The expression of O-linked glycosyltransferase GALNT7 in breast cancer is dependent on estrogen-, progesterone-, and HER2-receptor status, with prognostic implications.

GALNT7 is a glycosyltransferase enzyme transferring N-acetylgalactosamine to initiate O-linked glycosylation in the Golgi apparatus. Breast cancer is the most common cancer in women globally. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2; ERBB2) are important biomarkers in the prognosis and molecular subtyping of breast cancer. Here, we showed that ER-positive, PR-positive or HER2-positive breast tumors have higher expression of GALNT7 compared to ER-negative, PR-negative or HER2-negative breast tumors, respectively. We found that CpG-aggregated methylation of GALNT7 gene is decreased, and in parallel, its transcript levels are increased in breast cancer compared to healthy breast tissue. We observed that the difference in the expression of GALNT7 between negative and positive status of the receptors is the highest for HER2, followed by ER and PR, pointing that HER2 might be relatively more influential than ER and PR on the expression of GALNT7 in breast cancer. We reported that basal-like breast tumors have decreased expression of GALNT7 compared to non-basal-like tumors, and that high GALNT7 expression is associated with favorable relapse-free and distant metastasis-free survival in HER2 status-dependent manner in breast cancer patients. Moreover, we showed that GALNT7 expression in breast cancer is cell type- (epithelial vs stromal cells), tumor grade- and ethnicity-dependent. Combined, we propose that GALNT7 might contribute to different clinical outcomes depending on the receptor status in breast cancer, and that a better understanding of GALNT7 and its function in the context of breast cancer is needed.

MiR-30c facilitates natural killer cell cytotoxicity to lung cancer through targeting GALNT7.

BACKGROUND: MicroRNAs (miRNAs) have been reported to play important roles in regulating natural killer (NK) cell cytotoxicity to cancer cells. OBJECTIVE: This study aimed to investigate the effects and potential mechanism of miR-30c in regulating NK cell cytotoxicity to lung cancer cells. METHODS: Primary NK cells were derived from the peripheral blood of lung cancer and normal participants. Exosomes were isolated and validated via transmission electron microscopy and nanoparticle tracking analysis. The levels of miR-30c, polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) and proteins in PI3K/AKT pathway were determined using quantitative real-time polymerase chain reaction or western blot. Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) levels and the cytotoxicity of effector NK cells to target lung cancer cells were measured via enzyme linked immunosorbent assay, cell apoptosis or xenograft experiments. The relationship between miR-30c and GALNT7 was analyzed by luciferase activity, RNA pull-down and RNA immunoprecipitation assays. And a xenograft mice model was established to verify the effect of miR-30c in regulating NK cell cytotoxicity to lung cancer cells in vivo. RESULTS: NK cell-derived exosomes carrying miR-30c, and miR-30c level was significantly downregulated in primary NK cells of lung cancer patients. MiR-30c overexpression promoted TNF-α and IFN-γ secretion and enhanced the cytotoxicity of interleukin 2 (IL-2)-treated NK cells to lung cancer cells, while knockdown of miR-30c played an opposite effect in regulating the cytotoxicity of NK cells to lung cancer cells. GALNT7 was a target of miR-30c and was negatively regulated by miR-30c. Besides, miR-30c targeted GALNT7 to exert its function in regulating NK cell cytotoxicity. Furthermore, GALNT7 prompted the activation of PI3K/AKT pathway in NK cells. Additionally, miR-30c overexpression enhanced NK cell cytotoxicity to lung cancer cells and inhibited tumor growth in vivo. CONCLUSION: miR-30c enhanced NK cell cytotoxicity to lung cancer cells via decreasing GALNT7 and inactivating the PI3K/AKT pathway, suggesting that regulating miR-30c expression maybe a promising approach for enhancing NK cell-based antitumor therapies.

Long non-coding RNA CCDC144NL-AS1 promotes cell proliferation by regulating the miR-363-3p/GALNT7 axis in colorectal cancer.

Colorectal cancer (CRC) is a burdensome health concern worldwide. Long non-coding RNA (lncRNA) have emerged as vital roles in multiple cancers, including CRC. Increasing evidence has demonstrated that lncRNA CCDC144NL-AS1 acts crucial roles in tumor developments. Nevertheless, its role in CRC remains largely unknown. The level of CCDC144NL-AS1 expression was detected in 100 CRC tissues and paired adjacent tissues. The gain- and loss-of-function experiments were conducted to investigate the biological functions of CCDC144NL-AS1 in vitro and in vivo. The potential mechanism of CCDC144NL-AS1 exerting as competing endogenous RNAs (ceRNAs) was demonstrated by bioinformatics, luciferase reporter assay and in vitro experiments. CCDC144NL-AS1 was up-regulated in CRC tissues and cells. High CCDC144NL-AS1 was connected with the adverse clinicopathological features and worse prognosis of CRC. Furthermore, knockdown of CCDC144NL-AS inhibited the cell proliferation and led to the cell cycle G0-1/S arrest, whereas upregulated CCDC144NL-AS1 obtained the inverse results. Further study found that CCDC144NL-AS1 functioned as ceRNAs in regulating CRC proliferation. MiR-363-3p was the target of CCDC144NL-AS1, which sponges GALNT7 in regulating cell growth of CRC. The study demonstrated that the CCDC144NL-AS1/miR-363-3p/GALNT7 axis exerts on key roles in cell proliferation and presents an emerging target for CRC therapy and prognostic biomarker.

