RNA, Messenger ; biosynthesis ; genetics ; RNA, Viral ; chemistry ; genetics ; Ribonucleases ; biosynthesis ; genetics ; Virion ; chemistry ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

To detect the expression of telomerase subunits (human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA) in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect telomerase subunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25%, respectively. The former was significantly higher than the latter (chi2 = 26.4, P < 0.01). The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 91.7%, respectively and no significant difference was found between them (chi2 = 2.1, P > 0.05). The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100% and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.
Carrier Proteins/biosynthesis ; Carrier Proteins/genetics ; RNA/biosynthesis ; RNA/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms/*metabolism ; Telomerase/*biosynthesis ; Telomerase/genetics

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death in China. In recent years, therapies for oncogenedrivers and immune checkpoints have proved inspiring. Circular RNA (circRNA), which is a kind of RNA with covalent ring structure relating to stages and metastasis of cancer, has many special biological functions in physiological processes, diseases and so on. Thus, circRNA is expected to be a potential biomarker for cancer prediction and treatment in view of its high conservation and tissue-specific. However, function analysis and regulatory mechanism of circRNA in lung cancer come so far remains unclear and limited literatures are available. In this review, we highlight the research history, formation mechanism, biological function of circRNA and research progress in cancer, especially in lung cancer. We mean to provide theoretical evidences and new ideas for researches on circRNAs in lung cancer.
Animals ; Humans ; Lung Neoplasms ; genetics ; metabolism ; RNA ; biosynthesis ; genetics

Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4 degrees C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operates in the cell.
DNA Helicases ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; RNA, Transfer ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

OBJECTIVETo investigate inhibitory effect of short interfering RNA (siRNA) on the expression of lung resistance-related protein (LRP) in leukemia cells.

METHODSThe eukaryotic vectors of LRP, pcDNA3.0/LRP, were constructed. The transfection protocol of K562 cells grown in standard conditions consisted of different combinations of pcDNA3.0/LRP, pEGFP-C1 expressing mammalian enhanced green fluorescent protein (GFP), and their gene-specific siRNAs. RT-PCR and flow cytometry were employed to evaluate the mRNA and protein expression of LRP and fluoroscopy was performed for assay of GFP expression in the transfected cells.

RESULTSCompared with untreated K562 cells, pcDNA3.0/LRP-transfected cells showed increased LRP mRNA and protein expression and the positive cell percentage reached 30%. In the cells co-transfected with LRP gene-specific siRNA and pcDNA3.0/LRP, both LRP mRNA and protein expression decreased significantly to a level defined as negative results; the GFP expression showed no significant difference between the cells transfected with pEGFP-C1 and those co-transfected with LRP gene-specific siRNA and pEGFP-C1. LRP mRNA and protein expressions were also similar between the cells transfected with pcDNA3.0/LRP and those co-transfected with GFP gene-specific siRNA and pcDNA3.0/LRP.

CONCLUSIONSThe LRP gene-specific siRNA we designed is capable of degrading LRP mRNA and inhibiting the protein expression effectively and specifically, which shed light on the potential application of siRNA for gene-specific therapy to reverse LRP-induced multidrug resistance of leukemia cells.

Drug Resistance, Multiple ; genetics ; Genetic Therapy ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; K562 Cells ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection ; Vault Ribonucleoprotein Particles ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of RNA interference targeting human cytomegalovirus immediate early gene 1 (HCMV- IE1) on the gene expression in vitro.

METHODSAccording to the sequence of HCMV-IE1 gene, the small interfering RNA (siRNA) sequences were designed and introduced into the eukaryotic expression vector containing the U6 promoter. After verification by sequence analysis, the recombinant eukaryotic plasmid (pHCMV-IE1i) was transfected into HEL HCMVAD169 cells. The effectiveness of HCMV-IE1 gene silencing was investigated by fluorescence microscopy, flow cytometry and RT-PCR.

RESULTSSequence analysis confirmed successful construction of the recombinant eukaryotic expression plasmid pHCMV-IE1i. The expression of HCMV-IE1 was effectively suppressed by pHCMV-IE1i transfection in HEL cells as shown by fluorescence microscopy, flow cytometry (P < 0.05) and RT-PCR (P < 0.05).

CONCLUSIONThe expression of HCMV-IE1 can be effectively suppressed by RNA interference technique in vitro, which provides experimental data for prevention and treatment of HCMV infection.

Antigens, Viral ; biosynthesis ; genetics ; Cell Line ; Genes, Immediate-Early ; Genetic Vectors ; genetics ; Humans ; Immediate-Early Proteins ; biosynthesis ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

AIMTo construct the RNAi eukaryotic vector of inhibitory member of the prohibitin (PHB-1) gene and observe the interfering effect in HEK293 cell line after the vector transfection.

METHODSThe specific Mi RNA sequence was designed according to the PHB-1 sequence in GenBank, complementary single-strand DNA oligonucleotides were designed and synthesized, and annealed the single-stranded oligonucleotides to generate a double strands oligonucleotides , cloned the oligonucleotides into pcDNATM6.2-GW/EmGFP-MiR-PHB to obtain an entry clone and then sequence analysis was performed. The recombinant plasmid was transfected into HEK293 cell by liposome. PHB-1 expression was detected by Western blotting.

RESULTSThe DNA sequence of interest clone to the vector was constructed to generate an entry clone and an expression clone successfully, which were proved by sequence determination. Western blotting analysis demonstrated that PHB-1 MiR RNA expression construction could suppress the expression of PHB-1.

CONCLUSIONA RNAi eukaryotic vector containing prohibitin gene was successfully constructed.

Genetic Vectors ; genetics ; HEK293 Cells ; Humans ; MicroRNAs ; biosynthesis ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Repressor Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro.

METHODSA plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression.

RESULTSCotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines.

CONCLUSIONSThe study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.

Animals ; Fibrinogen ; biosynthesis ; genetics ; Gene Expression ; Mice ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

Total cDNA of human telomerase RNA(hTR) gene was cloned by means of RT-PCR and inverted into retroviral vector (pLNCX) to construct the mammalian cell expression plasmid. Then, by using lipofectin-mediated DNA transfection, the obtained expression plasmid was successfully transfected into human normal peripheral blood leukocyte. All data suggested that expression of transfected exogenous hTR gene can not reconstitute telomerase activity. Flow cytometry analysis and data from cell growth curve also indicated that expression of exogenous gene can not prolong the longevity of leukocyte, but rather inhibit the growth of leukocyte and induce its apoptosis. We conclude that expression of exogenous gene may block the coalition of telomerase RNA and its catalytic subunit(hTRT) and block the coalition of telomerase RNA template and telomere DNA, thus affecting telomerase activity and repressing cell proliferation.
Cells, Cultured ; Gene Expression ; Humans ; Leukocytes ; cytology ; enzymology ; metabolism ; RNA ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; Transfection

Carcinoma, Hepatocellular ; metabolism ; Glypicans ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; metabolism ; RNA, Messenger ; biosynthesis ; genetics