Molecular diagnostic approaches in detecting rearranged during transfection oncogene mutations in multiple endocrine neoplasia type 2

Different types of neuroendocrine cancer,including medullary thyroid cancer(MTC)and thyroid C-cell hyperplasia,are part of multiple endocrine neoplasia type 2(MEN2).A proto-oncogene mutation of the rearranged during transfection(RET)gene changes the way that receptor tyrosine kinases work.Multiple endocrine neoplasia,a pathological condition,involves these kinases.When the RET protooncogene changes,it can cause endocrine adenomas and hyperplasia to happen at the same time or one after the other.Pheochromocytoma,medullary thyroid carcinoma,and hyperparathyroidism,alone or in combination,are present in MEN2A patients.Some patients may also have skin lichen amyloidosis or Hirschsprung's disease.Patients with MEN2A often present with MTC.MTC is aggressive and has the worst prognosis,as most patients exhibit lymph node metastasis.MTC is one of the important causes of death in patients with MEN2A.RET mutation analysis aids in identifying MEN2A symptoms and monitoring levels of calcium,thyroid hormones,calcitonin,normetanephrine,fractionated metanephrines,and parathyroid hormone.The earlier diagnosis of MTC significantly improves survival and prompts better management of MEN2A.In this editorial,we will discuss the significance of molecular diagnostic approaches in detecting RET oncogene mutations in MEN2A.

γ-Ray-Radiation-Scissioned Chitosan as a Gene Carrier and Its Improved in vitro Gene Transfection Performance

Chitosan （CS） is expected to be an ideal gene carrier for its high biosafety. In this work, CS with low molecular weight were prepared through the γ-ray radiation on the acetic acid solution of CS. The CS chains were scissioned under the γ-ray radiation, and the molecu- lar weight （MW） of CS decreased with the absorbed dose. When the absorbed dose was above 30 kGy, the molecular weight of CS decreased about an order of magnitude. The γ-ray-radiation-scissioned CS can effectively bind with plasmid （pEGFP） through complex coacervation method, forming pEGFP/γ-ray-radiation-scissioned CS complex particles with a size of 200-300 nm. The complex particles have good stability and little cytotoxicity. The in vitro gene transfection efficiencies of the pEGFP/γ-ray-radiation-scissioned CS complex particles were investigated by fluorescence microscope and flow cytometry. The results showed that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS and the better transfection efficiency of the corresponding gene vector. This work provides a green and simple method on the preparation of CS-based gene vectors with high efficiency and biosafety.

Hydrodynamics based transfection in normal and fibrotic rats

AIM： Hydrodynamics based transfection （HBT）, the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated. METHODS： A lucJferase encoding plasmid was injected using the hydrodynamics based procedure into normal and thioacetamide-induced fibrotic Sprague Dawley rats. Scanning and transmission electron microscopy images were taken. The consequence of a dual injection of Ringer solution and luciferase pDNA was followed. Halofuginone, an anti collagen type I inhibitor was used to reduce ECM load in fibrotic rats prior to the hydrodynamic injection. RESULTS： Large endothelial gaps formed as soon as 10＇ following hydrodynamic injection; these gradually returned to normal 10 d post injection. Hydrodynamic administration of Ringer 10 or 30 m prior to moderate injection of plasmid did not result in efficient transfection suggesting that endothelial gaps by themselves are not sufficient for gene expression. Gene transfection following hydrodynamic injection in thioacetamide induced fibrotic rats was diminished coinciding with the level of fibrosis. Halofuginone, a specific collagen type I inhibitor, alleviated this effect. CONCLUSION： The hydrodynamic pressure formed following HBT results in the formation of large endothelial gaps. These gaps, though important in the transfer of DNA molecules from the blood to the space of Disse are not enough to provide the appropriate conditions for hepatocyte transfection. Hydrodynamics based injection is applicable in fibrotic rats provided that ECM load is reduced.

Obstructive Effects of Ultrasonic Microbubble Intensifier on CHG-5 Cell with Survivin Antisense Oligonucleotides Transfection

Objective： To study the effects on human glioma cell line CHG-5 by ultrasonic microbubble intensifier with survivin antisense oligonucleotides （ASODN） transfection. Methods： Antisense oligonucleotides targeting survivin mRNA was designed and synthesized. Four regimen groups were designed, group A： survivin antisense oligonucleotides transfected with ultrasonic microbubble intensifier combined with ultrasound irradiation, group B： survivin antisense oligonucleotides transfected with lipofectamine combined with ultrasound irradiation, group C： survivin antisense oligonucelotides with lipofectamine transfection, group D： blank control. The expression changes of surviving protein were measured by immunohistochemical staining and Western blotting, and MTT assay was used to measure the changes of proliferation. Results： Survivin protein expression in group A was decreased significantly in human glioma cell line CHG-5 than other groups（P〈0.05）, and the proliferating rate of CHG-5 in group A was also significantly inhibited（P〈0.05）. Conclusion： Ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is a promising method in gene transfection effectively and noninvasively.

