Pan-cancer analysis of ARNT2 and its oncogenic role in cervical cancer

Abstract

Objective: This study aims to elucidate the role of aryl hydrocarbon receptor nuclear transporter 2 (ARNT2) in cervical cancer (CC) and explore the potential mechanism by which ARNT2 promotes the progression of CC through the protein phosphatase 2A (PP2A)/Akt signaling pathway.

Methods: Bioinformatics tools were used to analyze the expression level of ARNT2 in cancer and its correlation with cancer prognosis. Western Blot and immunohistochemistry staining were used to detect the expression of ARNT2 protein in CC tissues and cells. ARNT2 was knocked down in SiHa and HeLa cells, respectively. Cell Counting Kit-8 assay and colony formation assay were used to detect changes in cell proliferation. Transwell assay and plate scratch assay were used to detect changes in cell migration and invasion. Western Blot assay was used to detect changes in the expression of PP2A/Akt signaling pathway after ARNT2 expression was downregulated. Finally, a CC xenograft tumor model was constructed to evaluate the effect of ARNT2 on SiHa cell tumorigenesis in vivo.

Results: ARNT2 is highly expressed in tumor tissues and cell lines. ARNT2 knockdown can significantly inhibit the proliferation, invasion and migration of SiHa and HeLa cells in vitro and in xenograft models. Further studies have shown that ARNT2 may promote tumor formation by regulating the PP2A/Akt pathway.

Conclusion: ARNT2 promotes the malignant biological behavior of CC cells through the PP2A/Akt signaling pathway, confirming its potential as a prognostic marker for CC.

Keywords: Aryl Hydrocarbon Receptor Nuclear Translocator; Cervical Cancer; Metastasis; Neoplasm Invasiveness.

Fig. 1. The ARNT2 expression level in human pan-cancer analyses. (A) The mRNA level of ARNT2 in TCGA. (B) ARNT2 expression in 33 tumor types and paracancer tissues determined by TIMER 2.0. (C) Analysis of ARNT2 expression in BRCA, CHOL, COAD, DLBC, KIRC, PCPG, READ, SKCM, and TGCT from the GEPIA database. (D) Analysis of protein expression levels of ARNT2 in Breast cancer, GBM and UCEC from UALCAN databases. The 33 cancer types included ACC, BLCA, BRCA, CESC, CHOL, COAD, DLBC, ESCA, GBM, LGG, HNSC, KICH, KIRC, KIRP, LAML, LIHC, LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, TGCT, THCA, THYM, UCEC, UCS, and UVM.

ACC, adrenocortical carcinoma; ARNT2, aryl hydrocarbon receptor nuclear translocator 2; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; HPV, human papillomavirus; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TCGA, The Cancer Genome Atlas; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; TIMER, Tumor Immune Estimation Resource; TPM, transcripts per million; UCEC, terine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma. ^*p<0.05, ^**p<0.01, ^***p<0.001.

Fig. 2. Human pan-cancer analysis of ARNT2 using the HPA database. (A) Expression levels of ARNT2 mRNA in the different organs of the human body. (B) Expression levels of ARNT2 protein in the different organs of the human body. (C) Expression levels of ARNT2 in different cell lines. (D) Localization of ARNT2 in cells and immunohistochemical results of cervical cancer.

ARNT2, aryl hydrocarbon receptor nuclear translocator 2; HPA, Human Protein Atlas; TPM, transcripts per million.

Fig. 3. Correlation of ARNT2 expression with cancer stage and prognosis from Gene Expression Profiling Interactive Analysis database. (A) Expression of ARNT2 in different tumor stages. Log2 (TPM+1) was applied for the log scale. (B) Association of ARNT2 expression with patient RFS and OS in pan-cancer.

ARNT2, aryl hydrocarbon receptor nuclear translocator 2; BRCA, breast invasive carcinoma; DFS, disease-free survival; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver hepatocellular carcinoma; OS, overall survival; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; SKCM, skin cutaneous melanoma; TPM, transcripts per million; UVM, uveal melanoma.

Fig. 4. ARNT2 is highly expressed in CC tissues. (A, I) Immunohistochemical staining of ARNT2 and Ki-67 in CC tissue. (C, K) Immunohistochemical staining of ARNT2 and Ki-67 in HSIL tissue. (E, M) Immunohistochemical staining of ARNT2 and Ki-67 in LSIL tissue. (G, O) Immunohistochemical staining of ARNT2 and Ki-67 in normal cervical tissue. The corresponding magnified images are (B), (D), (F), (H), (J), (L), (N), and (P). (Q) Immunohistochemical intensity score of ARNT2 staining in all specimens. (R) Immunohistochemical intensity score of Ki-67 staining in all specimens. (S, T) ARNT2 protein expression and statistical analysis in CC tissues and normal cervical tissues.

ARNT2, aryl hydrocarbon receptor nuclear translocator 2; CC, cervical cancer; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesions; N, normal; T, tumor. ^*p<0.05, ^**p<0.01, ^***p<0.001, ^****p<0.0001.

Fig. 5. Establishment of ARNR2 knockdown cervical cancer cell lines. (A, B) Relative protein expression and statistical analysis of ARNT2 in SiHa, HeLa, Caski, C33a, and HCerEpic. (C, E) Relative protein expression and statistical analysis of ARNT2 in SiHa after transfection. (D, F) Relative protein expression and statistical analysis of ARNT2 in HeLa after transfection. (G) Green fluorescent protein expression in SiHa and HeLa after transfection.

ARNT2, aryl hydrocarbon receptor nuclear translocator 2. ^*p<0.05, ^**p<0.01.

Fig. 6. Cervical cancer cells with ARNT2 knockdown exhibited attenuated tumorigenicity. (A, B) Cell counting kit-8 assay. (C, D) Colony formation assay and corresponding statistical plots. (E, F) Cell migration and invasion ability assays and corresponding statistical plots.

ARNT2, aryl hydrocarbon receptor nuclear translocator 2; OD, optical density. ^**p<0.01, ^***p<0.001, ^****p<0.0001.