Effect of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7.

OBJECTIVE: To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7. METHODS: Hepatocellular carcinoma Huh7 was cultured in vitro and lipidosome was used to transfect miR-520c-3p and miR-132, respectively or together. The effects of transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of Huh7 were detected by CCK8 and Annexin V staining and flow cytometry, and the expression level of the targeted gene of over-expressed miR-520c-3p and miR-132 was determined by Western blot and realtime PCR. RESULTS: Compared with the control group, the proliferation ability of Huh7 of the single transfected and co-transfected miR-520c-3p and miR-132 decreased significantly, and the apoptosis ratio increased distinctly (P < 0.05). Besides, the effect of the co-transfection group was better than that of the single transfection group. The protein levels of GPC3 (Glypican-3) and YAP (Yes-associated protein), the target genes transfected only by miR-520c-3p and miR-132, respectively, reduced obviously (P < 0.05), which was similar with the co-infected cells, but cells transfected by miR-132 only showed a decrease of YAP. CONCLUSIONS: The co-transfection of miR-520c-3p and miR-132 can target-regulate the expression of GPC3 and YAP, enhance the exhibition effect on proliferation of hepatocellular carcinoma Huh7 and induce cell apoptosis synergistically.

Change of the peripheral blood immune pattern and its correlation with prognosis in patients with liver cancer treated by sorafenib.

OBJECTIVE: To study the change of the peripheral blood immune pattern and its correlation with prognosis in patients with liver cancer after treated by sorafenib. METHODS: Patients with advanced liver cancer admitted in our hospital were enrolled and treated with sorafenib. After two months of the treatment, their peripheral blood was collected. The immune cell subset and cytokines level were determined by flow cytometry and luminex technology. According to the reaction expressed by patients towards sorafenib, patients were divided into the response group and the no response group. The changes of the peripheral blood immune pattern and its correlation with prognosis of patients in the two groups were compared. RESULTS: Before and after treatment of sorafenib, there was no significant difference in the ratios of T cells, NK cells and their subtypes in peripheral blood of patients between the two groups; while after treatment the ratio of B cells and regulatory B cells (Breg) of patients in the response group was significant higher than that of the no response group (P < 0.05), and the prognosis conditions of patients with decreased ratio of Breg cells were better than other patients after undergoing chemotherapy. The levels of plasma cytokines IL-6, IL-10, IL-12, IL17, FIL-3L, IFN-γ, TNF-α, MCP-1 and VEGF showed no significant differences. CONCLUSIONS: After treatment of sorafenib, the prognosis conditions of patients of advanced liver cancer with a reduced Breg ratio are better than patients with an unaltered or increased Breg ratio. The ratio of Breg in peripheral blood may be considered as early biological indicator for the prediction of the curative effects of sorafenib.

miR-542-3p inhibits the growth and invasion of colorectal cancer cells through targeted regulation of cortactin.

Colorectal cancer is one of the most common malignancies. Previous studies have reported that cortactin (CTTN) is often overexpressed in tumors and is associated with metastasis and poor prognosis of patients. The abnormal expression of microRNAs (miRNAs or miRs) is closely related to the development and progression of various types of cancer, including colorectal cancer. However, little is known about the miRNAs targeting cortactin. In the present study, prediction using biological software revealed that cortactin has binding sites for miR-542-3p. Transfection with miR-542-3p mimic demonstrated that miR­542-3p reduced the expression of cortactin in colorectal cancer cells. Dual luciferase reporter assays further demonstrated that miR-542-3p regulated cortactin in a targeted manner and that miR-542-3p expression was significantly downregulated in colorectal cancer cells. A cell proliferation assay and Transwell migration assay were undertaken: we noted that miR­542-3p inhibited the proliferation and invasion of colorectal cancer cells while promoting their apoptosis. By contrast, cortactin acted antagonistically. When co-transfected with miR-542-3p mimic and CTTN overexpression vector, the inhibitory effect of miR-542-3p was blocked. This indicates that miR-542-3p regulates CTTN in a targeted manner to modulate the growth and invasion of colorectal cancer cells. The present study thus provides new targets for the prevention and treatment of colorectal cancer.

Anti-tumor effect of LTA combined with 5-FU on H22 tumor bearing mice.

OBJECTIVE: To study the effect of lipoteichoic acid (LTA) and 5-FU on the expression of caspase-3, EGFR, TGF-α proteins of tumor tissue of H22 cancer bearing mice and its anti-tumor mechanism. METHODS: A total of 40 SPF grade Kunming mice were selected to establish H22 liver cancer model, and then the mice were divided into 4 groups at random with ten mice in each group. Group A was given saline lavage treatment, Group B was treated with 5-FU by intraperitoneal injection, Group C was treated with LTA by lump body injection; Group D was treated with LTA by lump body injection and 5-FU by intraperitoneal injection. Two weeks after the treatment, the mice in each group were executed and the tumor tissue was stripping and weighted, and the tumor growth inhibition ratio was calculated. Then the tumor tissue was processed for conventional embedding, sectioned to observe the expression of caspase-3, EGFR, TGF-α by immunohistochemical staining method. RESULTS: The tumor inhibitory rate o f Group D was significantly higher than Groups B and C (P < 0.05); B, the tumor inhibitory rate o f Group B had no statistical difference compared with Group C (P > 0.05). The IDO values of TGF-α, EGFR proteins in Groups B, C, D mice tumor tissue were significantly lower than that in group A (P < 0.05); while IDO value of caspase-3 in Groups B, C, D group mice tumor tissue was significantly higher than that in Group A (P < 0.05). The IDO value of TGF-α, EGFR in Group D mice tumor tissue were significantly lower than that in Groups B and C; While IDO value of aspase-3 in Group D was significantly higher than that in Groups B and C (P < 0.05). CONCLUSIONS: LTA combined with 5-FU can effectively inhibit the tumorigenesis of H22 tumor bearing mice, increase the caspase-3 protein expression, inhibit TGF-α and EGFR protein expression, further promote tumor cell apoptosis and play a synergistic antitumor effect.

