Percutaneous Robot-Assisted versus Freehand S2 Iliosacral Screw Fixation in Unstable Posterior Pelvic Ring Fracture.

OBJECTIVES: To assess the efficiency, safety, and accuracy of S2 (IS) screw fixation using a robot-assisted method compared with a freehand method. METHODS: This is a retrospective clinical study. We analyzed the patients treated with S2 IS screw fixation for unstable pelvic fractures from January 2016 to January 2019 in our institution. Sixty-three patients (17 men and 46 women) aged between 21 and 55 years (with an average age of 39.22 ± 9.28) were included in this study. According to the Tile classification, there were 26 (41.3%) type B fractures and 37 (58.7%) type C fractures. All patients were divided into robot-assisted (RA) group (38 patients) or the traditional freehand (FH) group (25 patients). In RA group, the S2 IS screws were implanted with a robot-assisted technique. And S2 IS screws were implanted with a traditional freehand technique in FH group. The screw-related complications were recorded during and after the surgery. The position of all screws and fracture reduction was assessed by postoperative CT scans according to the Gras classification. The number of guide wire attempts and the radiation exposure for S2 screw implantation during operation were also recorded. Finally, the Matta standard was used to evaluate the fracture reduction of the IS joint. RESULTS: A total of 89 IS screws were implanted into S2 iliosacral joint. Fifty-four screws were placed by RA (38 patients) and 35 screws were by FH (25 patients). There was no difference between the two groups with respect to demographic data. There was no screw-related complications or revision surgery in any group. In terms of screw placement, the excellent and good rate was 100% in the RA group, better than that in the FH group where it was only 85.7% (P < 0.001). The fluoroscopy time was 8.06 ± 3.54 s in RA group, which was much less than that in the FH group (27.37 ± 8.82 s, P < 0.001). The guide wire attempts in the RA group (0.685 ± 0.820) were much less than those in the FH group (5.77 ± 3.34) (P < 0.001). Both the fluoroscopy time per screw and the number of guide wire attempts in the RA group were much less than those in the FH group (P < 0.001). The overall postoperative excellent and good rate of Matta standard in RA and FH groups were 86.8% (34/4) and 90.0% (23/25), respectively (P = 0.750), and there was no statistical difference. CONCLUSION: The robot-assisted surgery is an accurate and minimally invasive technique. S2 IS screw implantation assisted by TiRobot to treat the posterior pelvic ring fractures, have a high success rate than the freehand technique. Percutaneous RA S2 IS screw fixation for unstable posterior pelvic ring injuries is safe and clinically feasible and has great clinical application value.

The CAG promoter maintains high-level transgene expression in HEK293 cells.

The vast majority of therapeutic recombinant proteins are produced in mammalian cell lines. However, proteins generated in nonhuman cell lines, such as Chinese hamster ovary (CHO) cells, are decorated with human-like glycan structures that differ from those of human cells, and these may induce immunogenic responses in human cells. Human embryonic kidney cells (HEK293F) are also extensively used as hosts for the expression of recombinant therapeutic proteins, but their utility is limited by the low expression of transgenes in these cells. Here, we investigated recombinant protein expression from eight frequently used promoters in transfected HEK293F cells. The expression levels and stability of the transgenes were evaluated by flow cytometry and qRT-PCR. The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate-early enhancer combined with the chicken beta-actin promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In addition, application of mild hypothermia (i.e., 33 °C) after transfection improved the positive effect of the CMV enhancer fused to the chicken beta-actin promoter (CAG promoter) on enhanced green fluorescent protein (eGFP) expression. Although the temperature sensitivity of the CMV promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. When eGFP was replaced with hepatitis B surface antigen, the CAG promoter still showed the highest transgene expression. In conclusion, our data show that the CAG promoter is a strong promoter for recombinant protein expression in HEK293F cells.

Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells.

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.

Guidance for Treatment of Pelvic Acetabular Injuries with Precise Minimally Invasive Internal Fixation Based on the Orthopaedic Surgery Robot Positioning System.

