Long non-coding RNA HOXA11-AS accelerates the progression of keloid formation via miR-124-3p/TGFßR1 axis.

Emerging evidence reveals the importance of long non-coding RNAs (lncRNAs) in the development and progression of keloid formation, whereas the underlying mechanisms are not well understood. In the present study, we investigated the biological effects and molecular mechanisms of lncRNA HOXA11-AS in keloid formation. First, the expression levels of HOXA11-AS, miR-124-3p, and transforming growth factor ß receptor type I (TGFßR1) were measured in both keloid tissues and human keloid fibroblasts (HKFs) using qRT-PCR and western blot analysis, respectively. Next, we adopted both gain- and loss-of-function strategies to explore the significance of HOXA11-AS. TUNEL, flow cytometry, DNA ladder, and tube formation assays were performed to measure cell apoptosis and angiogenesis, respectively. Besides, the potential binding relationship between HOXA11-AS and miR-124-3p, as well as miR-124-3p and TGFßR1 was identified using bioinformatic screening and verified by luciferase reporter assay. Furthermore, we explored the importance of miR-124-3p in HOXA11-AS-induced phenotypes and regulations on TGFß signaling or PI3K/Akt signaling. We found that HOXA11-AS and TGFßR1 were significantly up-regulated, while miR-124-3p was down-regulated both in keloid tissues or fibroblasts than in normal skin tissues or fibroblasts. Functionally, high expression of HOXA11-AS essentially inhibited cell apoptosis and promoted fibroblast-induced angiogenesis. Mechanistically, miR-124-3p was identified as a downstream effector to be involved in HOXA11-AS-mediated phenotypes through directly targeting TGFßR1, thus modulating PI3K/Akt signaling pathway. Taken together, our findings revealed that HOXA11-AS inhibits cell apoptosis and promotes angiogenesis through miR-124-3p/TGFßR1 axis, contributing to the progression of keloid formation, which might provide a novel target for keloid therapy.

Long non-coding RNA HOXA11-AS induces type I collagen synthesis to stimulate keloid formation via sponging miR-124-3p and activation of Smad5 signaling.

Keloid, characterized by exuberant collagen deposition and invasive growth beyond original wound margins, results from abnormal wound healing. A recent microarray analysis identified homeobox (HOX) A11 antisense (HOXA11-AS) as a keloid-specific long non-coding RNA, although its potential role in keloid formation remains elusive. In this study, hematoxylin-eosin, Masson, and immunohistochemical staining of type I collagen (ColI) revealed abnormal arrangement and hyperplasia of fibers in keloid tissues along with increased ColI level. qRT-PCR and Western blot showed that HOXA11-AS and ColI were significantly upregulated, while miR-124-3p was decreased in both keloid tissues and human keloid fibroblasts (HKFs). Knockdown of HOXA11-AS inhibited cell proliferation (by CCK-8 and immunofluorescence staining of Ki67) and cell migration (by wound healing and transwell assays). Mechanistic experiments verified that HOXA11-AS acted as a sponge of micro-RNA (miR)-124-3p and Smad5 was a target of miR-124-3p. miR-124-3p sufficiently reversed the regulatory effects of HOXA11-AS, and Smad5 was involved in miR-124-3p-mediated biological functions. Furthermore, HOXA11-AS induced ColI synthesis via sponging miR-124-3p-mediated Smad5 signaling, thus promoting keloid formation. Overall, our study implied that HOXA11-AS induces ColI synthesis to promoted keloid formation via sponging miR-124-3p-mediated Smad5 signaling, which might offer a novel target for developing the therapy of keloid formation.

MiR-139-5p inhibits the tumorigenesis and progression of oral squamous carcinoma cells by targeting HOXA9.

Our study sought to clarify the effects of microRNA-139-5p (miR-139-5p) in the tumorigenesis and progression of oral squamous cell carcinoma (OSCC) by regulating HOXA9. MiR-139-5p and HOXA9 expression in OSCC tissues, tumour adjacent tissues, OSCC cells and normal cells were tested by qRT-PCR. SAS and CAL-27 cell lines were selected in among four OSCC cell lines and then transfected with miR-139-5p mimics, pEGFP-HOXA9 and cotransfected with miR-139-5p mimics + pEGFP-HOXA9. We used MTT, colony formation, transwell and wound healing assays to analyse cell viability, proliferation, invasion and migration. The target relationship between miR-139-5p and HOXA9 was verified by luciferase reporter assay and Western blot, respectively. MiR-139-5p was down-regulated, whereas HOXA9 was up-regulated in OSCC tissues and cells. The proliferation, invasion and migration ability of SAS and CAL-27 cells in miR-139-5p mimics group were significantly weaker than those in the control group and the miR-NC group (P < 0.01). MiR-139-5p can negatively regulate HOXA9. The proliferation, invasion and migration of SAS and CAL-27 cells in the miR-139-5p mimics + pEGFP-HOXA9 group were not significantly different from those in the blank control and negative control groups (P > 0.05). Our results indicated that miR-139-5p could directly inhibit HOXA9, which might be a potential mechanism in inhibiting the proliferation, invasiveness and migration of OSCC cells.

