5′-GTAATACGGTTATCCACGCG-3′. The shRNA lentiviral vector targeting the SPERT gene (LV-SPERT-RNAi) and the control lentiviral vector (LV-shRNA-NC) were constructed and packaged by Shanghai Genechem Co., Ltd. (Shanghai, China). The lentiviral vector contained a GFP open reading frame that could quickly and directly display the efficiency of lentiviral infection, which helped us to judge the expression of exogenous knockdown sequences in cultured cells. In addition, there was also a FLAG-tag which could be recognized by the Anti-Flag antibody, so it was convenient to detect and identify the target protein containing FLAG by western blotting.

Detection of SPERT expression in CRC cell lines by RT-qPCR

The human CRC cell lines RKO, SW480 and HCT116 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were screened to determine SPERT expression. Total RNA was isolated from cells using TRIzol® reagent (Shanghai Pufei Biotech Co., Ltd., Shanghai, China) and reverse-transcribed into cDNA. SPERT gene expression (forward primer, 5′-TACTTCTCCCCATCCGCCTCC-3′ and reverse primer, 5′-GCGACGTGGTCCTTCTTCACC-3′) was quantified in these three cells lines using a Roche LightCycler® 480 Real-Time PCR platform (Roche Diagnostics, Indianapolis, IN, USA) with 40 cycles at 95°C for 5 sec and at 60°C for 30 sec, while GAPDH (forward primer, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse primer, 5′-CACCCTGTTGCTGTAGCCAAA-3′) served as the internal control. The relative quantity was calculated using the 2−ΔΔCq method. Detection of SPERT expression was repeated in triplicate in each cell line. Of the three cell lines, the cell line expressing the highest level of SPERT was selected for use in further experiments.

The same RT-qPCR method was used to quantify SPERT mRNA levels after transfection of the chosen cell line, RKO, with the shRNA lentiviral constructs (as described below).

RKO cell culture and transfection

Human CRC RKO cells were incubated in RPMI-1640 medium (Beyotime Institute of Biotechnology, Shanghai, China) supplemented with 10% fetal bovine serum (Ausbian, Adelaide, Australia) at 37°C in an atmosphere of 5% CO2. Log-phase cells were digested with trypsin (Sangon Biotech Co., Ltd., Shanghai, China), and prepared as cell suspensions with complete DMEM (Corning Life Sciences, Manassas, VA, USA) at a density of 3×104-5×104 cells/ml. Cells were then seeded into culture plates and grown until they reached 15–30% confluence. Subsequently, the cells were transfected with shSPERT or shCtrl at a multiplicity of infection (MOI) of 10, in the presence of Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). The medium was changed to RPMI-1640 at 8–12 h post-transfection, and the target gene expression was observed under an Olympus IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan) at 72 h post-transfection.

Western blot analysis

At 48 h post-transfection, total protein was extracted using RIPA lysis buffer and quantified using a BCA protein assay kit (both from Beyotime Institute of Biotechnology). Total protein was then separated by 10% SDS-PAGE, and transferred to PVDF membranes (EMD Millipore, Bedford, MA, USA), which were blocked and incubated at 4°C overnight. The blots were then incubated with mouse anti-FLAG monoclonal antibodies (1:2,000 dilution; cat. no. F1804; Sigma-Aldrich Trading Co. Ltd., Shanghai, China), washed three times (10 min each) with TBST, incubated with the secondary antibody (goat anti-mouse IgG; dilution, 1:2,000; cat. no. sc-2005; Santa Cruz Biotechnology, Co., Ltd., Shanghai, China) for 1 h, and washed three times (10 min each) with TBST. The membranes were viewed with an Odyssey fluorescence imaging system (LI-COR Biosciences, Lincoln, NE, USA), and immunoreactive protein bands were visualized with the Pierce™ ECL Western Blotting Substrate (Pierce, Rockford, IL, USA). ImageJ (version 1.46 release; National Institutes of Health, Bethesda, MD, USA) software was used for quantification of the western blots.

Celigo-based cell-counting and viability assay

Log-phase cells were digested with trypsin, resuspended in complete DMEM and counted. Cells were then seeded into cell culture plates at a density of 2,000 cells/well in a 100-µl system, and three replicate wells were established for each group. Cells were incubated at 37°C in an atmosphere of 5% CO2. Commencing at day 2 post-seeding, the cells were counted on a Celigo® Image Cytometer (Nexcelom Bioscience, Lawrence, MA, USA) daily for 3–5 successive days. The 5-day cell growth curve was plotted.

