Hsa-miR-218 inhibits growth of cervical cancer HeLa cells by targeting to LASP1

AIM: To study the effect of hsa-miR-218 on cervical cancer HeLa cell growth and the underlying molecular mechanism.METHODS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed. pmiR-218 was transfected into HeLa cells. The number of viable HeLa cells was counted by the method of Trypan blue exclusion. The inhibitory rate of cell activity was detected by WST-8 assay. The expression of LIM and SH3 protein 1(LASP1) at mRNA and protein levels was determined by real-time PCR and Western blot. The interaction between miR-218 and LASP1 was examined using a luciferase reporter assay.RESULTS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed successfully and confirmed by DNA sequencing. Over-expression of miR-218 inhibited the activity of HeLa cells with the inhibitory rates of 15%, 26% and 65% at 24 h, 48 h and 72 h, respectively. The difference between transfection group and blank control/negative control group was statistically significant. The luciferase activity was reduced when co-transfection with miR-218 mimics and LASP1-3,UTR plasmid. The relative expression of miR-218 was increased after transfection with pmiR-218. Over-expression of miR-218 down-regulated the LASP1 expression at mRNA and protein levels by 25% and 75% respectively. Compared with blank control group and negative control group, the difference was statistically significant(P<0.05).CONCLUSION: pmiR-218 effectively inhibits the growth of HeLa cells in a time-dependent manner. miR-218 targets to the 3,UTR of LASP1, thus down-regulating the expression of LASP1 in HeLa cells.     

核桃外果皮不同萃取部位对人宫颈癌HeLa细胞抑制作用研究

[目的]研究核桃外果皮不同萃取部位对人宫颈癌细胞的抑制作用,筛选核桃外果皮抗肿瘤活性部位.[方法]采用MTT法,选用人宫颈癌HeLa细胞,对核桃外果皮不同萃取部位进行抗肿瘤活性筛选.[结果]石油醚萃取部位、氯仿萃取部位、乙酸乙酯萃取部位、正丁醇萃取部位对HeLa细胞的IC50分别为158.81、40.43、130.49和137.67 mg/L,水相部位对HeLa细胞无抑制作用.[结论]核桃外果皮石油醚萃取部位、氯仿萃取部位、乙酸乙酯萃取部位、正丁醇萃取部位对HeLa细胞均有抑制作用,其中氯仿萃取部位对HeLa细胞具有较强的抑制作用,初步认定氯仿萃取部位为核桃外果皮的抗肿瘤活性有效部位.     

SBHL对HeLa细胞c-myc，H—ras和hTRT基因表达的研究

目的观察澳洲茄胺盐酸盐对HeLa细胞c-myc，H—ras和hTRT基因表达的影响．方法用鼠抗人c-mycMcAb，H-ras McAb和hTRTPcAb作为一抗，生物素化羊抗鼠IgG作为二抗，用免疫组化方法测定澳洲茄胺盐酸盐对HeLa细胞c-myc，H—ms和hTRT基因的表达．结果澳洲茄胺盐酸盐处理的HeLa细胞与对照组相比，c-myc，H-ras和hTRT基因的表达均明显降低（P〈0．05）．结论澳洲茄胺盐酸盐下调HeLa细胞c-myc和H-ras基因的表达，抑制细胞增殖，诱导分化；澳洲茄胺盐酸盐下调HeLa细胞hTRT基因的表达，抑制端粒酶活性，抑制细胞生长．     

苏云金芽孢杆菌抗癌的研究进展

最近研究发现某些非杀虫的苏云金芽孢杆菌（Bacillus thuringiensis,Bt)株系的伴孢蛋白对体外培养的人癌细胞（MOLT－4，HeLa)具有毒杀能力。这类蛋白不属于已知的任何一种Cry/Cyt蛋白，其抗癌活性需经蛋白酶水解活化，所导致的细胞病变有明显的核凝聚和细胞肿胀过程。这个发现可能导致Bt在医疗上的新应用。文章综述了国外一些研究的新进展以期对国内的Bt研究提供新的思路。     

Antiradical Capacity and Induction of Apoptosis on HeLa Cells by a <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Extract

Jamapa bean is a black Phaseolus vulgaris variety rich in condensed tannins, anthocyanins and flavonols with interesting biological activities. The objective of this work was to evaluate the antiradical capacity (ARC) of a Jamapa bean methanolic extract (BME) and some of the proanthocyanidin-rich fractions derived from it, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The effect of the BME on some proteins involved in apoptosis on HeLa cells was also evaluated. A strong correlation between proanthocyanidin concentration in BME and antiradical capacity was found, suggesting that these compounds contribute significantly to antiradical activity. BME was a better radical scavenger than butylated hydroxytoluene (45.6 and 33.9% ARC at 400 μM, respectively). Two proanthocyanidin-rich fractions obtained after a preliminary separation of the BME using Toyopearl (TP4 and TP6) exhibited a higher antiradical activity than the parent extract. The treatment of HeLa cells with 35 μg BME/ml/24 h increased the expression of Bax and Caspase-3, pro-apoptotic proteins (6.13 and 1.2 times for Caspase-3 and Bax, respectively). The mechanism of action of some proteins involved in apoptosis was also evaluated, and the results suggest that black Jamapa bean could be an important source of polyphenolic compounds with potential biological use as antioxidant and anticancer agents.     

