hsa_circ_0002980 prevents proliferation, migration, invasion, and epithelial-mesenchymal transition of liver cancer cells through microRNA-1303/cell adhesion molecule 2 axis.

BACKGROUND: Liver cancer (LC) is a rare malignancy. Circular RNA (circRNA) dysregulation is associated with LC metastasis. hsa_circ_0002980 was found to be unexpectedly downregulated in LC tissues; however, its specific function remains unclear. METHODS: hsa_circ_0002980 expression was confirmed using RT-qPCR. The effects of circ_0002980 on the proliferation, metastasis, and EMT-related proteins of LC cells were assessed using clone formation, flow cytometry, Transwell assays, and Western blotting. The relationship between circ_0002980 and miR-1303 or miR-1303 and CADM2 was analyzed using a dual-luciferase reporter assay. Thereafter, the influence of these three genes on LC cell progression was determined through rescue experiments. RESULTS: hsa_circ_0002980 expression was lower in LC. circ_0002980 overexpression inhibited the proliferation, migration, invasion, and EMT of LC cells. In addition, circ_0002980 specifically binds to miR-1303, and the accelerated effect of miR-1303 overexpression on LC progression was partially reversed by circ_0002980. Moreover, miR-1303 can also target CADM2, and CADM2-mediated prevention can also be attenuated by miR-1303 overexpression. CONCLUSIONS: In LC cells, circ_0002980 upregulation prevents cell proliferation, metastasis, and EMT by affecting the miR-1303/CADM2 axis. Therefore, this axis may be a novel therapeutic target in LC.

Identification of cuproptosis-related subtypes, cuproptosis-related gene prognostic index in hepatocellular carcinoma.

Cuproptosis is a novel form of cell death, correlated with the tricarboxylic acid (TCA) cycle. However, the metabolic features and the benefit of immune checkpoint inhibitor (ICI) therapy based on cuproptosis have not yet been elucidated in Hepatocellular carcinoma (HCC). First, we identified and validated three cuproptosis subtypes based on 10 cuproptosis-related genes (CRGs) in HCC patients. We explored the correlation between three cuproptosis subtypes and metabolism-related pathways. Besides, a comprehensive immune analysis of three cuproptosis subtypes was performed. Then, we calculated the cuproptosis-related gene prognostic index (CRGPI) score for predicting prognosis and validated its predictive capability by Decision curve analysis (DCA). We as well explored the benefit of ICI therapy of different CRGPI subgroups in two anti-PD1/PD-L1 therapy cohorts (IMvigor210 cohort and GSE176307). Finally, we performed the ridge regression algorithm to calculate the IC50 value for drug sensitivity and Gene set enrichment analysis (GSEA) analysis to explore the potential mechanism. We found that cluster A presented a higher expression of FDX1 and was correlated with metabolism, glycolysis, and TCA cycle pathways, compared with the other two clusters. HCC patients with high CRGPI scores had a worse OS probability, and we further found that the CRGPI-high group had high expression of PD1/PDL1, TMB, and better response (PR/CR) to immunotherapy in the IMvigor210 cohort and GSE176307. These findings highlight the importance of CRGPI serving as a potential biomarker for both prognostic and immunotherapy for HCC patients. Generally, our results provide novel insights about cuproptosis into immune therapeutic strategies.

microRNA-497 prevents pancreatic cancer stem cell gemcitabine resistance, migration, and invasion by directly targeting nuclear factor kappa B 1.