Epigenetic Regulation of the ERβ Gene on the Estrogen Signal Transfection Pathway in Colon Cancer Cells

We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant expression plasmid,pEGFP-C1-ERβ,was constructed and transfected into the Caco-2 colon cancer cell line,a line with low ERβ gene expression.The expression of ERβ mRNA and protein was detected 72h after transfection.RT-PCR was used to examine the expression levels of the progesterone recepror(PR)gene ...

Brain-derived neurotrophic factor gene transfection promotes neuronal repair and neurite regeneration after diffuse axonal injury

This study sought to assess the potential of brain-derived neurotrophic factor （BDNF） to promote neuronal repair and regeneration in rats with diffuse axonal injury, and to examine the accompanying neurobiological changes. BDNF gene transfection reduced the severity of the pathological changes associated with diffuse axonal injury in cortical neurons of the frontal lobe and increased neurofilament protein expression. These findings demonstrate that BDNF can effectively promote neuronal repair and neurite regeneration after diffuse axonal injury.

Protection of rat islet viability following heme oxygenase-1 gene transfection via adenoviral vector in vitro

Objective： To investigate the effect of Heme oxygenase-1 （HO-1） gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods：Recombinant adenovirus vector containing human HO-1 gene（Ad-HO-1 ） or enhanced green fluorescent protein gene（Ad-EGFP） was generated by using AdEasy system respectively. The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index（SI） was calculated. Glucose-stimulated insulin release was used ＇to assess islet viability. Results：Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index （SI） of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets （P 〈 0.05）. Conclusion： Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.

BASIC FIBROBLAST GROWTH FACTOR GENE TRANSFECTION TO ENHANCE THE REPAIR OF AVASCULAR NECROSIS OFTHE FEMORAL HEAD

Objective To explore a new method for the therapy of avascular necrosis of the femoral head. Method The recombinant plasmid pCD-rbFGF was mixed with collagen and was implanted in the necrotic femoral head. Expression of basic fibroblast growth factor (bFGF) was examined by RT-PCR and immunohistochemical method. Re-pair of the femoral head was observed by histological and histomorphometric analysis. Result Expression of bFGF was detected in the femoral head transfected with bFGF gene, indicating significant increase of angiogenesis 2 weeks after gene transfection and increased new bone formation 8 weeks after gene transfection on histom-orphometric analysis (P< 0.01). Conclusion Transfection of bFGF gene enhances bone tissue angiogenesis. Repair in osteonecrosis would be accelerated accordingly.

Optimization on cationic liposome-mediated cell transfection of plasmid DNA

Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by plasmid DNA.Methods:We studied the optimal condition for higher efficiency of cationic lipid-mediated cell transfection.Four experimental groups were set.Plasmid DNA and liposome were mixed in each groups at different ratios(μg:μL),1:2.5,1:3.5,1:4.0 and 1:5.0,respectively.LacZ gene functioned as reporter gene,measuring the transfection efficiency of the four groups using the method of X-gal staining.Results:When the ratio was 1:3.5,the cell transfection rate was the highest.While the ratio of 1:2.5 recommended by product manual achieve the lowest transfection rate.Their difference had statistical significance.Conclusion:In order to obtain a higher transfection efficiency,optimization on conditions of the ratio of plasmid DNA to liposome is necessary in cell transfection.

Ultrasound-targeted cationic microbubble-mediated gene transfection and inhibition of retinal neovascularization

AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit retinal neovascularization in mice.METHODS:CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy.Experiments assessing HRECs migration and vascular formation were per formed to evaluate gene therapy’s efficiency in vitro.A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl,Bcl-2,vascular endothelial growth factor(VEGF)and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction(q PCR).The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5,14,and 28 d after treatment.RESULTS:The gene expression of endostatin was the highest in the group of the CMBs.Besides,the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro.In vivo,retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups.CONCLUSION:The research illustrates ultrasoundtargeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.

