- PROX1-AS1 and PD-L1 were upregulated in CRC.
- PROX1-AS1 knockdown restrained CRC progression.
- PROX1-AS1 downregulation inhibited immune escape of CRC cells.
- PROX1-AS1 positively modulated PD-L1 via miR-520d.
- PROX1-AS1 regulated PD-L1 to promote CRC progression by targeting miR-520d.
As a kind of malignant gastrointestinal tumor with high morbidity and mortality rate worldwide, colorectal cancer (CRC) poses a threat to global public health . Despite improvements attained in CRC diagnosis, screening, and treatment, the overall survival rate of patients with advanced CRC remains low [2,3]. It is estimated that the global CRC burden will increase by 60% by 2030, including almost 2 million new CRC cases and 1 million CRC-associated deaths . Metastasis is a major cause of mortality for CRC patients [5,6]. Statistically, above 50% of CRC patients suffer from distant metastasis . Nowadays, increasing research have proven that immunotherapy may offer ideal therapeutic effects for patients with metastatic CRC .
Immunotherapy is a novel alternative for cancer treatment . Innately, tumor cells can maintain antigenicity and immunogenicity to escape from immune surveillance of tumor-specific T lymphocytes, which is known as ‘immune evasion . In tumor microenvironment (TME), T-lymphocyte activation is largely impaired . Hence, T-lymphocyte activation is promoted during immunotherapy to enhance their anti-tumor immune responses . As CD8+ T lymphocytes are key cytotoxic immune cells to kill tumor cells, they are preferred for tumor immunotherapy . It has also been reported that CRC cells could evade the immune monitoring of T lymphocytes [13,14]. However, the specific molecular mechanisms involved in CRC immune evasion remain poorly understood.
As a widely-recognized checkpoint for immune responses, PD-L1 could inhibit the activation of T lymphocytes, further leading to the evasion of tumor cells against the anti-tumor effects mediated by T lymphocytes . PD-L1 upregulation in cancer cells may lead to increased apoptosis or restrained activity of tumor-infiltrating T lymphocytes . Also, blocking the interaction between PD-L1 and its receptor PD-1 with certain antibodies has been increasingly regarded as a promising therapeutic treatment for immunotherapy in a variety of human cancers . Additionally, former studies revealed that PD-L1 was closely related to CRC immunotherapy and immune evasion . Here, the regulatory function of PD-L1 in CRC development and immune evasion was further studied.
Long non-coding RNAs (LncRNAs) are known as a group of RNAs (>200 nucleotides) without protein-coding ability . A large body of evidence has proven that lncRNAs are implicated in various physiological and pathological processes . As indicated by previous reports, lncRNA PROX1 antisense RNA 1 (PROX1-AS1) generally exerts carcinogenic functions in many malignancies. For example, PROX1-AS1 facilitated the progression of papillary thyroid carcinoma by accelerating proliferation, invasion, and migration . Besides, PROX1-AS1 sponged miR-519d to positively regulate SOX2 expression, thereby promoting resistance of retinoblastoma cells to chemotherapy . Also, PROX1-AS1 could upregulate the PD-L1 level through interaction with miR-877-5p to promote the progression of gastric cancer, another gastrointestinal cancer . Interestingly, a report by Chen et al. identified that PROX1-AS1 expression was extraordinarily upregulated in CRC samples , implying the involvement of PROX1-AS1 in the tumorigenesis of CRC. However, specific mechanisms by which PROX1-AS1 regulates CRC progression and immune evasion remain to be elucidated.
This work was intended to study the function of PROX1-AS1 and its possible regulatory network in CRC progression and immune evasion. By functional experiments, our findings demonstrated the promoting role of PROX1-AS1 and the in-depth involvement of PROX1-AS1/miR-520d/PD-L1 competitive endogenous RNA (ceRNA) network in CRC malignant development and immune evasion. Therefore, PROX1-AS1 might be a potential biomarker or target for the development of therapeutic strategies for CRC immunotherapy.
