CircSMYD4 regulates proliferation, migration and apoptosis of hepatocellular carcinoma cells by sponging miR-584-5p

Abstract Background There is evidence that circSMYD4 is differentially expressed in hepatocellular carcinoma (HCC), but its mechanism of action remains unclear. Therefore, this study aimed to explore the role of circSMYD4 in the occurrence and development of HCC and its specific molecular mechanism. Methods The expressions of related genes and proteins in the development of HCC were detected by real-time quantitative-PCR and Western blot. HCC cells treated with RNase R and Actinomycin D were used to examine the stability of circSMYD4. Bioinformatics analysis, RNA pull-down assay, luciferase assay and Spearman correlation analysis were performed to evaluate the interaction between circSMYD4 and miRNA. Cell Counting Kit-8, clone formation assay, wound healing assay, Transwell, flow cytometry, nude tumor formation experiment, and immunohistochemistry were employed to analyze the function of circSMYD4 in HCC. A rescue experiment was conducted to analyze the effect of miR-584-5p on the physiological functions of cells. Results CircSMYD4 was down-regulated in HCC tissues and cells, and was not easily affected by RNase R and Actinomycin D. The abundances of circSMYD4 and SMYD4 in the cytoplasm were significantly higher than in the nucleus. Up-regulation of circSMYD4 inhibited the proliferation, invasion and migration and promoted the apoptosis of HCC cells in vitro, while it inhibited tumor growth, promoted apoptosis-related proteins, and suppressed alpha-fetoprotein (AFP) levels in vivo. CircSMYD4 could be used as a miRNA sponge to target miR-584-5p. In addition, miR-584-5p overexpression partially reversed the regulatory effect of circSMYD4 on HCC. Conclusion CircSMYD4 prevents the development of HCC through regulating multiple signaling pathways such as metastasis and apoptosis by sponging miR-584-5p.

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Liver-specific LINC01146, a Promising Prognostic Indicator, Inhibits Malignant Phenotype of Hepatocellular Carcinoma Cells Both in Vitro and in Vivo

10.21203/rs.3.rs-1088533/v1 ◽

2021 ◽

Author(s):

Xiaoyun Ma ◽

Meile Mo ◽

Chao Tan ◽

Jennifer Hui Juan Tan ◽

Huishen Huang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Tumor Growth ◽

Tumor Formation ◽

The Cancer Genome Atlas ◽

Cell Counting ◽

Coagulation Cascade ◽

Migration And Invasion ◽

Hcc Cells

Abstract BackgroundLong non-coding RNAs (lncRNAs) have been proven to be involved in the development of hepatocellular carcinoma (HCC). We aimed to investigate the function of LINC01146 in HCC.MethodsThe expression of LINC01146 in HCC tissues was explored via the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, and was verified using quantitative real-time polymerase chain reaction (qRT-PCR) in our HCC cohort. Kaplan-Meier analysis was used to assess the relationship between LINC01146 and the prognosis of HCC patients. Cell Counting Kit 8, colony formation assays, transwell assays, flow cytometric assays, and tumor formation models in nude mice were conducted to reveal the effects of LINC01146 on HCC cells both in vitro and in vivo. Bioinformatic methods were used to explore the possible potential pathways of LINC01146 in HCC.ResultsLINC01146 was significantly decreased in HCC tissues compared with adjacent normal tissues and it was found to be related to the clinical presentations of malignancy and the poor prognosis of HCC patients. Overexpression of LINC01146 inhibited the proliferation, migration, and invasion, while promoting the apoptosis of HCC cells in vitro. On the contrary, downregulation of LINC01146 exerted the opposite effects on HCC cells in vitro. In addition, overexpression of LINC01146 significantly inhibited tumor growth, while downregulation of LINC01146 promoted tumor growth in vivo. Furthermore, the co-expression genes of LINC01146 were mainly involved in the “metabolic pathway” and “complement and coagulation cascade pathway”. ConclusionLINC01146 expression was found to be decreased in HCC tissues and associated with the prognosis of HCC patients. It may serve as a cancer suppressor and prognostic biomarker in HCC.