EFFECTS OF CARBOXYMETHLY DEXTRAN MAGNETIC NANOPARTICLES CARRIER SYSTEM ASSOCIATED WITH EXTERNAL MAGNETIC FIELDS ON KILLING TUMOR CELLS AND GENE TRANSFECTION

Objective: To investigate the preparation of the carboxymethly dextran iron oxide magnetic nanoparticles (CDMN) and the effects of CDMN carrier system associated with external magnetic fields on killing tumor cells and gene transfection in vitro. Methods: Epirubicin-CDMN (EPI-CDMN) and green fluorescent protein (GFP) plasmid-CDMN (GFP-CDMN) were prepared by the oxidation-reduction procedure and their characters were detected, respectively. The effects of EPI-CDMN associated with external pulsed electromagnetic fields (PEMFs) (10 mT) on killing human bladder cancer BIU-87 cells were studied by MTT assay and Annexin-V/PI double-labeled flow cytometry technique, respectively. And the transfection efficiency of GFP when CDMN were used as gene carrier associated with the external magnetic fields was evaluated under fluorescence microscope in vitro. Results: The diameter of EPI-CDMN and GFP-CDMN were about 8~10 nm and 5~9 nm, respectively, and saturation magnetization were 0.22 emu/g and 0.26 emu/g, respectively. EPI-CDMN associated with PEMFs could significantly inhibit the proliferation of BIU-87 cells and induce cells apoptosis, the growth inhibitory rate and apoptosis rate were (21.82±3.18)% and (16.79±3.37)%, respectively. The transfection efficiency of GFP-CDMN combined with PEMFs was significant higher than that of GFP-CDMN without PEMFs [(45.70±4.32)% vs (35.85±2.16)%, P<0.05]. Conclusion: It seemed that EPI-CDMN associated with external magnetic fields could significantly killed human bladder cancer BIU-87 cells and CDMN could effectively transfer GFP gene into tumors cells with the help of external magnetic fields which provided experimental basis for the magnetic targeting therapy of tumor.

Gene Transfection Mediated by Ultrasound and Pluronic P85 in HepG2 Cells

In order to assess whether gene transfection could be mediated by ultrasound in association with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irra- diated by ultrasound at 1 MHz, 0.4-2.0 W/cm2 and 50% duty cycle with plasmid encoding enhanced green fluorescent protein （EGFP） as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal transfection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm2 for 30 s. The transfection efficacy in ulstrasound＋P85 group was three times higher than in single ultrasound group [（17.63±1.07）% vs （5.57±0.56）%, P〈0.05]. The cell viability was about 81% and 62% in ultrasound group and ultrasound＋P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm2 for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound.

Combined Pluronic P85- and Ultrasound Contrast Agents-mediated Gene Transfection to HepG2 Cells

This study examined the effect of P85 （a pluronic block copolymer） and microbubble （MB） ultrasound contrast agents under ultrasound irradiation on gene transfection and expression. The pEGFP plasmids that can encode enhanced green fluorescent protein （pEGFP） served as a report gene and were mixed with different concentrations of MB/0.05% （w/v） P85. Then the plasmids were transfected into human hepatoma G2 （HepG2） cells. The HepG2 cells treated with MB/P85 or without treatment were exposed to ultrasound （US parameters： 1 MHz, 1.0 W/cm2, 20 s, 20% duty cycle）. Twenty-four hours later, the transfection efficiency was assessed by fluorescence microscopy and fluo-rescence activated cell sorting （FACS） analysis. The cell viability was evaluated by Trypan blue exclusion test. The results showed that the gene transfection efficiency in HepG2 cells under ultrasound irradiation was significantly higher than that without ultrasound irradiation. HepG2 cells in the MB or P85 group in the absence of ultrasound expressed less amount of green fluorescent protein. The expression efficiency reached （22.14±3.06）% and the survival rate was as high as （55.73±3.32）% in the 30% MB plus P85 group. It was concluded that MB and P85 in the presence of ultrasound can enhance gene transfection and expression.