miR-30b-5p inhibits proliferation, invasion, and migration of papillary thyroid cancer by targeting GALNT7 via the EGFR/PI3K/AKT pathway.

BACKGROUND: Papillary thyroid carcinoma (PTC) is a common endocrine tumor. Increasing evidence has shown that microRNA dysfunction is involved in the occurrence and development of cancer. The expression of MicroRNA-30b-5p (miR-30b-5p) was down-regulated in PTC; however, its role in the development of PTC is not clear. Hence, this study aimed to explore the role and mechanism of miR-30b-5p in the occurrence and development of PTC. METHODS: The qRT-PCR assay was used to detect the expression of miR-30b-5p in 60 cases of papillary thyroid carcinoma along with their matched non-cancerous tissues. This study explored the biological function of miR-30b-5p by the functional gain and loss experiments in vitro and vivo. The direct target gene of miR-30b-5p and its signaling pathway was identified through bioinformatics analysis, qRT-PCR, western blot, rescue experiments, and double luciferase 3'-UTR report analysis. RESULTS: This study demonstrated that the low expression of miR-30b-5p is related to poor clinicopathological features. Functionally, the overexpression of miR-30b-5p inhibited the proliferation, invasion, and migration of PTC cells. Bioinformatics and luciferase analysis showed that GALNT7 is the direct and functional target of miR-30b-5p. Moreover, miR-30b-5p inhibited the proliferation of PTC in vivo by inhibiting the expression of GALNT7. The studies on the mechanism have shown that GALNT7 promotes cell proliferation and invasion by activating EGFR/PI3K/AKT kinase pathway, which can be attenuated by the kinase inhibitors. CONCLUSIONS: Overall, miR-30b-5p inhibited the progression of papillary thyroid carcinoma by targeting GALNT7 and inhibiting the EGFR/PI3K/AKT pathway.

Prenatal Cadmium Exposure Alters Proliferation in Mouse CD4+ T Cells via LncRNA Snhg7.

Objective: Prenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA host gene 7 (lncSnhg7) in T cell proliferation. Methods: RNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. Finally, lncSnhg7 was knocked down in splenic CD4+ T cells with lentivirus to assess its effect on proliferation. Results: We identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. Knockdown on lncSnhg7 inhibits proliferation of CD4+ T cells. Conclusion: Prenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression and knockdown of lncSnhg7 inhibited proliferation suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

Screening siRNAs against host glycosylation pathways to develop novel antiviral agents against hepatitis B virus.

AIM: Hepatitis B virus (HBV) relies on glycosylation for crucial functions, such as entry into host cells, proteolytic processing and protein trafficking. The aim of this study was to identify candidate molecules for the development of novel antiviral agents against HBV using an siRNA screening system targeting the host glycosylation pathway. METHODS: HepG2.2.15.7 cells that consistently produce HBV were employed for our in vitro study. We investigated the effects of siRNAs that target 88 different host glycogenes on hepatitis B surface antigen (HBsAg) and HBV DNA secretion using the siRNA screening system. RESULTS: We identified four glycogenes that reduced HBsAg and/or HBV DNA secretion; however, the observed results for two of them may be due to siRNA off-target effects. Knocking down ST8SIA3, a member of the sialyltransferase family, significantly reduced both HBsAg and HBV DNA secretion. Knocking down GALNT7, which transfers N-acetylgalactosamine to initiate O-linked glycosylation in the Golgi apparatus, also significantly reduced both HBsAg and HBV DNA levels. CONCLUSIONS: These results showed that knocking down the ST8SIA3 and GALNT7 glycogenes inhibited HBsAg and HBV DNA secretion in HepG2.2.15.7 cells, indicating that the host glycosylation pathway is important for the HBV life cycle and could be a potential target for the development of novel anti-HBV agents.

miR-125a-5p post-transcriptionally suppresses GALNT7 to inhibit proliferation and invasion in cervical cancer cells via the EGFR/PI3K/AKT pathway.