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LncRNA PITPNA-AS1 as a Potential Diagnostic Marker and Therapeutic Target Promotes Hepatocellular Carcinoma Progression via Modulating miR-448/ROCK1 Axis

Frontiers in Medicine ◽

10.3389/fmed.2021.668787 ◽

2021 ◽

Vol 8 ◽

Author(s):

Qing-fang Wang ◽

Qing-lin Wang ◽

Ming-bo Cao

Keyword(s):

Hepatocellular Carcinoma ◽

Antisense Rna ◽

Diagnostic Marker ◽

Luciferase Assay ◽

Migration And Invasion ◽

Biological Functions ◽

Non Coding Rnas ◽

Hcc Cells

Background: Long non-coding RNAs are critical to hepatocellular carcinoma (HCC) developments. LncRNA PITPNA antisense RNA 1 (PITPNA-AS1) is a new regulator in several tumors. However, the mechanism by which PITPNA-AS1 mediates the tumorigenesis of HCC remains unclear.Methods: RT-qPCR was used to detect the level of PITPNA-AS1 in HCC specimens and cells. The biological functions of PITPNA-AS1 were explored by several functional experiments in vivo and in vitro. The binding relationship among PITPNA-AS1, miR-448 and ROCK1 were studied by Luciferase assay and pull-down assays.Results: We found that PITPNA-AS1 expressions were distinctly upregulated in both HCC specimens and cell lines. High PITPNA-AS1 levels were an unfavorable biomarker for patients with HCC. Functionally, knockdown of PITPNA-AS1 suppressed the proliferation, migration and invasion of HCC cells. Mechanistically, PITPNA-AS1 functioned as competing endogenous RNA to increase ROCK1 expressions via sponging miR-448.Conclusion: The newly identified PITPNA-AS/miR-448/ROCK1 axis promoted the oncogenicity of HCC cells. This novel axis is likely to be a promising HCC therapeutic aim.

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Long Non-coding RNA TMEM220-AS1 Suppressed Hepatocellular Carcinoma by Regulating the miR-484/MAGI1 Axis as a Competing Endogenous RNA

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.681529 ◽

2021 ◽

Vol 9 ◽

Author(s):

Cong Cao ◽

Jun Li ◽

Guangzhi Li ◽

Gaoyu Hu ◽

Zhihua Deng ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Western Blotting ◽

Pdz Domain ◽

Cell Counting ◽

Luciferase Reporter ◽

Qrt Pcr ◽

Low Levels ◽

Hcc Cells

Long non-coding RNAs (lncRNAs) have a considerable regulatory influence on multiple biological processes. Nevertheless, the role of TMEM220-AS1 in hepatocellular carcinoma (HCC) remains unclear. We used The Cancer Genome Atlas (TCGA) database to analyze the differentially expressed lncRNAs. qRT-PCR was used to verify the results for a large population. The in vitro effects of TMEM220-AS1 on HCC cells were determined using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), flow cytometry, and Transwell assays in HCC cells. We used qRT-PCR and western blotting to identify the epithelial-mesenchymal transition (EMT). Moreover, we performed bioinformatics analysis, western blotting, dual luciferase reporter gene assay, RNA pull-down, and RNA binding protein immunoprecipitation (RIP) to investigate the underlying molecular mechanisms of TMEM220-AS1 function. Finally, the function of TMEM220-AS1 was verified in vivo. The results showed that TMEM220-AS1 was expressed at considerably low levels in HCC. It was demonstrated that malignant phenotypes and EMT of HCC cells were promoted by the knock down of TMEM220-AS1 both in vivo and in vitro. TMEM220-AS1, which was detected primarily in the cytoplasm, functioned as an miRNA sponge to bind miR-484 and promote the level of membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1), thereby curbing the malignant phenotypes of HCC cells. In conclusion, low levels of TMEM220-AS1 promote proliferation and metastasis through the miR-484/MAGI1 axis in HCC.