OBJECTIVES: Cancer stem cells (CSCs) comprise a small population of cells in cancerous tumors and play a critical role in tumor resistance to chemotherapy. miRNAs have been reported to enhance the sensitivity of pancreatic cancer to chemotherapy. However, the underlying molecular mechanism requires better understanding. METHODS: Cell viability and proliferation were examined with CCK8 assays. Quantitative real-time polymerase chain reaction was executed to assess mRNA expression. StarBase database was used to select the target genes of miRNA, which were further affirmed by dual luciferase assay. Transwell assay was used to analyze cell invasion and migration. RESULTS: We proved that miR-497 could be obviously downregulated in pancreatic cancer tissues and CSCs from Aspc-1 and Bxpc-3 cells. In addition, inhibition of miR-497 evidently accelerated pancreatic CSC gemcitabine resistance, migration and invasion. Moreover, we revealed that nuclear factor kappa B 1 (NFκB1) was prominently upregulated in pancreatic cancer tissues and pancreatic CSCs, and NFκB1 was also identified as a direct target of miR-497. Furthermore, we demonstrated that overexpression of NFκB1 could also notably promote the viability, migration, and invasion of gemcitabine-treated pancreatic CSCs, but this effect could be partially abolished by miR-497 overexpression. CONCLUSIONS: Those findings suggest that miR-497 overexpression could suppress gemcitabine resistance and the metastasis of pancreatic CSCs and non-CSCs by directly targeting NFκB1.

The novel circular RNA HIPK3 accelerates the proliferation and invasion of hepatocellular carcinoma cells by sponging the micro RNA-124 or micro RNA-506/pyruvate dehydrogenase kinase 2 axis.

Circular RNAs (circRNAs) have been confirmed to be associated with the progression of various cancers, including hepatocellular carcinoma (HCC). However, the role and mechanism of circHIPK3 in HCC are still unclear. To investigate its function, circHIPK3 expression was first determined by RT-qPCR in HCC tissues or cells. Functionally, cell proliferation and invasion were investigated by CCK-8, EdU, or Transwell assays. In terms of understanding the mechanism, the interaction of the circRNA HIPK3/micro RNA 124 (miRNA 124) or micro RNA 506 (miRNA506) /PDK2 regulatory loop was verified by dual-luciferase reporter gene assay. In addition, a xenograft tumor model was established to confirm the impact of circHIPK3 on the growth of HCC cells in vivo. We found that circHIPK3 was upregulated in HCC patients and associated with clinical characteristics, while miR-124 and miR-506 were downregulated in HCC patients. Additionally, we proved that knock down of circHIPK3 remarkably suppressed the proliferation and invasion of HCC cells. Mechanistically, circHIPK3 directly bound to miR-124 or miR-506 and inhibited their expression, and PDK2 was a target gene of miR-124 or miR-506. Moreover, circHIPK3 overexpression reversed the inhibitory effect of miR-124 or miR-506 on HCC progression. miR-124 or miR-506 could also suppress tumorigenesis of HCC cells by PDK2. Furthermore, in vivo evidence confirmed that knock down of circHIPK3 inhibited tumor formation. We suggest that circHIPK3 can accelerate the proliferation and invasion of HCC cells by sponging miR-124 or miR-506 to upregulate PDK2, which is the underlying mechanism of circHIPK3-induced HCC progression.

Epigenetically silenced linc00261 contributes to the metastasis of hepatocellular carcinoma via inducing the deficiency of FOXA2 transcription.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. In recent decades, long non-coding RNAs (lncRNAs) have attracted increasing attention and have been reported to play important roles in human cancers, making them ideal candidates for precise disease assessment and treatment. Our previous study found that the loss of linc00261 was significantly correlated with the malignant biological behaviors of HCC, particularly MVI, and serves as an excellent independent prognostic factor for recurrence-free survival. In this study, our in-depth research demonstrated that linc00261 inhibits epithelial-mesenchymal transition (EMT) in liver cancer cells, thereby suppressing migration, invasion, and the formation of lung metastatic lesions. Moreover, linc00261 and its neighbor gene FOXA2 were positively correlated in HCC, the gain- and loss-of-function analyses indicated that linc00261 transcriptionally promotes the expression of FOXA2. Additionally, bioinformatic analysis and rescue assays confirmed that linc00261 partially suppresses migration, invasion, and EMT by upregulating FOXA2 expression. Molecular mechanism studies showed that linc00261 transcriptionally upregulates FOXA2 in cis by recruiting SMAD3. Finally, we identified EZH2 is responsible for linc00261 transcription repression via modulating trimethylation of H3K27 at Lys27 (H3K27Me3), both EZH2 and H3K27Me3 were negatively correlated with linc00261 expression in HCC. In conclusion, these findings demonstrated a crucial role of linc00261 in HCC metastasis, and that EZH2/linc00261/FOXA2 axis might reveal potential prognostic factors and be applied as therapeutic targets for HCC metastasis.

