RETRACTED ARTICLE: Inhibited MicroRNA-301 Restrains Angiogenesis and Cell Growth in Esophageal Squamous Cell Carcinoma by Elevating PTEN

Objective Esophageal squamous cell carcinoma (ESCC) is featured by early metastasis and late diagnosis. MicroRNA-301 (miR-301) is known to participate in diverse cancers. Nevertheless, effects of miR-301 on ESCC remain unexplored. Thus, we aim to explore the role of miR-301 in ESCC progression. Methods Expression of miR-301 and phosphatase and tensin homologue (PTEN) in ESCC tissues and cell lines was assessed. Next, the screened cells were treated with altered miR-301 or PTEN oligonucleotide and plasmid, and then, the colony formation ability, cell viability, migration, invasion, cell cycle distribution and apoptosis of ESCC cells were assessed. Moreover, tumor growth and microvessel density (MVD) were also assessed, and the targeting relationship between miR-301 and PTEN was affirmed. Results MiR-301 was upregulated, and PTEN was downregulated in ESCC tissues and cells. KYSE30 cells and Eca109 cells were selected for functional assays. In KYSE30 cells, inhibited miR-301 or overexpressed PTEN suppressed cell malignant behaviors, and silenced PTEN eliminated the impact of miR-301 inhibition on ESCC progression. In Eca109 cells, miR-301 overexpression or PTEN inhibition promoted cell malignant behaviors, and PTEN overexpression reversed the effects of miR-301 elevation on ESCC progression. The in vivo assay revealed that miR-301 inhibition or PTEN overexpression repressed ESCC tumor growth and MVD, and miR-301 elevation or PTEN reduction had contrary effects. Moreover, PTEN was targeted by miR-301. Conclusion Taken together, results in our study revealed that miR-301 affected cell growth, metastasis and angiogenesis via regulating PTEN expression in ESCC.


Introduction
Esophageal cancer (EC), the 8th most common cancer all over the world, is a critical malignancy with high mortality and poor prognosis [1]. Accounting for about 90% of the total EC cases, esophageal squamous cell carcinoma (ESCC) is the main form of EC in China [2]. Multiple causes including low socioeconomic status, smoking, alcohol consumption, poor nutrition intake, nitrosamine-rich or mycotoxin-contaminated food lead to the occurrence of ESCC [3]. In spite of the promoted clinical outcomes as well as administration, there is still a poor prognosis among the ESCC patients, accompanying a 5-year survival rate of 15-25% [4]. Hence, it is crucial to confirm oncogenes or tumor-repressive genes which could function as biomarkers in the development of ESCC to provide more effective therapeutic methods for ESCC patients.
MicroRNAs (miRNAs) are small non-coding RNAs playing an essential part in modulating gene expression [5] and are clarified to have the capacity of influencing tumor progression through regulating mRNA stability and ability of mRNAs [6]. An amount of miRNAs such as miR-4324 [7], miR-889-3p [8] and miR-9 [9] have been found to be associated with the process of ESCC. MiR-301 is a member of the miRNAs which is formed by the fam33a transcription unit situated at 17q22-23 in human genome. The overexpression of miR-301 has been previously identified, which reflected that it is implicated in human diseases [6,10]. However, the function mechanisms of miR-301 have not been uncovered in ESCC. Moreover, phosphatase and tensin homologue (PTEN) has been affirmed to be frequently disrupted in tumors and targeted by germ line mutations in cancer patients, which plays an inhibitive role of tumors [11]. It has been validated that the dysregulation of PTEN is correlated with ESCC development [12]. Interestingly, a recent research has revealed that miR-301 targets PTEN in non-small cell lung cancer [13]. However, this targeting relationship between miR-301 and PTEN in ESCC development remains to be unveiled. Our research focused on the effects of miR-301 and PTEN on ESCC progression, which remain largely unknown and are of novelty. We inferred that miR-301 may influence angiogenesis and cell growth in ESCC by modulating PTEN expression.

