Effects of let-7a microRNA and C-C chemokine receptor type 7 expression on cellular function and prognosis in esophageal squamous cell carcinoma

doi:10.21203/rs.3.rs-1712002/v1

Background

The C-C chemokine receptor type 7 (CCR7) plays an important role in chemotactic and metastatic responses in various cancers, including esophageal squamous cell carcinoma (ESCC). Several studies have reported that let-7a microRNA (miRNA) suppresses migration and invasion of various types of cancer cells by downregulating CCR7 expression.

Methods

The expression of CCR7 and let-7a was measured in the cell lines, tumor tissues, and peritumoral tissues of ESCC patients. KYSE cell lines were transfected with synthetic let-7a miRNA and a let-7a miRNA inhibitor, and their CCR7 expression and invasive ability were evaluated. A highly invasive cell line was established via an invasion assay, and CCR7 and let-7a expression was subsequently evaluated. In vivo, cancer cells overexpressing CCR7 were injected subcutaneously into mice and monitored for tumor growth and lymph node metastasis.

Results

A negative correlation between CCR7 and let-7a expression was observed in ESCC cell lines and patient tissue samples. Synthetic let-7a decreased CCR7 expression, while the let-7a inhibitor increased it. In vitro, the established highly invasive cancer cells with high and low levels of CCR7 and let-7a expression, respectively, exhibited a greater invasive ability than the wild-type cell line. In vivo, the highly invasive cells were associated with tumor growth and lymph node metastasis in mice. Patients in the high-CCR7/low-let-7a group had the worst prognosis with five-year recurrence free survival (5-RFS) at 37.5%, followed by the high-CCR7/high-let-7a (5-RFS: 60.0%) and low-CCR7 (5-RFS: 85.7%; p = 0.038) groups.

Conclusions

The expression of CCR7 was downregulated by let-7a miRNA in esophageal cancer cells. The decrease in let-7a expression led to the increased expression of CCR7 in ESCC cells, consequently increasing their invasive ability and malignancy, and resulted in a worse prognosis for ESCC patients.

Trial registration:

Retrospectively registered.

esophageal squamous cell carcinoma

C-C chemokine receptor type 7

let-7a microRNA

invasive ability

metastasis

Cancer incidence and mortality are rapidly increasing worldwide. In 2018, esophageal cancer ranked seventh and sixth in terms of incidence and overall mortality, respectively [1]. Although the survival rate of esophageal cancer is improving due to the development of multidisciplinary treatment, the five-year post-esophagectomy survival rate is approximately 50% [2] [3]. Therefore, there is an urgent need to investigate the mechanisms of cancer progression and metastasis to improve the prognosis of esophageal cancer patients.

Some studies showed that chemokines are deeply involved in tumor cell proliferation, infiltration, and metastasis [4, 5]. In a previous study, we demonstrated the significant clinicopathological relationship and functional causality between C-C chemokine receptor type 7 (CCR7) expression and lymph node metastasis in esophageal squamous cell carcinoma (ESCC) patients [6].

MicroRNAs(miRNAs) are non-coding small RNA molecules that can control the translation of mRNA and regulate several cancer-related genes [7–9]. Let-7a has been reported as one of the miRNAs that suppress various types of cancers[10–13]. In breast cancer, it has been shown to suppress the expression of CCR7 and reduce the migration and invasion ability of cancer cells. In gastric cancer, lack of expression of let-7a was demonstrated to increase CCR7 expression and be associated with cancer metastasis and poor prognosis [12]. However, it has not been confirmed whether the downregulation of let-7a miRNA expression is responsible for high CCR7 expression in ESCC tissues. Thus, in this study, we investigated the relationship between and regulatory mechanisms of CCR7 and let-7a miRNA expression in esophageal cancer cell lines, tumor tissues, and peritumor tissues of ESCC patients.

2.1 Tissue samples

Tissue samples were obtained during a biopsy from 17 ESCC patientss at the Keio University School of Medicine in Japan. Immediately after the biopsy, the tissue samples were frozen in liquid nitrogen and stored at -70°C until use.

2.2 Esophageal cancer cell lines

For this study, we used six established ESCC cell lines from the KYSE series (KYSE-350, 510, 590, 1260, 1440, and 2400), which were purchased from the Japanese Collection of Research Bioresources Cell Bank of the National Institutes of Biomedical Innovation, Health, and Nutrition.

