Biological Behaviors Of HepG2 Cells Research Articles

Effects of targeted-edited oncogenic insulin-like growth factor-1 receptor with specific-sgRNA on biological behaviors of HepG2 cells.

BACKGROUNDInsulin-like growth factor-1 receptor (IGF-1R) is over-expressed in hepatocellular carcinoma (HCC). However, the relationship between IGF-1R activation and HCC progression remains unidentified. AIMTo investigate the effects of editing IGF-1R on the biological features of HCC cells.METHODSImmunohistochemistry analyzed the expressions of IGF-1R and P-glyco protein (P-gp) in HCC tissues and their distal non-cancerous tissues (non-Ca). IGF-1R was edited with Crispr/Cas9 system, screened specific sgRNAs, and then transfected into HepG2 cells. CCK-8, scratch wound test detected cell proliferation, migration, invasion and transwell assays, respectively. Alterations of IGF-1R and P-gp were confirmed by Western blotting. Alterations of anti-cancer drug IC50 values were analyzed at the cell level. RESULTSThe positive rates of IGF-1R (93.6%, χ2 = 63.947) or P-gp (88.2%, χ2 = 58.448) were significantly higher (P < 0.001) in the HCC group than those (36.6% in IGF-1R or 26.9% in P-gp) in the non-Ca group. They were positively correlated between high IGF-1R and P-gp expression, and they were associated with hepatitis B virus infection and vascular invasion of HCC. Abnormal expressions of circulating IGF-1R and P-gp were confirmed and associated with HCC progression. Biological feature alterations of HCC cells transfected with specific sgRNA showed IGF-1R expression down-regulation, cell proliferation inhibition, cell invasion or migration potential decreasing, and enhancing susceptibility of HepG2 cells to anti-cancer drugs.CONCLUSIONEdited oncogenic IGF-1R was useful to inhibit biological behaviors of HepG2 cells.

Identification of alanine aminotransferase 1 interaction network via iTRAQ-based proteomics in alternating migration, invasion, proliferation and apoptosis of HepG2 cells.

Objective: To investigate the mechanism of alanine aminotransferase 1 (ALT1) in the progression of HCC, the differentially expressed proteins (DEPs) in the ALT1 interaction network were identified by targeted proteomic analysis.Methods: Wound healing and transwell assays were conducted to assess the effect of ALT1 on cellular migration and invasion. Cell Counting Kit-8 (CCK-8), colony formation, and flow cytometry assays were performed to identify alterations in proliferation and apoptosis. After coimmunoprecipitation processing, mass spectrometry with iso-baric tags for relative and absolute quantitation was utilized to explore the protein interactions in ALT1 knockdown HepG2 cells.Results: The results showed that ALT1 knockdown inhibits the migration, invasion, proliferation of HepG2 cells, and promotes apoptosis. A total of 116 DEPs were identified and the bioinformatics analysis suggested that the ALT1-interacting proteins were primarily associated with cellular and metabolic processes. Knockdown of ALT1 in HepG2 cells reduced the expression of Ki67 and epithelial cell adhesion molecule (EP-CAM), while the expression of apoptosis-stimulating protein 2 of p53 (ASPP2) was increased significantly. Suppression of the ALT1 and EP-CAM expression contributed to alterations in epithelial–mesenchymal transition (EMT) -associated markers and matrix metalloproteinases (MMPs). Additionally, inhibition of ALT1 and Ki67 also decreased the expression of apoptosis and proliferation factors. Furthermore, inhibition of ALT1 and ASPP2 also changed the expression of P53, which may be the signaling pathway by which ALT regulates these biological behaviors.Conclusions: This study indicated that the ALT1 protein interaction network is associated with the biological behaviors of HepG2 cells via the p53 signaling pathway.

Effects of Serum From Radiofrequency Ablation Patients Receiving General Anesthesia or Local Anesthesia on Hepatocellular Carcinoma Cancer Cell Malignancy: A Prospective Randomized Controlled Trial.

BackgroundWhether anesthesia methods affect malignant biological behavior of cancer remains unresolved. In this study, we aim to compare the effects of general anesthesia (GA) and local anesthesia (LA) on serum collected from primary hepatocellular carcinoma (HCC) patients presenting for radiofrequency ablation (RFA).MethodsFrom August 2020 to December 2020, a prospective, randomized, and controlled study was conducted at Renji Hospital, which is affiliated with Shanghai Jiaotong University School of Medicine. 25 qualified patients from 18 to 65 years of age undergoing RFA were enrolled in the study and randomly assigned into two groups: the GA group (n = 14) and the LA group (n = 11). Venous blood was drawn from all patients preoperatively and 1 hour postoperatively. The serum collected was then used for the culturing of HepG2 cells. The malignant biological behaviors of HepG2 cells, including invasion, migration and proliferation, were observed after 24 hours of exposure to patients’ serum. ELISA was used to compare expression levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and lymphokines (IFN-γ, IL-2) in patients’ serum from both groups.ResultsHepG2 cells cultured with postoperative serum obtained from patients who received GA, but not LA, were associated with significantly increased cell invasion, migration and proliferation, compared to preoperative serum from the same patient group. Expression levels of pro-inflammatory cytokines were significantly higher, and lymphokines significantly lower in postoperative serum from GA patients compared to the corresponding preoperative serum.ConclusionGA affects the serum milieu of patients with HCC, promoting the malignant biological behavior of a human HCC cell line.