Ethics Statement
Written informed consents were acquired from all patients prior to the study. The protocols of this study were approved by the Ethic Committee of The Second Hospital of Jilin University and based on the ethical principles for medical research involving human subjects of the Helsinki Declaration. Animal experiments were strictly consistent with the Guide to the Management and Use of Laboratory Animals issued by the National Institutes of Health. The protocol of animal experiments was approved by the Institutional Animal Care and Use Committee of The Second Hospital of Jilin University.

Study Subjects
One hundred and ten samples of ESCC tissues and adjacent normal tissues (> 5 cm from the tumor) were collected from ESCC patients (78 males and 32 females) that had accepted esophagectomy in the thoracic surgery department of The Second Hospital of Jilin University from January 2015 to December 2017. Among the 110 patients, there were 84 cases > 60 years, and 26 cases ≤ 60 years; tumor size: 65 cases ≥ 5 cm and 45 cases < 5 cm; 71 cases without lymph node metastasis (LNM) and 39 cases with LNM; the tumor, node and metastasis (TNM) stage: 60 cases were in the I + II stage, and 50 cases were in the III stage; tumor location: 13 cases were upper ESCC, and 97 cases were mid-lower ESCC. The patients were all diagnosed with ESCC and had not accepted radiotherapy or chemotherapy before. The tumors were completely excised, and the negative surgical margin has been affirmed by pathology. According to the staging criteria of ESCC that proposed by Union for International Cancer Control (UICC) in 2009 [14], the postoperative pathological staging of the patients was identified as the pT1-4N1-2(I-IIIb) stage. There was no significant complication in patients after the surgery, and perioperative deaths were excluded.

Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNAs in tissues and cells were extracted using Trizol kits (Invitrogen Inc., Carlsbad, CA, USA). The RNA concentration and quality were measured. RNA primers (Table 1) were designed and synthesized by TaKaRa Biotechnology Co., Ltd. (Liaoning China). The RNA was reversely transcribed into cDNA based on the instructions of Takara PrimeScript ™ RT reagent Kit with g DNA Eraser (Takara). We performed qPCR on the Light Cycler 480II (Roche) with the Power PCR SYBR green PCR master mix (Takara). U6 was used as the loading control of miR-301, and β-actin was utilized as the internal reference of PTEN. Data were analyzed using 2 −△△Ct method [15].

Western Blot Analysis
RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) was used to extract total protein in cells and tissues, and the protein was quantified by a BCA Protein Assay kit (Beyotime). The protein concentration of each sample was measured, and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed. The samples were transferred onto the nitrocellulose membranes, which were then blocked with 5% skim milk powder at 4 °C overnight. Afterward, the

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membranes were supplemented with primary antibodies PTEN and β-actin (both 1: 500 and from Santa Cruz Biotechnology Inc, CA, USA) for overnight incubation, then added with the respective secondary antibodies and incubated for 1 h. After immersed in the enhanced chemiluminescent reagent (Pierce Chemical Inc., Dallas, TX, USA) for 1 min, the membranes were exposed in dark environment and developed using LAS4000 mini chemiluminescence imager. The gray values were evaluated by an imaging system software with β-actin as the control; therefore, the final relative protein expressed was obtained. The protein bands were analyzed by ImageJ2x software.

Cell Counting Kit (CCK-8) Assay
Cells were seeded in a 96-well plate (1 × 10 3 cells/well) and incubated for different time periods. After incubated for 24 h, 48 h, 72 h, and 96 h, each well was supplemented with 10 μL CCK-8 solution (5 mg/mL), and then, the cells of each group were incubated at 37 °C without light exposure for 2 h. The optical density (OD) values at 450 nm were analyzed by a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Colony Formation Assay
Cells were seeded at 500 cells/well in 6-well plates after transfection and cultured for 14 d. Colonies were fixed with methanol, stained using 0.5% crystal violet and counted under the inverted microscope.

