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Abstract
Objective: This article aims to study the effect of phosphate and tension homolog deleted on chromosome ten (PTEN) knockdown on colon cancer progression and macrophage polarization in the cancer environment. Materials and Methods and Results: The expression of PTEN in colon cancer tissues and colon cancer cells was significantly lower than in precancerous tissues or CCD-18Co cells, and the decrease was most evident in SW620 cells. The expressions of phosphate (p)-p38, c-Jun N-terminal kinase (JNK), activator protein 1 (AP-1), B-cell lymphoma-2 (Bcl-2) protein in colon cancer tissues and cells were significantly higher than in precancerous tissues or CCD-18Co cells (P-values < 0.05). Bcl-2-associated X (Bax) and Caspase-3 expressions in colon cancer tissues and cells were significantly lower than in precancerous tissues or CCD-18Co cells (P-values < 0.05). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was applied to measure cell viability. Transwell evaluated the cell migration and invasion ability. Si-PTEN improved the proliferation, migration, and invasion of SW620 cells (P-values < 0.05). The expression levels of arginase-1 (Arg-1), CD163, CD206 in colon cancer tissues were significantly higher than in precancerous tissues (P-values < 0.05). The cell cycle, the number of M1 and M2 double-positive cells were assessed by flow cytometry. Si-PTEN reduced the expression of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and inducible nitric oxide synthase (iNOS), which upregulated the expression of Arg-1, CD206, CD163, p-p38, JNK, and AP-1 (P-values < 0.05). Conclusion: Si-PTEN promoted colon cancer progression and induced the polarization of M2 tumor-associated macrophages in the colon cancer cell environment.
Keywords: Colon cancer, macrophage M2 polarization, PTEN
How to cite this article: Introduction 
Colon cancer is currently one of the most prevalent cancers globally[1] and one of the leading causes of morbidity and mortality associated with cancer in the world.[2] Surgery is currently the primary treatment for colon cancer. Despite recent advances in clinical studies, colon cancer treatment is still uncertain due to its high prognostic recurrence risk and drug resistance. Accumulated evidence has shown that tumor immune cell infiltration is highly related to colon cancer, but the relevant mechanism remains unclear.[3]
According to previous reports, tumor-associated macrophages (TAMs) are the most numerous inflammatory cells in the tumor microenvironment, differentiating into anti-tumor M1 macrophages or tumor-promoting M2 macrophages under different signal stimulation.[4] Li Jinyuan et al.[5] pointed out that the number of M1 and M2 cells infiltrating the tumor tissue is directly related to the prognosis of colon cancer. Huang Yanjun et al.[6] have demonstrated that M2 TAM increased the expression of YAP1 (yes-associated protein 1) and associated carcinogenic pathway factors, thereby promoting colon tumorigenesis. Therefore, colon cancer progression is closely related to the M2 polarization of macrophages.
As a tumor suppressor factor, phosphate and tension homolog deleted on chromosome ten (PTEN) promotes colon cancer cells proliferation and invasion by activating downstream PI3K/AKT signals.[7] The meta-analysis also indicated that low expression of PTEN in kidney cancer is positively correlated with the risk of tumorigenesis.[8] PTEN tumor suppressor gene may play a huge role in different cancers, but its mechanism needs to be explored further.
A study has suggested that the PTEN/PI3K/AKT pathway mediates macrophage M2 polarization.[9] A pancreatic cancer study proposed that miR-301a-3p induces M2 polarization of macrophages by activating the PTEN/PI3K signaling pathway.[10] The PTEN/PI3K/AKT pathway could induce M2 polarization in various cells, but it is uncertain whether such a regulatory mechanism exists in colon cancer. This article aims to explore the potential mechanism of PTEN on macrophage polarization in colon cancer, which may provide a new strategy for colon cancer treatment in the future.
Materials and Methods Clinical samples
Thirty human colon cancer tissues and paired adjacent normal tissues (precancerous tissues) (at least 5cm from the cancer tissue) were provided by the Fourth Hospital of Changsha. These were collected from patients recruited from affiliated oncology hospitals from January 2018 to December 2019. All patients did not receive any adjuvant therapy before surgery. Pathologists diagnosed all samples as colon cancer and precancerous tissue. Tumors were classified according to WHO 2018 Classification. The colon cancer in the study was histological grade 3. All of these cancers were of poorly differentiated adenocarcinoma tissue. The collection of clinical samples followed ethical requirements. All patients enrolled in this study had obtained informed consent. The experimental procedure was approved by the ethics committee of the Fourth Hospital of Changsha. The EIC ID and date were CSSDSYY-LLSC-KYXM-2019-02-16.