MiR-376c-3p regulates the proliferation, invasion, migration, cell cycle and apoptosis of human oral squamous cancer cells by suppressing HOXB7.

PURPOSE: To test the influence of miR-376c-3p on the proliferation, invasion, migration, cell cycle and apoptosis of human oral squamous cancer cells (OSCC) and the relevant mechanism. METHODS: We applied qRT-PCR and Western blot to compare the expression level of miR-376c-3p and HOXB7 in SCC-4, SCC-9, SCC-15, SCC-25 OSCC cell lines and 49 paired OSCC and normal oral epithelial tissue specimens were included in our present study. Also we analyzed the relative relationship of expression level between miR-376c-3p and HOXB7 in cancer tissues. Luciferase assay was used to confirm the target relationship between miR-376c-3p and HOXB7. Besides, MTT, Transwell, wound healing, colony formation and flow cytometer experiments were applied to evaluate the proliferation, cell viability, apoptosis, invasion and migration of transfected OSCC. RESULTS: MiR-376c-3p was down-regulated while HOXB7 was up-regulated in OSCC tissues and cells than the normal ones. MiR-376c-3p directly targeted HOXB7 and reduced the expression of HOXB7. Overexpression of miR-376c-3p attenuated proliferation of SCC-9, SCC-15, SCC-24 and SCC-25 cells. Moreover, miR-376c-3p suppressed proliferation, viability, migration and invasion and induced G1/G0 arrest and cell apoptosis of SCC-25 cells. Besides, overexpression of HOXB7 efficiently abrogates these influences caused by overexpression of miR-376c-3p. CONCLUSION: MiR-376c-3p suppresses the fission, proliferation, migration and invasion and induces cell apoptosis of OSCC via targeting HOXB7.

[Modified two-stage surgery for total auriculoplasty with autogenous rib cartilage].

OBJECTIVE: To introduce a modified surgery for total auriculoplasty and the experience in one hundred and forty-six cases (155 ears). METHODS: The procedure was a two-stage operation. The first stage involved fabrication and grafting of a costal cartilage framework. A U-shaped skin incision was made on the posterior edge of the lobule and the remnant ear cartilage was removed completely. The area for the insertion of the cartilage framework was undermined. Skin flaps were sutured after insertion of the cartilage framework. The second-stage surgery was usually performed six months after the first-stage operation. The reconstructed auricle was elevated, and a costal cartilage block was fixed to the posterior part of the auricle. A temporoparietal fascia flap was then used to cover the costal cartilage block. Finally, the posterior aspect of the projected auricle was covered with a spit-thickness skin graft. RESULTS: The incisions healed in one hundred and forty-one patients (150 ears) after the first stage operation. Partial necrosis of the postauricular flap was observed in five cases (5 ears) after the first stage operation, but no exposure or absorption of the cartilage took place. The skin grafts survived in one hundred and thirty-nine cases (147 ears) after the second-stage surgery. Partial necrosis of the skin graft was observed in seven cases (8 ears), but healed after one-week of dressing changes. Ninety-four cases (97 ears) were followed up, but fifty-two cases (58 ears) were lost to follow up. The follow-up at six months to two years showed satisfactory contour and projection of the constructed ears. CONCLUSION: This two-stage surgery is simple and ideal for auricloplasty with few complications.

[Biologic effect of transforming growth factor-ß1 on urethra cells cultured in vitro].

OBJECTIVE: To investigate the effects of transforming growth factor-ß1 (TGF-ß1) on growth controlling and the expression of connective tissue growth factor mRNA(CTGF mRNA) in urethra epithelium cells and fibroblasts cultured in vitro. METHODS: Urethra epithelial cells and fibroblasts were cultured in vitro and identified. The fourth generation cells were divided into control group (cultured by cell medium without TGF-ß1) and experimental groups(cultured by cell medium containing TGF-ß1 1, 2, 4 and 8 µg/L), the vital force of cells were examined by MTT and cell counting, the expression of CTGF mRNA were examined by RT-PCR after 24 hours. RESULTS: The optical density and cell count decreased in experimental groups of urethra epithelium cells and increased in experimental groups of fibroblasts with the concentration of TGF-ß1 being heightened compared with the control group (P < 0.05). The expression of CTGF mRNA increased with the heightening concentration of TGF-ß1 in all experimental groups of urethra epithelium cells and fibroblasts by RT-PCR (P < 0.05). CONCLUSIONS: TGF-ß1 can inhibit the growth of urethra epithelium cells and promote the growth of fibroblasts in vitro, it can induce the expression of CTGF mRNA in two cells above-mentioned.