Transwell Assay
Cells (5 × 10 3 ) incubated in RPMI 1640 medium were seeded into apical chambers of Transwell devices with non-coated or matrigel-coated membrane (Corning, NY, USA). After 24 h, cells on apical chambers were removed, while cells remaining on the underside were fixed and then stained using 0.1% crystal violet. A microscope (Olympus Corporation, Tokyo, Japan) was used for counting on 3 random fields to calculate the number of cells.

Flow Cytometry
Cell cycle and apoptosis were evaluated by flow cytometry. Annexin V-fluorescein isothiocyanate (10 µL) and propidium iodide (PI; 5 µL, Sigma) were incubated with the cells (5 × 10 5 cells/well) in dark at 4 °C for 30 min. The percentage of apoptotic cells was calculated using a flow cytometer (BD Biosciences, CA, USA) with FlowJo version 10 software (FlowJo LLC, OR, USA).
To assess the cell cycle, cells (5 × 10 5 cells/well) were fixed with 75% ethanol overnight at 4 °C and stained with 5 µl PI/ribonuclease A (Sigma) at 4 °C for 30 min in the dark. Data were analyzed with a flow cytometer (BD Biosciences). Fluorescence signals (14,000) of each sample were collected and calculated using ModFit LT version 3.2 software (Verity Software House, Inc., ME, USA).

Subcutaneous Tumorigenesis in Nude Mice
Forty-two female BALB/c-nu nude mice (aging 4 w, weighing 16-24 g) were obtained from Experimental Animal Center Jilin University (Changchun, China). The nude mice were separated into 14 groups (n = 3). Nude mice of seven groups were, respectively, injected with KYSE-30 cells according to the cell grouping, and nude mice in the rest seven groups were separately injected with Eca109 cells based on the grouping. The concentration of transfected KYSE-30 and Eca109 cells was adjusted to 5 × 10 6 cells/100 μL. The nude mice were fixed and subcutaneously injected with corresponding ESCC cells under sterile conditions. The largest length (L) and width (W) of the tumors were measured every week and tumor volume (V) = 1/2 × L × W 2 . The nude mice were euthanized in the 5 th week of injection with the tumors resected, and the tumors were weighed and photographed. Tumor formation rate was calculated as the number of mice with subcutaneous tumor/total number of injected nude mice in the group × 100%. The injection time was taken as abscissa, and the tumor size was taken as the ordinate; thus, the tumor growth curve was graphed.

Immunohistochemical Staining
The tumor tissues of nude mice were fixed by 10% formaldehyde, embedded by paraffin and sectioned into 4 μm. Next, the sections were dried at 60 °C for 2 h, dewaxed by xylene, dehydrated by gradient ethanol and incubated with 50 μL 3% H 2 O 2 for 10 min. Afterward, the sections were soaked in 0.01 M citric acid buffer solution, boiled at 95 °C for 20 min, blocked by normal goat serum work solution at 37 °C for 10 min and appended with CD 34 (1: 100, Santa Cruz) at 4 °C overnight. Afterward, the sections were supplemented with HRP-labeled goat anti-rabbit/mice IgG polymer (ZSGB-Bio, Beijing, China), counterstained by hematoxylin, dehydrated and permeabilized and then sealed with neutral balsam. PBS was used to replace the primary antibodies as the NC. Measurement of microvessel density (MVD): the sections were observed under a low-magnification microscope. A endothelial cell or a endothelial cell cluster stained into brownish yellow and significantly distinguished with the surrounding tumor cells, and connective tissues were taken as a microvessel. The branch structure was also taken as a vessel if it was disconnected, while vessels with lumen size > 8 erythrocytes, muscular layer or thicker lumen were excluded. The numbers of microvessel of 3 high visual fields were recorded, and the average number was MVD of each case.

