A research of STEAP1 regulated gastric cancer cell proliferation, migration and invasion in vitro and in vivos

Abstract Six‐Transmembrane Epithelial Antigene of the Prostate 1 (STEAP1) is associated with the occurrence and development of cancer. This study aimed to clarify the role of STEAP1 in gastric cancer tumour growth and metastasis, as well as its molecular mechanism of action.Statistical methods were used for clinical data analysis. Protein expression was detected using immunohistochemistry(IHC). The mRNA and protein expression in the cell cultures were detected using reverse transcription‐polymerase chain reaction(RT‐PCR) and western blot analysis. Overexpression and silencing models were constructed using plasmid and lentivirus transfection. To detect cell proliferation in vitro, Cell Counting Kit‐8(CCK‐8), flow cytometry and colony formation assays were used; transwell and wound healing assays were used to detect cell migration and invasion;For in vivo experiments, nude BALB/c mice were used for detecting subcutaneous tumorigenesis and intraperitoneal implantation. In the results,we found STEAP1 was overexpressed in gastric cancer tissues and cell lines. Single‐factor and Cox analyses showed that STEAP1 gene expression level correlated with poor prognosis. Up‐regulation of STEAP1 increased cell proliferation, migration and invasion, which decreased after STEAP1 was knocked down. These changes were achieved via the activation of the AKT/FoxO1 pathway and epithelial‐mesenchymal transformation (EMT). The in vivo animal experiments showed that STEAP1 knock down, resulted in a decrease in the subcutaneous tumour and peritoneal tumour formation.

Tumour development involves many factors, which are controlled by many genes, including prostate transmembrane epithelial antigen.
Six-Transmembrane Epithelial Antigene of the Prostate (STEAP) was found as a prostate-specific cell surface antigen using suppression subtractive hybridization technique for the first time. 10,11 STEAP is highly expressed in spontaneous transgenic mouse prostate cancer models and human prostate cancer. In addition, it is also expressed in the pancreas, ovary, gastrointestinal tract, cervix, testis, bladder, Ewing sarcoma and melanoma cells. 10,12 There are four members in the STEAP protein family, STEAP1-4. The main focus of our study is STEAP1.
Gene STEAP1 is located in the 7q21.13 region of the human chromosome; it is 10.4 kb long and contains four introns and five exons. The transcription of the gene STEAP1 can produce two different kinds of mRNAs: a 1.4 kb and a 4 kb mRNAs. However, only the 1.4 kb mRNA can be processed into a mature protein, which contains 339 amino acids with molecular weight of 36 KD, 10,12 while the 4 kb mRNA contains a 2399 BP large intron, which is not translated into a mature protein. 13 Data has shown that gene STEAP1 is closely related to communication between the adjacent cells, and it seemed to be beneficial for the occurrence and development of tumours. 14 Its structural prediction and the location at the cell-cell contacts indicated that gene STEAP1 product may be a transporter or channel. 10,15 Some previous research on different kinds of cancer have found that STEAP1 was observed in tumour tissue but not in normal tissue. The expression of STEAP1 was closely related to the malignant phenotype of cancer cells. 13,[16][17][18][19][20] However, in Lee's study, they found no correlation between expression of STEAP1 and the clinicopathological factors. 21 These conflicting results indicated that the roles of STEAP1 were varied depending on different cancer types. In our study, we will discuss the influence of STEAP1 on the proliferation, invasion and inflammatory reactions in gastric cancer. None of the patients received preoperative chemotherapy or radiotherapy, and all of them were proven to have gastric cancer by pathology. The cancer tissue was fixed with formalin and preserved in paraffin. All pathological data were complete, and the postoperative follow-up was sufficient. All patients were approved by the ethics committee of China Medical University to participate in the study and provided written informed consent.