BACKGROUND: The carcinogenesis and progression of cervical cancer is a complex process in which numerous microRNAs are involved. The purpose of this study is to investigate the role of miR-125a-5p in progression of cervical cancer. METHODS: RT-qPCR was used to detect the expression of miR-125a-5p and GALNT7 in cervical cancer tissues and cell lines. Then, the miR-125a-5p mimic, miR-125a-5p inhibitor, GALNT7 siRNA, or/and pcDNA-GALNT7 were respectively transfected into HeLa and Caski cervical cancer cells, and Cell Counting kit-8 assay, Transwell assay and flow cytometry analysis were respectively used to observe cell proliferation, invasion and apoptosis. Subsequently, luciferase reporter gene assay was employed in confirming the target relationship between miR-125a-5p and GALNT7. MiR-125a-5p mimic or/and pcDNA-GALNT7 were transfected into the cervical cancer cells at the absence of epidermal growth factor (EGF) or not, and the pcDNA-GALNT7 was transfected into the cervical cancer cells at the absence of inhibitors of multiple kinases or not. Furthermore, the effect of miR-125a-5p on tumor growth was also studied using a xenograft model of nude mice. RESULTS: MiR-125a-5p was down-regulated in both cervical cancer tissues and cell lines and it inhibited cell proliferation and invasion of cervical cancer cells. MiR-125a-5p directly targeted and post-transcriptionally downregulated GALNT7 that was strongly upregulated in cervical cancer tissues and cell lines. Similar to the effect of miR-125a-5p mimic, silencing GALNT7 inhibited proliferation and invasion of cervical cancer cells. In addition, miR-125a-5p overexpression could counteract both GALNT7- and EGF-induced cell proliferation and invasion. GALNT7 promoted cell proliferation and invasion by activating the EGFR/PI3K/AKT kinase pathway, which could be abated by the inhibitors of the kinases. Moreover, the role of miR-125a-5p inhibited tumor formation in cervical cancer by suppressing the expression of GALNT7 in vivo. CONCLUSION: In conclusion, miR-125a-5p suppressed cervical cancer progression by post-transcriptionally downregulating GALNT7 and inactivating the EGFR/PI3K/AKT pathway.

Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx.

The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.

Long non-coding RNA-SNHG7 acts as a target of miR-34a to increase GALNT7 level and regulate PI3K/Akt/mTOR pathway in colorectal cancer progression.

BACKGROUND: Colorectal cancer (CRC) arises in a multistep molecular network process, which is from either discrete genetic perturbation or epigenetic dysregulation. The long non-coding RNAs (lncRNAs), emerging as key molecules in human malignancy, has become one of the hot topics in RNA biology. Aberrant O-glycosylation is a well-described hallmark of many cancers. GALNT7 acts as a glycosyltransferase in protein O-glycosylation, involving in the occurrence and development of CRC. METHODS: The microarrays were used to survey the lncRNA and mRNA expression profiles of primary CRC cell line SW480 and metastatic CRC cell line SW620. Cell proliferation, migration, invasion, and apoptosis were assayed. Xenograft mouse models were used to determine the role of lncRNA-SNHG7 in CRC in vivo. In addition, CNC analysis and competing endogenous analysis were used to detect differential SNHG7 and relational miRNAs expression in CRC cell lines. RESULTS: SNHG7 expression showed a high fold (SW620/SW480) in CRC microarrays. The CRC patients with high expression of SNHG7 had a significantly poor prognosis. Furthermore, SNHG7 promoted CRC cell proliferation, metastasis, mediated cell cycle, and inhibited apoptosis. SNHG7 and GALNT7 were observed for co-expression by CNC analysis, and a negative correlation of SNHG7 and miR-34a were found by competing endogenous RNA (ceRNA) analysis. Further results indicated that SNHG7 facilitated the proliferation and metastasis as a competing endogenous RNA to regulate GALNT7 expression by sponging miR-34a in CRC cell lines. SNHG7 also played the oncogenic role in regulating PI3K/Akt/mTOR pathway by competing endogenous miR-34a and GALNT7. CONCLUSION: The CRC-related SNHG7 and miR-34a might be implicated in CRC progression via GALNT7, suggesting the potential usage of SNHG7/miR-34a/GALNT7 axis in CRC treatment.

MiR-154 inhibits the growth of laryngeal squamous cell carcinoma by targeting GALNT7.