Statistical Analysis
All statistical analyses were carried out using SPSS version SPSS 21.0 software (IBM Corp. Armonk, NY, USA) and presented with Graphpad Prism Software 6.0. Data were expressed as mean ± standard deviation. Differences between two independent groups were tested with Student's t test. One-way ANOVA was performed for comparing three or more groups. P value < 0.05 was indicative of statistically significant difference.

MiR-301 is Highly Expressed, While PTEN is Poorly Expressed in ESCC Tissues and Cells
MiR-301 and PTEN expression in ESCC tissues and adjacent normal tissues was assessed using RT-qPCR and Western blot analysis to reveal their roles in ESCC, and it was found that (Fig. 1a-c) miR-301 was upregulated, while PTEN was downregulated in ESCC tissues. The patients were divided into the low and high expression groups according to the median value of miR-301 or PTEN expression to analyze the correlation between miR-301 or PTEN expression and clinicopathological characteristics of ESCC patients. The results indicated that miR-301/PTEN expression was not related to age, gender, tumor size, location and differentiation, while was correlated with TNM stage and LNM of ESCC patients (Table 2).
Then, expression of miR-301 and PTEN in 4 ESCC cell lines and HEECs was determined using RT-qPCR and Western blot analysis. We found that ( Fig. 1d-

PTEN is Targeted by miR-301
A bioinformatic software (http://www.micro rna.org/) predicted that PTEN was the target gene of miR-301 (Fig. 2a). It was further confirmed by dual luciferase reporter gene assay that the luciferase activity was significantly decreased in ESCC cells co-transfected with PTEN-wt vector and miR-301 mimic compared with those co-transfected with PTEN-mut vector and miR-301 mimic, implying that miR-301 could particularly bind to PTEN (Fig. 2b, c).
RT-qPCR and Western blot analysis were used to assess miR-301 and PTEN expression in transfected cells, and it was found that in KYSE-30 cells (Fig. 2d-

Inhibited miR-301 or Overexpressed PTEN Restricts Viability of ESCC Cells; Elevated miR-301 or Reduced PTEN Promotes Viability of ESCC Cells
Cell viability of ESCC cells was assessed using colony formation and CCK-8 assays. The results revealed that in KYSE-30 cell line (Fig. 3a-c), transfection of miR-301 inhibitor or oe-PTEN repressed colony formation ability and cell viability; transfection of silenced PTEN eliminated the impact of miR-301 inhibitor on ESCC cell viability; in Eca109 cell line ( Fig. 3d-f ), transfection of miR-301 mimic or si-PTEN promoted colony formation ability and cell viability; PTEN overexpression reversed the promotive role of miR-301 elevation in colony formation ability and viability of Eca109 cells. These results suggested that miR-301 knockdown or PTEN overexpression repressed the viability of ESCC cells, which were promoted by miR-301 elevation or PTEN inhibition.

Inhibited miR-301 or Overexpressed PTEN Represses Migration and Invasion of ESCC Cells; Elevated miR-301 or Reduced PTEN Induces Migration and Invasion of ESCC Cells
Migration and invasion abilities of ESCC cells were assessed using Transwell assay. The results suggested that in KYSE-30 cell line (Fig. 4a, b), the cell migration and invasion abilities were restrained by inhibition of miR-301 or overexpression of PTEN; the suppressive role of miR-301 inhibitor in cell migration and invasion abilities was reversed by si-PTEN. In Eca109 cell line (Fig. 4c, d), the cell migration and invasion abilities were promoted after the transfection of miR-301 mimic or si-PTEN; overexpressed PTEN reversed the impact

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of miR-301 mimic on cell migration and invasion abilities. The above findings implied that the migration and invasion of ESCC cells were inhibited by miR-301 repression or PTEN elevation, whereas were facilitated by miR-301 upregulation or PTEN downregulation.