Effects of rearranged during transfection mutation on calcitonin and procalcitonin expression in sporadic medullary thyroid carcinoma

Objective The aim of this study was to investigate the effects of rearranged during transfection(RET)mutation on the expressions of calcitonin(CTn)and procalcitonin(PCT)in sporadic medullary thyroid carcinoma(SMTC).Methods RET mutation was detected by polymerase chain reaction direct sequencing in 64 cases of SMTC,and the expression levels of CTn and PCT in SMTC tissues were detected using the immunohistochemical streptavidin-perosidase(SP)method.The effect of RET mutations on the expression of CTn and PCT along with its relationship with clinicopathological parameters were analyzed.Results The expression rates of CTn and PCT in SMTC tissues were 90.6%(58/64)and 67.2%(43/64),respectively.CTn and PCT expression were found to be associated with tumor size and lymph node metastasis(P<0.05)but not with gender,age,or tumor capsule invasion(P>0.05).There was a significant correlation between CTn and PCT expression(r=0.269,P=0.041),and the intensity of positive CTn expression was positively correlated with RET mutation(r=0.507,P=0.000).However,PCT expression was not associated with RET mutation(r=0.188,P=0.136).Conclusion High expression of CTn and PCT was associated with the progression of medullary carcinoma,and the intensity of CTn expression was associated with RET mutation.PCT may provide valuable information for the diagnosis and prognosis of SMTC.

Enhancement of the transfection efficiency of DNA into Crocus sativus L. cells via PEI nanoparticles

Over the past decade, several natural and synthetic cationic polymers have been utilized for gene delivery into cells. Among them, polyethylenimine(PEI) was used for gene therapy successfully. The present study investigated the effect of PEI and ultrasound waves on ssD NA delivery into saffron cells. Gel retardation, dynamic light scattering(DLS) and scanning electron microscopy(SEM) assays were employed to determine the physicochemical properties of PEI/f-DNA polyplex(complex of PEI and fluorescently labeled DNA). Moreover, the cytotoxicity of PEI, PEI/f-DNA polyplex and ultrasound were investigated on saffron cells at different concentrations. The gel retardation results indicated that the formation and neutralization of the PEI/f-DNA polyplex were completed at N/P=5. The particle size distribution of the polyplexes was from 50 to 122 nm. The experimental results revealed that the cytotoxicity of the PEI/f-DNA polyplex was lower than that of PEI alone, hence the cells showed both dose-and exposure duration-dependent responses. Furthermore, the viability of saffron cells declined extremely after 5 and 10 min sonication but this reduction was not significant at 2 min exposure duration. The results also indicated that the combined utilization of ultrasound and PEI nanoparticles increased the transfection efficiency of saffron cells up to two times higher than those obtained by PEI or ultrasound separately.

Cationic Liposome-mediated bcl-xl Gene Transfection into Human Keratocytes

The efficiency and safe range of LipofectamineTM2000 (LF2000)/bcl-xl applied in human keratocytes, the optimal ratio of LF2000/bcl-xl and the bcl-xl gene expression in human keratocytes were investiaged. By using trypan-blue staining, the effects of LF2000 and bcl-xl on the survival rate of the cultured human keratocytes were measured respectively. By using semi-quantitative RT-PCR, the efficiency and the expression of LF2000-mediated bcl-xl transfection into keratocytes were examined. The results showed that the survival rate of human keratocytes had no signficant change in the presence of LF2000 (20 μg/ml) or bcl-xl (10 μg/ml) for 24 h. LF2000 could effectively mediate the transfection of exogenous gene bcl-x1 into human keratocytes. The best transfection efficiency could be obtained when the ratio of bcl-xl/LF2000 was 1:8. One day after transfection, the positive cells for bcl-x1 could be detectable, and the positive rate reached the peak on the post-transfection day 3 (48.3 %), then gradually decreased. Fifteen days after transfection, there were few positive cells. It was suggested that LF2000 could effectively transfer the exogenous gene bcl-xl into human keratocytes without obvious toxicity during a concentration range. LF2000/bcl-xl may be likely to play an important role in gene therapy of human keratocytes.

Effects of Transfection of ICAP-1α and Its Mutants on Adhesion and Migration of 2H-11 Cells