| Immunohistochemistry of human gastric cancer
To fix gastric cancer tissue samples, 10% formalin was used, then the tissue was paraffin-embedded and cut into 4-μm slices. The xylene and alcohol were used for dewaxing and rehydrating.
Endogenous peroxidase activity was blocked by using hydrogen peroxide (30%), the citrate buffer (pH 6.0) was used to boil the sections fro 3 minutes in a pressure cooker. Next, normal goat serum was used for incubating the sections to reduce the nonspecific binding. Finally, the tissue sections were incubated (4°C, Two pathologists examined all tumour slides randomly. We evaluated STEAP1 staining intensity as follows: scored 0 (negative), 1 (weakly negative), 2 (weak positive) and 3 (strong positive). The percentage scores of positive cells per single field vision were as follows: scored 1 (0%-25%), 2 (26%-50%), 3 (51%-75%) and 4 (76%-100%). We multiplied the two scores above and obtained a final score ranging from 0 to 12. Tumour samples with a score < 6 were considered as negative expression; on the contrary, the score ≥ 6 was considered as positive expression.

| Animals
Twenty-four BALB/c nude female mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were raised in the animal experimental centre of China Medical University. The twenty-four nude mice were randomly divided into four groups. Two groups were used for hypodermic injection, and the other two groups were used for intraperitoneally injected.In the subcutaneous tumorigenesis experiment,
Lipofectamine 3000 reagent (Thermo Fisher Scientific, Inc) was used for plasmid transfection. The cells were harvested 48 hours after transfection. Western blot analysis and RT-PCR were used to check the transfection efficiency.

| RT-PCR
TRIzol reagent was used to extracted total RNA. The reverse transcription kit PrimeScript RT was purchased from Takara. The primer designs were provided by Huada Gene Co., and the sequences were shown in Table 1.

| Western blot analysis
We used RIPA lysis buffer(purchased from Beyotime company) to lysis cells and obtain proteins. Proteins were separated using sodium IgG secondary antibodies were used to incubate the membrane at room temperature for 60 minute. Finally, the ECL was used to visualize and detect the proteins by using BioImaging Systems (UVP Inc, Upland, CA, USA).

| Colony formation assay
For the colony formation assay, SGC-7901 and MGC-803 cells were transfected with plasmid or shRNA for 36 hours and plated into 6-well cell plates (1000 cells/well). The cells were cultured in a 37°C incubator for 2-3 weeks, fixed with alcohol for 30 minutes and stained with Trypan Blue for 20 minutes at room temperature.
The colonies with more than 50 cells were counted. Finally, an HD camera was used to obtain the images.The experiment was repeated three times.

| Flow cytometry
We used a flow cytometry assay to detect the cell cycle stage. SGC-7901 and MGC-803 cells were transfected with plasmid or shRNA and plated into 6-well plates (1 × 10 5 cells/well). After 24 hours, the cells were harvested using 0.25% trypsin in 1.5 mL Eppendorf tubes.
Then, the cells were stained with propidium iodide (PI, 500 μ/tube, KeyGEN, Nanjing, China) at 37°C in the dark for 30 minutes. Finally, the cells were analysed using a FACSCalibur flow cytometer (Becton Dickinson, USA). The experiment was repeated three times.

| Transwell assay
Cell migration experiments were performed using a 24-well transwell chamber with a pore size of 8 μm (Costar). A total of 5 × 10 4 cells in serum-free DMEM were placed in the upper chamber, and DMEM with 10% FBS was added to the lower chamber. After more than 10 hours, the migration experiment was terminated, and the cells were observed in the medium below. Then, the cells on the membrane in the bottom chamber were fixed with 75% alcohol for 30 minutes and stained with Trypan Blue at room temperature for 20 minutes. Images were obtained using an inverted microscope. In addition, the transwell chamber was also used for cell invasion experiments. For these experiments, in addition to the above steps, Matrigel (1:9 dilution, BD Bioscience) was added to the upper chamber to observe the change in cell invasion ability.The experiment was repeated three times.

| Wound healing assay
A wound healing assay was used to observe the migration of cells.
In this study, 1 × 10 5 cells were seeded into 6-well plates for every group. After the cells had covered the entire plate, a pipette tip was used to make a scratch in the cell monolayer, and phosphate buffer saline (PBS) was used to wash the floating cells three times.
Subsequently, we used serum-free DMEM instead of the former medium. Finally, an inverted microscope (Olympus, Japan) was used to take images at 0 hour and 96 hours. The difference in scratch distance between the two phases can reflect the difference in the cell migration ability.The experiment was repeated three times.