[In situ quantitation of activity of cultured urethral epithelial cells in vitro].

OBJECTIVE: To resolve the tough problem of how to observe the growing cells in an opaque vector. METHODS: The urethral epithelial cells from a young male New Zealand rabbit were inoculated, and were primarily cultured in vitro and subcultured for 3 passages. Then, the urethral epithelial cells were cultured in the collagen-chitosan complex for 3, 7, 14 and 21 days. The cells were dyed with 6-carboxyfluorescein diacetate-acetoxymethyl ester and propidium iodine, respectively. Then, Interactive Laser Cytometer was used to detect the growing cells. RESULTS: The urethral epithelial cells grew and proliferated very well in the collagen-chitosan complex vector. After the urethral epithelial cells grew in the collagen-chitosan complex vector for 3 and 7 days, the fluorescent density amount of the surviving cells were (1.09 +/- 0.13) x 10(8) and (2.04 +/- 0.13) x 10(8), respectively. However, after 14 and 21 days, the fluorescent density amount of the surviving cells was (0.55 +/- 0.09) x 10(8) and (0.47 +/- 0.03) x 10(8), respectively. There was a significant difference when compared with the amount of the surviving cells at 3 and 7 days (P < 0.05). CONCLUSION: Using Interactive Laser Cytometer for measurement of the green and red fluorescent densities of different waves, the activity of the cultured urethral epithelial cells in vitro can be rapidly measured with the in situ quantitation method. This method solves a difficult problem of observing the growing cells in an opaque vector. The dynamic growing state of the engineering tissues can be observed.

[Combined superficial temporal fascial flap with free dermis-fat graft to reconstruct hemifacial atrophy].

OBJECTIVE: To introduce a method to reconstruct hemifacial atrophy (Romberg's disease). METHODS: Through a temporal incision, the compound grafts of pedicled superficial temporal fascial flap and free dermis-fat were inserted into the cheek to correct soft tissue depression on the face. The dermis-fat was harvested from gluteal crease site. RESULTS: 6 cases were treated with this technique. 3 to 10 months' follow-up showed satisfactory results and few resorption of the compound grafts. CONCLUSIONS: The mentioned technique is simple and reliable in reconstructing bulk defects of the face.

[Repairing the defects in the chest, back and axilla with a split-breast flap].

OBJECTIVE: To evaluate a method to repair the defects after the secondary tumor excision and radiation ulcer in the chest, back and axilla. METHODS: Eight patients, with the defects after the secondary tumor excision and the radiation ulcer in the chest, back and axilla, were undergoing the treatment. A "T" shape incision or up-side-down "T" shape incision was designed above the breast or along the inframammary fold below breast, just close to the defect. A split-breast flap was raised above the pectoralis major or deep fascia. The defect was then repaired with a rotating and advancing way. RESULTS: Eight patients were repaired in one stage. Blood circulation of the flaps was abundant except one with distal edge necrosis. The ptosis breast was corrected and the fullness of the chest wall was also achieved. But, the Nipple of the opposite health breast was lost the original position to the lateral or medial. CONCLUSIONS: The above-mentioned technique may be an efficient method to repair the defects after the secondary tumor excision and radiation ulcer in the chest, back and axilla. It is adapt to the old patients whose health is worse, but it is not good for the young patients resulted from the injury breast.

[Urethral epithelium culture by using L929 cells as trophoderm in vitro].

OBJECTIVE: To study the technique and method of urethral epithelium culture in vitro, so as to lay the groundwork for reconstructing a tissue engineering urethra and to provide an experimental model of urethral mucosa in physiological, pathological, toxicological and microbiological study. METHODS: The urethral mucosa from a young male New Zealand hare that had just been out of milk, was digested into single cell liquid with Dispase II and mixed enzyme, and the fibroblast were removed. After being seeded, the cells were cultured by using L929 cells as trophoderm. The medium was changed regularly and the cells were subcultured when they grew to mix together 80% to 90%. The cultured cells were analyzed with histochemistry, immunohistochemistry dyeing and flow cytometry examination. We observed the ultrastructure of cells with scanning electron microscope and transmission electron microscope. RESULTS: The primary cultured cells fused when they had been cultured for about ten days. They were the same in size like road rocks. The cultured cells were all epithelial cells without fibroblasts and were diploid cells. The cells could be subcultured 11-13 generations, and could survive 50-60 days. CONCLUSION: The urethral epithelium of young New Zealand hare can be cultured in vitro and maintain the ability to proliferate within a certain time. The study result not only sets a role in reconstructing a tissue engineering urethral mucosa, but also provides an experimental model for the research of urethral mucosa in vitro.

