1.Preparation of polyphenol-mediated copper ion coating on titanium surface and antibacterial and antioxidant properties
Zhenju GUAN ; Yonglin XIE ; Shougang XIANG ; Chengdong ZHANG ; Xiaolong LI ; Xingping LI ; Chao PU ; Bo ZHANG ; Xuwei LUO ; Dongqin XIAO
Chinese Journal of Tissue Engineering Research 2025;29(10):1997-2005
BACKGROUND:Titanium implants are widely used in clinical practice because of their high strength and good biocompatibility.However,during implantation,bacterial infection and tissue damage environment produce a large number of reactive oxygen species,which can easily lead to delayed tissue healing and surgical failure.Consequently,the development of titanium implants with antimicrobial and antioxidant properties becomes paramount. OBJECTIVE:Considering the potent antimicrobial attributes of copper ions and the remarkable antioxidant qualities of polyphenols,we proposed the fabrication of polyphenol-mediated copper ion coatings on titanium surfaces.These coatings were subsequently assessed for their in vitro antimicrobial and antioxidant properties. METHODS:Nanostructures were generated on the titanium surface using the alkali thermal method.The titanium was immersed in a solution containing tannic acid and copper ions to achieve polyphenol-mediated copper ion coatings.The surface morphology and water contact angle were detected.The loading and release of copper ions were examined using atomic absorption spectroscopy.Staphylococcus aureus was inoculated on the surface of pure titanium sheet(blank group),alkali heat treated titanium sheet(control group),and polyphenol mediated copper ion modified titanium sheet(experimental group)to observe the bacterial survival status.Osteoblast precursor cells MC3T3-E1 were co-cultivated on the surface of three groups of titanium sheets to assess their antioxidant properties and bioactivity. RESULTS AND CONCLUSION:(1)Scanning electron microscopy showed that the polyphenol-mediated copper ion modified titanium sheet had rod-like nanostructures and no cracks on the surface.The surface hydrophilicity of copper ion modified titanium sheet mediated by polyphenol was close to that of pure titanium sheet.Atomic absorption spectrometry results showed a 51%increase in the loading capacity of copper ions after polyphenol mediation,with a uniform release of copper ions.(2)The antibacterial rates of titanium sheets in the blank group,control group,and experimental group were 0%,21.65%,and 93.75%,respectively.The live/dead staining and CTC staining showed that the live bacteria on the surface of titanium plates in the blank group were the most,and the live bacteria on the surface of titanium plates in the experimental group were the least.(3)The results of live/dead staining and CCK-8 assay showed that the three groups of titanium sheets had good cytocompatibility,and the titanium sheets in the experimental group were more conducive to the proliferation of MC3T3-E1 cells.Active oxygen fluorescence probe detection exhibited that compared with the other two groups,the fluorescence intensity of active oxygen on the surface of the experimental group was significantly reduced.The results of alkaline phosphatase and alizarin red S staining showed that the osteogenic differentiation and extracellular matrix mineralization of MC3T3-E1 cells on the surface of titanium sheets in the experimental group were stronger than those in the other two groups.(4)These results show that the polyphenol-mediated copper ion coating has strong antibacterial and antioxidant properties and promotes osteogenic differentiation.