MicroRNA-214 suppresses cell proliferation and migration and cell metabolism by targeting PDK2 and PHF6 in hepatocellular carcinoma.

MiR-214 has been reported to act as a tumor suppressor or oncogene involved in various malignancies. However, the biological functions and molecular mechanisms of miR-214 in hepatocellular carcinoma (HCC) still remain unclear. Previous studies suggest that pyruvate dehydrogenase kinase 2 (PDK2) and plant homeodomain finger protein 6 (PHF6) may be involved in some tumor cell proliferation and migration. Therefore, we studied the relationship between PDK2/PHF6 and miR-214. The expression of miR-214, PDK2, and PHF6 was determined by quantitative real-time polymerase chain reaction in HCC tissues and cell lines. The Luciferase reporter assay was used to confirm the interaction between miR-214 and PDK2/PHF6. Cell proliferation, apoptosis, and migration were evaluated by cell counting kit-8 assay, flow cytometry, and transwell assay, respectively. The expressions levels of α-smooth muscle actin (α-SMA) and E-cadherin were detected via immunofluorescence assay. Here, we found that the expression of miR-214 decreased in HCC and was negatively correlated with PDK2 and PHF6. Moreover, PDK2 and PHF6 were the direct targets of miR-214 in HCC cells. Functional analysis showed that knockdown of PDK2 or PHF6 as well as miR-214 overexpression significantly suppressed cell proliferation and migration in HCC cells. Furthermore, we found that the suppression of cell proliferation and migration through PDK2 or PHF6 knockdown could be partially reversed by miR-214 down-regulation. Moreover, we demonstrated a decrease of mesenchymal cell marker α-SMA and increase of the epithelial marker E-cadherin after miR-214 overexpression, PDK2 knockdown or PHF6 knockdown, respectively, which also suggested that cell proliferation and migration were suppressed. Additionally, lactate and pyruvic acid production experiments confirmed miR-214 could suppress the HCC cell lactate and pyruvic acid levels by down-regulating PDK2/PHF6. In conclusion, MiR-214 may act as a tumor suppressor gene, presenting its suppressive role in cell proliferation and migration of HCC cells by targeting PDK2 and PHF6, and might provide a potential therapy target for patients with HCC.

MALAT1 functions as a competing endogenous RNA to regulate SMAD5 expression by acting as a sponge for miR-142-3p in hepatocellular carcinoma.

BACKGROUND: Long non-coding RNAs are involved in the pathology of various tumors, including hepatocellular carcinoma. The expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is increased in numerous types of tumors and is involved in tumor cell proliferation, migration, invasion and apoptosis. MALAT1 level was reported to be upregulated in hepatocellular carcinoma tissues, but its roles and the specific molecular mechanisms are still unclear. METHODS: The expression of MALAT1 and miR-142-3p in hepatocellular carcinoma tissues, cell lines and adjacent non-tumor tissues was assessed by Q-PCR. The putative-binding sites between MALAT1 and miR-142-3p were predicted by bioinformatics analysis. The expression of MALAT1 in HepG2 and SMMC-7721 cells was knocked down by transfection with MALAT1 siRNAs. Cell viability was assessed by the Cell Counting Kit-8 (CCK-8) assay after the indicated transfection in HepG2 and SMMC-7721 cells. Cell proliferation was assessed by EdU assay, and cell apoptosis was explored by flow cytometry. The migration and invasion potency of HepG2 and SMMC-7721 cells was assessed by the cell migration assay and matrigel invasion assay. Protein level of vimentin, E-cadherin and SMAD5 were assessed by Western blot. RESULTS: Overexpressed MALAT1 acts as a competing endogenous RNA sponge for miR-142-3p in hepatocellular carcinoma. The knockdown of MALAT1 inhibited the proliferation, migration, invasion, and epithelial cell-to-mesenchymal transition (EMT), and promoted apoptosis of hepatocellular carcinoma cells via miR-142-3p. MiR-142-3p inhibited cell proliferation, migration, invasion and EMT, and promoted the cell apoptosis by targeting SMAD5 in hepatocellular carcinoma. MALAT1 promoted tumor growth by regulating the expression of miR-142-3p in vivo. CONCLUSION: MALAT1 promoted cell proliferation, migration, and invasion of hepatocellular carcinoma cells by antagonizing miR-142-3p.