MTT assay

Log-phase cells were digested with trypsin, resuspended in complete DMEM and counted. Cells were then seeded into cell culture plates, and three replicate wells were established for each group. Cells were incubated at 37°C in an atmosphere of 5% CO2. At day 2 post-seeding, 20 µl MTT solution (5 mg/ml; GenView Corp., Houston, TX, USA) was added to each well and incubated for 4 h, before the culture solution was completely removed, leaving the formazan crystals on the bottom of the well. Subsequently, 100 µl DMSO (Shanghai Shiyi Chemicals Reagent Co., Ltd., Shanghai, China) was added to dissolve the formazan crystals. Following vibration for 2–5 min, the optical density (OD) was measured at 490 nm using a Tecan Infinite M200 Pro Microplate Reader (Tecan Group, Ltd., Männedorf, Switzerland).

Caspase-Glo® 3/7 assay

Cells were seeded into 96-well plates, and incubated at 37°C with 5% CO2 for 3–5 days. Following cell counting, the cell density was adjusted to 1×104 cells/ml at room temperature. shSPERT- and shCtrl-transfected cells were transferred to a new 96-well plate, with 100 µl medium/well. Wells containing blank medium served as the negative controls. Subsequently, 100 µl of Caspase-Glo reagent (Promega Corp., Madison, WI, USA) was added to each well, and the well content was gently mixed with a plate shaker at 100–200 × g/min for 30 min. Following incubation at 18–22°C for 0.5–3 h, the signal intensity was measured.

Flow cytometry

Log-phase cells were harvested, digested with trypsin, and resuspended in complete DMEM. The cell suspension and supernatant were then transferred to a 5-ml centrifuge tube and centrifuged at 300 × g/min for 5 min. Three replicate wells were assigned for each group (≥5×105 cells). The supernatant was then discarded, and the sediment was washed in 4°C precooled D-Hanks' Balanced Salt Solution (pH 7.2–7.4). Subsequently, the cells were washed with 1X binding buffer and centrifuged at 300 × g/min for 3 min. The supernatant was discarded, and the sediment was resuspended in 200 µl of 1X binding buffer, and stained with 10 µl of Annexin V-APC (eBioscience, San Diego, CA, USA) at room temperature in darkness for 10–15 min. To each tube, 500 µl of 1X binding buffer was added, and the cells were then subjected to flow cytometry. All measurements were repeated in triplicate.

Detection of SPERT-related biological pathways

SPERT-related biological pathways were detected with the PathScan® Signaling Antibody Array Kit (Cell Signaling Technology, Inc., Danvers, MA, USA) following the manufacturer's instructions. Briefly, the cells were lysed, and incubated in 75 µl of 1X antibody mixture on a horizontal shaker for 1 h. The antibody mixture was removed, and the cells were washed four times with 1X wash buffer on a horizontal shaker (5 min each time). Then, the cells were incubated in 75 µl of 1X HRP-conjugated streptavidin on a horizontal shaker for 0.5 h. Following removal of HRP-conjugated streptavidin, the cells were washed four times with 1X wash buffer on a horizontal shaker (5 min each time). Finally, the cell slides were immersed in 1X wash buffer, visualized and analyzed. Cell slides were immersed in 1X washing buffer. Exposure buffer was formulated with 9 ml ddH20, 0.5 ml LumiGLO (Cell Signaling Technology, Danvers, USA) and 0.5 ml peroxide, and the cell slides were incubated with exposure buffer, then exposed within 1–2 sec using a visualization system (Clinx ChemiScope 5300; Clinx Science Instruments, Co., Ltd., Shanghai, China). The resulting images were analyzed using the visualization system aforementioned, and raw data were further analyzed manually.

Statistical analysis

All measurement data are presented as the mean ± standard deviation (SD). Differences in the means between groups were tested for statistical significance with the Student's t-test, and comparison of proportions was conducted with a Chi-square test. The association of SPERT expression with clinicopathological characteristics was examined with a Mann-Whitney U test. All statistical analyses were performed using SPSS statistical software version 17.0 (SPSS, Inc., Chicago, IL, USA), and a P-value of <0.05 was considered to indicate a statistically significant difference.