Cell culture
Normal human colon cells CCD-18Co (CL-0591) and human colon cancer cells SW480 (CL-0223), Caco-2 (CL-0050), SW620 (CL-0225), HCT116 (CL-0096) were purchased from Wuhan Procell Life Technology Co. Ltd. The cells were cultured in a 37°C incubator with 5% CO2 and 95% relative humidity for two to three days. After the cells adhered to the wall and grew to 80% confluence, trypsin digestion, they were divided into two for passage.
Cell transfection
RIBOBIO (Shanghai, China) provided siRNA (si-PTEN) and siRNA NC sequences for PTEN. According to the manufacturer's instructions, the oligonucleotides were transfected into SW620 cells at a final concentration of 100 nM using Lipofectamine 2000 (Invitrogen, Shanghai, China). Cells were collected 48 h after transfection for subsequent detection.
Cell co-culture
THP-1 monocytes were cultivated in RPMI 1640 medium containing 10% FCS 10 mM Hepes. After reaching 80% confluence, the medium added 150 nM phorbol 12-myristate 13-acetate to induce differentiation for 24 h. In addition, 20 ng/ml of IFN-γ and 10 pg/ml of LPS were used to induce M1 differentiation, while 20 ng/ml of interleukin 4 and 20 ng/ml of interleukin 13 for induction M2 differentiation after re-cultivation in serum-free RPMI medium for 24 h. The RPMI culture group was used as a control. The experimental group was cultured with cell supernatants grouped by SW620, si-NC, and si-PTEN. After 24 h, the cells were collected for further testing.
qRT-PCR
Trizol reagent (ThermoFisher, 15596026, USA) was used to extract total Ribonucleic Acid (RNA) from an appropriate amount of tissues (colon cancer tissues or precancerous tissues) or cells (CCD-18Co cells or human colon cancer cells). The cells' density was 5 × 106 cells/ml. mRNA reverse transcription kit (CW2569, CWBIO, China) was applied to synthesize cDNAs. The Ultra SYBR Mixture (CW2601, CWBIO, China) was used for polymerase chain reaction (PCR) amplification. Forty cycles were magnified. The fluorescent quantitative PCR system was Thermo Fisher (PIKOREAL96). All PCR tests were performed in triplicates. The 2-ΔΔCt method reflects the relative expression level. Actin was used as an internal reference. The primer sequences of PTEN and other mRNAs (Sangon Biotech, Shanghai, China) are in [Table 1].
Western blot
An appropriate amount of tissues (colon cancer tissues or precancerous tissues) or cells (CCD-18Co cells or SW620 cells) was added to RIPA lysate (P0013B, Beyotime, China). The protein concentration was determined according to the instructions of the BCA protein quantification kit. The same quality of protein in each group was transferred to Blot Bis-Tris gels. After electrophoresis, the protein was transferred to the membrane. The membrane was immersed in 5% skimmed milk and sealed at room temperature for 1 h. Samples of each group were incubated at an appropriate amount of primary antibody or internal control for 90 min at room temperature, including PTEN (ab109454, Rabbit, 1:2000, abcam, UK), p38 (14064-1-AP, Rabbit, 1:1000, proteintech, USA), phosphate (p)-p38 (ab4822, Rabbit, 1:1000, abcam, UK), c-Jun N-terminal kinase (JNK) (ab179461, Rabbit, 1:1000, abcam, UK), activator protein 1 (AP-1) (22114-1-AP, Rabbit, 1:500, proteintech, USA), B-cell lymphoma-2 (Bcl-2) (12789-1-AP, Rabbit, 1:2000, proteintech, United States), Bcl-2-associated X (Bax) (60267-1-Ig, Mouse, 1:4000, proteintech, United States), Caspase-3 (19677-1-AP, Rabbit, 1:1000, proteintech, USA), arginase-1 (Arg-1) (16001-1-AP, Rabbit, 1:10000, proteintech, USA), CD163 (16646-1-AP, Rabbit, 1:1000, proteintech, USA), CD206 (18704-1-AP, Rabbit, 1:1000, proteintech, USA), tumor necrosis factor-alpha (TNF-α) (60291-1-Ig, Mouse, 1:3000, proteintech, USA), interleukin-1beta (IL-1β) (16806-1-AP, Rabbit, 1:1000, proteintech, USA), inducible nitric oxide synthase (iNOS) (ab3523, Rabbit, 1:500, abcam, UK), and GAPDH (10494-1-AP, Rabbit, 1:5000, proteintech, USA). HRP Goat-anti-mouse IgG (SA00001-1, 1:5000, proteintech, USA) or HRP Goat-anti-rabbit IgG (SA00001-2, 1:6000, proteintech, USA) was selected as the secondary antibody. The membrane was incubated with them at room temperature for 90 min. The chemical imaging agent exposed the membrane. The protein level of each sample was evaluated with Image 6.0 software.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
5 mg/ml MTT working solution was added into each group of cells at 37°C, 5% CO2 for 4 h. The absorbance (OD) value was analyzed at 490 nm by a Bio-Tek microplate reader. The average value was taken to draw a histogram.