The peptide encoded by a novel putative lncRNA HBVPTPAP inducing the apoptosis of hepatocellular carcinoma cells by modulating JAK/STAT signaling pathways

When the hepatitis B virus (HBV) enters target cells, there are complex trans-regulatory mechanisms involved in the interactions between the virus and the target cells. In the present study, a new gene screened from the hepatoblastoma cell line HepG2 using suppression subtractive hybridization, referred to as lncRNA HBVPTPAP, was used to study the trans-regulation of HBV DNA polymerase. According to the structural characteristics of the full-length sequences, it was classified as long non-coding RNA. However, a unique and complete open reading frame (ORF) was still present. Therefore, to further identify the lncRNA HBVPTPAP gene's encoding potential, this study used several online tools to analyze and verify its encoding polypeptide authenticity. On that basis, the effects of the lncRNA HBVPTPAP gene on the biological behaviors of HepG2 cells and its molecular regulatory mechanism were investigated. It was found that the lncRNA HBVPTPAP subcellular was mainly located in the cytoplasm, and possibly activated the downstream JAK/STAT signaling pathway through the interaction between the encoding polypeptide and PILRA intracellular domain. Then, the mitochondrial apoptosis pathway may have been initiated to induce apoptosis. These results provided a basis for further study of the biological functions of the lncRNA HBVPTPAP gene.

Plantamajoside inhibits the proliferation and epithelial-to-mesenchymal transition in hepatocellular carcinoma cells via modulating hypoxia-inducible factor-1α-dependent gene expression.

As a potential antitumor herbal medicine, plantamajoside (PMS) benefits the treatment of many human malignances. However, the role of PMS in the progression of hepatocellular carcinoma (HCC) and the related molecular mechanisms is still unknown. Here, we proved that the cell viabilities ofHepG2 cells were gradually decreased with the increasing concentrations of CoCl2 and/or PMS via cell counting kit-8 assay. Meanwhile, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and western blot assays were used to further confirm that PMS inhibited the CoCl2 -induced cell proliferation in HepG2 cells via suppressing the Ki67 and proliferating cell nuclear antigen expressions. We also performed wound-healing and transwell assays and demonstrated that PMS inhibited CoCl2 -induced migration and invasion in HepG2 cells via suppressing the epithelial-mesenchymal transition (EMT) process. In addition, the use of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole further proved that PMS inhibited the malignant biological behaviors of HepG2 cells under hypoxic condition by suppressing the hypoxia-inducible factor-1α (HIF-1α) expression. Besides, we further confirmed that PMS suppressed the growth and metastasis of implanted tumors in vivo. Given that PMS suppressed the proliferation and EMT induced by CoCl2 in HCC cells via downregulating HIF-1α signaling pathway, we provided evidence that PMS might be a novel anti-cancer drug for HCC treatment.

Circ_0009910 promotes proliferation and metastasis of hepatocellular carcinoma cells through miR-335-5p/ROCK1 axis.

CircRNAs serve an essential role in regulating the development and progression of various tumors. The aim of this study was to examine the role and mechanism of circ_0009910 in hepatocellular carcinoma (HCC). RT-qPCR was used to detect the expression of circ_0009910 and miR-335-5p in tissues and cell lines of HCC. The proliferation, migration, and invasion of HCC cells were examined using 5-ethynyl-2-deoxyuridine (EdU), colony formation, and Transwell assay, respectively. Dual-luciferase reporter gene assay was performed to verify the interaction between miR-335-5p and circ_0009910 or ROCK1. Western blot was applied to detect the protein levels. Furthermore, the antitumor effect of circ_0009910 knockdown was examined by establishing xenograft tumor model of HCC in vivo. Circ_0009910 was upregulated in HCC tissues and cell lines. Knockdown of circ_0009910 significantly inhibited the proliferation, migration, and invasion of HepG2 cells and suppressed tumor growth and metastasis in vivo. Moreover, circ_0009910 directly targeted miR-335-5p, as well as for ROCK1 was a direct target gene of miR-335-5p. Mechanically, simultaneous over-expression of miR-335-5p and circ_0009910 or ROCK1 could restore the biological behaviors of HepG2 cells, which were inhibited by miR-335-5p. Circ_0009910-silenced suppressed the growth and metastasis of HCC cells through upregulating the inhibitory effect of miR-335-5p on ROCK1.