[Autologous buccal mucosal graft for urethral reconstruction].

OBJECTIVE: To search for a new method for urethra reconstruction using autologous buccal mucosal graft while lacking of local skin. METHODS: Since 1998, a total of 25 patients with complex hypospadias have been treated using buccal mucosal grafts for urethral reconstruction. The reconstructed urethra was anastomosed with the meatus half year later. RESULTS: All the reconstructed urethra survived without contracture or stricture except one infection, which healed with no adverse consequence. CONCLUSION: The key points for operation success is rich capillary network, thick epidermis and thin lamina propria of the buccal mucosa. Buccal mucosa is an excellent tissue for urethral reconstruction.

[Complication of polyacrylamide hydrogel injection for facial plasty].

OBJECTIVE: To investigate the causes of complications of polyacrylamide hydrogel injection for facial plasty and reliable treatments. METHODS: Eight patients were included in the study. Some of them were examined by MRI. All the patients received surgical treatments. RESULTS: The injected polyacrylamide hydrogel was found in the superficial layer of the superficial temporal fasica, the loose connective tissue below the deep temporal fascia, the subcutaneous tissue or the orbicularis muscle. Polyacrylamide hydrogel injected into the superficial layer of the superficial temporal fascia could spread to the face along the SMAS. Polyacrylamide hydrogel injected into the loose connective tissue below the deep temporal fascia could spread down to the cheek. The patients' symptoms were relieved with the operation. Satisfactory results were obtained. CONCLUSION: Polyacrylamide hydrogel injection does not adapt to facial plasty. The reliability of polyacrylamide hydrogel injection for facial plasty is in doubt.

[Early repair of full layer eyelid defect caused by chemical burn].

OBJECTIVE: To investigate the optimal time and method of the early repair of the full layer eyelid defect caused by chemical burn. METHODS: Free nasal septum mucosal cartilage flap with muscle flap, skin grafting, or skin flap were performed in 18 cases (19 eyelids) with chemical burn within 4 postburn weeks. Eyelid reconstruction and corneal transplantation were performed at the same time in 4 patients. RESULTS: All the reconstructed eyelids and transplanted cornea survived. The incidence of severe complications, such as exposure keratitis, corneal ulcer and eyeball perforation decreased. CONCLUSION: Full layer eyelid defect caused by chemical burn should receive early reconstruction and repair, including timely reconstruction of eyelid for the sake of protecting the eyesight and of alleviating the inflammatory reactions, and the corneal transplantation should be done at the same time to avoid corneal perforation. Nasal septum mucosal cartilage flap could be ideal for the eyelid reconstruction.

[Evaluation of the facial artery and vein using color Doppler imaging].

OBJECTIVE: The purpose of this study was to provide the guidelines with respect to the location of the facial vessels, observe the potential reversed flow of the facial artery, and reemphasize the value of color Doppler ultrasound studies in flap planning. METHODS: A study was carried out to investigate the location and dynamics of the facial artery and vein using color Doppler ultrasonography in 12 adults. RESULTS: The facial artery and the vein were located together at the lower border of the mandible. Around the oral commissure and under the nasal ala, they run apart from each other at variable distances. The reverse flow was observed in the 12 patients after the blood flow of the facial artery was blocked by applying pressure manually at the lower border of the mandible. CONCLUSIONS: The divergence of the facial vein from the artery is important information in planning of axial pattern flaps. Observation of the reversed flow confirms the possibility of safe elevation of a retrograde flow-arterialized flap based on the distal portion of the facial artery.

[A study of oral epithelial cells cultured with L929 cells as seed cells in vitro].

OBJECTIVE: To purpose of study aimed at investigating the technique of culturing oral epithelia in vitro and to set up an experimental model for further reconstructing oral mucosa in vitro. METHODS: The oral mucosa was taken from young New Zealand rabbits, and the mucosa was digested with enzyme and suspended in liquid to form cellular suspension. Being seeded, the cells were cultured motionlessly. The medium was changed regularly and the cells were subcultured. RESULTS: The cultured cells were all epithelial cells without fibroblasts, and they were proved to be diploid cells. The cells were subcultured in 1-13 generations which survived for 50-60 days. CONCLUSION: The oral epithelium of young New Zealand rabbit can be cultured in vitro, maintaining the ability to proliferate in a certain period. It is a pilot study to reconstruct oral tissue in vitro.