ABSTRACT
Objective To investigate the potential biological effect of long non-coding RNA( lncRNA) HOXA transcript at the distal tip( HOTTIP) on proliferation, migration and invasion of cervical cancer cells.Methods HOTTIP small interference RNA(siRNA) was transfected into HeLa and C-33A cervical cancer cell lines, with negative siRNA as a control.qPCR assay was performed to confirm the knock-down of the level of HOTTIP.CCK8 assay and colony-forming unit (CFU) assay were performed to evaluate the effect of HOTTIP knock-down on HeLa and C-33A cell proliferation.Wound healing assay was performed to evaluate the effect of HOTTIP knock-down on HeLa and C-33A cell proliferation and migration.Tumor invasion assay was used to evaluate the effect of HOTTIP knock-down on HeLa and C-33A cell invasion. Results The expression level of HOTTIP was efficiently knocked down by siRNA 48 h post transfection.The results of CCK8 assay and CFU assay showed that HOTTIP knock-down significantly decrease of cervical cancer cell proliferation. Wound healing assay result indicated that HOTTIP knock-down obviously suppressed cervical cancer cell proliferation and migration.Tumor invasion assay results demonstrated that HOTTIP knock-down significantly suppressed cervical cancer cell invasion.Conclusion HOTTIP levels in HeLa and C-33A cervical cancer cell lines can be efficiently knocked down with the siRNA strategy, and the HOTTIP knock-down can significantly suppress the tumor characteristics of cervical cancer cells, including the ability of proliferation, migration and invasion.
ABSTRACT
To generate a mWAP-hLF hybrid locus that the transcription of human lactoferrin (hLF) genomic sequence is directed by the up & down stream regulatory sequence of murine whey acidic protein (mWAP) gene locus, we describe here a successive three-step 'Gap-repair' method. First, a gap-repair vector based on pBR322 vector backbone by inserting six joint homologous arms was constructed. Then using 'Gap-repair 'method mediated by Red recombination system of lambda-prophage in Escherichia coli, in the first step, the 8 kb 3' flanking region of the mWAP gene was subcloned from the Bacterial artificial chromosome which harbors the mWAP gene locus(mWAP BAC) into the gap-repair vector; in the second step, the 29 kb hLF genomic sequence from the ATG code to the TAA code was subcloned from the hLF BAC; in the third step, the 12 kb 5' flanking region of the mWAP gene was subcloned from the mWAP BAC. Finally, all these three DNA fragments were automatically combined together without any gap in the gap-repair vector, and a 49 kb mWAP-hLF hybrid locus that the hLF genomic sequence was flanked by the 5' & 3' flanking region of mWAP gene locus was constructed. The result was confirmed by PCR, restriction enzyme digestion and sequencing. Our method provide a new way for the construction of large mammary-gland expression vector.
Subject(s)
Animals , Humans , Mice , Bioreactors , DNA Repair , Genetics , Genetic Engineering , Methods , Hybridization, Genetic , Lactoferrin , Genetics , Mammary Glands, Animal , Metabolism , Mice, Transgenic , Milk Proteins , GeneticsABSTRACT
Objectives To investigate and evaluate the effect of Nutrison-5Fu on immune function of perioperative patients with progressive gastrointestinal cancer,and simultaneously we also recommended the methods of application of Nutrison-5Fu.Methods Seventy eight cases were equally divided at random into three groups,26 cases in each group.⑴Nutrison-5Fu group:the patients were treated with Nutrison-5Fu.⑵Chemotherapy group:the patients only received 5-Fu.⑶Control group:there were not any chemotherapy and immunetherapy during the perioperation.The values of CD 3,CD 4,CD 8 and NKCC were determined in all cases.Results Values of CD 3,CD 4,CD/CD 8 ratio and NKCC in most patients were distinctly low,but the condition rapid improved by giving them Nutrison-5Fu.On the contrary,the values declined further after chemotherapy alone.Conclusion The treatment of Nutrison-5Fu not only can repress the development of the tumor,but also can improve the dystrophic status and immune function of the patients with cancer.