Pelvic acetabular fracture is a common kind of fracture, mostly caused by high energy injuries. It is associated with high mortality and disability rates. The aim of surgical treatment of pelvic acetabular fractures is to restore the symmetry and stability of the pelvic ring structure and the anatomical structure of acetabular. Open reduction internal fixation is often used for the treatment of such fractures, but open surgery is in cases of serious injury, more bleeding, and high risk of infection. With the development of minimally invasive technology and the concept of the bone channel structure, the percutaneous lag screw technique for the treatment of pelvic and acetabular fractures has been applied in clinical practice and has proven to be effective. However, the anatomical structure of the pelvis and acetabulum is complex, and there are many important nerves and vessels adjacent to it. Traditional fluoroscopy screw placement is prone to screw malposition, and even minor angle changes may lead to screw perforation and damage of nerve vessels. The problem of radiation exposure is also noteworthy. Robotic-assisted surgery can be used to carry out screw position planning through preoperative imaging, intraoperative real-time tracking, and mechanical arm assistance to ensure that the screw placement position is consistent with the planning. In this way, robotic-assisted surgery can be used to accurately insert lag screws, and can reduce surgical risk and radiation exposure. This guide uses the TiRobot system as an example to describe the application of robot surgery in detail, aiming at standardizing the application of robots in orthopaedic surgery.

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells.

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2'-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.

Evaluation of a Bi-Planar Robot Navigation System for Insertion of Cannulated Screws in Femoral Neck Fractures.

OBJECTIVE: To evaluate the bi-planar robot navigation system for insertion of cannulated screws in femoral neck fractures. METHOD: Between January 2016 and December 2016, 60 patients with femoral neck fractures were separately treated using percutaneous cannulated screws assisted by the bi-planar robot navigation system (robot group) and conventional freehand surgery (freehand group). The fluoroscopy time, the number of drilling attempts, and the operation time were recorded during operations; the dispersion and parallelism of the cannulated screws on the posteroanterior and lateral images were measured after operations. Patients were followed up for 12-24 months and the Harris scores and the final results of the two groups were compared. RESULTS: During bi-planar robot navigation system-assisted surgery, the fluoroscopy time for acquisition of images was 2.3 seconds on average, and the time for planning screws during the operation was 2.8 min on average. The average fluoroscopy time during the placement of the guide pin was 5.7 seconds and 14.14 seconds (P = 0.00), respectively. The average time of the placement of the cannulated screws was 12.7 min and 19.4 min (P = 0.00), respectively, in the robot group and the freehand group. In the robot group, only one guide pin was replaced during the operation, and the average number of adjustments for each guide pin was 2.39 in the freehand group. The screw parallelism and dispersion measured by postoperative imaging in the robot group were significantly superior to those in the freehand group. From postoperative CT it was evident that there were 5 cases of screws exiting the posterior cortex in both groups. During the follow-up phase, 1 case of femoral head necrosis and 5 cases of femoral neck shortening of more than 10 mm occurred in the robotic navigation group; 3 cases of femoral head necrosis, 1 case of fracture nonunion, and 2 cases of shortening of more than 10 mm occurred in the freehand group. At 18 months after surgery, the average Harris scores of the patients were 85.20 and 83.45, respectively, with no significant difference. CONCLUSION: Using bi-planar robot navigation system-assisted placement of femoral neck cannulated screws can significantly reduce the time of intraoperative fluoroscopy, drilling attempts, and operation time. The placed screws are superior to the screws placed freehand in relation to parallelism and dispersion. However, it is still necessary for surgeons to have a good reduction of the femoral neck fracture before surgery and to be proficient in the operation of the robot navigation system. In summary, the bi-planar robot navigation system is an effective assistant instrument for surgery.

Two human MARs effectively increase transgene expression in transfected CHO cells.

Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR-3 and MAR-7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real-time PCR. The results showed that the expression level of eGFP of cells transfected with MAR-containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR-7 was higher than that of MAR-3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR-3 and MAR-7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.

Retraction Note: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.

This paper has been retracted at the request of the authors.

[Effects of Different Promoters and MAR Combinations on Transgene Expression of Recombinant CHO Cells].