2.3 Cell culture

Cells were maintained and cultured in a Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C and 5% CO₂ atmospheric content. Cells were collected and prepared as single-cell suspensions in phosphate-buffered saline.

2.4 Transfection

The sequence of double-stranded RNA (dsRNA) used in transfection experiments as scrambled small interfering RNA was 5′-UCACAACCUCCUAGAAAGAGUAGA-3′, the synthetic let-7a-5p miRNA sequence was 5′-UGAGGUAGUAGGUUGUAUAGUU-3′, and the inhibitor let-7a-5p miRNA sequence was 5′-UGAGGUAGUAGGUUGUAUAGUU-3′. These RNAs were synthesized by Applied Biosystems (Tokyo, Japan). The cell lines were transfected with dsRNA using the reagents Lipofectamine RNAiMAX and Lipofectamine 2000 (Invitrogen, Tokyo, Japan), according to the reagent manufacturer’s instructions. The cell lines were harvested 2 d after transfection, and various analyses were performed.

2.5 Isolation and quantitative expression of let-7a

Isolation of let-7a miRNA from the cell lines was conducted using the mirVana™ miRNA Isolation Kit. The RNA concentration was quantified via the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Tokyo, Japan). The complementary DNA (cDNA) was synthesized by the reverse transcription of let-7a miRNA. A quantitative reverse transcription polymerase chain reaction (RT-PCR) was conducted using ViiA™ 7 (Applied Biosystems). Thermal cycling consisted of an initial cycle of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles for 15 s at 95°C and 60 s at 60°C. Relative let-7a expression was quantified using the 2^−ΔΔCt method.

2.6 Isolation of RNA and synthesis of single-stranded cDNA

Isolation of total RNA from cell lines was conducted using an RNeasy® Micro Kit (QIAGEN, Tokyo, Japan). The RNA concentration was quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). Then, synthesis of cDNA from total RNA was performed using an RNA-to-cDNA kit (Applied Biosystems). The quality and quantity of the cDNA samples were evaluated via standard electrophoresis and a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific).

2.7 RT-PCR analysis

The quantitative RT-PCR analysis was performed via ViiA™ 7 (Applied Biosystems) and the Fast SYBR® Green Master Mix (Applied Biosystems). Glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) and CCR7 expression were evaluated using PCR primes. Thermal cycling consisted of an initial 20 s at 95°C, followed by 40 cycles for 1 s at 95°C and 20 s at 60°C, using GAPDH as an internal control. The relative amount of CCR7 expression in KYSE cell lines was calculated using the 2^−ΔΔCt method with the KYSE-350 expression level was defined as 1.

2.8 Let-7a transfection

A synthetic let-7a and a let-7a inhibitor were used for let-7a transfection. We diluted 15 µL of the Lipofectamine® RNAiMAX reagent (Life Technologies) in 2 mL of Opti-MEM® (Life Technologies). Then, we added 2 mL of diluted let-7a miRNA (300 pmol) to diluted Lipofectamine® RNAiMAX and incubated the mixture for 5 min at room temperature. Next, we plated the transfection reagent onto a 10 cm plate, which was incubated for 10–20 min in a CO₂ incubator at 37°C. A suspension containing of 5 × 10⁵ cells in an antibiotic-free medium was added to the plate, which was incubated for 3 days in a CO₂ incubator at 37°C.

2.9 Invasion assay

To create highly invasive cell lines, we used Corning BioCoat Matrigel Invasion Chambers containing a polyester membrane with 8 µm pores and a thin layer of a Matrigel Basement Membrane Matrix. We prepared suspensions containing of 2 × 10⁴ cells/mL of culture medium for 24-well chambers and incubated it in the Invasion Chambers for 72–96 h in a humidified tissue culture incubator at 37°C in a 5% CO2 atmosphere. After incubation, the non-invading cells were removed from the upper surface of the membrane. The cells on the lower surface of the membrane were collected and reseeded to the chamber. After repeating this process six times, a highly invasive cell population was obtained and isolated using a cell dissociation solution, solubilized, and stained using a staining solution. By measuring the absorbance of the solution, the number of infiltrated cells were quantified.

2.10 Development of lymph node metastasis model

Lymph node metastasis model was conducted through the following steps: 1) a xenograft tumor was created subcutaneously by injecting 1 × 10⁷ wild-type and invasive KYSE-510 cells, which were visualized with Green Fluorescent Protein (GFP) transfection, into five-week-old mice; 2) after tumor growth (4–6 weeks after injection), they were resected, and cut into small pieces (approximately 2 mm3) and were transplanted to the elbows of different five-week-old mice; 3) the accessory axillary lymph nodes were removed and examined 8 weeks after transplantation.