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miR-186-5p Prevents Hepatocellular Carcinoma Progression Through Targeting METTL3 to Regulate m6A-Mediated Stabilization of FSTL5

10.21203/rs.3.rs-598803/v1 ◽

2021 ◽

Author(s):

DengYong Zhang ◽

FangFang Chen ◽

ShuoShuo Ma ◽

YongChun Zhou ◽

Wanliang Sun ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Rna Stability ◽

The Cancer Genome Atlas ◽

Cell Counting ◽

Migration And Invasion ◽

Dependent Manner ◽

Report Analysis ◽

Hcc Cells

Abstract Background: Hepatocellular carcinoma (HCC) processes in multi-steps which involves the sophisticated interactions of genetics, epigenetics, and transcriptional changes. According to before investigations, methyltransferase-like 3 (METTL3)-mediated m6A modification regulates the development of various cancers by regulating gene stability. However, the studies focusing on miRNA’s regulatory effect of N6-methyladenosine (m6A) modification on HCC progression are still limited. Methods: Immunochemistry (IHC) staining detected the histopathological changes in the tumor tissues. Cell Counting Kit-8 (CCK-8), clone formation, and transwell assay investigated the changes in cancer cell proliferation, invasion, and migration. The RNA m6A level was confirmed by methylated RNA immunoprecipitation. The RNA stability assay indicated the half-life (t1/2) of RNA in HCC cells. The prognosis of the indicated patients’ cohort was analyzed using the cancer genome atlas (TCGA) datasets. Luciferase report analysis was used to study the potential binding between microRNA (miRNA) and mRNA. A mice tumor transplant model was further established to study the changes in tumor progression. Results: Follistatin-like 5 (FSTL5) was found to be significantly downregulated in HCC, and it inhibited the further progression of HCC. The RNA stability analysis indicated that the mRNA t1/2 gene of HCC cells was shortened. Besides, METTL3 reduced the stability of FSTL5 mRNA in a m6A-YTH domain family 2(YTHDF2)-dependent manner. Functional experiments revealed that the downregulated METTL3 inhibited the HCC progression by up-regulating FSTL5 in vitro and in vivo. Luciferase report analysis confirmed that miR-186-5p directly targeted the METTL3. Additionally, miR-186-5p inhibited the proliferation, migration, and invasion of HCC cells by downregulating METTL3. We identified that miR-186-5p prevented the HCC progression by targeting METTL3 to regulate m6A-mediated FSTL5 stabilization. Conclusions: The miR-186-5p/METTL3/YTHDF2/FSTL5 axis perhaps point out a new direction for the targeted therapy of HCC.

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Silencing of KNTC1 inhibits hepatocellular carcinoma cells progression via suppressing PI3K/Akt pathway

10.21203/rs.3.rs-32661/v2 ◽

2020 ◽

Author(s):

Hui Tong ◽

Junjie Xie ◽

Xiaohui Liu ◽

Chenghong Peng ◽

Baiyong Shen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Tumor Regression ◽

Annexin V ◽

Tumor Formation ◽

Migration Ability ◽

Migration Assay ◽

In Vivo Experiments ◽

Hcc Cells

Abstract Background: Kinetochore associated 1 (KNTC1) encodes a kinetochore component in Rod-Zwilch-ZW10 (RZZ) complex which is essential for the segregation of sister chromatids during mitosis and participates in the spindle checkpoint. Recent research demonstrated that kinetochore proteins may be potential biomarkers and may contribute to the development of human malignancies. Here, we sought to identify the biological significances of KNTC1 in hepatocellular carcinoma (HCC).Methods: KNTC1 expression was studied in HCC tissues by immunohistochemistry. Lentivirus delivered short hairpin RNA (shRNA) was performed to generate KNTC1 knockdown HCC cell lines. The effects of KNTC1 on HCC cells proliferation, migration, apoptosis and tumor formation was analyzed by MTT assay, colony formation assay, wound-healing assay, transwell migration assay, annexin V assay in vitro and in nude mouse models in vivo.Results: Our immunohistochemistry experiment showed that KNTC1 was highly expressed in HCC tissues and correlated with terrible prognosis, indicating that KNTC1 acts a pivotal role in HCC development. Furthermore, shRNA KNTC1 (Lv‑shKNTC1) was applied to infect BEL-7404 and SK-HEP-1 to identify roles of KNTC1 on HCC. Lv‑shKNTC1 cells showed reduced proliferation ability, increased apoptosis and decreased migration ability. In vivo experiments suggested that xenografts grow significantly slower upon the silencing of KNTC1. Mechanistically, the protein levels of PIK3CA, p-Akt, CCND1, CDK6 are all down-regulated in Lv-KNTC1 SK-HEP-1 cells. Therefore, KNTC1 may affect the biological activity of HCC cells through PI3K/Akt signaling pathway. Conclusions: In summary, the key finding of this report highlighted the significance of KNTC1 in tumor regression of HCC, demonstrating KNTC1 as an innovative target for adjuvant treatment of HCC.