MicroRNAs are critical regulators of the development and progression of laryngeal squamous cell carcinoma (LSCC). However, the role of microRNA-154 (miR-154) in the development and progression of LSCC has not been clarified. We found that down-regulated miR-154 expression in LSCC tissues was associated with poorer prognosis in LSCC patients. MiR-154 over-expression inhibited the proliferation, clonogenicity, and migration of LSCC cells and induced cell cycle arrest, which were reversed by miR-154 inhibition. MiR-154 targeted GALNT7 expression by reducing GALNT7-regulated luciferase activity in LSCC cells while up-regulating GALNT7 mRNA transcription in LSCC tissues and cells. GALNT7 silencing significantly attenuated the proliferation, clonogenicity, and migration of LSCC cells and induced cell cycle arrest. Finally, intravenous treatment with lentivirus for miR-154, but not scrambled control miRNA, significantly restrained the growth of implanted LSCC Hep-2 tumors and decreased the tumor mass by reducing GALNT7 expression in mice. Therefore, miR-154 may serve as a novel prognostic marker and therapeutic target for LSCC.

miR-214 inhibits invasion and migration via downregulating GALNT7 in esophageal squamous cell cancer.

Previous studies verified that miR-214 is of great significance in the invasion and migration of a variety of cancers. It has been demonstrated that UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7(GALNT7) is a putative target of miR-214. We performed this study to figure out how miR-214 and GALNT7 play their roles in the invasion and migration of esophageal squamous cell carcinoma (ESCC). The expression of miR-214 was significantly downregulated in tumors compared to the corresponding non-tumor tissues while GALNT7 showed an opposite tendency. The low expression of miR-214 and the high expression of GALNT7 were found positively correlated with poor tumor differentiation (P = 0.004), tumor invasion (P = 0.013), and lymph node metastasis (P = 0.012) in ESCC patients. Functional study demonstrated that overexpression of miR-214 or knockdown of GALNT7 could weaken invasive and migratory ability in Eca109, TE1, and KYSE150. Moreover, tumorigenicity assay showed us mice injected with cells containing miR-214 mimic or GALNT7 small interfering RNA formed substantially smaller tumors than that in miR-214 inhibitor group. Consequently, we concluded that miR-214 shows potential to be a diagnostic marker and therapeutic target in ESCC.

GALNT7, a target of miR-494, participates in the oncogenesis of nasopharyngeal carcinoma.

GalNAc-transferase-7 (GALNT7) is essential for the regulation of cell proliferation and has been implicated in tumorigenesis. However, the role of GALNT7 in the development and progression of nasopharyngeal carcinoma (NPC) remains unclear. Our previous study showed that GALNT7 was a putative target of miR-494, which was confirmed by luciferase reporter assay. In the present study, we demonstrated that in vitro knockdown of GALNT7 significantly inhibited the proliferation, colony formation, migration, and invasion of NPC-derived cells. In vivo tumorigenicity assay showed that miR-494 and GALNT7-small interfering RNA (siRNA) reduced tumor growth in nude mice. Taken together, our results provided new evidence for an oncogenic role of GALNT7 in NPC.

Functional elucidation of miR-494 in the tumorigenesis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma has very high incidence and high mortality worldwide. MiRNA is related to the tumorigenesis and metastasis of a variety of tumors. In the present study, we verify that the expression of miR-494 in NPC tissues and NPC-derived cells was down-regulated, respectively. The proliferation, colony formation, migration, and invasion of NPC-derived cells were suppressed, while the cell apoptosis was promoted, when miR-494 was over-expressed in these cells. GALNT7 and CDK16 were confirmed to be the direct targets of miR-494. These results suggested that miR-494 play an inhibitory role in the tumorigenesis of NPC.

Mature miR-17-5p and passenger miR-17-3p induce hepatocellular carcinoma by targeting PTEN, GalNT7 and vimentin in different signal pathways.

To study the physiological role of a single microRNA (miRNA), we generated transgenic mice expressing the miRNA precursor miR-17 and found that the mature miR-17-5p and the passenger strand miR-17-3p were abundantly expressed. We showed that mature miR-17-5p and passenger strand miR-17-3p could synergistically induce the development of hepatocellular carcinoma. The mature miR-17-5p exerted this function by repressing the expression of PTEN. In contrast, the passenger strand miR-17-3p repressed expression of vimentin, an intermediate filament with the ability to modulate metabolism, and GalNT7, an enzyme that regulates metabolism of liver toxin galactosamine. Hepatocellular carcinoma cells, HepG2, transfected with miR-17 formed larger tumors with more blood vessels and less tumor cell death than mock-treated cells. Expression of miR-17 precursor modulated HepG2 proliferation, migration, survival, morphogenesis and colony formation and inhibited endothelial tube formation. Silencing of PTEN, vimentin or GalNT7 with their respective siRNAs enhanced proliferation and migration. Re-expressing these molecules reversed their roles in proliferation, migration and tumorigenesis. Further experiments indicated that these three molecules do not interact with each other, but appear to function in different signaling pathways. Our results demonstrated that a mature miRNA can function synergistically with its passenger strand leading to the same phenotype but by regulating different targets located in different signaling pathways. We anticipate that our assay will serve as a helpful model for studying miRNA regulation.