2.11 Statistical analysis

Statistical analysis was done with IBM SPSS statics 26.0. Continuous variables are expressed as means ± SD and compared with the Student’s t-test. The recurrence-free survival was counted from the time of surgery to the event, any recurrence, or death, whichever came first. The RFS curves were estimated with the Kaplan–Meier method and compared by the log-rank test.

Cox’s proportional hazard model was used to perform the univariate analysis. A p-value of 0.05 was considered statistical significance.

3.1 Expression of CCR7 and let-7a in esophageal cancer cell lines

Using quantitative RT-PCR analysis, CCR7 mRNA expression was confirmed in all esophageal cancer cell lines (Fig. 1A). The level of let-7a miRNA expression was also determined using quantitative RT-PCR analysis (Fig. 1B). KYSE-510 and 1440 exhibited high CCR7 and low let-7a expression, whereas KYSE-590 and 1260 had low CCR7 and high let-7a expression. The expression level of the CCR7 and let-7a was described based on the expression in KYSE-350 cell that was defined as 1.

3.2 Relationship between let-7a and CCR7 expression in ESCC cells

As determined via quantitative RT-PCR analysis, the expression of CCR7 was downregulated by the transfection of let-7a in five of the six KYSE cell lines (Fig. 2A). The decrease in CCR7 expression was most prominent in KYSE-1440, in which CCR7 expression was the highestin the wild type.

3.3. Downregulation of let-7a increases CCR7 expression in KYSE-590 cells and evaluation of invasion ability

The inhibitory effect of let-7a was determined by using synthetic anti-let-7a oligonucleotides in KYSE-590 esophageal cancer cells, which expressed a low level of CCR7 and a high level of let-7a in the wild type. After transfection with synthetic anti-let-7a oligonucleotides, the level of CCR7 expression increased (p < 0.001; Fig. 2B). Transfection with anti-let-7a oligonucleotides also increased the invasive ability of KYSE-590 (p = 0.061; Fig. 2C).

3.4 Development of highly invasive cell lines and lymph node metastasis model

After six courses of the invasion assay, the invasive ability of the highly invasive lines increased (KYSE-510: p = 0.004, Fig. 3A–B; KYSE-590: p = 0.001, Fig. 4A–B). In the invasive type, CCR7 expression increased (KYSE-510: p = 0.04, Fig. 3C; KYSE-590: p = 0.203, Fig. 4C), but let-7a expression decreased relative to the wild type (KYSE-510: p < 0.001, Fig. 3D; KYSE-590: p = 0.001, Fig. 4D).

Because only the KYSE-510 cell line was injected into mice and could be used in the in vivo experiment, we used the highly invasive KYSE-510 cells for the mouse model. The implanted primary tumor of invasive KYSE-510 was both larger (6.5 ± 3.51 vs. 13.0 ± 3.31 mm, p = 0.025) and heavier (0.158 ± 0.161 vs. 0.481 ± 0.263 g, p = 0.070) than that of wild-type KYSE-510 cells (Fig. 5A–D). Similarly, the percentage of positive lymph node metastasis of the wild and invasive types, labeled with GFP, was 0% (0/5) and 25% (1/4), respectively (Fig. 5E–F).

3.5 Relationship between CCR7 and let-7a expression in ESCC patients

Tissue samples were obtained from 17 ESCC patients during a biopsy, and their characteristics are listed in Table 1. Figure 6A shows the expression of CCR7 and let-7a in cancer tissue relative to that in normal tissue, defined as 1, based on quantitative RT-PCR. The graph uses a logarithmic notation, thus presenting negative values when the CCR7 and let-7a expression ratio is less than 1. In 11 ESCC patients, CCR7 expression was higher in malignant tissues than normal tissues (positive value). In contrast, in a different grouping of 11 ESCC patients, let-7a expression was more downregulated in malignant tissues than normal tissues (negative value). Nine ESCC patients exhibited a negative correlation between CCR7 and let-7a expression.