Effect of Alkylresorcinols on Autophagy, Migration, and Invasion of HepG2 Cells.

Alkylresorcinols are phenolic lipids that mainly exist in the cortex of grains, and they exhibit anticancer activity against various cancer cells in vitro. However, the underlying action mechanisms are still unclear. In our study, the influence of alkylresorcinols C19:0 and C21:0 (ARs) upon migration, invasion, and autophagy in human hepatoblastoma HepG2 cells was evaluated. Results showed that ARs at 80 and 160 µg/mL significantly suppressed cells proliferation, migration, and invasion, downregulated the expression of proteins RhoA and MMP-7 associated with migration and invasion. ARs at 160 µg/mL, the rate of LC3 puncta was appreciably increased. After autophagy was blocked by 3-MA or CQ, the expression of LC3II was significantly increased in 3-MA+ARs group and p62 was significantly decreased in CQ+ARs group. The results indicate that ARs may promote autophagic flow. ARs (80, 160 µg/mL) significantly inhibited the expression of proteins p-mTOR, p-PI3K, and p-Akt related to the PI3K/Akt pathway. The results of the present study suggest that ARs can activate autophagy and suppresses the biological behaviors of HepG2 cells by inhibiting the activation of MMP-7, Rho/Rho-associated protein kinase, and activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. PRACTICAL APPLICATION: The anticancer mechanism of ARs in wheat bran was studied, which provided a basis for the development of anticancer functional auxiliary food with wheat bran as raw material. It is of great practical significance to promote the effective utilization of grain processing by-products and improve the economic benefits of the grain industry.

Effect of silencing NEK2 on biological behaviors of HepG2 in human hepatoma cells and MAPK signal pathway.

To investigate the expression level of NEK2 in 40 tissue specimens of primary liver cancer and to search for clues whether the effect of NEK2 depletion plays a role on biological behaviors of HepG2 cells and the relevant molecular mechanism are the objectives of this study. Real-time PCR and immunohistochemistry assessed expression level of NEK2 in specimens of cancerous tissues and carcinoma-adjacent tissues. The NEK2 expression level in HepG2, Huh7, SMMC, and 7402 cells was detected by real-time PCR and western blot to screen experimental cell line. To assess the expression levels of NEK2 mRNA and protein, an effective siRNA transfected into the HepG2 cells was designed. CCK8 and colony-forming assays were performed to verify short-term and long-term proliferative activities, respectively. Capacity of apoptosis and cell cycle changes were assessed by flow cytometry. Ability of transference and invasion was measured by Transwell Chambers. Western blot approach was used to determine the protein expression levels. There was significantly high expression level of NEK2 in cancerous tissues compared to adjacent tissues. The expression of NEK2 was higher in HepG2 cells than other cell lines. Real-time PCR and western blot shown there were obviously down-regulated NEK2 expression in the NEK2-siRNA group compared to control groups. The capacity of amplification and invasion was inhibited distinctly, and FCM revealed the apoptosis rate was increased and G1 phase was arrested in NEK2-siRNA group. Western blot indicated that low expression of NEK2 in HepG2 cells could increase the expression levels of Bax, caspase-3, P21, and TIMP-1, but significantly suppressed the c-myc, c-jun, Bcl-2, cyclinD1, CDK4, MMP2, and MMP9 expression levels and the phosphorylation levels of ERK, JNK, and P38 compared with the control groups. Our findings demonstrated that NEK2 could be a valuable carcinogenic factor and a promising therapeutic target for primary liver cancer; NEK2 may regulate proliferation, apoptosis, and other biological behaviors of HepG2 cells via MAPK signal pathway.

Effect of silencing RRM2 by specific small interference RNA on biological behaviors of human hepatoma cancer cell line HepG2