This study examined the effect of integrin cytoplasmic domain-associated protein 1α (ICAP-1α) and its mutatants T38A and I138A on the adhesion, migration and tube formation of 2H-11 cells.rAAV-ICAP-1α, rAAV-T38A and rAAV-I138A were constructed.After infection, the expression of ICAP-1α and p-ERK1/2, p-c-Jun protein was measured by Western blotting.Adhesion ability was evaluated by using MTT.Cell migration was determined by using Boyden chamber method.Tube formation test was conducted on Matrigel.The results showed that in ICAP-1α, T38A and I138A groups, ICAP-1α protein expression was increased.In T38A and I138A groups, phospho-ERK1/2, phospho-c-Jun protein expressions were significantly increased as compared with the control group and the GFP group.ICAP-1α group protein expression was obviously decreased when compared with the control group and the GFP group.Cell adhesion ratio was 0.1429±0.0080 in control group, 0.1434±0.0077 in GFP group and the ratio in T38A and I138A groups increased to 0.3210±0.0082 and 0.3250±0.0079, respectively.In ICAP-1α group, the ratio was decreased to 0.1005±0.0073.In T38A and I138A groups, the number of migrating 2H-11 cells was increased to 31.45±3.20 and 33.10±5.40 against 18.51±2.80 in control group and 20.47±3.12 in GFP group.In ICAP-1α group, the number was decreased to 12.06±1.72.The number of tube-like structures was increased to 20.41±2.54 in T38A and to 22.26±3.07 in I138A groups as compared to those of control group 12.45±1.84 and GFP group 13.63±2.71.In ICAP-1α group, the number of tube-like structures was decreased to 8.32±1.24.It was suggested that rAAV-T38A and rAAV-I138A transfection can substantially increase 2H-11 cell adhesion, migration and angiogenisis, while rAAV-ICAP-1α can greatly inhibit the effect.These effects might be correlated with ERK1/2 and c-Jun protein phosphorylation.

ALK5 transfection of bone marrow mesenchymal stem cells to repair osteoarthritis of knee joint

Previous studies have suggested that the transforming growth factor-β receptor ALK5 is crucial for articular chondrogenesis by bone marrow mesenchymal stem cells. Here, the wild-type ALK5 plasmids were mutated by overlapping extended PCR and transfected into bone marrow mesenchymal stem cells. The knee joint osteoarthritis mouse model was constructed by cutting oft＂ the anterior cruciate ligament and divided into three groups： saline group, bone marrow mesenchymal stem cells and ALK5-transfected bone marrow mesenchymal stem cells group. HE staining showed that the articular cartilage lesions were more serious of saline group compared with that of mesenchymal stem cell group, and this trend was more pronounced as time goes on. Immunohistochemical staining showed that although the expression level of type II collagen in all three groups down-regulated gradually upon time, its expression in ALK5-transfected bone marrow mesenchymal stem cells group was significantly enhanced compared with the other two groups. Micro-CT also suggested that ALK5 transfection of mouse bone marrow mesenchymal stem cells would promote repairing the knee cartilage lesions with arthritis of the mice. Although the osteoarthritis mechanism underlying a variety of factors work together, and the appropriate proportion of ALKS/ALK1 was also emphasized for the treatment of osteoarthritis. This work therefore demonstrated that ALK5 transfection of bone marrow mesenchymal stem cells could be a promising stem cell therapy for repair of cartilage lesions.

Editor's Choice—Adenovirus-mediated gene transfection of stem cells

Neural stem cells transplantation plays an important role in repair and cell replacement therapy for nervous system degenerative diseases. However, the self-repair effects are minimal because of a limited absolute number, as well as the local microenvironment, post-transplantation. A combined treatment utilizing stem cell transplantation and gone therapy can exert a dual effect involving stem cells and neurotrophic factors. The adenovirus carrier is

Optimization of Culture Medium and Transfection Method for Head and Neck Squamous Cell Carcinoma Organoids

[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term suspension transfection for organoid in matrigel. [Methods] Advanced DMEM/F12 medium, GlutaMax and HEPES buffer, nicotinamide, N-acetylcysteine, B27, A83-01, EGF, Y-27632 and Primocin primary cell antibiotics were prepared. On this basis, fibroblast growth factor 10(FGF10), Neuregulin 1, Noggin and R-spondin-1 were added in turn to prepare the selection medium, and the organoid diameter was used as the evaluation index to evaluate the effect of organoid medium. Using lentivirus, mCherry red fluorescent protein was transfected into HNSCC—PDO in different ways, and the transfection effect was evaluated by the fluorescence intensity of organoid sphere. [Results] Nrg1 Noggin and R-Spondin-1 promoted the growth of head and neck squamous cell carcinoma sphere(P<0.05) while FGF10 did not significantly promote the growth of head and neck squamous cell carcinoma sphere(P>0.05). Compared with direct transfection, short-term suspension transfection had higher transfection efficiency for HNSCC—PDO in matrigel. [Conclusions] R-Spondin-1 Nrg1 and Noggin may be the key cytokines in culture of HNSCC—PDO whereas FGF10 played an insignificant role in this study. Short-term suspension transfection could improve the transfection efficiency of lentivirus to HNSCC—PDO.