3.6 Relationship between prognosis and CCR7 and let-7a expression

Recurrence was observed in six patients, and the most predominant sites were the lymph nodes. Using the median value, patients were split into two respective groups with low and high CCR7 expression; patients with high CCR7 expression were further divided into two respective groups with low and high let-7a expression. The high-CCR7/low-let-7a group (5-RFS: 37.5%) had the worst prognosis, followed by the high-CCR7/high-let-7a (5-RFS: 60.0%) and low-CCR7 group (5-RFS: 85.7%; p = 0.038, Fig. 6B). Cox proportional hazards analysis identified the high-CCR7/low-let-7a group as a significant prognostic factor (Table 2).

This study showed that synthetic let-7a and the let-7a inhibitor respectively decreased and increased CCR7 expression in KYSE cells, demonstrating a negative correlation between CCR7 and let-7a expression. It can thus be inferred that the downregulation of let-7a miRNA is a factor in increased CCR7 expression in esophageal cancer cells and the development of malignant tumors.

High CCR7 expression is related to lymphatic tumor metastasis and prognosis in gastric cancer and colorectal cancer [14, 15]. Most relevant to this study, elevated CCR7 expression caused by a deficiency in let-7a expression is closely linked to the metastasis and progression of cancer [12]. Furthermore, CCR7 is correlated with lymphatic metastasis and poor prognosis in different types of breast cancer, and let-7a has been found to suppress breast cancer cell migration and invasion through the downregulation of CCR7 expression [10, 16]. This study confirmed that the negative correlation between CCR7 and let-7a expression in ESCC, which strongly suggests that let-7a suppresses the expression of CCR7.

In normal human cells, CCR7 is mainly expressed in differentiated lymphocytes and on the surface of dendritic cells and is thought to mediate lymphocyte migration [17]. It has also been reported that the invasive ability of cancer cells is affected by the expression of CCR7 [10, 16]. In this study, the invasive ability of the ESCC increased after transection with synthetic anti-let-7a through increased and decreased CCR7 and let-7a expression. And after invasion assay, the expression of CCR7 and let-7a in invasive type respectively increased and decreased in comparison with the wild type. Therefore, it can be inferred that the respective increase and decrease in CCR7 and let-7a expression are factors in the invasive ability of ESCC. We also used the highly invasive cell line for in vivo experiments on mice. The implanted primary tumor of the invasive type was larger in size and mass than that of the wild type. Similarly, the percentage of positive lymph node metastasis of the wild and invasive types, labeled with GFP, was 0% and 25%, respectively. Therefore, the highly invasive cell line was more likely to cause and accelerate lymph node metastasis in vivo.

We also investigate the expression of CCR7 and let-7a miRNA in the ESCC patient’s tissue and demonstrate that ESCC tissues tend to have higher CCR7 and lower let-7a miRNA expression than normal tissues. Furthermore, we found that the expression level of CCR7 and let-7a miRNA can stratify the prognosis of the ESCC patients. Patients in the two high-CCR7 groups had a worse prognosis than those in the low-CCR7 group, with the low-let-7a group exhibiting a worse prognosis than the high-let-7a group. These results suggest that during carcinogenesis, the suppression of CCR7 expression diminishes owing to the decreased expression of let-7a and that these changes may affect the prognosis of ESCC patients. Supporting this finding, He et al. [18] found that plasma levels of let-7a miRNA were significantly lower in ESCC patients than healthy participants.

This study has three main limitations. First, while this study confirmed the negative correlation between CCR7 and let-7a, it did not account for the potential influence of the expression of other oncogenes controlled by let-7a. Second, in the mouse experiment, we demonstrated the in vivo malignancy of the synthesized highly invasive cell line but did not measure CCR7 or let-7a expression in metastatic lymph nodes. However, the relationship between CCR7 expression and lymph node metastasis in ESCC cells has been examined in a previous study [6]. Third, the number of cell lines and patient tissue samples used in this study was relatively small; therefore, additional studies are required to validate these results.

The expression of CCR7 was found to be downregulated by let-7a miRNA in ESCC cells. The decrease in let-7a expression led to an increase in the expression of CCR7 in ESCC cells, which consequently acquired increased invasive ability and malignancy that resulted in a worse prognosis for ESCC patients.