Downregulation of PHF6 Inhibits Cell Proliferation and Migration in Hepatocellular Carcinoma.

Background: The plant homeodomain finger 6 (PHF6) was originally identified as single gene mutated in Börjeson-Forssman-Lehmann syndrome, which was reported to be a tumor suppressor in T-cell acute lymphoblastic leukemia. However, the biological function of PHF6 in hepatocellular carcinoma (HCC) has been poorly characterized. Materials and Methods: In this study, we first determined the mRNA levels of PHF6 in HCC tissues and adjacent normal tissues using quantitative real-time PCR. Then the expression of PHF6 was knocked down in HCC cell lines (HepG2, SMMC-7721, and Bel-7402) by siRNA transfection. A series of functional experiments, including EdU proliferation assay, colony formation assay, and Transwell assay, were performed in HCC cells. Western blot analysis was used to detect the expression of PHF6, E-cadherin, and Vimentin. Results: We found that PHF6 was significantly elevated in HCC tissues and positively correlated with TNM stage, differentiation, and lymph node metastasis. Silencing PHF6 significantly inhibited cell proliferation, colony formation, and migration in HCC cells. Furthermore, silencing PHF6 obviously increased E-cadherin and decreased Vimentin expression. Conclusions: These findings suggest that PHF6 plays a positive role in the growth of HCC cells, and targeting PHF6 could serve as a promising therapeutic strategy for human HCC.

Loss-of-function of miR-142 by hypermethylation promotes TGF-ß-mediated tumour growth and metastasis in hepatocellular carcinoma.

OBJECTIVES: Hypermethylation-induced epigenetic silencing of tumour suppressor genes (TSGs) are frequent events during carcinogenesis. MicroRNA-142 (miR-142) is found to be dysregulated in cancer patients to participate into tumour growth, metastasis and angiogenesis. However, the tumour suppressive role of miR-142 and the status of methylation are not fully understood in hepatocellular carcinoma (HCC). METHODS: Hepatocellular carcinoma tissues and corresponding non-neoplastic tissues were collected. The expression and function of miR-142 and TGF-ß in two HCC cell lines were determined. The miRNA-mRNA network of miR-142 was analysed in HCC cell lines. RESULTS: We found that the miR-142 expression was reduced in tumour tissues and two HCC cell lines HepG2 and SMMC7721, which correlated to higher TNM stage, metastasis and differentiation. Moreover, miR-142 was identified to directly target and inhibit transforming growth factor ß (TGF-ß), leading to decreased cell vitality, proliferation, EMT and the ability of pro-angiogenesis in TGF-ß-dependent manner. Interestingly, the status of methylation of miR-142 was analysed and the results found the hypermethylated miR-142 in tumour patients and cell lines. The treatment of methylation inhibitor 5-Aza could restore the expression of miR-142 to suppress the TGF-ß expression, which impaired TGF-ß-induced tumour growth. CONCLUSION: These findings implicated that miR-142 was a tumour suppressor gene in HCC and often hyermethylated to increase TGF-ß-induced development of hepatocellular carcinoma.