Objective To investigate the effect of small interference RNA (siRNA) silencing RRM2 on the biological behaviors of human hepatoma cancer cell line HepG2 and and the molecular mechanisms.Methods RRM2 expression was knocked down in human hepatoma cancer cell line HepG2 by RRM2 siRNA.The expression level of RRM2 was detected in human hepatoma cancer cell line HepG2 and human normal liver cell line HL-7702 at mRNA and protein levels by real-time polymerase chain reaction (PCR) or Western blotting.The cell proliferation was measured by cell counting Kit-8 (CCK-8).The migration was observed by using Transwell cell migration system.The expression levels of B lymphocytes/leukemia-2 (bcl-2),bax and cytochrome C (Cyt-C) proteins were detected by using Western blotting.Ten male BALB/C nude mice were subcutaneously injected with siRNA-RRM2/HepG2,siRNA-NC/HepG2 or NEG/HepG2 cells respectively to study the growth of tumor in each group.Results siRNA-RRM2 could down-regulate the expression of RRM2 in HepG2 remarkably,and siRNA-RRM2 could inhibit both the proliferation [(41.20 ± 2.13) ％ vs (91.35 ± 10.22) ％] and migration ability of HepG2 cells (21.20 ±1.28 vs 132.50 ±7.88) as compared with the negative control group.siRNA-RRM2 could down-regulate the expression of bcl-2 and up-regulate the expression of bax and Cyt-C.Additionally,the volume of xenograft tumors in siRNA-RRM2 group was decreased significantly as compared with control group.The volume of xenograft tumors in siRNA-RRM2 group was (171.13 ± 28.12) mm3 and that in siRNA-NC group was (1487.41 ± 168.20) mm3 at 8th week.Conclusion siRNA-RRM2 can down-regulate the expression of RRM2 in the HepG2 cells and may play an important role in the regulation of biological behaviors of HepG2 cells,and also inhibit the tumor growth of nude mice model. Key words: RNA interference ; RRM2 gene ; Carcinoma,hepatocellular

Effect of short hairpin RNA of Bcl-xL gene on biological behaviors of HepG-2 cells

To investigate the inhibitory effect of short hairpin RNA (shRNA) targeting the Bcl-xL gene on the apoptosis and proliferation of hepatocellular carcinoma HepG-2 cells. We constructed Bcl-xL shRNA, and transfected it into HepG-2 cells, then detected Bcl-xL gene expression on mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry, respectively. Cell proliferation was detected by MTT assay. Cell apoptosis and Bcl-2 and Bax expression were detected by flow cytometry. The expression of Bcl-xL was obviously inhibited by RNA interference. The inhibition rates of Bcl-xL mRNA and protein were 86.6 and 70.2%, respectively. Bcl-xL shRNA inhibited cell proliferation (P < 0.05), whereas promoted apoptosis (P < 0.01). After transfection of Bcl-xL shRNA in HepG-2 cells, the expression of Bcl-2 did not change, but Bax increased significantly. Bcl-xL shRNA effectively inhibits Bcl-xL gene expression and proliferation of HepG-2 cells and promotes apoptosis.

MicroRNA expression alteration after arsenic trioxide treatment in HepG‐2 cells

More and more microRNA (miRNA) are found to be involved in tumor genesis and progress. Arsenic trioxide has been an effective chemotherapeutic drug in cancer therapy for many years. In this study, we aimed to find the miRNA involved in the mechanisms of arsenic trioxide treatment in cancer therapy. We detected the expression profile of miRNA by miRNA microarray and quantitative real-time polymerase chain reaction. Cell viability assay, flow cytometry analysis, prediction of miRNA targets, Western blot analysis and luciferase reporter assay were carried out to determine the role of one selected miRNA, namely mir-29a, in affecting the biological behaviors of HepG-2 cells. Among the 677 human miRNA in the microarray, five miRNA were upregulated and four were downregulated in HepG-2 cells treated with arsenic trioxide compared to their controls. If only changes above two folds were considered, four miRNA were identified, namely miR-24, miR-29a, miR-30a and miR-210, which were all upregulated. Among them, miR-29a showed a positive therapeutic effect in liver cancer cells by inhibiting cell growth and inducing cell apoptosis, and PPM1D was confirmed to be the target gene of miR-29a. Furthermore, a synergy effect was detected between miR-29a and arsenic trioxide. Arsenic trioxide altered miRNA expression profile in HepG-2 cells. Among the altered miRNA, miR-29a seemed to take a role in the mechanism of arsenic trioxide in liver cancer therapy. The synergy effect between miR-29a and arsenic trioxide may offer this drug a new chance in cancer therapy by decreasing its dose and toxic side-effects.

MicroRNA‐224 is upregulated in HepG2 cells and involved in cellular migration and invasion

MicroRNAs are a class of small non-coding RNAs that negatively regulate the expression of their target genes. The aim of the present study was to explore the effects of microRNA on biological behaviors of HepG2 cells and further analyze its characteristics. We detected different expression profiles of miRNAs in HepG2 and L02 cell lines by microRNA microarray. Northern blot, quantitative real-time polymerase chain reaction, methylthiazolyl tetrazolium, fluorescence-activated cell sorting, scratch wound, transwell migration and Matrigel invasion assays and western blot were carried out to determine whether or not microRNA-224 (miR-224) can influence the biological behaviors of HepG2 cells. MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224. Taking invasion and migration as a breakthrough, a close relationship between the expression of miR-224 and its proteins such as PAK4 and MMP9, which were involved in the invasion of tumor, was found. Overexpression of miR-224 was involved in the malignant phenotype of HepG2 cells, and it may be an important factor in regulating the migration and invasion of HepG2 cells.