Methylation regulation of MUC6 correlates with metastasis of gastric cancer

Background: The aim of this study was to investigate the mechanism of the downregulation of MUC6 and its influence on GC metastasis. Methods: The expression of MUC6 was examined in cancer tissues and their corresponding adjacent normal tissues in 40 gastric adenocarcinoma patients. The investigation of methylation level of MUC6 promoter region in gastric cell lines and gastric specimen tissues was performed through immunohistochemistry and/or quantitative polymerase chain reaction (qPCR)s. MUC6 was knocked down in GES-1 cell lines and overexpressed in SGC7901 cell lines; the effects of MUC6 knockdown and overexpression on cell migration and invasion were examined using Transwell migration assay. The effects of demethylation and methylation on MUC6 expression were examined using Western blot, qPCR, or double luciferase report experiment. Results: The expression of MUC6 in GC tissues was significantly lower than that in normal paracancerous tissues. While the cells migration and invasion abilities were decreased significantly after overexpression of MUC6, these abilities increased significantly after the knocking down of MUC6. The methylation levels of MUC6 in GC tissues and GC cell lines (MGC803, MKN45, AGS, SGC7901, and BGC823) were significantly higher than those in paracancerous tissues and gastric epithelial cells. The promoter methylation could significantly reduce the binding of MUC6 promoter region to the related transcription factors. The expression of MUC6 increased with the concentration of demethylated drugs and the time of action. Conclusion: The expression of MUC6 was regulated by methylation of its promoter, and this methylation of MUC6 promoter may lead to significant downregulation of MUC6 in GC and promote the metastasis of GC. Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression.


Abstract
Background: The aim of this study was to investigate the mechanism of the downregulation of MUC6 and its influence on GC metastasis. in the lower layer of the gastric mucosa of the stomach [1][2][3][4] . MUC6 is known to be a marker of gastric foveolar and antral mucous glandular cells, reflecting gastric phenotypes and acts as a significant physiological barrier against various aggressions of the underlying epithelia [5] . It is also reported to be significantly correlated with epithelial growth factor receptor 2, which is related to the invasive behavior of gastric cancer (GC) [6] . Nevertheless, MUC6 is a relatively less-investigated molecule; thus, not much is documented about its expressional changes in GC, the mechanism of such changes, and the effect of these changes on the metastasis of GC. Few studies have reported the downregulation of MUC6 expression in GC [5,[7][8][9] , which may be associated with carcinogenesis, malignant potential, progression, clinical behaviors, and poor prognosis of GC [10][11][12][13] . These were not independent studies and did not directly focus on MUC6. Thus, till date, the mechanism of the downregulation of MUC6 and the effect of MUC6 change on the occurrence and progression of GC have not been fully explained.

Methods
Some of the earlier studies have recorded that mucins may be positioned at a hotspot for methylation in the genome [14] and that methylation modifications played a significant role in regulating mucin genes in epithelial cancer cells [15] . A few studies indicated that the repression of MUC2 expression in colon carcinoma cells was linked to methylation of its promoter [16] . However, de novo expression of MUC2 was exhibited to be triggered by promoter demethylation or hypomethylation in pancreatic and gastric carcinoma cells [17,18] . Nevertheless, data regarding MUC6 are unavailable. The above-mentioned studies motivated us to study the relationship between MUC6 and GC using the TCGA database and the software Gene Expression Profiling Interactive Analysis. The expression of MUC6 in GC tissue was found to be significantly lower than that in normal tissues and to be closely associated with the metastasis of tumors (Supplementary Figure 1). Retrieved MethHC database indicated that the methylation level of MUC6 promoter region in GC tissues was higher than that in the normal tissues, and a significant difference between the two levels was found (Supplementary Figure 2). Thus, MUC6 is assumed to promote the metastasis of GC through methylation of its promoter region, resulting in the downregulation of its expression. The effect of downregulation of MUC6 on the biological behavior of GC cells and the mechanism of downregulation of MUC6 expression are the focus of this study. The above hypothesis was investigated through clinical samples, MUC6 overexpression and knockdown, and MUC6 methylation, and demethylation.

Patients and tissue samples
All tissue samples were supplied by the Department of Gastroenterology, Huamei Hospital, University of Chinese Academy of Sciences between January, 2017 and October, 2018. The study was approved by the Ethics Committee of Huamei Hospital, University of Chinese Academy of Sciences. All participating patients were informed regarding the procedure, and written informed consent was obtained from them. Each patient's surgical sample was divided into two sections. One section was fixed in formalin and embedded in paraffin and then its histopathology was investigated. The other section was immediately refrigerated to be used for MUC6 detection. None of the patients underwent chemotherapy and radiotherapy prior to the tissue harvest.

Immunohistochemistry (IHC)
Tumor tissues were fixed in 4% formalin and embedded in paraffin.
Slices of thickness of 4µm were prepared and baked in a 65°C thermostat for 6-12 hours), and IHC was performed. Sections were dewaxed using xylene and rehydrated using gradient alcohol, were blocked using endogenous peroxidase, and inactivated with 3%

Hematoxylin-eosin(H&E) staining
Followed by xylene dewaxing and gradient alcohol rehydrating, sections were immersed in hematoxylin dyeing solution for 5 minutes at room temperature. The sections were then washed under running water for 1 minute, immersed in 1% hydrochloric acid alcohol solution for several seconds, and then under tap water and returned to blue stain. They were then immersed in the eosin dye for 3-5 minutes, and the excess dye on the slide was washed off with tap water. After dehydrating for 0.5 minutes with 80% ethanol, 95% ethanol I, 95% ethanol II, absolute ethanol I, and absolute ethanol II, the sections were treated with xylene I and II for 3 minutes to make them clear and transparent. Finally, the sections were sealed with neutral gum.