3′-UTR

3-untranslated region

CCR7

C-C chemokine receptor type 7

cDNA

complementary DNA

dsRNA

double-stranded RNA

ESCC

esophageal squamous cell carcinoma

GAPDH

glyceral-dehyde-3-phosphate dehydrogenase

GFP

Green Fluorescent Protein

miRNA

microRNA

RFS

Recurrence free survival

RT-PCR

reverse transcription polymerase chain reaction

Ethics approval and consent to participate: This study was approved by the Institutional Review Board of Keio University (approval number: 20210340). All participants provided informed, written consent for their participation in the study. All procedures were performed in accordance with the Helsinki Declaration

Availability of data and materials:

The datasets KYSE-350, 510, 590, 1260, 1440, and 2400 for this study can be found and are available in the JCRB cell bank(https://cellbank.nibiohn.go.jp/english)

https://cellbank.nibiohn.go.jp/~cellbank/en/search_res_det.cgi?RNO=JCRB1428

https://cellbank.nibiohn.go.jp/~cellbank/cgi-bin/search_res_det.cgi?ID=6561

https://cellbank.nibiohn.go.jp/~cellbank/cgi-bin/search_res_det.cgi?ID=6572

https://cellbank.nibiohn.go.jp/~cellbank/en/search_res_det.cgi?RNO=JCRB1434

https://cellbank.nibiohn.go.jp/~cellbank/cgi-bin/search_res_det.cgi?ID=6557

https://cellbank.nibiohn.go.jp/~cellbank/cgi-bin/search_res_det.cgi?ID=6542

The datasets of miRNAs are available in the miRbase (https://www.mirbase.org/)

https://www.mirbase.org/cgi-bin/mature.pl?mature_acc=MIMAT0000062

https://www.mirbase.org/cgi-bin/mature.pl?mature_acc=MIMAT0000039

Competing interests: The authors declare that they have no competing interests.

Funding: No financial support to declare.

Authors’ contributions: MY and KF designed the study. And MY is the first author of this manuscript. KF also checked the manuscript and advised the first author on the preparation of the manuscript; MS contributed to collection of data and approved the version to be submitted; IT contributed to collection of data and approved the version to be submitted; RN conceived the study and supervised all experiments performed; HK conceived the study and supervised all experiments performed; HT and YK supervised the study as principal investigator. All participants contributed commentary on and corrected the manuscript. All authors read and approved the final manuscript.

Acknowledgments: The authors would like to thank Mrs. Satsuki Fukuhara of the Keio University School of Medicine for valuable experimental advice.

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Table 1. Background characteristics and pathological findings of esophageal cancer patients.

Variables	esophageal cancer patients n=17
Gender
Male	14
Female	3
Age (median, range)	69 (35-75)
UICC TNM 8th
Pathological T factor
T1	3
T2	1
T3	13
Pathological N factor
N0	6
N1	4
N2	7
Pathological Stage
Stage I	1
Stage II	7
Stage III	9
UICC Union for International Cancer Control

UICC, Union for International Cancer Control TNM Classification of Malignant Tumors (8th Edition)

Table 2. Univariate Cox proportional hazards analysis

	Univariate
Variables	HR	95% CI	p-value
Age			0.547
<70	1.000
>70	1.644	0.326-8.306
Pathological T factor			0.381
T1-2	1.000
T3-4	2.639	0.302-23.083
Pathological N factor			0.831
negative	1.000
positive	0.831	0.152-4.552
CCR7 high / Let-7a low			0.028
None	1.000
Yes	6.476	1.222-34.309

HR, hazard ratio; CI, Confidence Intervals

No competing interests reported.

Supplementary.xls

Effects of let-7a microRNA and C-C chemokine receptor type 7 expression on cellular function and prognosis in esophageal squamous cell carcinoma

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Trial registration:

Figures

1. Background

2. Methods

2.1 Tissue samples

2.2 Esophageal cancer cell lines

2.3 Cell culture

2.4 Transfection

2.5 Isolation and quantitative expression of let-7a

2.6 Isolation of RNA and synthesis of single-stranded cDNA

2.7 RT-PCR analysis

2.8 Let-7a transfection

2.9 Invasion assay

2.10 Development of lymph node metastasis model

3. Results

3.1 Expression of CCR7 and let-7a in esophageal cancer cell lines

3.2 Relationship between let-7a and CCR7 expression in ESCC cells

3.3. Downregulation of let-7a increases CCR7 expression in KYSE-590 cells and evaluation of invasion ability

3.4 Development of highly invasive cell lines and lymph node metastasis model

3.5 Relationship between CCR7 and let-7a expression in ESCC patients

3.6 Relationship between prognosis and CCR7 and let-7a expression

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1