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Liver X receptor inhibits the growth of hepatocellular carcinoma cells via regulating HULC/miR-134-5p/FOXM1 axis

10.21203/rs.2.22627/v1 ◽

2020 ◽

Author(s):

Yan Zhang ◽

Jintao He ◽

Teng Yang ◽

Wenhui He ◽

Shan Jiang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cyclin D1 ◽

Gene Promoter ◽

Liver X Receptor ◽

Cell Counting ◽

Luciferase Reporter ◽

In Vivo Experiments ◽

Hcc Cells

Abstract Background Highly upregulated in liver cancer (HULC), the specifically overexpressed long non-coding RNA (lncRNA) in human hepatocellular carcinoma (HCC), can promote the growth and metastasis of HCC cells. Therefore, it will be benefit to HCC treatment by effectively downregulating HULC. Liver X receptor (LXR), a member of nuclear receptor superfamily, exerts anti-tumor effects on various human malignancies including HCC. However, it is unclear whether the anti-HCC function of LXR is involved in the regulation of HULC. Methods Quantitative real-time PCR and Western blot were used to separately examine RNA and protein levels in HCC cells. Cell counting kit-8 assay was used to detect the growth of HCC cells in vitro . Dual-luciferase reporter assays were performed to analyze the regulation of forkhead box M1 (FOXM1) by miR-134-5p and the regulation of miR-134-5p by HULC. Xenograft models were engaged to evaluate the growth of HCC cells in vivo . Results In this study, we found that activation of LXR could inhibit the growth of HCC cells by downregulating HULC. Mechanistically, LXR decreased HULC via suppressing its gene promoter activity. Moreover, HULC and FOXM1 were highly expressed while miR-134-5p was lowly expressed in HCC tissues, and the level of HULC was positively correlated with that of FOXM1 while negatively correlated with that of miR-134-5p. Additionally, miR-134-5p downregulated FOXM1 by targeting 3′-untranslated region (UTR) of its mRNA, and HULC upregulated FOXM1 and its downstream target molecule cyclin D1 through sequestrating miR-134-5p. Furthermore, activation of LXR increased miR-134-5p while decreased FOXM1 by reducing HULC in HCC cells. The in vivo experiments showed that activation of LXR repressed the growth of HCC xenografts, and decreased HULC, FOXM1 and cyclin D1 while increased miR-134-5p in the xenografts. Conclusions Our results for the first time reveal that LXR can inhibit the growth of HCC cells by regulating HULC/miR-134-5p/FOXM1 axis. The novel pathway LXR/HULC/miR-134-5p/FOXM1 may serve as a promising target in HCC treatment.

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MiR-3150b inhibits hepatocellular carcinoma cell proliferation, migration and invasion by targeting GOLPH3

Journal of Investigative Medicine ◽

10.1136/jim-2019-001181 ◽

2019 ◽

Vol 68 (3) ◽

pp. 770-775

Author(s):