Results

Associations of SPERT expression with clinicopathological characteristics

All data were obtained from highly reliable genetic disease databases, and the gene list was finally obtained following random condensation (Table I). In fact, we screened ~6,000 disease-related genes in different tumor types, but to ensure the originality and innovation of the candidate genes, we selected the gene SPERT through the four screening conditions aforementioned. We first analyzed high-throughput RNA-sequencing data of the colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) cohorts of TCGA, and found that SPERT expression was significantly increased in CRC tissues compared with peri-cancer tissues (P<0.0001) (Table II and Fig. 1). Then, we analyzed the relationship between SPERT expression and various clinicopathological parameters of patients with CRC. The Mann-Whitney U test revealed that SPERT expression was associated with N, M and pathological stages in patients with CRC (all P<0.01) (Table III and Fig. 2).

Table I.

Gene list for analysis based on The Cancer Genome Atlas dataset.

Table I.

Gene list for analysis based on The Cancer Genome Atlas dataset.

Gene IDGene nameNo. of transcriptsPublications in PubMedNovoseek disease relationships for the geneMalaCards disease relationships for the gene
220082SPERT31300

Table II.

SPERT expression in colorectal cancer and peri-cancer specimens in The Cancer Genome Atlas dataset.

Table II.

SPERT expression in colorectal cancer and peri-cancer specimens in The Cancer Genome Atlas dataset.

No. of samples

IDGene symbolFCP-valueTotalWith unchanged SPERT expressionWith upregulated SPERT expressionWith downregulated SPERT expression
220082SPERT100.9752.94E-56505450

[i] Fold-change (FC) is calculated as SPERT expression in colorectal cancer specimens divided by SPERT expression in peri-cancer specimens.

Table III.

Associations of SPERT expression with clinicopathological features in human colorectal cancer patients.

Table III.

Associations of SPERT expression with clinicopathological features in human colorectal cancer patients.

SPERT expression, n

Clinicopathological featuresLowHighTotal, nP-value
N
  N0197155352<0.001
  N1/2110155265
Total307310617
M
  M0243215458<0.001
  M1  27  61  88
Total270276546
Pathological stage
  Stage I  56  491050.001
  Stage II131  98229
  Stage III  87  92179
  Stage IV  28  62  90
Total302301603

[i] N, local lymph node involvement; M, distal metastasis.

SPERT expression in various human CRC cell lines

In order to fully demonstrate the effect of SPERT on CRC cells, we determined the cell line with the highest content of SPERT and selected this for use in subsequent experiments. RT-qPCR was used to quantify SPERT expression in the different cell lines (RKO, SW480 and HCT116 cells), and the results revealed that SPERT was most prominent in RKO cells, as revealed in Fig. 3. Therefore, RKO cells were selected for the subsequent experiments.

SPERT expression following shRNA transfection

Fluorescence microscopy revealed that >80% of RKO cells were successfully infected with LV-SPERT-RNAi or LV-shRNA-NC at 72 h post-transfection, indicating a high success rate of lentiviral vector infection (Fig. 4). RT-qPCR detected decreased SPERT mRNA expression in shSPERT-transfected RKO cells than in shCtrl-transfected cells (0.437±0.040 vs. 1.001±0.046, respectively, P<0.01), and the transfection efficiency was 90.3% (Fig. 5A). Consistently, western blotting detected a reduction of 82.25±0.25% in SPERT expression in the shSPERT-transfected RKO cells than in the shCtrl-transfected cells (P<0.01) (Fig. 5B and C). These results confirmed that RNAi could effectively reduce the endogenous expression of the target gene.

Effect of SPERT knockdown on RKO cell growth and proliferation

shSPERT-transfected RKO cells exhibited a decline in number on days 1–5 post-transfection, while the number of shCtrl-transfected cells increased, indicating that shRNA-induced knockdown of SPERT inhibited RKO cell proliferation (Fig. 6A and B). The MTT assay revealed a clear decrease in the proliferation of shSPERT-transfected RKO cells relative to shCtrl-transfected RKO cells (P<0.01), demonstrating that shRNA-induced knockdown of SPERT suppressed RKO cell proliferation (Fig. 6C).

Effect of SPERT knockdown on RKO cell apoptosis

The Caspase-Glo 3/7 assay detected an increase in the caspase-3/7 activity and the number of apoptotic cells in the shSPERT-transfected RKO cells than in the shCtrl-transfected cells (P<0.05) (Fig. 7C). Additionally, flow cytometry detected a higher apoptotic rate in the shSPERT-transfected RKO cells than in the shCtrl-transfected cells (20.65±0.26 vs. 5.93±0.06%, respectively, P<0.05) (Fig. 7A and B). These findings demonstrated that shRNA-induced knockdown of SPERT promoted RKO cell apoptosis.