2.Growth differentiation factor-5 modified by bisphosphonate promotes osteogenic differentiation of MC3T3-E1 cells
Lisi LI ; Chengdong ZHANG ; Xiaolong LI ; Ziyu YE ; Chao PU ; Zaijun YANG ; Feng SHI ; Dongqin XIAO
Chinese Journal of Tissue Engineering Research 2024;28(3):373-379
BACKGROUND:As a member of bone morphogenetic proteins,growth differentiation factor-5 shows promising potential in the application of cartilage and bone repair.The affinity of growth differentiation factor-5 onto bone tissue determines protein use efficiency,so it is of great significance to prepare growth differentiation factor-5 with bone targeting capability. OBJECTIVE:To modify growth differentiation factor-5 using bisphosphonates and investigate the effects of modified protein on the growth of preosteoblasts in mice. METHODS:Pamidronate disodium/growth differentiation factor-5 complex was prepared using chemical crosslinking to couple growth differentiation factor-5 with pamidronate disodium.The functional groups and structures of the complex were characterized using Fourier transform infrared spectroscopy and circular dichromatography.To determine the bone targeting in vitro,the binding of the modified growth differentiation factor-5 with calcium phosphate and in vitro release amount of growth differentiation factor-5 were measured with an ELISA kit.Growth differentiation factor-5(control group)and the pamidronate disodium/growth differentiation factor-5 complex(experimental group)were co-cultured with preosteoblasts MC3T3-E1.Individually cultured cells were blank controls.The effect of the complex on cell proliferation and differentiation was evaluated. RESULTS AND CONCLUSION:(1)The infrared spectroscopy and circular dichromatography results indicated that the bisphosphonate/growth differentiation factor-5 complex was successfully prepared without significant changes in the protein secondary structure.In vitro protein adsorption results showed that growth differentiation factor-5 adsorption on calcium phosphate was increased by about one time after coupling with a bisphosphonate.In the presence of cysteine,growth differentiation factor-5 could be released from the bisphosphonate/growth differentiation factor-5 complex.(2)CCK-8 assay results showed that the absorbance value of the experimental group cultured for 4 and 7 days was higher than that of the control group and blank control group(P<0.000 1).After 7 days of culture,the expression of alkaline phosphatase in the experimental group was significantly higher than that in the control group and blank control group(P<0.000 1).After 13 days of culture,the content of calcium nodules in the experimental group was significantly higher than that in the control group and the blank control group(P<0.000 1).The results of qRT-PCR showed that the mRNA expression of alkaline phosphatase,osteocalcin and Runx2 in the experimental group was higher than that in the control group and the blank control group after 7 days of culture(P<0.01,P<0.001,P<0.000 1).(3)These findings exhibit that bisphosphonate modification can enhance the binding capacity of growth differentiation factor-5 to calcium phosphate as well as improve its biological activity.
3.Neonatal linear IgA bullous dermatosis
Guangwen YIN ; Mengmeng GENG ; Yan CUI ; Shoumin ZHANG ; Dongqin LI ; Xiuyong CHENG
Chinese Journal of Dermatology 2023;56(2):146-148
A 10-day-old male infant presented with skin erythema and blisters for 6 days. Skin examination showed scattered or confluent erythema all over the body, tense blisters of varying sizes on the normal skin or an erythematous base, and some blisters were ulcerated and erosive; bloody bullae and erythematous erosive patches could be seen on the oral mucosa. Histopathological examination revealed subepidermal blisters, and there were some neutrophils and a few eosinophils in the blisters. Direct immunofluorescence assay showed homogeneous linear IgA and granular C3 deposits along the basement membrane zone, without IgG deposits. The diagnosis of neonatal linear IgA bullous dermatosis was confirmed. After comprehensive treatments including nutritional support and anti-infection treatment, skin erythema and blisters subsided, and the mucosal damage was attenuated. The telephone follow-up 16 months after discharge showed that the infant was in good general condition with normal growth and development, and the oral mucosal lesions had subsided and healed, without new skin lesions.