Yi Zhang ◽

Jianjun Wang ◽

Hongling Su

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Carcinoma Cell ◽

Cell Counting ◽

Luciferase Assay ◽

Migration And Invasion ◽

Invasion And Migration ◽

Hepatocellular Carcinoma Cell ◽

And Migration ◽

Hcc Cells

BackgroundIn this study, we aimed to explore the potential involvement of miR-3150b in hepatocellular carcinoma (HCC) carcinogenesis.MethodsThe expression of miR-3150b and Golgi phosphoprotein 3 (GOLPH3) was determined in HCC cell lines. Cell proliferation, migration and invasion were estimated by Cell Counting Kit-8, wound healing and Transwell assays. The association between miR-3150b and GOLPH3 was verified by luciferase assay.ResultsMiR-3150b was downregulated, while GOLPH3 was remarkably upregulated in HCC cells. Furthermore, miR-3150b inhibited HCC cell proliferation, migration and invasion. MiR-3150b directly targeted and negatively regulated GOLPH3.ConclusionMiR-3150b suppressed HCC cell proliferation, invasion and migration by targeting GOLPH3.

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LncRNA BCYRN1/miR-490-3p/POU3F2, served as a ceRNA network, is connected with worse survival rate of hepatocellular carcinoma patients and promotes tumor cell growth and metastasis

Cancer Cell International ◽

10.1186/s12935-019-1081-x ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 4

Author(s):

Shichao Ding ◽

Yanfeng Jin ◽

Qingzhi Hao ◽

Yanmeng Kang ◽

Ruiping Ma

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Counting ◽

Luciferase Assay ◽

Dual Luciferase Assay ◽

Relative Expression ◽

Invasion And Migration ◽

Qrt Pcr ◽

Relative Expression Level ◽

Tcga Dataset ◽

Hcc Cells

Abstracts Backgrounds LncRNA Brain Cytoplasmic RNA 1 (BCYRN1) has been certified to modulate cancer cells growth and aggressiveness in several tumors. However, research about function of BCYRN1 in hepatocellular carcinoma (HCC) is limited. Therefore, our research intends to explore the function of BCYRN1 in HCC. Methods HepG2 and BEL-7402 cell lines were employed for later function experiments. Differently expression levels of BCYRN1, miR-490-3p, and POU class 3 homeobox 2 (POU3F2) were determined on the base of TCGA dataset including 375 HCC patients and 50 normal. 370 cases of patients, which have fairly complete clinical data, were utilized for survival analysis of BCYRN1, miR-490-3p, or POU3F2 by Kaplan–Meier method. Relative expression pattern of BCYRN1 was examined by quantitative real time polymerase chain reaction (qRT-PCR), and relative expression level of POU3F2 was assessed by qRT-PCR and western blot. Cell biological behaviors were analyzed by cell counting kit-8, cloning formation, and transwell assays. Bioinformatics software and dual luciferase assay were applied to predict and confirm the targeted relationship between BCYRN1 and miR-490-3p, as well as miR-490-3p and POU3F2. Further associations among BCYRN1, miR-490-3p, and POU3F2 were analyzed by rescue assays. Results Our results exhibited that BCYRN1 was over expressed in HCC samples, which was connected with unfavorable prognosis in HCC patients. In addition, a series of experiments exhibited that overexpression of BCYRN1 significantly expedited HCC cells growth, clone formation, and movement abilities, and vice versa. Moreover, targeted relationships between BCYRN1 and miR-490-3p, as well as miR-490-3p and POU3F2 were affirmed by dual luciferase assay. Furthermore, POU3F2 expression was negatively connected with the expression of miR-490-3p and positively associated with BCYRN1 expression. Whilst, either overexpression of miR-490-3p or knockdown of POU3F2 could remarkably inhibit the increasing trends of proliferation, clone formation, invasion, and migration abilities induced by BCYRN1 in HCC cells. Conclusions BCYRN1, served as a competing endogenous RNA, up-regulated the expression of POU3F2 to promote the development of HCC through sponging miR-490-3p, supplying novel molecular targets and underlying prognostic biomarkers for HCC therapy.