Materials and methods
Thirty-eight pairs of CRC and normal samples were obtained from CRC patients and tissues were kept in liquid nitrogen prior to total RNA extraction. Each patient enrolled signed the informed consent. Our study was permitted by the ethics committee of Affiliated Hospital of Shandong University of Traditional Chinese Medicine.
Cell culture and transfection
Human normal colon epithelial cells (FHC), human CRC cells (LoVo, HCT-8, SW-620, RKO, HT-29, and HCT-116), and mouse colon carcinoma cells (CT-26) were procured from BeNa Culture Collection (China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (10% fetal bovine serum [FBS]).
Small interfering RNA (siRNA) against PROX1-AS1 (siPROX1-AS1), siNC, miR-520d mimic, NC mimic, miR-520d inhibitor, NC inhibitor, pcDNA3.1-PD-L1 (oe-PD-L1), and blank vector (Vector) constructed by GenePharma (Shanghai, China) were severally transfected into cells via Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, California, USA) and cultured for 48 h before subsequent tests.
TRI Reagent (Sigma-Aldrich, Saint Louis, Missouri, USA) was applied to isolate RNA from tissues and cells. Next, reverse transcription was conducted via PrimeScript RT Master Mix (Takara). With GAPDH or U6 as internal control, SYBR Green PCR Kit was applied for qPCR assay. The gene expresion was calculated by 2−ΔΔCt method .
The transfected ESCC cells were transferred to 96-well plates and cultured for 48 h. Cell counting kit-8 (CCK-8) reagent (10 μL/well) was added at the indicated time. Then, optical absorbance at 450 nm was measured with a microplate reader.
Transwell chambers (Corning Inc., New York, USA) with upper chamber covered with Matrigel or not were used for invasion or migration assay. DMEM plus 10% FBS was supplemented to the bottom chamber. In brief, 2 × 104 HCT-8 or HT-29 cells were inoculated to the upper chamber. Next, the cells were cultivated for another 24 h at 37°C in 5% CO2. Cells on the membranes were fastened, stained, and finally counted under a microscope (Olympus, Tokyo, Japan).
Isolation and lactate dehydrogenase test for CD8+ T lymphocytes
Via Dynabeads CD8 Positive Isolation Kit (Invitrogen, Thermo Fisher Scientific, California, USA), CD8+ T lymphocytes were separated from peripheral blood mononuclear cells from healthy volunteers. After coculture with transfected HCT-8 or HT-29 cells for 24 h, the CD8+ T lymphocytes were determined for cytotoxicity by lactate dehydrogenase (LDH) Cytotoxicity Assay (Invitrogen, Thermo Fisher Scientific, California, USA).
To analyze the percentage of CD8+ T lymphocytes, Canto II Flow Cytometer (BD Bioscience, California, USA) was applied to separate and determine CD8+ T lymphocytes after coculture with transfected HCT-8 or HT-29 cells.
To analyze the apoptosis of CD8+ T lymphocytes, Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) was applied. Briefly, the collected cell pellets were resuspended in Annexin V binding buffer, and stained via Annexin V-FITC and PI in the dark for 30 min. Next, the cells were detected by a flow cytometer (BD Biosciences).
Dual-luciferase reporter assay
Wildtype or mutated binding sequences of PROX1-AS1 and PD-L1 3′-UTR for miR-520d were amplified and inserted into pmirGLO-control basic luciferase vector (Promega, Madison, Wisconsin, USA) to generate PROX1-AS1-WT, PD-L1-WT, PROX1-AS1-MUT, or PD-L1-MUT luciferase reporter vectors.CRC cells were cotransfected with these vectors and NC mimic or miR-520d mimic and cultured for 48 h. Then, luciferase activity of transfected CRC cells from each group was detected with Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA).
Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (Millipore, Billerica, Massachusetts, USA) was utilized for RNA immunoprecipitation (RIP) assay. CRC cell lysate was incubated with magnetic beads conjugated with antibodies against IgG or Ago2 (Millipore, Billerica, Massachusetts, USA). The immunoprecipitated RNA was measured with quantitative reverse transcription-PCR (RT-qPCR).
Six male BALB/c mice were kept in a specific pathogen-free environment with free access to food and water. To construct xenograft models based on CT-26 cells, BALB/c mice were subcutaneously injected with CT-26 cells (5 × 105) transfected with siNC or siPROX1-AS1 (three mice per group). Tumors were measured every 7 days for tumor volume. Twenty-eight days later, all mice were sacrificed. The xenografted tumors were excised for weight and gene expression determination.
GraphPad Prism 6.0 software was employed for statistical analysis in this study. All the results were presented as mean ± SD. The correlation between genes was analyzed via Pearson’s correlation analysis. One-way analysis of variance or Student’s t-test was applied for the determination of data differences. P < 0.05 was considered significant. Every experiment was conducted three times.
High PROX1-AS1 and PD-L1 expression levels in colorectal cancer
To explore the clinical role of PROX1-AS1 in patients with CRC in detail, we detected PROX1-AS1 levels in 38 pairs of CRC tissues and non-tumor tissues via RT-qPCR. The data showed that PROX1-AS1 enrichment was markedly increased in CRC tissues (Fig. 1a). Conformably, RT-qPCR also indicated that PROX1-AS1 abundance was markedly upregulated in CRC cells (LoVo, HCT-8, SW-620, RKO, HT-29, and HCT-116), relative to human normal colon epithelial cell line (FHC) (Fig. 1b). As indicated in Table 1, high PROX1-AS1 expression was significantly correlated with lymph node metastasis, distant metastasis, and high tumor-node-metastasis stage. Besides, PD-L1 level was also elevated in CRC tissues and cells (Fig. 1c and d). Furthermore, a positive relation between PROX1-AS1 and PD-L1 expressions was observed in CRC tissues (Fig. 1e). Therefore, high PROX1-AS1 and PD-L1 levels might be related to CRC progression.
Table 1 -
The association between PROX1-AS1 expression and clinical features of colorectal cancer patients
||Low (n = 19)
||High (n = 19)
|Lymph node metastasis
PROX1-AS1 knockdown impedes malignant cellular phenotypes in colorectal cancer
Next, functional assays were applied to explore the regulatory function of PROX1-AS1 in CRC progression in vitro. The transfection efficiencies of siPROX1-AS1 in CRC cells were detected by RT-qPCR (Fig. 2a). Moreover, PROX1-AS1 depletion suppressed CRC cell viability and growth (Fig. 2b and c). Their migrative and invasive capabilities were also reduced by PROX1-AS1 silencing (Fig. 2d and e). To summarize, PROX1-AS1 interference led to suppression of CRC progression both in vitro.
PROX1-AS1 downregulation enhances cytotoxicity and inhibits apoptosis in CD8+ T lymphocytes
After HCT-8 or HT-29 cells were co-incubated with CD8+ T lymphocytes, the cytotoxicity of CD8+ T lymphocytes to CRC cells was reinforced by PROX1-AS1 silencing (Fig. 3a). After PROX1-AS1 blockage, the percentage of CD8+ T lymphocytes was elevated (Fig. 3b), whereas CD8+ T lymphocyte apoptosis was inhibited (Fig. 3c and d). Collectively, PROX1-AS1 knockdown exerted an inhibitory effect on immune escape of CRC cells.
