UALCAN online (http://ualcan.path.uab.edu/analysis.html) is biological analysis tool. Data were obtained from a wide range of sources, including RNA sequencing expression data from tumor and normal samples from the TCGA database. All data were standardized for subsequent analysis. UALCAN database was used to verify the differential expression of core genes between esophageal cancer and normal tissues, and P<0.05 was considered to be statistically significant. LinkedOmics database (http://www.linkedomics.org) was employed to analyze the correlation of RNF6 genes and the functional enrichment analysis of co-expressed genes.

Human esophageal cancer cell lines Eca-109 and KYSE-150 (provided by Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Conventional subculture was conducted in a 5% CO₂ incubator at 37°C with saturated humidity.

The cells were inoculated in six-well plates and cultured at 37°C and 5% CO₂ for 12 h. When the cell fusion reached 80%, a transfection experiment was carried out following the operation instructions of the transfection reagent. The transfection reagents containing pcDNA.3.1-RNF6 (3 μg), pcDNA.3.1 (3 μg), and RNF6 siRNA or unrelated control siRNA (3 μg) were mixed with 200 μL of RPMI-1640 medium without serum and added into each well. The transfection efficiency was detected by QRT-PCR after incubation at 37°C with 5% CO₂ for 48 h. Human RNF6 (si-RNF6) sequences were as follows: sense 5′-CCCGAACAAUGGAGAGUUUTT-3′ and antisense 5′-AAACUCUCCAUUGUUCGGGTT-3′. Negative scramble control sequences (si-NC) were as follows: sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense 5′-ACGUGACACGUUCGGAGAATT-3′.

Cell lysate was added to each group of cells, and total cell RNA was extracted. PCR amplification was performed after reverse transcription into cDNA. Reaction conditions were as follows: 95°C for 3 min; 95°C for 5 s, 56°C for 10 s, and 72°C for 25 s for 40 cycles; and 65°C for 5 s. The relative quantification method (18) was applied to determine differences in the mRNA expression of RnF6 in each group using the following primers: RNF6-Forward: 5′-AGAAGATGGCAGCAAGAGCG-3′, RNF6-Reverse: 5′-TCAAGTCAGGCTGAGATGCTAGT-3′; GAPDH-Forward: 5′-GAAGGTGAAGGTCGGAGTC-3′; and GAPDH-Reverse: 5′-GAAGATGGTGATGGGATTTC-3′. GAPDH was selected as the internal reference gene, and 2^-ΔΔCt was used to represent the mRNA expression fold relationship of RNF6 in each group.

Cells were seeded into six-well plates at a cell density of 5×10 (5) per well. FBS-free medium was added, and a straight line parallel to the middle part of each hole was drawn with 1 mL sterile elongated spear head in the same direction. Scratches were observed under a mirror, photographed, and marked. The scratches of each group were observed under a microscope at 0 and 24 h.

Each Transwell membrane was spread with 30 μL of matrix glue and left overnight. On the next day, the cell suspension was added and the membrane was inoculated. A medium without FBS was added to the upper layer, and a medium containing 10% FBS was added to the lower layer. After incubation for 24 h, the cells of the upper compartment were removed. 0.1% crystal violet was dyed at room temperature for 20min. The cells were counted under a microscope.

In brief, 2×10 (5) Eca-109 and KYSE-150 cells were inoculated on six-well plates (two cover slides were placed in each well). The culture medium was discarded, and the plates were washed with PBS for three times and fixed with 4% paraformaldehyde. The cover glass was removed after successive treatment with 3% H₂O₂-methanol and 0.2% Triton X-100. TUNEL staining was performed following the In Situ Cell Death Detection Kit instructions. The number of green fluorescent cells was observed under an inverted fluorescence microscope (Leica, Germany), and the apoptotic cell rate was calculated.

Cells were collected and washed with PBS once. Total proteins were extracted from cell samples using NP-40 lysis buffer containing 40μg/mL protease inhibitor and phosphatase inhibitor. The protein concentration was determined using the BCA protein determination kit (Beyotime Biotechnology Co., LTD. Shanghai, China) according to the manufacturer’s instructions. The same amount of 40 μg protein was electrophoretic in SDS-PAGE 10% gel. It was then transferred to the PVDF membrane (Millipore, USA). After being closed with 5% skim milk for 1h, they were incubated with primary antibody E-cadherin (Abcam, 1:1000) and N-cadherin (Abcam, 1:1000) at 4°C overnight. TBST film washing 3 times for 10min each time. The secondary antibody conjugated with horseradish peroxidase was incubated at room temperature for 1h. TBST washed the film 3 times, 10min each time. Protein bands were observed by ECL assay kit.

SPSS 22.0 software was employed for statistical analysis, and values were expressed as (mean ± SEM). T test was used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. Kaplan–Meier method was applied for survival analysis. P<0.05 was considered statistically significant.

TCGA data analysis showed that RNF6 expression significantly increased in human esophageal cancer group compared with the paracancer control group ( Figure 1A , P<0.01). Figure 1B depicts RNF6 expression in different tumor stages. Compared with that in normal cells, RNF6 was highly expressed in Stage1 cells. Figure 1C shows RNF6 expression in ESCA based on tumor histology. RNF6 was highly expressed in adenocarcinoma and squamous cell carcinoma. Survival analysis revealed that patients with high RNF6 expression (n=82) had a shorter survival than patients with low RNF6 expression (n=93) ( Figure 1D , P<0.05).