4.A clinical analysis of 74 cases of bullous pemphigoid with stroke
Xiaoyan JIAO ; Guangwen YIN ; Dongqin LI ; Yakun YIN ; Bingjie CAI ; Hui FANG
Chinese Journal of Dermatology 2023;56(4):330-334
Objective:To investigate differences in clinical characteristics between bullous pemphigoid (BP) patients with stroke and those without, and their relationship with the prognosis of stroke.Methods:A retrospective analysis was performed on medical records of 330 BP inpatients in the First Affiliated Hospital of Zhengzhou University from September 2012 to April 2020. These patients were divided into BP + stroke (ST) group and BP - ST group according to whether they were accompanied by stroke, and clinical manifestations and relevant laboratory examination results were compared between the two groups. According to the stroke outcome score assessed by modified Rankin Scale (mRS), patients in the BP + ST group were further divided into good-prognosis ST group (mRS ≤ 2 points) and poor-prognosis ST group (mRS > 2 points), and subgroup analysis was conducted. Correlations between measurement data (such as age, disease course and laboratory examination results) and mRS scores were analyzed.Results:In the BP - ST group (256 cases), 151 were males and 105 were females, and their age ranged from 19 to 92 (66.8 ± 13.6) years; in the BP + ST group (74 cases), 45 were males and 29 were females, and their age ranged from 48 to 92 (74.6 ± 9.6) years; Compared with the BP - ST group, the BP + ST group showed older age ( t = -5.57, P < 0.001), shorter disease course of BP ( Z = -3.07, P = 0.002), and higher anti-BP180 IgG antibody levels (215.0 [157.2, 283.1] U/ml vs. 155.0 [63.9, 279.8] U/ml; Z = -2.12, P = 0.034). The distribution of skin lesions significantly differed between the two groups ( χ2 = 10.51, P = 0.015), and the BP + ST group showed a significantly lower proportion of patients with generalized lesions ( P<0.05), but a higher proportion of patients with lesions on the limbs ( P<0.05). Subgroup analysis showed significant differences in the patients′ age, BP course, lesion distribution and anti-BP180 IgG antibody levels among the good-prognosis ST group, poor-prognosis ST group and BP - ST group ( F = 10.83, P<0.001; Z = 17.24, P<0.001; χ2 = 15.57, P = 0.026; Z = 6.29, P = 0.043, respectively). There was no significant difference in the age between the good-prognosis ST group and poor-prognosis ST group (adjusted P = 1.000), but the patients were significantly older in the two above groups than in the BP - ST group (adjusted P = 0.001, 0.007, respectively) ; the poor-prognosis ST group showed significantly shorter BP courses (adjusted P = 0.016, < 0.001, respectively) and a higher proportion of patients with lesions on the limbs (both P < 0.05) compared with the good-prognosis ST group and BP - ST group, and significantly higher serum anti-BP180 IgG antibody levels compared with the BP - ST group (226.2 [163.6, 285.8] U/ml vs. 155.0 [63.9, 279.8] U/ml; adjusted P = 0.037). There were no significant differences in the gender distribution, lesional morphology, percentages and counts of peripheral blood eosinophils, serum total IgE levels, and anti-BP230 IgG antibody levels between the BP + ST group and BP - ST group (all P > 0.05), or among the good-prognosis ST group, poor-prognosis ST group and BP - ST group (all P > 0.05). Correlation analysis in the BP + ST group showed a significantly negative correlation between the BP course and mRS scores ( r = -0.33, P = 0.004), and a significantly positive correlation between the anti-BP180 IgG antibody levels and mRS scores ( r = 0.34, P = 0.032) . Conclusion:There were differences in the patients′ age, BP course, lesion distribution, and anti-BP180 IgG antibody levels between the BP patients with stroke and those without, and the differences were more obvious between the poor-prognosis ST group and BP - ST group.
5.Chronic disseminated paracoccidioidomycosis: the first case reported in China
Xiaoyan JIAO ; Guangwen YIN ; Dongqin LI ; Xiaogai LI ; Zhongqin MENG ; Lili LOU ; Yakun YIN ; Yan ZHENG
Chinese Journal of Dermatology 2023;56(5):415-420