| Statistical analysis
GraphPad Prism 7.0 was used for image editing. SPSS 21.0 statistical software was used for data analysis.The data of three repeated experiments were input to analysis and expressed as the means ± SEMs. The chi-square test was used to examine possible correlations between STEAP1 expression and clinicopathological factors. Survival rates were calculated using Kaplan-Meier analysis. The log-rank test was used for single-factor analysis. Cox risk proportion model was used for multifactor analysis, and a value of P < .05 was considered statistically significant.

| STEAP1 was highly expressed in gastric cancer tissue and closely connected with OS
The GEPIA database showed that the STEAP1 gene was more highly expressed in gastric cancer than in the normal tissue ( Figure 1A).
We detected 212 cases of gastric cancer tissues and 60 cases of paracancerous tissues using IHC and scored them. The results showed that STEAP1 was highly expressed in cancer tissues and was mainly localized to the membrane and cytoplasm in cells ( Figure 1B). The positive expression rate of STEAP1 was 54.7% (116/212). However, it was expressed at low levels or was negative in paracancerous tissues ( Figure 1C). The 5-year OS in the high expression group was 25.9%, which was significantly lower than 60.7% in patients with low expression group (P < .001, Fig. d). Figure 1E shows the detailed score    Table 2). Cox multifactor analysis showed that the independent factors influencing the prognosis of patients included STEAP1 expression (P < .001), T stage (P = .005) and N stage (P < .001) ( Table 3). In the study of the relationship between the expression of STEAP1 and clinicopathological factors, high expression of STEAP1 was closely related to Borrmann type (P = .009) and N stage (P < .001) ( Table 4).

| Screening of the experimental cell lines and knockdown virus transfection
We detected the STEAP1 mRNA level in GES-1, AGS, SGC-7901 and MGC-803 cells using RT-PCR ( Figure 1G). The results showed that STEAP1 was more highly expressed in SGC-7901 and MGC-803 cells.
The western blot analysis yielded the same conclusion ( Figure 1H). showed that shRNA2 had the highest knockdown efficiency ( Figure 1I).
Subsequently, we transfected NC virus and STEAP1-shRNA2 into SGC-7901 and MGC-803 cells. The results showed successful transfection and knockdown using RT-PCR and western blot analysis ( Figure 1J-k).

| STEAP1 gene regulates the cell cycle via the Akt/FoxO1 pathway to influence cell proliferation
The CCK-8 assay results showed that the absorbance in the sh-STEAP1 group was lower than that in the NC group at 48, 72 and 96 hours in both the SGC-7901 and MGC-803 cell lines ( Figure 2A). In another group of comparisons, we found that  In the colony formation assay, we found that when STEAP1 was knocked down, the colony number was lower than that in the NC group ( Figure 2C), while when STEAP1 was overexpressed, the colony number was higher than that in the empty vector group ( Figure 2D). The colony formation assay results also indicated that the STEAP1 gene can influence cell proliferation. Next, we

| STEAP1 regulates cell migration and invasion via EMT
The transwell assay results showed that the number of migrating cells in the sh-STEAP1 group was lower than that in the NC group in both SGC-7901 and MGC-803 cells ( Figure 3A). The number of migrating cells in the STEAP1 plasmid group was higher than that in the empty vector group ( Figure 3B). The results of the wound healing assay also provided consistent conclusions. In SGC-7901 cells, the migration distance between 0 and 96 hours in the NC group was more obvious than that in the sh-STEAP1 group. The migration distance of the empty vector group was shorter than that of the STEAP1 plasmid group ( Figure 3C). This result was also identified in MGC-803 cells. The migration distance of the NC group was longer than that of the sh-STEAP1 group, and the migration distance of the empty vector group was shorter than that of the STEAP1 plasmid group ( Figure 3D). The results of the above two experiments indicated that when STEAP1 was knocked down, the cell migration ability was decreased, whereas it increased after the overexpression of STEAP1. We also used a transwell assay and placed Matrigel into the upper chamber for detecting the effect of the STEAP1 gene on cell invasion. The results showed that the number of invading cells in the sh-STEAP1 group was lower than that in the NC group in both SGC-7901 and MGC-803 cells ( Figure 3E). The number of invading cells in the STEAP1 plasmid group was higher than that in the empty vector group ( Figure 3F). These results indicated that the STEAP1 gene had an effect on cell invasion. When STEAP1 was knocked down, the cell invasion ability was decreased and increased after overexpressing STEAP1. Finally, we detected cell migration-and invasionrelated proteins using western blot analysis. The results showed that Vimentin, N-cadherin, MMP-2 and MMP-9 were down-regulated and E-cadherin was up-regulated after we down-regulated the gene STEAP1 ( Figure 3G). In contrast, when STEAP1 was overexpressed, Vimentin, N-cadherin, MMP-2 and MMP-9 were relatively up-regulated, and E-cadherin was down-regulated ( Figure 3H).