OBJECTIVE: To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells. METHODS: The expression vector was constructed by the combination of beta globin MAR (gMAR) with the human cytomegalovirus immediate-early promoter (CMV-IE) and simian virus 40 (SV40) promoter. These vectors were transfected into CHO cells,after 48 h,the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines,and the expression level of eGFP in CHO cells was analyzed by flow cytometry. The relative copy numbers of eGFP were analyzed by qPCR. RESULTS: Without gMAR expression vector,the expression of eGFP which was driven by CMV-IE promoter was stronger than that of SV40 promoter; gMAR could increase the expression level of eGFP driven by CMV-IE promoter,but did not show any enhancement in SV40 promoter. The expression level of eGFP which containing gMAR on both sides was stronger than that of gMAR on one side driven by CMV-IE promoter; After G418 screening,the expression level of eGFP containing gMAR driven by SV40 promoter wasunstable,the fluorescence gradually weakened,therefore,we only analyzed the expression vector stably expressing the eGFP gene driven by CMV-IE promoter by flow cytometry and qPCR. Compared with the expression vector without gMAR containing CMV-IE promoter,flow cytometry showed that the expression levels of eGFP on one and both sides with gMAR were increased by 9.85-fold and 12.94-fold,respectivley; The result of qPCR showed that the copy number of the eGFP gene without gMAR was set to 1,the copy number of the eGFP gene in the expression vector driven by CMV-IE with gMAR on one side and both sides were 3.68-fold and 9.25-fold,respectively. CONCLUSION: The activity of CMV-IE promoter is stronger than that of SV40 promoter. gMAR can enhance the expression levels of transgene,which may be related to the increase of gene copy number.

Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells.

Low-level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR-6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR-6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP-B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP-B, DHFR intron MAR element and MAR-6. Additionally, as expected, the three MAR-containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non-MAR-containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP-B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.

Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.

Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human ß-interferon and ß-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.

Cell Compatibility of an Eposimal Vector Mediated by the Characteristic Motifs of Matrix Attachment Regions.

The characteristic sequence of ß-interferon matrix attachment regions (MARs) can mediate transgene expression via episomal vectors in Chinese hamster ovary (CHO) cells. However, the host cells were from hamster ovaries, which are not suitable target cells for gene therapy. In this study, we aimed to evaluate the suitability of 12 different human cell lines as target cells for gene therapy. We transfected the cells with episomal vectors and obtained colonies stably expressing the vector products after G418 screening. Therefore the stably transfected cells were split into two and further cultured either in the presence or the absence of G418. Flow cytometry was used to observe the positive rate of cell transfection and level of green fluorescent protein (GFP) expression. Plasmid rescue assays, fluorescence in situ hybridization (FISH), and fluorescence quantitative polymerase chain reaction (PCR) were used to investigate the presence and gene copy numbers of plasmid in mammalian cells. The results showed that transfection efficiency and transgene expression levels in A375, Eca-109, and Changliver cells were high. In contrast, transgene silencing was observed in BJ, HSF, and A431 cells, and low expression of the transgene was observed in the other six cell lines. In addition, the plasmid was present in the episomal state in A375, Eca-109, and Chang-liver cells with relatively low copy numbers even under nonselective conditions. Thus, our results provide the first evidence showing transgene expression of an episomal vector mediated by the characteristic motifs of MARs for maintenance of the longterm stability of episomes in different types of cells.

Overexpression of suppressor of IKBKE 1 is associated with vincristine resistance in colon cancer cells.

In a previous study, the suppressor of IKBKE 1 expression level was confirmed to be higher in vincristine (VCR)-resistant HCT-8 (HCT-8/V) colon cancer cells than in non-VCR-resistant HCT-8 cells. In the current study, IKBKE 1 expression in VCR-resistant colon cancer cells was investigated further. HCT-8 and HCT-8/V human colon cancer cells were used, and polymerase chain reaction (PCR) primers were designed to amplify the IKBKE 1 gene. Fluorescence reverse transcription-quantitative PCR (RT-qPCR) was performed to detect differences in IKBKE 1 expression between sensitive and drug-resistant colon cancer cell lines. Western blotting was performed to further observe IKBKE 1 expression. Based on the RT-qPCR and western blot results, IKBKE 1 expression was observed to be markedly higher in the HCT-8/V cells, and this difference was significant (P<0.05). Thus, IKBKE 1 expression was identified to be associated with the resistance of colon cancer cells to VCR.