SPERT-related biological pathways

To evaluate the SPERT-associated biological pathways, we used the PathScan® Signaling Antibody Array Kit from Cell Signaling Technology. The PathScan Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) uses glass slides as the planar surface and is based upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 19 signaling molecules that are involved in the regulation of the stress response and apoptosis. Target-specific capture antibodies are spotted in duplicate on nitrocellulose-coated glass slides. Each kit contains two slides allowing for the interrogation of 32 different samples and the generation of 608 data points in a single experiment. As shown in Fig. 8, the levels of phosphorylated p44/42 MAPK (ERK1/2), Akt, Bad, HSP27, p38 MARK and Chk2, as well as PARP and caspase-3 expression were elevated in shSPERT-transfected RKO cells compared with shCtrl-transfected cells (P<0.05, P<0.01), indicating that shRNA-induced knockdown of SPERT may suppress RKO cell proliferation and promote cell apoptosis via these proteins.

Discussion

CRC is one of the most common gastrointestinal cancers (1). Currently, surgery remains the primary treatment for CRC (15), and neoadjuvant and adjuvant radiochemotherapy have been shown to be effective in preventing post-surgical recurrence and improving the survival rate in patients with CRC (16,17). In addition, the introduction of targeted therapy has been shown to increase the median survival time from 3.6–6 to 24–28 months in patients with metastatic CRC (1820).

CRC is a genetically heterogeneous disease (21). To date, the roles of APC, p53 and KRAS in the pathogenesis of CRC have been demonstrated; however, the contribution of other genes to the pathogenesis of CRC remains unclear (9,10). Systems biology studies have shown gene instability, microsatellite instability and methylation abnormalities in CRC (22). However, TCGA datasets provide valuable bases for clinical diagnosis, treatment and precision medicine for CRC (2325).

In this study, the colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) datasets in the TCGA were used, and RNA-Seq and RNA-SeqV2 were employed to analyze gene expression in paired COAD and READ samples. SPERT was selected from the screening. There was a significant difference in SPERT expression between cancer and peri-cancer specimens in patients with CRC, and SPERT gene expression was significantly associated with lymph node metastasis, distal metastasis and pathological stages. Our data indicated that SPERT was involved in the progression of CRC, and may serve as an indicator for clinicopathological staging in patients with CRC.

In the present study, knockdown of SPERT was found to suppress human CRC RKO cell proliferation and promote cell apoptosis. It is therefore hypothesized that SPERT overexpression may be involved in the development, progression and metastasis of CRC. SPERT, which is located on human chromosome 13q14.13, encodes the SPERT protein (also known as CBY2 and Nurit), which contains 338 amino acids and has a molecular weight of 51,570 Da. SPERT belongs to the Chibby protein family and has a quaternary structure of homodimers. It is a highly conserved gene in mammals, it is expressed in humans, Rhesus monkeys, mice and rats, but it is not expressed in Drosophila melanogaster or Caenorhabditis elegans (26). However, the function of the SPERT protein has remained unknown until now. It is reported that the SPERT protein contains a leucine zipper motif and two coiled-coil regions, and the leucine zipper motif is involved in homodimerization or oligomerization, which is associated with the regulation of oncogene expression (27). It is known that SPERT proteins may form dimers in mice, and the C-terminal coiled-coil domain mediates protein dimerization, which may link with Nek1 kinase (26,28). Nek1, a member of the NIMA-related kinase family, is aberrantly expressed in multiple cancer types, and aberrant Nek1 expression was reported to cause abnormal regulation of the entire cell cycle and abnormal cell proliferation, thereby resulting in cancer development and progression (29,30). Further studies to examine the interplay between the SPERT protein and Nek1 appear justified.

