3.1 PA can promote the biological behavior of EC cells by up-regulating the expression of KLF7
Ishikawa cells were treated with different contents PA respectively, the CCK-8 results showed that 20μM and 50μM PA had no toxic effect, and the ability of cell proliferation was significantly enhanced after 48h treatment (Figure 1A). Treated with 20μM and 50μM PA for 24h, the colony formation, invasion, migration and scratch healing ability of Ishikawa cells were significantly enhanced (Figure 1B-D), and the mRNA and protein expression levels of GPR40, GPR120 and KLF7 were significantly increased (Figure 1E,F).
In order to determine whether PA promotes the biological behavior of EC cells by up-regulating KLF7. In this study, firstly, we demonstrated that up or down regulation of KLF7 significantly promoted or inhibited the proliferation, invasion and migration of EC cells(Figure 2A-E, Figure S1A-E). The results of the rescue experiment showed that down-regulation of KLF7 could significantly reverse the promoting effect of PA on the proliferation, invasion and migration of Ishikawa cells(Figure 2F-J).
3.2 PA can up-regulate the expression of KLF7 through GPR40/120 and promote the biological behavior of EC cells
We treated Ishikawa cells with 50μM PA and 10μM GPR40 antagonist GW1100 at the same time. The results showed that antagonism of GPR40 significantly reversed the promotion of KLF7 expression by PA (Figure 3A,B). In addition, antagonizing GPR40 while treated with PA could also significantly reverse the promoting effect of PA on the proliferation, invasion and migration of Ishikawa cells (Figure 3C-F). Consistent with the above results, the GPR120 antagonist AH7614 also significantly reversed the up-regulation of KLF7 expression by PA and also alleviated the promotion of biological behavior of Ishikawa cells by PA (Figure 3G-L).
3.3 HEY1 may be a key downstream gene for KLF7 to promote the progression of EC
In order to screen the key downstream genes of transcription factor KLF7 in promoting the progression of EC, RNA-Seq was performed in tumor tissues and paracancerous tissues of patients with EC, Ishikawa cells with up/down regulation of KLF7 and their corresponding control groups. As shown in Figure 4A-E, four differentially expressed genes, such as IRX3, HEY1, RHBDF2 and FAM171A24, were screened after intersection of the differentially expressed genes in the above groups. After further verification in Ishikawa cells, it was found that the mRNA expression of HEY1 and RHBDF2 was significantly increased after up-regulation of KLF7, while the mRNA expression of IRX3, HEY1 and RHBDF2 decreased significantly after down-regulation of KLF7 (Figure 4F-G). Among them, the change of HEY1 expression level, which is closely related to the occurrence and development of tumor, is the most significant.
After prediction by JASPER software, it was found that there was a KLF7 binding site in the promoter region of HEY1 gene. Double luciferase reporter gene experiment showed that overexpression of KLF7 could significantly promote the activity of HEY1 promoter (Figure 4H). It has been suggested that HEY1 can induce the occurrence of EMT in tumor cells by inhibiting the expression of E-Cadherin.30,31 In this study, we also demonstrated that the expression of KLF7 in EC cells was positively correlated with the expression of HEY1 (Figure 4I,K) and negatively correlated with the expression of E-Cadherin (Figure 4J,K).
3.4 Under the state of obesity, the tumorigenesis ability of EC cells in mice was enhanced, and the expression of GPR40/120, KLF7 and HEY1 in tumor tissue increased
In order to explore the effect of high level of PA on the tumorigenesis of EC cells in obese mice, 4-week-old female BALB/c nude mice were fed with normal diet (10%fat Kcal%,ND) and high fat diet (55% fat+5% PA Kcal%,HFD) respectively. After 4 weeks of feeding, the body weight and Lee's index of mice in HFD group were significantly higher than those in ND group (Figure 5A,B). At this time, Ishikawa cells were injected subcutaneously into the right axilla of mice and fed in groups continuously. After 6 weeks, the contents of TG, TC, FFAs and GLU in serum of mice in HFD group were significantly higher than those in ND group, and the content of PA in serum in HFD group was also significantly higher than that in ND group (Figure 5C,D). As shown in Figure 5E-G, the subcutaneous tumorigenic rate of Ishikawa cells in HFD group (100%) was higher than that in ND group (50%), and the weight and volume of subcutaneous tumor tissue in HFD group were significantly higher than those in ND group. Western Blot results showed that the protein expression levels of GPR40/120, KLF7 and HEY1 in EC tissue of HFD mice were significantly higher than those in ND mice, and the protein expression level of E-cadherin was significantly lower than that in ND mice (Figure 5H).
3.5 Up/down regulation of KLF7 can significantly enhance/weaken the tumorigenic ability of EC cells and promote/inhibit the expression of HEY1 in tumor tissues
Ishikawa cells with stably overexpressing KLF7 plasmid (LV-Ad-KLF7,n=6) and no-load control plasmid (LV-NC,n=6) were injected subcutaneously into the right axilla of female BALB/c nude mice(feeding with normal diet). After 6 weeks, as shown in Figure 6A-E, the weight and volume of subcutaneous tumor tissue in Ad-KLF7 group were significantly higher than those in NC group. Over-expression of KLF7 significantly promoted the expression of HEY1 in tumor tissues (Figure 6F).
In addition, Ishikawa cells with stably interfered with KLF7 plasmid (si-NC,n=5) and ineffective interference fragments (si-NC,n=5) were injected subcutaneously into the right axilla of female BALB/c nude mice(feeding with high-fat diet)(Figure 6G,H). After 6 weeks, as shown in Figure 6I-K, the weight and volume of subcutaneous tumor tissue in si-KLF7 mice were significantly lower than those in NC mice. Down-regulation of KLF7 expression significantly suppressed HEY1 expression in tumor tissues (Figure 6L).
3.6 The expression level of KLF7 and HEY1 in tumor tissues of EC individuals were significantly higher than that of non-cancer individuals.
We collected the general data, serum and endometrial tissues of 29 non-cancerous individuals with endometrial benign lesions and 50 patients with endometrial carcinoma. As shown in Supplementary Table 2, the BMI and lipid levels in serum of EC individuals were significantly higher than those of non-cancer individuals. It was found that the detection rate of overweight/obesity in EC patients was significantly higher than that in patients with benign endometrial lesions (Table 1). Then immunohistochemical results showed that the protein expression level of KLF7 (Figure S2A,C) and HEY1 in endometrial tissue of patients with EC was obviously higher than that of non-cancerous individuals (Figure S2B,D). Moreover, the expression of KLF7 was significantly and positively correlated with the expression level of HEY1 in the tumor tissues of endometrial cancer patient (Figure S2E).
Table 1. Comparison of obesity detection rate of benign lesions and EC
Group
|
Normal (BMI<24)
|
Overweight and obesity (BMI≥24)
|
Total
|
Benign lesions
|
20(58.82%)
|
9(20%)
|
29
|
EC
|
14(41.18%)
|
36(80%)***
|
50
|
Total
|
34
|
45
|
79
|
X2 test. ***P<0.001