GNA14 stimulation of KLF7 promotes malignant growth of endometrial cancer through upregulation of HAS2

Abstract

Background: Endometrial cancer (UCEC) is one of the most common gynecological malignancies. We previously found that overexpression of G protein α subunit 14 (GNA14) promoted UCEC growth. Krüppel-like factor 7 (KLF7) acts as an oncogene in various cancer types, whereas the connection between GNA14 and KLF7 in UCEC is unclear. We herein explored the involvement of GNA14/KLF7 in UCEC development.

Methods: Clinical relevance of GNA14, KLF7 and HAS2 in UCEC was analyzed from TCGA and by immunohistochemical staining. Knockdown and overexpression of indicated genes were conducted by transfecting the cells with siRNAs and lentivirus, respectively. mRNA and protein expression was detected by qRT-PCR and Western blot. CCK8, colony formation, cell cycle, apoptosis, transwell and wound healing were performed to check cell biology function in vitro. Tumor growth in nude mice was conducted to check in vivo function. RNA sequencing was used to determine dys-regulated genes.

Results: We demonstrated that GNA14 stimulated the expression of KLF7 in UCEC cells. There was a positive correlation between GNA14 and KLF7 in normal and UCEC tissues. In vitro, KLF7 promoted cell proliferation, colony formation, cell cycle progression, and migration of UCEC cells. Apoptosis was inhibited by KLF7. Xenografted tumorigenesis of UCEC cells was suppressed by KLF7 knockdown. Furthermore, RNA sequencing results showed that KLF7 regulated the expression of a large amount of genes, among which hyaluronan synthase 2 (HAS2) was downregulated in KLF7 knockdown cells. Based on TCGA database and immunoblotting assays, KLF7 positively regulated HAS2 in UCEC cells and tissues. Lastly, knockdown of HAS2 reversed the oncogenic role of KLF7 on UCEC cell proliferation, migration, and xenografted tumor development.

Conclusion: Taken together, we reveal that GNA14/KLF7/HAS2 signaling cascade exerts tumor promoting function during UCEC development.

Keywords: Cancer development; Endometrial cancer; GNA14; HAS2; KLF7.

Fig. 1

GNA14 upregulates KLF7 in UCEC. a and b The correlation between GNA14 expression and KLF7 expression was analyzed in normal samples (A, R = 0.59, p = 0.035) and UCEC samples (B, R = 0.2, p = 0.0081) from TCGA database. c IHC staining of GNA14 and KLF7 in UCEC (n = 20) and normal (n = 15) samples. Left, representative images of IHC staining. Right, quantification results of GNA14 and KLF7 in cancer and normal tissues. Spearman correlation between KLF7 and GNA14 was analyzed in cancer and normal samples based on IHC staining. d and e qRT-PCR (d) and immunoblotting (e) analysis of GAN14 and KLF7 in GAN14 knockdown and overexpressed Hec-1-A and KLE cells. GAPDH acts as the internal control. **p < 0.01

Fig. 2

KLF7 promotes UCEC cell proliferation. a Immunoblotting analysis of KLF7 in siCtrl, siKLF7–1 and siKLF7–2 UCEC cells. b and c CCK8 analysis of cell proliferation in siCtrl, siKLF7–1 and siKLF7–2 KLE and Hec-1-A cells. d-f Colony formation was assessed in siCtrl, siKLF7–1 and siKLF7–2 KLE, Hec-1-A, and Hec-1-B cells. g Immunoblotting analysis of KLF7 in Ctrl and KLF7 overexpressed KLE cells. h CCK8 analysis of cell proliferation in Ctrl and KLF7 overexpressed KLE cells. i Colony formation was assessed in Ctrl and KLF7 overexpressed KLE cells. ***p < 0.001. Results are mean ± SEM (n = 3)

Fig. 3

KLF7 regulates cell cycle and apoptosis in UCEC cells. a-c Apoptosis was examined by PI/Annexin V staining in siCtrl, siKLF7–1 and siKLF7–2 Hec-1-A and Hec-1-B cells. a, apoptosis results. b and c, quantitative results. d-g Cell cycle was examined by PI staining in siCtrl, siKLF7–1 and siKLF7–2 Hec-1-A and Hec-1-B cells. d and f, cell cycle results. e and g, quantitative results. h and i Apoptosis was examined by PI/Annexin V staining in Ctrl and KLF7 overexpressed KLE cells. h, apoptosis results. i, quantitative results. j and k Cell cycle was examined by PI staining in Ctrl and KLF7 overexpressed KLE cells. j, cell cycle results. k, quantitative results. *p < 0.05. **p < 0.01. ***p < 0.001. Results are mean ± SEM (n = 3)

Fig. 4

KLF7 promotes UCEC cell migration. a-f Migration was determined by wound healing assay in siCtrl, siKLF7–1 and siKLF7–2 Hec-1-A (a and b), Hec-1-B (c and d), and KLE (e and f) cells. g and h Transwell detection of migration in siCtrl, siKLF7–1 and siKLF7–2 Hec-1-B cells. i and j Migration was determined by wound healing assay in Ctrl and KLF7 overexpressed KLE cells. *p < 0.05. **p < 0.01. ***p < 0.001. Results are mean ± SEM (n = 3)

Fig. 5

Molecule profiling in UCEC cells after KLF7 knockdown. a siCtrl and siKLF7 UCEC cells were subjected to RNA sequencing. Differentially expressed genes were identified following the criterion: fold change> 1.5 and p < 0.05. a Upregulated genes were shown as red. Downregulated genes were shown as blue. Gray represented unchanged genes. b GESA analysis of signaling pathway after KLF7 knockdown. c qRT-PCR detection of HAS2 in KLE cells after KLF7 knockdown and overexpression. d Correlation between KLF7 expression and HAS2 expression was analyzed in UCEC tissues base on TCGA database. **p < 0.01. Results are mean ± SEM (n = 3)

Fig. 6

KLF7 upregulation of HAS2 promotes UCEC cell proliferation and migration. a-d Immunoblotting analysis of HAS2 and cell viability were checked in Ctrl and HAS2 overexpressed KLE cells (a), in siCtrl, siKLF7 and siKLF7 + HAS2 KLE cells (b), in siCtrl and siHAS2 KLE cells (c), and in Ctrl, KLF7 and KLF7 + siHAS2 KLE cells (d). e CCK8 analysis of proliferation in Ctrl, KLF7 and KLF7 + siHAS2 KLE cells. f Colony formation was assessed in Ctrl, KLF7 and KLF7 + siHAS2 KLE cells. g Migration was assessed by wound healing in Ctrl, KLF7 and KLF7 + siHAS2 KLE cells. *p < 0.05. **p < 0.01. ***p < 0.001. Results are mean ± SEM (n = 3)

Fig. 7

KLF7 promotes tumorigenesis of UCEC cells through upregulation of HAS2. Equal number of shCtrl and shKLF7 Hec-1-B cells were subcutaneously implanted into female nude mice. Tumors were photographed after sacrifice. a Macroscopic images of tumors derived from shCtrl and shKLF7 Hec-1-B cells in nude mice. b Tumor weight was measured. c Immunoblotting analysis of KLF7 and HAS2 in shCtrl and shKLF7 tumor tissues. Equal number of Ctrl, KLF7, KLF7 + shHAS2 Hec-1-B cells were subcutaneously implanted into female nude mice. Tumors were photographed after sacrifice. d Macroscopic images of tumors derived from Ctrl, KLF7, KLF7 + shHAS2 Hec-1-B cells in nude mice. e Tumor weight was measured. *p < 0.05. **p < 0.01