Scratch test results showed that after the stable overexpression of RNF6 ( Figure 2A ), the Eca-109 and KYSE-150 cells showed significantly enlarged cell scratch healing area and enhanced migration ability ( Figure 2B , P<0.01). Transwell invasion assay revealed that the number of tumor cells below the Transwell compartment membrane significantly increased in the overexpressed RNF6 group, and the invasion ability of cells was significantly enhanced (P<0.01, Figure 2C ). Figure 2D illustrates the changes of E-cadherin and N-cadherin levels after RNF6 upregulation (Eca-109 and KYSE-150 cells). RNF6 overexpression inhibit E-cadherin but promoted N-cadherin. Apoptosis was detected by TUNEL after RNF6 overexpression. Compared with that of the control group, the apoptosis degree of RNF6 overexpressed group was significantly decreased (P<0.01, Figure 2E ).

RNF6 expression in the siRNF6 group was significantly lower than that in the non-spe group ( Figure 3A , P<0.01). In vitro scratch test showed that the migration ability of siRNF6 group was significantly weakened and the cell healing area was small ( Figure 3B ). Transwell assay showed that the number of invasive cells was reduced in the siRNF6 group ( Figure 3C ). qRT-PCR analysis of E-cadherin and N-cadherin showed that E-cadherin was up-regulated in the in siRNF6 group compared with that in the control group. Meanwhile, the expression of N-cadherin decreased ( Figure 3D ). TUNEL results showed that the apoptosis rate of siRNF6 group was significantly increased compared with that of the control group (P<0.01, Figure 3E ).

Figures 4A, B show the heatmap analysis of RNF6 co-expressed genes, respectively. The functions of RNF6 co-expressed genes were correlated with tumor proliferation, invasion and metastasis, and EMT. Functional enrichment analysis of RNF6 co-expressed genes revealed that RNF6 mainly affected cell communication, cellular component remodeling, and cell proliferation. It also affected the cytoskeleton, extracellular matrix, and microfilament microtubule composition ( Figure 4C ). Figures 4D–F show that RNF6 co-expressed genes affected spindle localization, transcription elongation factor complex, and microtubule organizing center localization of esophageal cancer cells. One of the hallmarks of EMT is a change in morphology, from pebble-like to fusiform cells. Cell polarity loss, skeletal changes, infiltration and migration ability increased. In addition, the EMT process can be triggered by a variety of signals, such as transcription factors and other oncogenic genes. Therefore, RNF affects the EMT of esophageal cancer.

The results confirmed that RNF6 can promote the invasion and migration of esophageal cancer cells. Further study was conducted to determine the regulatory mechanism and whether TGF-β signaling pathway can achieve the above functions. qRT-PCR results showed that Eca-109 and KYSE-150 cells with RNF6 overexpression exhibited significantly up-regulated Snail mRNA expression. After treatment with TGF-β specific inhibitor SB525334, the Snail expression was down-regulated ( Figure 5A ). After RNF6 overexpression in Eca-109 and KYSE-150 cells, E-cadherin was significantly decreased and N-cadherin was up-regulated. After SB525334 treatment, E-cadherin was up-regulated and N-cadherin expression was down-regulated ( Figures 5B, C ). Compared with those in the RNF6 + DMSO group, the migration and invasion abilities of Eca-109 and KYSE-150 cells in the RNF6 + SB525334 group were significantly reduced ( Figures 5D, E , P <0.01).

Compared with that in the control group, the Snail mRNA of Eca-109 and KYSE-150 cells in the TGF-β group was up-regulated. Compared with that in the TGF-β + si-NC group, the Snail mRNA in the TGF-β + si-RNF6 group was significantly reduced ( Figure 6A ). After TGF-β treatment, E-cadherin was down-regulated and N-cadherin was up-regulated, suggesting that TGF-β promoted EMT in ESCC. Compared with TGF-β, E-cadherin was up-regulated and N-cadherin was down-regulated in the TGF-β + si-RNF6 group ( Figures 6B, C ). Compared with those in the TGF-β + si-NC group, the migration and invasion abilities of Eca-109 and KYSE-150 cells in the TGF-β + si-RNF6 group were significantly reduced ( Figures 6D, E , P <0.01). Figures 6F, G show the co-expression correlation between RNF6 and TGF-β and c-Myb. RNF6 exhibited a positive correlation with TGF-β and c-Myb and its co-expression in esophageal cancer.

Compared with that in the control group, c-Myb mRNA increased in the TGF-β group ( Figure 7A ). qPCR was conducted to analyze c-Myb mRNA levels in Eca-109 and KYSE-150 cells treated with RNF6 overexpression or SB525334 (TGF-β inhibitor). c-Myb mRNA was significantly up-regulated in Eca-109 and KYSE-150 cells after the stable overexpression of RNF6. After treatment with TGF-β specific inhibitor SB525334, c-Myb mRNA was down-regulated ( Figure 7B ). Compared with that in the control group, the c-Myb mRNA of Eca-109 and KYSE-150 cells in the TGF-β group was up-regulated. Compared with that in the TGF-β + si-NC group, the c-Myb mRNA in the TGF-β + si-RNF6 group was significantly decreased ( Figure 7C ). TCGA was used to analyze the relative levels of TGF-β in paracancer normal tissues and esophageal cancer tissues. TCGA data analysis results showed that TGF-β expression was significantly increased in human esophageal cancer compared with that in the paracancer control group ( Figure 7D ). Figure 7E reveals the molecular mechanism of RNF6 in promoting EMT in esophageal cancer.