To report the first case of chronic disseminated paracoccidioidomycosis in China. A 49-year-old male patient presented with papules and nodules of the skin for 1 year, and papules and ulcers on the oral mucosa for 2 months. Skin examination showed the edema of the left foot, multiple crusting ulcers on the sole of the left foot, ulcers with a granular base in the interdigital regions between the third and fourth toes as well as fourth and fifth toes of the left foot, accompanied by punctate hemorrhage and exudation; there were multiple papules, nodules, and plaques on the dorsum and medial side of the left foot and the left knee, with ulcers and crusts in the center; 2 papules were observed on the left wrist, and 1 papule on the left upper lip with a crusted surface; red plaques with ulcers and punctate hemorrhage were observed on the gingival mucosa, buccal mucosa, labial mucosa, and palate, and the lesions mainly occurred on the left side. Ultrasonography of superficial lymph nodes showed bilateral cervical and supraclavicular lymph node enlargement, which was more obvious on the left side. Computed tomography of the chest and abdomen showed diffuse miliary nodular shadows, and cordlike, cloudy flocculent and nodular high-density shadows in both lungs, as well as obvious thickening of the left adrenal gland in the abdomen. Yeast cells were observed by immunofluorescent staining of biopsy tissues from the oral mucosa and left lower limb. Histopathological examination of biopsy tissues from the oral mucosa and left lower limb showed granulomatous inflammation, and refractive double-membrane yeast cells could be observed inside or outside the multinucleated giant cells, without or with a single bud or multiple buds; periodic acid-Schiff staining and hexamine silver staining of the above biopsy tissues were positive. Fungal culture of the left lower limb lesion in Sabouraud dextrose agar medium at 25℃ and 37℃ both yielded fungal hyphae. Metagenomics sequencing of the oral mucosal tissue and alveolar lavage fluid indicated the infection with Paracoccidioides brasiliensis. The diagnosis of chronic disseminated paracoccidioidomycosis was confirmed. After 1-month oral treatment with itraconazole capsules at a dose of 400 mg/d, the lesions on the skin and oral mucosa markedly improved, and computed tomography imaging of the lung and left adrenal gland also showed obvious improvement. The dose of itraconazole was reduced to 200 mg/d after 3 months. The patient′s condition further improved during a 10-month follow-up.
6.Clinical analysis of 25 cases of herpes zoster during pregnancy or the perinatal period
Xiaoyan JIAO ; Guangwen YIN ; Yakun YIN ; Dongqin LI
Chinese Journal of Dermatology 2022;55(10):881-884
Objective:To investigate clinical features, treatment and prognosis of herpes zoster during pregnancy or the perinatal period.Methods:Clinical data were collected from female inpatients with herpes zoster during pregnancy or the perinatal period in the First Affiliated Hospital of Zhengzhou University from January 2011 to December 2020, and clinical characteristics, treatment and prognosis were retrospectively analyzed.Results:A total of 25 patients were included in the study, including 22 pregnant patients at 10 - 38 gestational weeks (1 in the first trimester, 13 in the second trimester, and 8 in the third trimester) and 3 patients during the first postpartum week; their age ranged from 22 to 37 years, and the disease course varied from 2 to 9 days. Skin lesions were located on the head and face in 8 cases, on the chest and back in 5, on the waist and abdomen in 7, on the upper limbs in 1, on the lower limbs in 3, as well as on the perineum in 1; 3 patients presented with disseminated herpes zoster. The patients all presented with erythema, papulovesicles and blisters, which were accompanied by bloody bullae in 4 patients, by bullae in 3, as well as by erosions and exudations in 6; 5 patients had fever, 7 had mild pain, 6 had moderate pain, and 11 had severe pain. Sixteen pregnant patients and 3 postpartum patients received antiviral therapy (oral or intravenous infusion of acyclovir, vidarabine monophosphate, foscarnet sodium-sodium chloride injection) , 24 patients were treated with neurotrophic drugs, 1 pregnant patient and 3 postpartum patients received analgesic treatment, and other treatments included anti-infective therapy, wet compress, infrared radiation, etc. Preterm delivery occurred in 1 pregnant patient, other pregnant patients had full-term deliveries, and no abnormalities were observed in neonates. Sequelae occurred in 3 pregnant patients, including pain in 1 and pruritus in 2.Conclusion:Among the 25 patients, the treatment of herpes zoster during pregnancy or the perinatal period showed no obvious effect on their fetuses or newborns.
7.Parameter transfer learning based on shallow visual geometry group network and its application in motor imagery classification.