Inhibited miR-301 or Overexpressed PTEN Induces Cell Cycle Arrest and Apoptosis of ESCC Cells; Elevated miR-301 or Reduced PTEN Suppresses Cell Cycle Arrest and Apoptosis of ESCC Cells
Flow cytometry was used to detect the cell cycle transition and apoptosis of cells after transfection, and the results indicated that in KYSE-30 cell line (Fig. 5a-d), transfection of miR-301 inhibitor or oe-PTEN promoted the apoptotic rate and increased cells in G0/G1 phase, while decreased that in S and G2/M phases; the alteration of apoptosis and cell cycle arrest induced by miR-301 inhibitor could be reversed by si-PTEN. According to the results of flow cytometry, we have found that in Eca109 cell line (Fig. 5e-h), transfection of miR-301 mimic or si-PTEN inhibited apoptotic rate, decreased cells in G0/G1 phase and increased that of the S phase and G2/M phases; PTEN overexpression reversed the effect of miR-301 mimic on the apoptotic rate and cell cycle arrest of Eca109 cells. We concluded from these results that downregulated miR-301 or upregulated PTEN promoted cell cycle transition and apoptosis in ESCC cells, while inhibited miR-301 or silenced PTEN exerted opposite effects.

Inhibited miR-301 or Overexpressed PTEN Restrains Tumor Growth and Angiogenesis In Vivo in ESCC; Elevated miR-301 or Reduced PTEN Increases Tumor Growth and Angiogenesis In Vivo in ESCC
The growth and changes of ESCC tumors in nude mice were observed in each group. Tumor growth was assessed, and the results implied that in KYSE-30 cell line (Fig. 6a-e), nude mice injected with miR-301 inhibitor or oe-PTEN reduced tumor volume and weight; the repressive role of miR-301 inhibitor in tumor growth was abolished by si-PTEN. In Eca109 cell line (Fig. 6f-j), the tumor volume and tumor weight were advanced in nude mice injected with miR-301 mimic or si-PTEN; overexpression of PTEN reversed the effect of miR-301 mimic on tumor growth. Meanwhile, the expression of CD34 in xenografts from nude mice was assessed using immunohistochemical staining and the findings showed that (Fig. 7a-d)  inhibitor + si-NC group; a P < 0.05 versus the mimic-NC group; b P < 0.05 versus the si-NC group; c P < 0.05 versus the miR-301 mimic + oe-NC group, N = 3. Data were expressed as mean ± standard deviation, and ANOVA was used for comparisons among multiple groups

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after miR-301 downregulation or PTEN upregulation; silenced PTEN reversed impact of miR-301 inhibition on MVD. In Eca109 xenografts, MVD was increased after miR-301 upregulation or PTEN downregulation; the enhancement of MVD induced by upregulated miR-301 could be abolished by overexpressed PTEN. These data indicated that miR-301 inhibition or PTEN overexpression repressed tumor growth and angiogenesis in ESCC, while miR-301 elevation or PTEN silencing had reverse effects.