PD-L1 upregulation assuages the suppression of malignant phenotypes and immune escape of colorectal cancer cells mediated by PROX1-AS1 knockdown
Given that PROX1-AS1 enrichment was in positive correlation with PD-L1 level in CRC tissues, the regulatory relationship between PROX1-AS1 and PD-L1 in CRC progression was analyzed. RT-qPCR showed that PD-L1 level in CRC cells was significantly decreased after PROX1-AS1 depletion (Fig. 4a), evincing the positive regulatory effect of PROX1-AS1 on PD-L1 expression in CRC cells. Then, PD-L1 was overexpressed in CRC cells by oe-PD-L1, with transfection efficiency validated by RT-qPCR (Fig. 4b). Next, functional rescue experiments were performed in HCT-8 cells transfected with siNC, siPROX1-AS1, or siPROX1-AS1+oe-PD-L1. The experiment results exhibited that the suppressive impacts of PROX1-AS1 knockdown on the cell viability (Fig. 4c), growth (Fig. 4d and e), migration (Fig. 4f and g), and invasion (Fig. 4h and i) of CRC were partially reverted by PD-L1 overexpression. Furthermore, PD-L1 upregulation reduced the cytotoxicity and percentage of CD8+ T lymphocytes (Fig. 4j and k) but induced apoptosis in CD8+ T lymphocytes (Fig. 4l and m), thereby attenuating the activation of CD8+ T lymphocytes mediated by PROX1-AS1 deletion. Taken together, the function of PROX1-AS1 in CRC was closely relevant to its positive regulatory effect on PD-L1.
Association between PROX1-AS1, miR-520d, and PD-L1
According to online prediction provided by DIANA and StarBase websites, both PROX1-AS1 and PD-L1 had the binding sequences for miR-520d (Fig. 5a). Moreover, Li et al. found miR-520d could restrain gastric cancer progression , signifying its tumor-inhibiting role in cancers derived from the digestive system. As indicated by RT-qPCR, miR-520d enrichment was much lower in CRC tissues and cells (Fig. 5b and c). Therefore, miR-520d was selected for subsequent experiments. Then, a luciferase reporter assay was conducted in CRC cells to certify the relationship between miR-520d and PROX1-AS1 or PD-L1. As indicated in Fig. 5d, miR-520d overexpression efficiency was validated. It was shown that the luciferase activity of the PROX1-AS1-WT or PD-L1-WT group was suppressed by miR-520d overexpression; however, miR-520d upregulation exhibited no impact on the activity of PROX1-AS1-MUT or PD-L1-MUT group (Fig. 5e). According to the results of RIP assay, PROX1-AS1, miR-520d, and PD-L1 pulled down with anti-Ago2 were enriched in CRC cells, contrasted with the anti-IgG group (Fig. 5f), further affirming the association between miR-520d and PROX1-AS1 or PD-L1. RT-qPCR assay manifested that PROX1-AS1 knockdown resulted in a remarkable increase in miR-520d enrichment in CRC cells (Fig. 5g). Besides, miR-520d upregulation could markedly decrease PD-L1 expression (Fig. 5h). Next, the suppressive effect of miR-520d inhibitor was verified in CRC cells (Fig. 5i), and miR-520d inhibition abolished the decline in PD-L1 level induced by PROX1-AS1 deletion (Fig. 5j). In summary, the above results demonstrated that PROX1-AS1 could regulate PD-L1 expression via competitively binding to miR-520d.
PROX1-AS1 regulates PD-L1 to promote colorectal cancer cell proliferation, metastasis, and immune evasion through interaction with miR-520d
To investigate if miR-520d was implicated in the regulatory function of PROX1-AS1 in CRC in vitro, rescue assays were performed. The results indicated that the suppression of CRC cell viability, growth, migration, and invasion caused by PROX1-AS1 interference was partly abated after miR-520d inhibition (Fig. 6a–d). Furthermore, miR-520d downregulation eliminated the influence of PROX1-AS1 deletion on the cytotoxicity, cell percentage, and apoptosis of CD8+ T lymphocytes (Fig. 6e–h). Altogether, PROX1-AS1 played promoting role in CRC progression and immune escape in vitro by modulating PD-L1 expression via miR-520d.