Hsa-miR-132 inhibits proliferation of hepatic carcinoma cells by targeting YAP.

MicroRNAs and Yes-associated protein (YAP) play an important role in the occurrence and development of hepatic carcinomas. However, the interaction between microRNAs and YAP was seldom elucidated. In this study, we showed that miR-132 could target YAP gene by using dual-luciferase reporter system. Further quantitative polymerase chain reaction analysis and western blotting showed that miR-132 could significantly reduce the expression of YAP at mRNA and protein levels. Results of annexin V-fluorescein isothiocyanate, 5-ethynyl-2'-deoxyuridine staining and transwell assays showed that miR-132 significantly promoted the cell apoptosis and effectively inhibited the proliferation and invasion of hepatoma cells. These results indicated that miR-132 could inhibit the growth of hepatoma cells by targeting YAP gene and reducing its expression level. Taken together, results from this study would help us to understand the mechanisms for occurrence and development of hepatic carcinoma and provide new targets for diagnosis and treatment of liver cancer.

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7.

OBJECTIVES: To build GPC3 gene short hairpin interference RNA (shRNA) slow virus vector, observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth, and provide theoretical basis for gene therapy of liver cancer. METHODS: Hepatocellular carcinoma cell line Huh-7 was transfected by a RNA interference technique. GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR. Targeted GPC3 gene sequences of small interfering RNA (siRNA) PGC-shRNA-GPC3 were restructured. Stable expression cell lines of siRNA were screened and established with the help of liposomes (lipofectamine(TM2000)) as carrier transfection of human liver cell lines. In order to validate siRNA interference efficiency, GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot. The absorbance value of the cells of blank group, untransfection group and transfection group, the cell cycle and cell apoptosis were calculated, and effects of GPC3 gene on Huh-7 cell proliferation and apoptosis were observed. RESULTS: In the liver cancer cell lines Huh-7, GPC3 gene showed high expression. PGC-shRNA-GPC3 recombinant plasmid was constructed successfully via sequencing validation. Stable recombinant plasmid transfected into liver cancer cell lines Huh-7 can obviously inhibit GPC3 mRNA expression level. CONCLUSIONS: The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

MicroRNA-520c-3p inhibits hepatocellular carcinoma cell proliferation and invasion through induction of cell apoptosis by targeting glypican-3.

AIM: Glypican-3 (GPC3) is a membrane-associated heparan sulfate proteoglycan involved in regulation of cell proliferation, cell survival, cell migration and differentiation process. MicroRNAs (miRNAs) are single-stranded, non-coding functional RNAs that are important in many biological processes. GPC3 and miRNAs have been found to play essential roles in the development and progression of hepatocellular carcinoma (HCC). However, little information about the relationship between GPC3 and miRNAs is available nowadays. Therefore, this study aims to examine the relationship between GPC3 and miRNAs. METHODS: Dual-luciferase reporter assay was used to validate the direct target of GPC3. Fluorescence quantitative PCR and Western blotting were used to examined the gene expression at mRNA and protein levels. Cell apoptosis was evaluated by flow cytometric analysis and Annexin V-FITC staining. Invasion of cells was evaluated by Transwell matrigel assay. RESULTS: The results showed that miR-520c-3p could specifically target GPC3 in HCC cells. GPC3 protein levels decreased with unchanged transcription efficiency after miRNA transfection, and there was negative correlation of miR-520c-3p expression in HCC in relate to GPC3 protein levels. Moreover, miR-520c-3p not only induced HCC cell apoptosis, but also inhibited the growth and invasion of the cells. Interestingly, overexpression of GPC3 could effectively reverse apoptosis induced by miR-520c-3p transfection in HCC. CONCLUSIONS: Taken together, these results supported that miR-520c-3p may decrease GPC3 protein levels to inhibit proliferation of HCC cells. Therefore, GPC3 could be a new target for genetic diagnosis and treatment of HCC.

Knockdown of GPC3 inhibits the proliferation of Huh7 hepatocellular carcinoma cells through down-regulation of YAP.

Glypican-3 (GPC3), a membrane-associated heparan sulfate proteoglycan, is frequently upregulated in hepatocellular carcinoma (HCC). Yes-associated protein (YAP) is also found over-expressed in HCC and has been identified as a key effector molecule in Hippo pathway, which could control the organ size in animals through the regulation of cell proliferation and apoptosis and plays an important role in the development of malignant tumors. Studies have reported that GPC3 and YAP might collaborate to regulate the development of HCC. To elucidate the role of GPC3 in the development of HCC and its relationship with YAP, siRNA technique was employed to knock down GPC3 in Huh7 HCC cells. Moreover, recombinant human YAP-1 was used to examine the effects of GPC3 on Huh7 cells. The results of flow cytometric analysis and Annexin-V-FLUOS apoptosis assay showed that knockdown of GPC3-induced apoptosis in Huh7 cells, resulting in inhibition of cell proliferation as examined by EdU incorporation assay, migration, and invasion. GPC3 knockdown also suppressed the expression of YAP in mRNA and protein levels, as examined by fluorescence quantitative PCR and Western blot analysis. Moreover, addition of recombinant human YAP-1 effectively rescued the cells from apoptosis triggered by GPC3 knockdown. Taken together, our findings suggest that GPC3 regulates HCC cell proliferation with the involvement of Hippo pathway.