Objective To investigate the inhibitory effects and the mechanism of autophagy gene beclin 1 on cervical cancer HeLa cells. Methods The eukaryotic expression vector and short hairpin RNA (shRNA) expression vector of beclin 1 were transfected via lipofectamine into HeLa cells. Experimental cells were classified into 5 groups: pcDNA3. 1 ( + )-beclin 1 group, pSUPER-beclin 1 group, pcDNA3.1 ( + )group, pSUPER group and HeLa group. Real time-PCR and western blot were used for detecting expression of mRNA and protein of beclin 1 and caspase-9 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis, and autophagy of HeLa, while proliferation was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The ultrastructural analysis of autophagic vacuoles was under the electron microscope. Five groups cells were seeded subcutaneously on nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed, and immunohistochemistry was used to detect the protein expression of beclin 1 in tumor tissue. Results ( 1 ) The mRNA expression of beclin 1 and caspase-9: pcDNA3. 1 ( + )-beclin 1 group were 994.72 ±468.76 and 12. 88 ±2. 71, pSUPER-beclin 1 group were 0. 18 ± 0. 63 and 0. 11 ± 0. 08, pcDNA3. 1 ( + ) group were 0. 57 ± 0. 12 and 4. 28 ± 3. 25,pSUPER group were 0. 67 ± 0. 29 and 2. 77 ± 1.27, and HeLa group were 0. 74 ± 0. 25 and 3.67 ± 3.78,respectively. The eukaryotic expression vector pcDNA3. 1 ( + ) -beclin 1 significantly improved the expression of mRNA of beclin 1 and caspase-9 in HeLa cells( P ＜0. 05 ), and the shRNA expression vector inhibited the expression of mRNA of beclin 1 and caspase-9 ( P ＜ 0.05 ). (2) The cell proliferations: pcDNA3.1 ( + ) -beclin 1 vector significantly inhibited the growth of HeLa cells, while pSUPER-beclin 1 vector significantly improved the growth of HeLa cells(P ＜0. 05). (3) The rate of apoptosis: pcDNA3.1 ( + )-beclin 1 group was (28.2 ±2.3)%, pcDNA3. 1( + ) group was(14.6 ±4.6)%,pSUPER-beclin 1 group was(5.7 ±2. 0) %, pSUPER group was( 16. 2 ± 3.1 ) %, and HeLa group was( 11.2 ± 3. 0) %. The pcDNA3. 1 ( + ) -beclin 1 vector significantly increased the apoptosis rate, while the pSUPER-beclin 1 vector significantly decreased the apoptosis rate(P ＜0. 05 ). (4)The activity of autophagy: more autophagy cells were identified in pcDNA3.1( + )-beclin 1 group; the rate of autophagy of five group were( 10. 3 ± 1.5)% in pcDNA3. 1 ( + ) -beclin 1 group, ( 3.6 ± 0. 8 ) % in pcDNA3. 1 ( + ) group, ( 1.2 ± 0. 3 ) % in pSUPER-beclin 1 group, (3.2 ± 1.2)% in pSUPER group and (2.2 ± 1. 1)% in HeLa group, there was statistical significances between test groups and control groups( P ＜ . 05 ). (5)Carcinogenic activity of HeLa cells in nude mice: the duration of tumorigenesis was the longest in pcDNA3.1 ( + )-beclin 1 group and the shortest in pSUPER-beclin 1 group among all groups. The tumor size began to grow larger from 7th day after injection in pSUPER-beclin 1 group than in control groups( P ＜ 0. 05 ). The tumor size was smaller from 21st day after injection in pcDNA3.1( + )-beclin 1 group than in control groups(P ＜0. 05). From 28th day after injection,the tumor weigh was (0. 52 ± 0. 08 )g in pSUPER-beclin 1 group, apparently more than HeLa group (0. 37 ±0. 12) g and pSUPER group (0. 34 ± 0. 24 ) g ( P ＜ 0. 05 ). While in pcDNA3. 1 ( + )-beclin 1 group the tumor weighed (0. 18 ±0. 12) g, which was lower than HeLa group and pcDNA3. 1 ( + ) group (0. 34 ± 0. 18 ) g ( P ＜ 0. 05 ) . Conclusions Autophagy gene beclin 1 overexpression can inhibit proliferation and growth of HeLa cells in vitro and in vivo. Beclin 1 not noly participate in the regulation of autophagy signaling, but also play an important role in the regulation of endogenous apoptosis signaling through caspase-9. So it might be one of the new strategies for gene therapy of cervical carcinoma.

The shRNA expression vectors were constructed and transfected via lipofectamine into HeLa cells. Real time-ploymerase chain reaction (RT-PCR) and Western blot were used for detecting the expression of mRNA and protein of Beclin1 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis and cell cycle of HeLa, and proliferation was analyzed by MTT assay. The expression of caspase-9 in transfection cells was also detected by RT-PCR and Western blot. The constructed vectors significantly inhibited the expressin of mRNA and the protein of Beclin1 in HeLa cells. The growth of transfected cells was promoted, and less apoptosis cells were identified in these cells. After transfection of the constructed vectors into HeLa cells, the expression of caspase-9 was effectively inhibited. All of these indicate that autophagy and apoptosis are two types of programmed cell death, that autophagy gene Beclin 1 plays an important role in these two types, and that defect of autophagy and apoptosis may be important in tumor genesis.
Apoptosis ; genetics ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; genetics ; Beclin-1 ; Caspase 9 ; metabolism ; Down-Regulation ; Genetic Vectors ; HeLa Cells ; Humans ; Membrane Proteins ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection

The mitogen-activated protein kinase (MAPK) pathway controls intestinal epithelial barrier permeability by regulating tight junctions (TJs) and epithelial cells damage. Heme oxygenase-1 (HO-1) and carbon monoxide (CO) protect the intestinal epithelial barrier function, but the molecular mechanism is not yet clarified. MAPK activation and barrier permeability were studied using monolayers of Caco-2 cells treated with tissue necrosis factor α (TNF-α) transfected with FUGW-HO-1 or pLKO.1-sh-HO-1 plasmid. Intestinal mucosal barrier permeability and MAPK activation were also investigated using carbon tetrachloride (CCl 4) administration with CoPP (a HO-1 inducer), ZnPP (a HO-1 inhibitor), CO releasing molecule 2 (CORM-2), or inactived-CORM-2-treated wild-type mice and mice with HO-1 deficiency in intestinal epithelial cells. TNF-α increased epithelial TJ disruption and cleaved caspase-3 expression, induced ERK, p38, and JNK phosphorylation. In addition, HO-1 blocked TNF-α-induced increase in epithelial TJs disruption, cleaved caspase-3 expression, as well as ERK, p38, and JNK phosphorylation in an HO-1-dependent manner. CoPP and CORM-2 directly ameliorated intestinal mucosal injury, attenuated TJ disruption and cleaved caspase-3 expression, and inhibited epithelial ERK, p38, and JNK phosphorylation after chronic CCl 4 injection. Conversely, ZnPP completely reversed these effects. Furthermore, mice with intestinal epithelial HO-1 deficient exhibited a robust increase in mucosal TJs disruption, cleaved caspase-3 expression, and MAPKs activation as compared to the control group mice. These data demonstrated that HO-1-dependent MAPK signaling inhibition preserves the intestinal mucosal barrier integrity by abrogating TJ dysregulation and epithelial cell damage. The differential targeting of gut HO-1-MAPK axis leads to improved intestinal disease therapy.