PROX1-AS1 inhibition impairs colorectal cancer tumor growth in vivo
To further affirm the effect of PROX1-AS1 in CRC in vivo, xenograft models were established based on CT-26 cells stably transfected with si-NC or si-PROX1-AS1. It was found that both tumor volume and tumor weight were substantially impeded in the si-PROX1-AS1 group relative to those in si-NC group (Fig. 7a–c). Moreover, PROX1-AS1, miR-520d, and PD-L1 levels in harvested tumor tissues were detected by RT-qPCR. It was revealed that PROX1-AS1 and PD-L1 levels were remarkably decreased while miR-520d level was markedly increased in si-PROX1-AS1 group in comparison with those in si-NC group (Fig. 7d–f). In sum, PROX1-AS1 depletion might depress CRC tumor growth in vivo via modulating miR-520d and PD-L1 expression.
CRC is a cause of cancer-related mortality worldwide . Immunotherapy is a novel but effective therapeutic method for patients with metastatic CRC . However, immune escape of tumor cells from T-cell surveillance may cause malignant growth and metastasis, thereby leading to immunotherapy failure . Accumulated evidence indicates that some functional lncRNAs are associated with immune escape and play carcinogenic roles in CRC. For example, Huang et al. demonstrated that exosome-induced SNHG10 upregulation promoted immune evasion of CRC cells from natural killer cells . Xian et al. discovered that exosomal lncRNA KCNQ1OT1 facilitated malignant biological activities and immune escape in CRC via modulating PD-L1 ubiquitination . Xu et al. uncovered that lncRNA HCG18 induced chemoresistance to cetuximab and inactivated CD8+ T lymphocytes in CRC through the miR-20b-5p/PD-L1 cascade . In our work, it was uncovered that PROX1-AS1 levels were lifted in CRC. Also, experiment results manifested that CRC cell growth and mobility were markedly repressed by PROX1-AS1 knockdown. Besides, the animal experiment demonstrated that PROX1-AS1 depletion could dramatically inhibit CRC tumor growth in vivo. Furthermore, PROX1-AS1 silencing increased cytotoxicity and the percentage of CD8+ T lymphocytes and decreased the apoptotic rate of CD8+ T lymphocytes. Therefore, it was substantiated that PROX1-AS1 played an oncogenic role in CRC initiation and progression and facilitated the immune escape of CRC cells by inactivating CD8+ T lymphocytes.
As an essential regulator of immunosuppression in human malignancies, PD-L1 can suppress the activation of T lymphocytes and reinforce the immune resistance of tumor cells . Herein, PD-L1 level was substantially higher in CRC, and PROX1-AS1 could upregulate the PD-L1 level in CRC cells. PD-L1 upregulation counterbalanced the impacts of PROX1-AS1 knockdown in CRC cells, implying PROX1-AS1 played a promoting role in CRC development and immune escape through positively regulating PD-L1 expression.
There are a variety of studies on the regulatory functions of lncRNA-mediated ceRNA mechanism in tumor development and immune evasion in different human cancers . As an anti-tumor microRNA (miRNA), miR-520d exerted an anti-oncogenic function in a number of human cancers, including gastric cancer , breast cancer , hepatocellular carcinoma , and ovarian cancer . Via prediction and screening, miR-520d was chosen as a target of PROX1-AS1. In our study, miR-520d levels declined in CRC tissue samples and cells. Further experiments confirmed the binding relation between miR-520d and PROX1-AS1 or PD-L1. Additionally, it was also found that PROX1-AS1 absorbed miR-520d to modulate the PD-L1 level in CRC cells. Moreover, miR-520d suppression also abolished the inhibitory function of PROX1-AS1 depletion in CRC cells. Hence, it was proven that PROX1-AS1 exerted facilitating effects in CRC progression and immune escape through upregulating PD-L1 via targeting miR-520d.
The current study, for the first time, provided evidence that PROX1-AS1 functioned as a promoter in tumor progression as well as an immune escape in CRC via miR-520d/PD-L1 pathway. This work provided new perspectives for further research on PROX1-AS1 in the field of immunotherapy for CRC patients.
Conflicts of interest
There are no conflicts of interest.
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