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FAM21C Promotes Hepatocellular Carcinoma Invasion and Metastasis by Driving Actin Cytoskeleton Remodeling via Inhibiting Capping Ability of CAPZA1

Frontiers in Oncology ◽

10.3389/fonc.2021.809195 ◽

2022 ◽

Vol 11 ◽

Author(s):

Yao Lu ◽

Deng Huang ◽

Baolin Wang ◽

Bowen Zheng ◽

Jialong Liu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Actin Cytoskeleton ◽

Malignant Progression ◽

High Expression ◽

Invasion And Migration ◽

Cytoskeleton Remodeling ◽

And Migration ◽

Hcc Cells

Hepatocellular carcinoma (HCC) is characterized by a high incidence of metastasis. The dynamic remodeling of the actin cytoskeleton plays an important role in the invasion and migration of HCC cells. In previous studies, we found that CAPZA1, a capping protein, can promote EMT of HCC cells by regulating the remodeling of the actin filament (F-actin) cytoskeleton, thus promoting the invasion and migration of HCC cells. In this study, we found that FAM21C may have a regulatory effect on CAPZA1, and we conducted an in-depth study on its potential regulatory mechanism. First, we found that FAM21C is highly expressed in HCC tissues and its high expression could promote the malignant progression of HCC. Meanwhile, the high expression of FAM21C promoted the invasion and migration of HCC cells in vitro and in vivo. Further, FAM21C interacted with CAPZA1, and their binding inhibited the capping capacity of CAPZA1, thus promoting the invasion and migration of HCC cells. This effect of FAM21C was abolished by mutating the CP-interacting (CPI) domain, the CAPZA1 binding site on FAM21C. In conclusion, high expression of FAM21C in HCC tissues can promote malignant progression of HCC and its potential mechanism involves FAM21C inhibition of CAPZA1 capping capacity by binding to CAPZA1, which drives F-actin cytoskeleton remodeling, and thus promotes invasion and migration of HCC cells.

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Silencing of KNTC1 inhibits hepatocellular carcinoma cells progression via suppressing PI3K/Akt pathway

10.21203/rs.3.rs-32661/v3 ◽

2020 ◽

Author(s):

Hui Tong ◽

Junjie Xie ◽

Xiaohui Liu ◽

Chenghong Peng ◽

Baiyong Shen ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Tumor Regression ◽

Annexin V ◽

Tumor Formation ◽

Migration Ability ◽

Migration Assay ◽

In Vivo Experiments ◽

Hcc Cells

Abstract Background: Kinetochore associated 1 (KNTC1) encodes a kinetochore component in Rod-Zwilch-ZW10 (RZZ) complex which is essential for the segregation of sister chromatids during mitosis and participates in the spindle checkpoint. Recent research demonstrated that kinetochore proteins may be potential biomarkers and may contribute to the development of human malignancies. Here, we sought to identify the biological significances of KNTC1 in hepatocellular carcinoma (HCC).Methods: KNTC1 expression was studied in HCC tissues by immunohistochemistry. Lentivirus delivered short hairpin RNA (shRNA) was performed to generate KNTC1 knockdown HCC cell lines. The effects of KNTC1 on HCC cells proliferation, migration, apoptosis and tumor formation was analyzed by MTT assay, colony formation assay, wound-healing assay, transwell migration assay, annexin V assay in vitro and in nude mouse models in vivo.Results: Our immunohistochemistry experiment showed that KNTC1 was highly expressed in HCC tissues and correlated with terrible prognosis, indicating that KNTC1 acts a pivotal role in HCC development. Furthermore, shRNA KNTC1 (Lv‑shKNTC1) was applied to infect BEL-7404 and SK-HEP-1 to identify roles of KNTC1 on HCC. Lv‑shKNTC1 cells showed reduced proliferation ability, increased apoptosis and decreased migration ability. In vivo experiments suggested that xenografts grow significantly slower upon the silencing of KNTC1. Mechanistically, the protein levels of PIK3CA, p-Akt, CCND1, CDK6 are all down-regulated in Lv-KNTC1 SK-HEP-1 cells. Therefore, KNTC1 may affect the biological activity of HCC cells through PI3K/Akt signaling pathway.Conclusions: In summary, the key finding of this report highlighted the significance of KNTC1 in tumor regression of HCC, demonstrating KNTC1 as an innovative target for adjuvant treatment of HCC.

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