[Effect of Intron Orientation on the Expression of Transgene Imposed by MAR Expression Vector in Stably Recombinant CHO Cells].

OBJECTIVE: To determine the effect of intron orientation on the transgene expression level imposed by matrix attachment region (MAR) expression vector. METHODS: The MAR of ß-globin was amplified by PCR, and then cloned into MAR expression vectors. An intron sequence was digested with restriction enzyme, ligated to the MAR expression vector in reverse orientation, and then transfected into Chinese hamster ovary (CHO) cells. The transfected stable cells were screened by G418. The level of chloramphenicol acetyltransferase (CAT) gene expression was analyzed by ELISA method. RESULTS: The transgene expression levels of CHO cells with the two expression vectors with a positive intron or without MAR were higher than that of CHO cells with an expression vector with reverse intron (P < 0.05). MAR did not improve transgene expression with reverse intron presence. CONCLUSION: Different orientation of intron can affect transgene expression in recombinant CHO cells. The transgene expression level can be increased using positive intron and MAR.

Impact of Different Promoters on Episomal Vectors Harbouring Characteristic Motifs of Matrix Attachment Regions.

We previously demonstrated that the characteristic sequence of matrix attachment regions (MARs) allows transgenes to be maintained episomally in CHO cells. In the present study, six commonly used promoters from human cytomegalovirus major immediate-early (CMV), simian vacuolating virus 40 (SV40), Rous sarcoma virus, Homo sapiens ubiquitin C, phosphoglycerate kinase, and ß-globin, respectively, were evaluated to determine their effects on transgene expression and stability in CHO cells stably transfected via the episomal vector harbouring characteristic MAR motifs. The CHO cells were transfected with vectors and then screened using G418, after which the stably transfected cells were split into two and further cultured either in the presence or absence of G418. Of the six promoters, the CMV promoter yielded the highest transgene expression levels and the highest transfection efficiency, whereas the SV40 promoter maintained transgene expression more stably during long-term culture than the other promoters did. The CMV and SV40 promoter-containing vectors were furthermore episomally maintained and conferred sustained eGFP expression in the cells even under nonselective conditions. On the basis of these findings, we conclude that the CMV promoter performs best in terms of yielding both high expression levels and high levels of stability using this episomal vector system.

Molecular characterization of a human matrix attachment region that improves transgene expression in CHO cells.

Chinese hamster ovary (CHO) cells offer many advantages for recombinant gene expression, including proper folding and post-translational modification of the recombinant protein. However, due to positional effects resulting from the neighboring chromatin, transgenes are often expressed at low levels in these cells. While previous studies demonstrated that matrix attachment regions (MARs) can be utilized to increase transgene expression by buffering transgene silencing, the mechanism by which this occurs is poorly understood. We therefore performed a deletion analysis of the human ß-globin MAR sequence to characterize the regions that are necessary to enhance transgene expression in CHO cells. Our results indicate that of the six ß-globin MAR fragments tested (MAR-1-6; nucleotides 1-540, 420-1020, 900-1500, 1380-1980, 1860-2460, and 2340-2999, respectively), MAR-2, followed by MAR-3, was the most effective region for promoting stable and elevated transgene expression. Meanwhile, bioinformatic analyses demonstrated that these fragments encode a MAR-like motif and several transcription factor binding sites, including special AT-rich binding protein 1 (SATB1), CCAAT-enhancer-binding proteins (C/EBP), CCCTC-binding factor (CTCF), and Glutathione (GSH) binding motifs, indicating that these elements may contribute to the MAR-mediated enhancement of transgene expression. In addition, we found that truncated MAR derivatives yield more stable transgene expression levels than transgenes lacking the MAR. We concluded that the MAR-mediated transcriptional activation of transgenes requires a specific AT-rich sequence, as well as specific transcription factor-binding motifs.

Treating Colon Cancer Cells with FK228 Reveals a Link between Histone Lysine Acetylation and Extensive Changes in the Cellular Proteome.