Journal of Biomedical Engineering 2022;39(1):28-38
Transfer learning is provided with potential research value and application prospect in motor imagery electroencephalography (MI-EEG)-based brain-computer interface (BCI) rehabilitation system, and the source domain classification model and transfer strategy are the two important aspects that directly affect the performance and transfer efficiency of the target domain model. Therefore, we propose a parameter transfer learning method based on shallow visual geometry group network (PTL-sVGG). First, Pearson correlation coefficient is used to screen the subjects of the source domain, and the short-time Fourier transform is performed on the MI-EEG data of each selected subject to acquire the time-frequency spectrogram images (TFSI). Then, the architecture of VGG-16 is simplified and the block design is carried out, and the modified sVGG model is pre-trained with TFSI of source domain. Furthermore, a block-based frozen-fine-tuning transfer strategy is designed to quickly find and freeze the block with the greatest contribution to sVGG model, and the remaining blocks are fine-tuned by using TFSI of target subjects to obtain the target domain classification model. Extensive experiments are conducted based on public MI-EEG datasets, the average recognition rate and Kappa value of PTL-sVGG are 94.9% and 0.898, respectively. The results show that the subjects' optimization is beneficial to improve the model performance in source domain, and the block-based transfer strategy can enhance the transfer efficiency, realizing the rapid and effective transfer of model parameters across subjects on the datasets with different number of channels. It is beneficial to reduce the calibration time of BCI system, which promote the application of BCI technology in rehabilitation engineering.
Algorithms
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Brain-Computer Interfaces
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Electroencephalography/methods*
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Humans
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Imagination
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Machine Learning
8.Effect of extracorporeal shock wave combined with meloxicam tablets on joint function and serum levels of MMP-9 and resistin in elderly patients with osteoarthritis
Hui WU ; Dongqin XIAO ; Minggang GUO ; Liang LI ; Xuwei LUO
Journal of Chinese Physician 2021;23(2):203-206,210
Objective:To explore the effect of extracorporeal shock wave combined with meloxicam tablets on joint function and serum matrix metalloproteinase-9 (MMP-9) and resistin levels in elderly patients with osteoarthritis.Methods:80 cases of elderly patients with osteoarthritis in Nanchong Central Hospital from May 2016 to March 2019 were selected as the research objects, and they were randomly divided into two groups, 40 cases in each group. The control group was given conventional oral medicine (meloxicam + potassium glucosamine sulfate tablets), and the observation group was given meloxicam tablets combined with extracorporeal shock wave therapy. The total effective rate, incidence of adverse reactions, and the scores of osteoarthritis symptoms (WOMCA), joint function (Lysholm), serum MMP-9 and resistin before and after 8 weeks' treatment were compared between the two groups.Results:After treatment, the WOMCA score of the observation group was lower than that of the control group, and Lysholm score was higher than that of the control group ( P<0.05); the total effective rate of the observation group (95.00%) was higher than of the control group (77.50%) ( P<0.05); the serum resistin and MMP-9 levels of the observation group were lower than those of the control group after treatment ( P<0.05); there was no significant difference in the incidence of adverse reactions between the observation group (15.00%) and the control group (17.50%) ( P>0.05). Conclusions:Extracorporeal shock wave combined with meloxicam tablets in the treatment of elderly patients with osteoarthritis can significantly improve the clinical symptoms, promote the recovery of joint function, reduce the levels of serum resistin and MMP-9, and further improve the treatment effect with high safety.