To date, the exact role of SPERT in CRC remains unclear. In this study, the PathScan® Signaling Antibody Array Kit was employed to detect changes in key signaling molecules involved in stress and apoptosis signaling pathways. Our findings revealed upregulation of phosphorylated p44/42 MAPK (ERK1/2), Akt, Bad, HSP27, p38 MARK and Chk2, in addition to elevated PARP and Caspase-3 expression in shSPERT-transfected RKO cells, indicating that these proteins are involved in the progression of CRC, in which MAPK and PI3K/Akt signaling plays a predominant role. Bad is the downstream target of the MAPK and PI3K/Akt signaling pathways. MAPK regulates the function of the proapoptotic protein Bad through the phosphorylation of serine 112, while PI3K/Akt regulates its function by phosphorylation of serine 136. The dissociation of phosphorylated Bad from anti-apoptotic factor Bcl-2 leads to an increase in the activity of free Bcl-2, which inhibits apoptosis (31). Caspase-3 and HSP27 are the downstream target proteins of the p38 MAPK/HSP27 signaling pathway, and the most important substrate of caspase-3 is the poly(ADP-ribose) polymerase PARP. PARP cleavage is closely related to cell apoptosis and is one of the markers of cell apoptosis and caspase activation (32). MAP kinase cascades mediate Hsp27 phosphorylation, MAPKAPK2 and MAPKAPK3 regulate the function of the heat shock protein 27 (HSP27) by phosphorylating it on distinct sites, Ser-15, Ser-78 and Ser-82. Downstream, HSP27 also blocks the activation of caspase-9 and subsequent activation of caspase-3, thereby inhibiting the remaining of the proteolytic caspase cascade (33,34).

It has been revealed that both the MAPK and PI3K/Akt pathways, which are associated with cancer initiation and progression, serve critical roles in CRC progression and greatly contribute to intestinal epithelial cell differentiation (3539). Ras mutations have been detected in 35–45% of patients with CRC (4043), while 9–11% of patients with CRC harbor BRAF mutations (4446). KRAS and BRAF have been demonstrated to contribute to CRC progression (43,45). ERK1 and ERK2, which are located downstream of the Ras oncogene protein, bind to cell-surface receptor tyrosine kinases and G-protein-coupled receptors, resulting in Ras and BRAF activation (47) and subsequent MEK1/MEK2 and ERK1/ERK2 phosphorylation (48). The Ras/Raf/MEK/ERK cascade has been revealed to be involved in the mediation of cell growth signals, cell survival and cancer invasion (49), and is strongly associated with the differentiation, pathological stage and prognosis of cancers, which may be used to guide the selection of targeted drugs for use in clinical cancer therapies (50). Our findings revealed that knockdown of SPERT significantly increased the levels of phosphorylated p44/42 MAPK (ERK1/2) and p38 MAPK; this may explain why SPERT knockdown suppressed RKO cell proliferation and promoted cell apoptosis. Further studies are required to investigate the detailed mechanisms underlying the SPERT-mediated effects on the MAPK and PI3K/Akt signaling pathways.

In summary, the results of the present study demonstrated that SPERT expression was significantly upregulated in CRC specimens, and knockdown of SPERT suppressed CRC cell growth and promoted apoptosis via the MAPK and PI3K/Akt signaling pathways. SPERT may serve as an oncogene for CRC, and may be a promising biomarker for predicting the poor prognosis of CRC. In addition, SPERT may be a potential target for the treatment of CRC.

Acknowledgements

Not applicable.

Funding

The present study was supported in part by a grant from the National Natural Science Foundation of China (no. 81272465).

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

SZC and LZZ conceived and designed the study. LZZ performed the experiments and wrote the paper. SZC reviewed and edited the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors state that they have no competing interests.

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August-2018
Volume 40 Issue 2

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Zheng LZ and Zheng LZ: shRNA‑induced knockdown of the SPERT gene inhibits proliferation and promotes apoptosis of human colorectal cancer RKO cells. Oncol Rep 40: 813-822, 2018
APA
Zheng, L., & Zheng, L. (2018). shRNA‑induced knockdown of the SPERT gene inhibits proliferation and promotes apoptosis of human colorectal cancer RKO cells. Oncology Reports, 40, 813-822. https://doi.org/10.3892/or.2018.6455
MLA
Zheng, L., Chen, S."shRNA‑induced knockdown of the SPERT gene inhibits proliferation and promotes apoptosis of human colorectal cancer RKO cells". Oncology Reports 40.2 (2018): 813-822.
Chicago
Zheng, L., Chen, S."shRNA‑induced knockdown of the SPERT gene inhibits proliferation and promotes apoptosis of human colorectal cancer RKO cells". Oncology Reports 40, no. 2 (2018): 813-822. https://doi.org/10.3892/or.2018.6455