The therapeutic value of FK228 as a cancer treatment option is well known, and various types of cancer have been shown to respond to this drug. However, the complete mechanism of FK228 and the affect it has on histone lysine acetylation and the colon cancer cell proteome are largely unknown. In the present study, we used stable isotope labeling by amino acids in cell culture (SILAC) and affinity enrichment followed by high-resolution liquid chromatograph-mass spectrometer (LC-MS)/MS analysis to quantitate the changes in the lysine acetylome in HCT-8 cells after FK228 treatment. A total of 1,194 lysine acetylation sites in 751 proteins were quantified, with 115 of the sites in 85 proteins being significantly upregulated and 38 of the sites in 32 proteins being significantly downregulated in response to FK228 treatment. Interestingly, 47 histone lysine acetylation sites were identified in the core histone proteins. We also found a novel lysine acetylation site on H2BK121. These significantly altered proteins are involved in multiple biological functions as well as a myriad of metabolic and enzyme-regulated pathways. Taken together, the link between FK228 function and the downstream changes in the HCT-8 cell proteome observed in response to FK228 treatment is established.

Expression profile analysis of long non-coding RNA associated with vincristine resistance in colon cancer cells by next-generation sequencing.

Vincristine (VCR) is widely used in tumor treatment. However, long-term use of this drug can make tumor cells resistant to it. Furthermore, the mechanisms underlying resistance development are unclear. The aim of this study was to investigate the long non-coding RNAs (lncRNAs) associated with colon cancer drug resistance using next-generation sequencing. A cDNA library of HCT-8 VCR-resistant colon cancer cell was established through PCR amplification. Using HiSeq 2500 sequencing and bioinformatic methods, we identified lncRNAs showing different expression levels in drug-resistant and non-resistant cells, and constructed expression profiles of the lncRNA differences. The pretreatment of data was quality controlled using FastQC software. Transcription of lncRNA was calculated using Fragments Per Kilobase of transcript per Million fragments mapped (FPKM). To reveal the potential functions of these lncRNAs, we applied GO analysis to study the differentially expressed lncRNAs. Total transcript number was higher in resistant cells than in non-resistant colon cancer cells, and high-quality transcripts constituted the major portion of the total. In addition, 121 transcripts showed significantly different expression in VCR-resistant and non-resistant cells. Of these, we observed 23 up-regulated and 20 down-regulated lncRNAs (fold change >10.0). This is the first report of the expression profile of lncRNA of VCR-resistant colon cancer cells. Abnormal lncRNA expression was associated with VCR resistance in colon cancer cells and these expression differences may play a key role in VCR resistance of these cells.

The effect of recombinant lentiviral vector encoding miR-145 on human esophageal cancer cells.

miR-145, a newly identified microRNA molecule, is hypothesized to function as a tumor suppressor, but this activity has not been investigated in esophageal l carcinoma (EC). The aim of this study was to investigate the effect of miR-145 on the biological features of EC cells. miR-145 was obtained using PCR technology and cloned into the lentiviral vector, pLVX-IRES-ZsGreen1, to construct the resulting vector, pLVX-IZ-miR-145. The vector was packaged, the viral titer was tested, and ECA109 cells were infected with the optimal viral titer. Cells that were stably transfected with miR-145 were screened. Flow cytometry was used to analyze enhanced green fluorescence protein gene expression, and to measure cell apoptosis and cell cycle. miR-145 expression was detected by real-time fluorescent quantitative PCR. Furthermore, cell proliferation was assayed using CCK-8 assay. The pLVX-IZ-miR-145 vector was successfully constructed, and the viral titer achieved up to 5.0 × 10(8) TU/mL. The transfection efficiency was 90 %. Compared to the control group, the expression level of miR-145 in the transfected group was significantly higher (185-fold, P < 0.05). miR-145 overexpression significantly inhibited esophageal cancer cell proliferation (P < 0.05). Moreover, the number of cells at the G2/M stage, as well as the cell apoptotic rate, in the miR-145-transfected group was significantly increased (P < 0.05). Our study reveals that overexpression of miR-145 inhibits cell proliferation, increases apoptosis, and influences the cell cycle progression of EC cell.