9.LncRNA LEF1-AS1 regulates proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells by targeting miR-612: an in vitro experimental study
Yunpeng ZHENG ; Xuyang LI ; Bingjie CAI ; Dongqin LI ; Guangwen YIN
Chinese Journal of Dermatology 2020;53(6):415-423
Objective:To evaluate the effects of long non-coding RNA (lncRNA) LEF1-AS1 on proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells, and to explore their mechanisms.Methods:Cutaneous squamous cell carcinoma SCC13 cells were divided into si-LEF1-AS1 group transfected with lncRNA LEF1-AS1 interference oligonucleotides (si-LEF1-AS1) , si-NC group transfected with lncRNA LEF1-AS1 nonsense oligonucleotides (si-NC) , miR-612 group transfected with miR-612-overexpressing oligonucleotides, miR-NC group transfected with miR-612 nonsense oligonucleotides (miR-NC) , si-LEF1-AS1+anti-miR-612 group transfected with si-LEF1-AS1 and oligonucleotides against miR-612, and si-LEF1-AS1+anti-miR-NC group transfected with si-LEF1-AS1 and miR-612 nonsense oligonucleotides. Quantitative reverse transcription (qRT) -PCR was performed to determine the relative expression of miR-612 in SCC13 cells, cell counting kit-8 (CCK8) assay to evaluate cellular proliferative activity, flow cytometry to detect cell apoptosis, Transwell assay to assess migratory and invasive abilities of SCC13 cells, and Western blot analysis to determine protein expression of cyclin-dependent kinase 1 (cyclinD1) , cyclinD1 inhibitor p21, Bcl-2 family protein (Bcl-2) , Bcl-2 related X protein (Bax) , matrix metalloproteinase 2 (MMP-2) and MMP-9. The online bioinformatics database LncBase predicted v.2 was employed to predict the complementary sequence between lncRNA LEF1-AS1 and miR-612, and luciferase reporter gene plasmids were constructed by using the complementary/non-complementary sequence, which were co-transfected with miR-612-overexpressing oligonucleotides (miR-612 overexpression group) or miR-NC (overexpression control group) into SCC13 cells in order to verify the binding ability of lncRNA LEF1-AS1 to miR-612. Statistical analysis was carried out by using t test for comparison between two groups, one-way analysis of variance for comparison among multiple groups, and least significant difference (LSD) - t test for multiple comparisons. Results:Compared with the miR-NC group, miR-612 group showed significantly decreased cellular proliferative ability, number of migratory cells and invasive cells (all P < 0.05) , but a significantly increased apoptosis rate ( P < 0.05) . The relative expression of miR-612 ( F = 150.78, P < 0.001) , cellular proliferative activity at 24, 48, 72 hours (all P < 0.05) , apoptosis rate and number of migratory and invasive cells (all P < 0.05) significantly differed among the si-LEF1-AS1 group, si-NC group, si-LEF1-AS1+anti-miR-612 group and si-LEF1-AS1+anti-miR-NC group. Compared with the si-NC group, the si-LEF1-AS1 group showed significantly increased expression of miR-612 and apoptosis rates, but significantly decreased cellular proliferative activity at 48, 72 hours, and number of migratory and invasive cells (all P < 0.05) ; compared with the si-LEF1-AS1+anti-miR-NC group, the si-LEF1-AS1+anti-miR-612 group showed significantly decreased expression of miR-612 and apoptosis rates, but significantly increased cellular proliferative activity at 48, 72 hours, and number of migratory and invasive cells (all P < 0.05) . Western blot analysis showed that the relative protein expression of cyclinD1, p21, Bcl-2, Bax, MMP-2 and MMP-9 significantly differed among the si-LEF1-AS1 group, si-NC group, si-LEF1-AS1+anti-miR-612 group and si-LEF1-AS1+anti-miR-NC group (all P < 0.001) ; compared with the si-NC group, the si-LEF1-AS1 group showed significantly increased protein expression of cyclinD1, Bcl-2, MMP-2 and MMP-9, but significantly decreased protein expression of p21 and Bax (all P < 0.05) ; compared with the si-LEF1-AS1+anti-miR-NC group, the si-LEF1-AS1+anti-miR-612 group showed significantly increased protein expression of cyclinD1, Bcl-2, MMP-2 and MMP-9, but significantly decreased protein expression of p21 and Bax (all P < 0.05) . After co-transfection with complementary sequences, the fluorescence activity was significantly lower in the miR-612 overexpression group than in the overexpression control group ( t = 21.19, P < 0.001) ; after co-transfection with non-complementary sequences, no significant difference was observed in the fluorescence activity between the miR-612 overexpression group and overexpression control group ( t = 0.28, P = 0.78) . Conclusion:lncRNA LEF1-AS1 regulates the proliferation, apoptosis, migration and invasion of cutaneous squamous cell carcinoma cells, likely by targeting miR-612.