p15基因真核表达重组体及人宫颈癌细胞转染的研究

将含人p15cDNA质粒pBS-SK-p15以EcoRI和XhoI进行双酶切，再与真核表达载体pCR^TM3经相同酶切点连接，转化感受态细菌，筛选，鉴定得到可在真核细胞中表达人p15基因的重组质粒PCR-hp15。以脂质体介导法转染人宫颈癌细胞，用G418筛选，得到转染的人宫颈癌细胞，转染细胞的恶变程度有所改善。     

Expression and purification of re-constructed human CRP and observation of its internalization into HeLa cells

AIM: To construct prokaryotic expression vector for human C-reactive protein (CRP), to acquire the functional fusion protein purified from BL21(DE3) transformed with vector pET14b/EGFP-hCRP, and to observe the internalization of the fusion protein his-EGFP-CRP into tumor cell line HeLa. METHODS: CRP gene sequence was amplified with the vector p91023/CRP as template by PCR, and was inserted into vector pET14b/MCS-EGFP-(N)36 to construct prokaryotic expression vector. The E. coli cells BL21(DE3) transformed with the re-constructed vector pET14b/EGFP-hCRP was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the expressed protein his-EGFP-CRP were purified with affinity chromatography method and refolded with gradient filtration. The HeLa cells were observed under the fluorescence microscopy after the addition of purified renature protein. RESULTS: The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated the construction of vector pET14b/EGFP-hCRP was correct; the SDS-PAGE showed that the transformed E. coli cells could be induced to express the fusion protein his-EGFP-CRP and the purification of proteins were successful. We could found fluorescent signal around the cell membranes, in the cytoplasm and nuclei in the observation of the HeLa cells incubated with his-EGFP-CRP. CONCLUSION: The prokaryotic expression vector for human CRP linked with his and EGFP coding sequence is successfully constructed. The fusion protein his-EGFP-CRP is purified and refolded. The reconstructed protein expressed by prokaryotic cells adheres to the membrane of tumor cell HeLa and is internalized into the cytoplasm and nuclei of the cells.     

Expression and significance of miR-193b in cervical cancer

AIM: To investigate the expression of microRNA-193b(miR-193b) in the cervical tissues, and further to explore the effect of silencing miR-193b on diamminedichloroplatinum(DDP)-treated HeLa cell viability. METHODS: The expression levels of miR-193b in different cervical tissues were examined by qPCR. After transfection of miR-193b-inhibitor, the cell migration was determined by Transwell assay, the sensitivity of HeLa cells to DDP was measured by MTT assay, the protein levels of phosphate and tension homology deleted on chromsome ten(PTEN), protein kinase B(Akt), p-Akt and p-glycoprotein(P-gp) were determined by Western blot. RESULTS: The mRNA level of miR-193b was significantly increased in the cervical cancer tissues compared with normal cervical tissues(P<0.05). Knockdown of miR-193b obviously inhibited migration and enhanced sensitivity to DDP of HeLa cells(P<0.05). Additionally, after transfection of miR-193b-inhibitor, the expression of PTEN was increased, whereas the protein levels of p-Akt and P-gp were decreased(P<0.05). CONCLUSION: miR-193b is highly expressed in the cervical cancer tissues. Inhibition of miR-193b augments the sensitivity to DDP of HeLa cells, at least in part, through PTEN-PI3K/Akt signaling pathway.     

Role of autophagolysosomal inhibitor NH4Cl in apoptosis of human cervical cancer HeLa cells induced by vitamin K3

AIM：To investigate the role of autophagolysosomal inhibitor ammonium chloride (NH4Cl) in the apoptosis of human cervical cancer HeLa cells induced by vitamin K3(VK3). METHODS：The HeLa cells were divided into 4 groups：control group, 30 μmol/L VK3 group, 10 mmol/L NH4Cl group and 30 μmol/L VK3+10 mmol/L NH4Cl group. The viability of HeLa cells in each group was measured by MTT assay. The changes of lysosomal membrane in HeLa cells were detected by acridine orange (AO) staining. Autophagic vacuoles in the cells were observed by staining with monodansylcadaverine (MDC). Intracellular acidification in HeLa cells was detected by BCECF-AM flow cytometry analysis. The cells were stained with Hoechst 33342 to determine the chromatin condensation and apoptotic rate. RESULTS：Compared with control group,no effect of NH4Cl on the viability of HeLa cells was observed. In VK3 group, the cell viability decreased. AO staining appeared no change, and the autophagic vacuoles and the cytoplasmic acidification were observed. Nuclear chromatin condensation and cell apoptosis in the cells were also detected. Compared with VK3 group, combination of NH4Cl and VK3 enhanced the lysosomal permeability, the formation of autophagic vacuoles and the cytoplasmic acidification, and exacerbated the apoptosis. CONCLUSION：Autophagolysosomal inhibitor NH4Cl exacerbates apoptosis in HeLa cells, indicating that autophagy exerts protective effect on the process of injury in HeLa cells induced by VK3.