Discussion
EC is a kind of invasive malignancy in the gastrointestinal tract [16]. As the major type of EC, ESCC is a malignant tumor occurring in esophageal epithelial cells [17]. The miRNAs, known as small non-coding RNAs, have been demonstrated to function as a significant roles in leading molecules in the silencing of RNA [18]. Our research was designed to explore the effects of miR-301 and its target gene PTEN on ESCC progression, and we have found that the inhibited miR-301 could suppress angiogenesis and cell growth in ESCC by elevating PTEN. MiR-301 expression was assessed, and we found that miR-301 was highly expressed in ESCC cells in comparison with HEEC, and the higher expression of miR-301 has also been found in ESCC tissues in contrast to the adjacent normal tissues. Similar to this result, Li et al. have identified that miR-301 presented high expression in myocardial infarction tissues [19]. In addition, we have elucidated that PTEN was targeted by miR-301, and the target relation has been pointed out by an extant literature [20]. We have also discovered that PTEN, which has been affirmed to be targeted by miR-301, was downregulated in both ESCC tissues and cells. Similarly, a previous research has unearthed that PTEN was poorly expressed in ESCC compared with non-tumor esophageal epithelial tissue [21]. Furthermore, Ma et al. have illuminated that PTEN expression was degraded in Eca109 cell line [22], which has also been selected for a series of experiments in this research. These studies provide evidence for the high expression of miR-301 and low expression of PTEN in ESCC.
Another important outcome in this research indicated that the inhibited miR-301 could repress the colony formation ability as well as the cell proliferation of ESCC cells via enhancing the PTEN expression, and elevated miR-301 or reduced PTEN had contrary effects. Similarly, Han et al. have elucidated that the downregulation of miR-301 mediated by luteolin has the ability to restrain the cell proliferation in prostate cancer [6]. A recent literature has revealed that the overexpression of PTEN suppresses the proliferation of pancreatic cancer cells [23], and a same result has been summarized in a study focusing on prostate cancer [24]. Besides, we have also unearthed that the downregulation of miR-301 or the elevation of PTEN could inhibit migration and invasion of ESCC cells, and elevated miR-301 or reduced PTEN exhibited the opposite trends. In accordance with this outcome, Shi et al. have supported that inhibited miR-301 attenuated migration and invasion of breast cancer cells [10], and it has been reported that the migration and invasion of ESCC cells could be repressed by the inhibition of miR-130b and the elevation of PTEN [25]. These publications helped verifying the oncogenic role of miR-301 and tumor-repressive effect of PTEN in diverse human cancers. Another result in our research was that inhibited miR-301 overexpressed PTEN to promote cell apoptosis and induce cell cycle arrest at the G0/G1 phase in ESCC cells, and elevated miR-301 or reduced PTEN had the inverse results. Similarly, it has been uncovered by a recent literature that activated PTEN induces cell cycle arrest and apoptosis in ESCC [26]. Consistently, Tian et al. have found in their study that the elevation of PTEN inhibited the angiogenesis by reducing the expression of vascular endothelial growth factor in hepatocellular carcinoma [27]. Based on the above data, the roles of miR-301 and PTEN in cell apoptosis and angiogenesis in diverse diseases were further confirmed. Consequently,    & P < 0.05 versus the oe-NC group; # P < 0.05 versus the miR-301 inhibitors + si-NC group; a P < 0.05 versus the mimic-NC group; b P < 0.05 versus the si-NC group; c P < 0.05 versus the miR-301 mimic + oe-NC group, n = 3 mice. Data were expressed as mean ± standard deviation, and ANOVA was used for comparisons among multiple groups

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we concluded that the downregulation of miR-301 could restrain the tumor growth in ESCC through the high expression of PTEN, and the similar conclusion has also been unveiled in breast cancer [10] and prostate cancer [28]. On the contrary, miR-301 elevation or PTEN reduction induced the tumor growth in ESCC. It could be concluded that miR-301 and PTEN participated in the in vivo cancer cell growth.

Conclusion
In this study, we have shown that the repression of miR-301 prohibits angiogenesis, cell proliferation, migration and invasion but promotes apoptosis in ESCC cells by upregulating PTEN. This research may further the understanding on potential molecular mechanisms of ESCC and provide novel targets for ESCC treatment.

Ethics approval and consent to participate
This study was approved and supervised by the animal ethics committee of The Second Hospital of Jilin University. The treatment of animals in all experiments conforms to the ethical standards of experimental animals.

Consent for publication
Not applicable. & P < 0.05 versus the oe-NC group; # P < 0.05 versus the miR-301 inhibitors + si-NC group; a P < 0.05 versus the mimic-NC group; b P < 0.05 versus the si-NC group; c P < 0.05 versus the miR-301 mimic + oe-NC group, n = 3 mice. Data were expressed as mean ± standard deviation, and ANOVA was used for comparisons among multiple groups