10.lncRNA DLX6-AS1 regulates the proliferation, migration and invasion of a cutaneous squamous cell carcinoma cell line A431 by targeting miR-16-5p/NUCKS1
Yunpeng ZHENG ; Bingjie CAI ; Xuyang LI ; Dongqin LI ; Guangwen YIN
Chinese Journal of Dermatology 2020;53(8):607-615
Objective:To investigate effects of long non-coding growth stasis specific protein 6 antisense RNA1 (lncRNA DLX6-AS1) on the proliferation, migration and invasion of a cutaneous squamous cell carcinoma cell line A431, and to explore the underlying mechanisms.Methods:A dual-luciferase reporter system was used to verify the targeting relationship between lncRNA DLX6-AS1 and miR-16-5p, as well as between miR-16-5p and nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) mRNA. Cultured A431 cells were divided into several groups: si-DLX6-AS1 group and DLX6-AS1-NC group transfected with lncRNA DLX6-AS1 inhibitor and its negative control respectively; anti-miR-16-5p group and anti-miR-NC group transfected with miR-16-5p inhibitor and its negative control respectively; si-NUCKS1 group and NUCKS1-NC group transfected with NUCKS1 inhibitor and its negative control respectively; si-DLX6-AS1+ anti-miR-16-5p group transfected with lncRNA DLX6-AS1 inhibitor followed by miR-16-5p inhibitor, and si-DLX6-AS1+ anti-miR-NC group transfected with lncRNA DLX6-AS1 inhibitor followed by anti-miR-NC; si-DLX6-AS1+ anti-miR-16-5p+ si-NUCKS1 group transfected with lncRNA DLX6-AS1 inhibitor, miR-16-5p inhibitor and NUCKS1 inhibitor, and si-DLX6-AS1+ anti-miR-16-5p+ NUCKS1-NC group transfected with lncRNA DLX6-AS1 inhibitor, miR-16-5p inhibitor and NUCKS1-NC. After the above treatment, real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to measure the mRNA expression of lncRNA DLX6-AS1, miR-16-5p and NUCKS1 in A431 cells, Western blot analysis to determine the protein expression of NUCKS1, Cyclin D1 antibody, matrix metalloproteinase (MMP) 2 and MMP9, cell counting kit-8 (CCK8) assay to detect cell survival rate, and Transwell assay to evaluate cell migratory and invasive abilities. Two-independent-sample t test was used for comparisons between two groups. Results:Dual-luciferase reporter assay showed targeted binding of lncRNA DLX6-AS1 to miR-16-5p, as well as of miR-16-5p to NUCKS1. Compared with the DLX6-AS1-NC group, the si-DLX6-AS1 group showed significantly increased miR-16-5p expression in A431 cells (3.01 ± 0.31 vs. 1.02 ± 0.10, t = 18.33, P < 0.001) , but significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 (all P < 0.05) , and significantly decreased cell survival rate and numbers of migratory and invasive cells (all P < 0.05) . Compared with the NUCKS1-NC group, the si-NUCKS1 group showed significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 in A431 cells (all P < 0.05) , and significantly decreased cell survival rate and numbers of migratory and invasive cells (all P < 0.05) . After inhibition of lncRNA DLX6-AS1 expression, the si-DLX6-AS1+ anti-miR-16-5p group showed significantly decreased miR-16-5p expression in A431 cells (0.34 ± 0.04) compared with the si-DLX6-AS1+ anti-miR-NC group (1.00 ± 0.12, t = 15.65, P < 0.05) , but significantly increased protein expression of Cyclin D1, MMP2 and MMP9, cell survival rate and numbers of migratory and invasive cells compared with the si-DLX6-AS1+ anti-miR-NC group (all P < 0.05) . After inhibition of lncRNA DLX6-AS1 expression and knockdown of miR-16-5p, the si-DLX6-AS1+ anti-miR-16-5p+ si-NUCKS1 group showed significantly decreased protein expression of NUCKS1, Cyclin D1, MMP2 and MMP9 in A431 cells, as well as cell survival rate and numbers of migratory and invasive cells, compared with the si-DLX6-AS1+ anti-miR-16-5p+ NUCKS1-NC group (all P < 0.05) . Conclusion:lncRNA DLX6-AS1 can regulate the proliferation, migration and invasion of A431 cells by targeting miR-16-5p/NUCKS1, suggesting that lncRNA DLX6-AS1 may be a potential molecular target for the treatment of cutaneous squamous cell carcinoma.

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