Flow cytometry
The processed cell suspension of each group was collected. The cells were fixed with 75% ethanol, then stained by Propidium iodide (PI) for 30 min in the dark. The cells were then transferred to a flow cytometer (A00-1-1102, Beckman, USA) test tube. Cells were collected by a scatter plot to analyze the percentage of each cell cycle on the PI fluorescence histogram. On the other hand, a multicolor flow cytometry method was established to identify and distinguish circulating macrophages. Studies have shown that M1 and M2 macrophages could be evaluated by surface markers CD86 and CD206.[11],[12],[13] Therefore, in this study, we detected CD86 and CD206 by flow cytometry. Briefly, each group added fluorescent dye-conjugated antibodies according to experimental needs, including CD11b (11-0118-42, mouse, eBioscience, USA) + CD86 (12-0869-42, mouse, eBioscience, USA), CD11b + CD206 (17-2069-42, mouse, eBioscience, USA.), or the above three indicators to incubate for 30 min in the dark. The cells of each group were transferred to the corresponding channel (FITC, PE, or APC) of the flow cytometer for detection.
Migration
100 μL cell suspension was added to the upper chamber of the small chamber, and the lower chamber was added to the complete medium containing 10% FBS for 48 h. The culture medium in the chamber was discarded. The cells on the upper chamber were wiped with a wet cotton swab. The cells were fixed for 20 min and stained with 0.5% crystal violet for 5 min. The cells were observed and pictures were taken under an inverted microscope.
Invasion
Matrigel glue was used to prepare Transwell cells. The basement membrane was hydrated. The following steps were the same as the migration.
Statistics and analysis
Data are expressed as mean ± standard deviation. Each experiment was repeated three times independently. Student's t-test and one-way analysis of variance were commonly used to compare the data between two or three groups using Graph Pad Prism 8.0 statistical software. P values < 0.05 were considered statistically significant.
Results The expression of PTEN in clinical colon cancer samples and colon cancer cells and the polarization of M2 macrophages in the tumor environment
First, the expression of PTEN and M2 macrophages related markers in colon cancer tissues and cells were evaluated. The median values of PTEN mRNA expression in colon cancer tissues and precancerous tissues were 0.26 and 0.94, respectively. The median values of PTEN mRNA expression in CCD-18Co cells, SW480 cells, Caco-2 cells, SW620 cells, and HCT116 cells were 1.02, 0.45, 0.33, 0.11, and 0.17, respectively. The molecular level of PTEN expression in colon cancer tissues or cells was significantly lower than that in precancerous tissues or CCD-18Co cells (P-values = 0.001, P values < 0.05, respectively). Among them, PTEN expression was the weakest in SW620 cells [Figure 1]a and [Figure 1]b. Therefore, SW620 cells were selected for subsequent studies. The median values of PTEN, p-38, p-p38, JNK, AP-1, Bax, Bcl-2, and Caspase-3 proteins expression in colon cancer tissues were 0.14, 0.51, 0.33, 0.64, 0.42, 0.06, 0.56, and 0.16, respectively, which were 0.38, 0.49, 0.06, 0.21, 0.14, 0.27, 0.31, and 0.35 in precancerous tissues. The median values of PTEN, p-38, p-p38, JNK, AP-1, Bax, Bcl-2, and Caspase-3 proteins expression in SW620 cells were 0.08, 0.53, 0.40, 0.37, 0.34, 0.1, 0.48, and 0.14, respectively, which were 0.33, 0.52, 0.05, 0.18, 0.07, 0.28, 0.29, and 0.29 in CCD-18Co cells. The expression of p-p38, JNK, AP-1, and Bcl-2 protein in colon cancer tissues or cells was