Western blot (WB) protocol
The total cell protein was extracted as per the following steps: Samples from each group were collected into Eppendorf tubes, and 200 ml Western and Input pyrolysates were added into each tube (PMSF was added before using, and the final concentration was 1 mM

Quantitative polymerase chain reaction (qPCR) protocol
The total RNA was prepared with the help of a UGUAUUCAGUAGUCGUUCUUUGUU, and siRNA-3: GCACAUAAUAAGAAACAGUAG. The control siRNA sequence was a random small fragment with the same length.

Methylation
The

Cell invasion
Matrix gel was removed from the refrigerator at −20°℃ and was  demographic data are given in Table 1.

MUC6 expression levels in GC and paracancerous tissues
IHC and qPCR were performed in 40 cases of GC and corresponding normal adjacent tissues. By way of either IHC (p<0.05) or qPCR (p<0.01), the results indicated that the expression of MUC6 in GC tissues was significantly lower than that in normal paracancerous tissues (Figures 1 and 2).

SGC7901 cells with the lowest expression of MUC6 for transfection
were selected (Supplementary Figure 3). After the transfection of MUC6 plasmid, the expression of MUC6 in SGC7901 cells was found to increase significantly to a value higher than that in the control group, which was confirmed by qPCR (p<0.001; Figure 3A) and WB detection (p<0.001; Figure 3B). The migration (p<0.01) and invasion (p<0.05) abilities of SGC7901 cells were observed to be decreased significantly after overexpression of MUC6 ( Figure   4).  Figure 6).

Methylation level of MUC6 promoter region
The methylation of MUC6 promoter region was detected by way of qPCR in 5 GC patients in the GC and adjacent tissues. The methylation level of MUC6 in paracancerous tissues was found to be significantly lower than that in GC tissues (Figure 7).
The methylation levels of MUC6 promoter region in several GC and epithelial cell lines were detected through qPCR. The methylation levels of MGC803, MKN45, AGS, SGC7901, and BGC823 were recorded to be significantly higher than those of normal GES-1 ( Figure 8).

Effect of promoter methylation and demethylation on MUC6 expression
The pGL3-MUC6-Promoter plasmid was methylated by M.SssI methyltransferase, and the protective degree of M.SssI methylation was verified by restriction endonuclease digestion with BstUI.
Compared with pGL3, the fluorescence activity of MUC6 promoter region after methylation was observed to decrease significantly (p<0.001), suggesting that BstUI enzyme could cut plasmid DNA without M. SssI modification, but not that modified by M. SssI

Discussion
For the first time, as per our knowledge, the methylation of MUC6 promoter region was shown to cause a significant downregulation of MUC6 in GC and that the downregulation of MUC6 may promote the metastasis of GC. Earlier studies focused on detecting the expression of MUC6 in GC and normal tissues [6] . A decrease in MUC6 expression in GC has been elucidated [9,19] , and MUC6 gene polymorphism appears to be associated with the tendency of GC [20] .
Varied expression of MUC6 gene in GC has been described in the literature [11] . The expression of MUC6 was highly correlated with the progression of GC [13] and was found to be repressed by methylation in KATO-III cells [21,22] . However, these studies only draw conclusions based on clinical specimen detection, and there was no direct evidence of basic tests, lack of systematic and complete demonstration. Therefore, regulation of MUC6 gene expression in gastric cells and the effect of downregulation of MUC6 expression on metastasis of GC are yet to be further explored.
The expression of MUC6 in GC and adjacent tissues was investigated, and it was indicated to be significantly lower in GC than in the adjacent tissues, which was consistent with the findings of earlier studies [9,10]  expression showed aggressive behavior [10] and was significantly correlated with the factor such as the depth of invasion, venous invasion, stage, and poorer patient prognosis [23] , which mutually correlated with our results. Therefore, at the cellular level, the hypothesis that MUC6 downregulation promotes the metastasis of GC was confirmed. Earlier studies have concluded that regional hypermethylation occurring preferentially at promoter CpG islands has a significant role in carcinogenesis [24][25][26] and results in the inactivation of tumor suppressor genes [27,28] . A high number of CpG sites throughout MUC6 promoter had been identified [15] , which became the material basis of methylation affecting MUC6.
It may be concluded that our results completely correlate with those of earlier findings [22] , that is, methylation of promoter region leads to downregulation of MUC6 expression. Thus, the hypothesis that promoter methylation caused MUC6 downregulation and promoted GC metastasis was verified at the molecular level.
The limitation of this study was that only in vitro experiments revealed that the downregulation of MUC6 promoted the metastasis of GC, and no evidence was reported from animal experiments. In addition, although we observed that methylation in the MUC6 promoter region was related to metastasis of GC, we did not provide any information on the related sites of methylation.
These will be investigated in our next study.

Conclusion
To conclude, the expression of MUC6 was evidently regulated by methylation of its promoter, and the methylation of MUC6    qPCR showed that MUC6 in GC tissues was significantly downregulated compared with that in adjacent tissues (**p<0.01).