| In vivo animal experiments
Twelve BALB/c nude mice were used to study tumour formation and were randomly divided into two groups. We subcutaneously injected 3 × 10 6 of SGC-7901 NC cells per mouse in the first group, which was called the NC group. The mice in the other group were subcutaneously injected with 3 × 10 6 of SGC-7901 and sh-STEAP1 cells per mouse, which was called sh-STEAP1 group. The tumour sizes were measured every 2 days from 4 to 14 days after the injection and the results are shown in Figure 4C. Twelve tumour specimens were removed from the mice on the 14th day. The results showed that the tumour size in the NC group was larger than that in the sh-STEAP1 group ( Figure 4A). Then, we carried out paraffin embedding, sectioning and IHC experiments with the tumour tissue. The results showed that the expression of Ki67, IL-1β and IL-6 in the NC group was higher than that in the sh-STEAP1 group, while cleaved caspase-3 expression was lower than that in the sh-STEAP1 group ( Figure 4B). In the intraperitoneal tumorigenesis experiment, similar to the subcutaneous tumorigenesis experiment, twelve mice were divided into the NC and sh-STEAP1 groups, and 3 × 106 cells per mouse were injected by intraperitoneal injection. Three weeks later, the mice were sacrificed to observe the number of intraperitoneal tumours, including the mesentery, on the wall of the intestine.The number of tumours in the abdominal cavity of the two groups were statistically analysed and the difference was statistically significant ( Figure 4D). Figure 4E showed the general distribution of tumours of NC group..We used haemostatic forceps to clamp the two sides of the intestine to expand the mesentery, we found a huge number of tumours in the mesentery arranged like beads ( Figure 4E.). However, tumours were rare or absent in the sh-STEAP1 group ( Figure 4F). We enlarged the image of one case in NC group when the abdominal cavity of nude mice was just opened. We can clearly see that the tumours covered the abdominal cavity, and several larger tumours on the mesenteric and intestinal wall were marked at the arrow. ( Figure 4G).

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ZHANG et Al.  Our results also showed that when STEAP1 was down-regulated, P-AKT, CDK4 and Cyclin D1 were relatively down-regulated, and P27 and P-FoxO1 were up-regulated ( Figure 2G). When STEAP1 was up-regulated, P-AKT, CDK4 and Cyclin D1 were relatively upregulated, and P-FoxO1 and P27 were down-regulated. P-AKT was up-regulated and P-FoxO1 was down-regulated ( Figure 2H).

| D ISCUSS I ON
These results indicated that STEAP1 can regulate the cell cycle via the Akt/FoxO1 pathway to influence cell proliferation. The results of the transwell and wound healing assays showed that when we overexpressed STEAP1, cell migration and invasion increased. In contrast, when STEAP1 was knocked down, the two abilities above decreased ( Figure 3A-F)

| Conclusions
In conclusion, STEAP1 was overexpressed in gastric cancer and closely connected with OS. STEAP1 can regulate the cell cycle via the Akt/FoxO1 pathway to influence cell proliferation. STEAP1 may affect cell migration and invasion via EMT induction.

ACK N OWLED G EM ENTS
This work was supported by National Natural Science Foundation of China (No.81602522). We would like to thank Editage (www.edita ge.com) for English language editing.

CO N FLI C T S O F I NTE R E S T
There is no competing interests.