significantly higher than that in precancerous tissues or CCD-18Co cells, and the expressions of PTEN, Bax, and Caspase-3 were opposite (P-values < 0.0001). The expression of p38 protein in colon cancer tissue or cells was slightly higher than in precancerous tissue or CCD-18Co cells, which was not statistically significant (P-values > 0.05) [Figure 1]c and [Figure 1]d. The median values of Arg-1, CD163, and CD206 mRNA expression in colon cancer tissues were 8.24, 2.14, and 2.72, respectively, which were 0.98, 1.06, and 1.06 in precancerous tissues. The median values of Arg-1, CD163, and CD206 proteins expression in colon cancer tissues were 0.49, 0.54, and 0.65, respectively, which were 0.11, 0.08, and 0.16 in precancerous tissues. The expression levels of Arg-1, CD163, and CD206 molecules and proteins in colon cancer tissues were significantly higher than those in precancerous tissues (P-values < 0.05, P values = 0.046, P values = 0.008, respectively) [Figure 1]e. It could be seen from the above experimental results that PTEN was low expressed, and macrophage M2 type markers were highly expressed in colon cancer tissues and cells.
Figure 1: PTEN and macrophage M2 type markers were highly expressed in colon cancer tissues and cells. (a-b) PTEN mRNA expression in colon cancer tissues and cells. (c-d) PTEN, p38, p-p38, JNK, AP-1, Bax, Bcl-2, and Caspase-3 protein expression in colon cancer tissues and cells in each group. (e) The molecular and protein expression of Arg-1, CD163, and CD206 in colon cancer tissues and precancerous tissue. The data were expressed as mean ± standard deviation. The results were reproducible in three independent experiments. Normal was the normal colon cell CCD-18Co, and Cancer was the colon cancer cell SW620. *P-values < 0.05 vs. precancerous tissue group or CCD-18Co group or Normal groupSi-PTEN enhanced the proliferation, invasion and migration of colon cancer cells
Then, the si-PTEN and siRNA NC vectors were constructed and transfected into SW620 cells to evaluate the effect of PTEN on colon cancer. The median values of PTEN mRNA expression in the SW620 group, si-NC group, and si-PTEN group were 1.00, 1.05, and 0.27, respectively. Si-PTEN reduced PTEN mRNA expression in SW620 cells with a P value of 0.001 [Figure 2]a. The median values of cell proliferation in the SW620 group, si-NC group, and si-PTEN group were 1.43, 1.45, and 2.11, respectively. Similarly, si-PTEN promoted the proliferation of SW620 cells (P-values < 0.0001) [Figure 2]b. The median values of the G2 + S phase in the SW620 group, si-NC group, and si-PTEN group were 53.98%, 53.36%, and 56.68%, respectively. In addition, Si-PTEN upregulated the proportion of cells in the G2 + S phase (P-values > 0.05) [Figure 2]c, which showed that si-PTEN plays a role in promoting cell proliferation. The median values of p38, p-p38, JNK, AP-1, Bax, Bcl-2, and Caspase-3 proteins expression in the SW620 group, si-NC group, and si-PTEN group were in [Table 2]. Si-PTEN improved the expression of p38, p-p38, JNK, AP-1, and Bcl-2 protein in SW620 cells and inhibited Bax and Caspase-3 (P-values < 0.05) [Figure 2]d, [Figure 2]e. The median values of migration cells in the SW620 group, si-NC group, and si-PTEN group were 0.94, 0.94, and 1.25, respectively. The median values of invasion cells in the SW620 group, si-NC group, and si-PTEN group were 0.45, 0.45, and 0.92, respectively. Si-PTEN also accelerated the migration and invasion of SW620 cells (P-values < 0.0001) [Figure 2]f. In summary, si-PTEN played an active role in promoting the proliferation, migration, and invasion of colon cancer cells.
Table 2: The median values of p-38, p-p38, JNK, AP-1, Bax, Bcl-2, and Caspase-3 proteins expressionSi-PTEN inhibited the proliferation of M1 macrophages
To determine the possible mechanism of si-PTEN promoting the development of colon cancer, the effect of si-PTEN on the proliferation of M1 macrophages was evaluated through a co-culture cell system. The median values of CD86 expression in the Control group, SW620 group, si-NC group, and the si-PTEN group were 28.20%, 20.73%, 19.64%, and 12.25%, respectively. It could be seen from the results of flow cytometry that the SW620 group slowed down the polarization of M1 macrophages in THP-1 cells compared to the control group (P-values < 0.0001). Si-PTEN increased the slowing effect (P-values < 0.0001) [Figure 3]a. Inducible nitric oxide synthase (iNOS) is an intracellular marker of M1 macrophages.[11],[12],[13] The median values of iNOS, TNF-α, and IL-1β mRNA and proteins expression in the Control group, the SW620 group, the si-NC group, and the si-PTEN group were in [Table 3]. The real-time reverse transcription-PCR (qRT-PCR) results showed that the expressions of iNOS, TNF-α, and IL-1β in the SW620 group were significantly lower than those in the control group (P-values = 0.002, P values = 0.006, P values < 0.0001, respectively). Si-PTEN exacerbated this effect (P-values = 0.02, P values = 0.03, P values = 0.03, respectively) [Figure 3]b. The above indicators had the same result at the protein level (P-values < 0.05) [Figure 3]c. Experimental results showed that si-PTEN had an inhibitory effect on M1-type macrophages.
Figure 3: Si-PTEN inhibited the proliferation of M1 macrophages. (a) Flow cytometry staining for CD11b and CD86, evaluating the number of double-positive cells. (b) TNF-a, IL-1β, and iNOS mRNA expression in each group of cells. (c) TNF-a, IL-1β, and iNOS protein expression in each group of cells. The data were expressed as mean ± standard deviation. The results were reproducible in three independent experiments. * P values < 0.05 vs control group, # P < 0.05 vs si-NC group
Table 3: The median values of iNOS, TNF-α and IL-1β mRNA, and proteins expressionSi-PTEN improved the proliferation of M2 macrophages
To determine the effect of si-PTEN on the proliferation of M2 macrophages, flow cytometry, qRT-PCR, and western blot experiments were used for evaluation. The median values of CD206 expression in the Control group, the SW620 group, the si-NC group, and the si-PTEN group were 5.65%, 11.23%, 11.48%, and 19.21%, respectively. Compared with the control group, THP-1 cells differentiated into M2 type faster in the SW620 group (P-values < 0.0001). Si-PTEN enhanced the proliferation of M2 macrophages (P-values < 0.0001) [Figure 4]a. Arg-1 is an intracellular marker of M2 macrophages.[11],[12],[13] The median values of Arg-1, CD163, and CD206 mRNA and proteins expression in the Control group, the SW620 group, the si-NC group, and the si-PTEN group were in [Table 4]. The expression of Arg-1, CD163, and CD206 mRNA in the SW620 group was higher than in the control group (P-values = 0.0002, P values = 0.04, P values > 0.05, respectively). Compared with the si-NC group, knockdown PTEN accelerated the expression of the above indicators (P-values < 0.0001, P values < 0.0001, P values = 0.0002, respectively) [Figure 4]b. The above indicators had the same result at the protein level (P-values < 0.05) [Figure 4]c. Based on the above, it was concluded that si-PTEN could promote the proliferation of M2 macrophages. Therefore, we speculated that si-PTEN induced M2 polarization of macrophages in the tumor environment, which may be closely related to the promotion of colon cancer development mentioned above.
Figure 4: Si-PTEN enhanced the proliferation of M2 macrophages. (a) Flow cytometry staining CD11b and CD206. (b) Arg-1, CD206, and CD163 mRNA expression in cells of each group. (c) Arg-1, CD206, and CD163 protein expression of cells in each group. The data were expressed as mean ± standard deviation. The results were reproducible in three independent experiments. *P values < 0.05 vs. control group, #P values < 0.05 vs. si-NC group
Table 4: The median values of Arg-1, CD163, and CD206 mRNA, and proteins expressionSi-PTEN enhanced the ratio of M2/M1 in the tumor environment
To further explore the effect of si-PTEN on the macrophages polarization in colon cancer, the ratio of M2/M1 and Arg-1/iNOS was evaluated. The median values of CD86, CD206, Arg-1, and iNOS expression in the Control group, the SW620 group, the si-NC group, and the si-PTEN group were in [Table 5]. Si-PTEN decreased the proportion of M1 macrophages and increased the proportion of M2 macrophages in the tumor environment of SW620 cells (P-values < 0.0001) [Figure 5]a. The expression level of Arg-1 molecules in the SW620 group was higher than that of the control (P-values > 0.05). Si-PTEN improved the level of Arg-1 molecules in the tumor environment (P-values < 0.0001). The level of iNOS obtained the opposite result [Figure 5]b. The protein level and molecular level of the above two indicators remain consistent (P-values < 0.0001) [Figure 5]c. The results showed that si-PTEN induced M2 polarization of macrophages in the colon cancer environment, which may be related to the inhibition of the progression of colon cancer.
Figure 5: Si-PTEN increased the ratio of M2/M1 in the tumor environment. (a) Flow cytometry staining for CD11b, CD86, and CD206 to evaluate the proportion of M1 and M2 macrophages in each group. (b) Expression of Arg-1 and iNOS in each group of cells at the molecular level. (c) The expression of Arg-1 and iNOS in each group of cells at the protein level. The data were expressed as mean ± standard deviation. The results were reproducible in three independent experiments. *P values < 0.05 vs. control group, #P values < 0.05 vs. SW620 group Discussion Accumulated research results have shown that tumor development is closely related to the tumor microenvironment[14],[15],[16],[17] and immunotherapy.[18],[19],[20],[21] According to reports, BMP4 in bladder cancer cells is beneficial to the polarization of peripheral blood monocytes/macrophages to M2 type, leading to high expression of CD163, and promoting the development of bladder cancer.[22] In a clinical study, the percentage of M1 macrophages in breast cancer patients was significantly reduced, while M2 macrophages were significantly improved.[23] Our research results showed that the expression levels of M2 markers Arg-1, CD163, and CD206 in colon cancer tissue cells were significantly higher than those in precancerous tissues. The same conclusion was reached at the cellular level, which is consistent with previous studies. Zhang Yuelong et al.[24] have indicated that miR-410-3p promotes prostate cancer progression by down-regulating PTEN. In this article, SW620 cells were transfected with a si-PTEN vector. Xenograft tumor models were constructed by different treatment SW620 cells. We found that si-PTEN promoted cell proliferation, migration, and invasion. Si-PTEN speeded up DNA synthesis in SW620 cells. Si-PTEN also enhanced the expression of p38, p-p38, JNK, AP-1, and Bcl-2 in SW620 cells and tumor tissue while decreasing Bax and Caspase-3 expression in SW620 cells. Si-PTEN increased tumor volume and tumor size. Based on the above, we have determined that si-PTEN accelerated colon cancer progression, but the potential pathways of action need to be explored further. Wang Xiaofeng et al.[10] have suggested that miR-301a targets the PTEN/PI3K pathway to mediate the polarization of M2 macrophages. In our experiment, the polarization of M1 and M2 macrophages of THP-1 cells was observed through flow cytometry experiments. We found that si-PTEN inhibited the proliferation of M1 macrophages and enhanced the proliferation of M2 macrophages. The qRT-PCR and western blot results manifested that si-PTEN inhibited the expression of iNOS, TNF-α, and IL-1β in cells but had the opposite effect on the expression of Arg-1, CD163, and CD206. More importantly, si-PTEN improved the ratio of M2/M1 in THP-1 cells. In the coming time, we will continue to determine the relationship between M2 macrophages and colon cancer. In the process of PTEN exhaustion to promote the development of colon cancer, the role of PI3K/AKT pathway is also the focus of the next work.
Conclusion In short, current research manifests that si-PTEN promotes the progression of colon cancer. This process may be related to the induction of M2-type polarization of macrophages. Our findings suggest a potential mechanism affecting colon cancer, which may provide a new treatment strategy for colon cancer.
Ethics approval and consent to participate
All patients provided written consent before participating in the study. This study was approved by the ethics committee of the Fourth Hospital of Changsha.
Human and animal rights
All clinical samples were tested following the Principles of the Declaration of Helsinki. This article did not include any studies of animals by any of the authors.
Consent for publication
All authors have read and approved the final manuscript. We further confirm that all have approved the order of authors listed in our manuscript.
Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Acknowledgments
We thank the Science Foundation of Hunan Province (Project No: 2018JJ6067) and the Fourth Hospital of Changsha for all the support.
Financial support and sponsorship
This work was supported by Science Foundation of Hunan Province (Project No: 2018JJ6067).
Conflicts of interest
There are no conflicts of interest.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_786_21
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