Effects Of HOTAIR On Proliferation And Differentiation Of ESCs As Well As On Healing Of Deep Second-degree Burn Wounds In Mice

Objective:Current studies have shown that HOTAIR(lnc RNA)can maintain proliferation and multidirectional differentiation potential of stem cell,but effect of HOTAIR on ESCs has not been studied.Currently,studies including our research group have shown that HOTAIR is closely related to epidermal tissue development and wound repair.Base on the above,we plan to further detect the effect of HOTAIR on ESCs by using biphasic regulation system of lentivirus,cell function experiment in vitro and q PCR.Meanwhile,we plan to explore the effect of ESCs transplantation of HOTAIR overexpression on wound repair.It will provide new ideas and theoretical basis for further searching for new targets in clinical treatment.Methods:1.To obtain the wound tissues of deep second-degree burn patients and their normal skin tissues.The total RNA was extracted by Trizol.qRT-PCR was used to observe HOTAIR expression levels in wound tissues and skin tissues.2.To obtain cutaneous tissues from the trunk of female BALB/c rats,and the ESCs were isolated by trypsinization and type?collagen adhesion.The second passage of cells in logarithmic growth phase were selected in the following experiments(1)?(5).(1)The morphology of cells in culture was observed under inverted microscope of 200-fold.Flow cytometry were employed to detected the changes of cells markers(CD14,CD34,CD44 and CD45).(2)qRT-PCR was used to observe HOTAIR expression levels in lentivirus-transfected ESCs.The above cells were subsequently grouped according to m RNA expression levels of HOTAIR:pc-HOTAIR,pc-vector group,sh-HOTAIR group,sh-NC group and the con-ESCs.(3)MTT assay were employed to test the optical density(OD)values of the cells at24 h,48 h,72 h and 96 h after one batch of cells were cultured.(4)ESCs were cultured for 10 d,images of the stained cells were then obtained and the percentage of BrdU-positive cells was calculated.(5)qRT-PCR were employed to observe expression of CK10 at 24 h,48 h,72 h and 96 h after one batch of cells were cultured.One batch of cells were cultured for 10 days and the m RNA expressions of NANOG in the cells were observed by qRT-PCR.The number of samples of each batch of cells at each time point is 3.3.BALB/c rats no matter male and female were selected,and a circular,deep second-degree burn cutaneous wound of 10 mm in diameter on the dorsal skin of each rat was made by 100°C electric copper plate tip.The above rats were selected in the following experiments(1)?(2).(1)12 injured rats were selected,and three rats were taken on post burn day 0,3,7 and 14 according to the random number table.qRT-PCR was employed to evaluate the expression levels of HOTAIR during burn wound healing.(2)According to the random number table,80 BALB/c rats were divided into four groups:i)HOTAIR-overexpressing ESCs(the pc-HOTAIR group);ii)ESCs with blank lentivirus transfection(the pc-vector group);iii)ESCs without lentivirus transfection(the con-ESCs group);and iv)physiological saline solution without ESCs(the control group).The above four groups were immediately and respectively injected 20?l phosphate buffer solution(PBS)containing ESCs(0.8×10~6cells,HOTAIR-overexpressing ESCs or not)and 20?l PBS after wound,one times a day,and continued for 3 days.According to the random number table,five rats in each group were taken to observe and record the wound healing status on post injection day(PID)0(immediate),3,7 and 14 and the wound healing rates on PID0,3,7 and 14 were calculated.Then five rats of each group were respectively sacrificed on on PID 0,3,7 and 14 for harvesting the tissue of wounds.The skin structure was observed by hematocylin-eosin staining.The histological scores,which were given to each slide,were calculated at days.Date were processed with analysis of variance of factorial design,LSD-t test,and Bonferroni correction.Results:1.The expression of HOTAIR on the wound tissue of deep second-degree burn patients(25 cases)was(29.28±2.07),which was significantly higher than(1.11±0.11)of the normal skin tissue of the above patients(P<0.05).2.Cells in culture are distributed in slow-glowing clusters.The cell clusters in culture were large and round where cells are mainly cells with large and round nucleus and few cytoplasm.These results accorded with the morphological characteristics of ESCs.3.The above cells showed strongly positive for CD44((95.1±2.06)%).Expressions of CD45,CD34 and CD14 were negative((0.22±0.03)%,(0.17±0.05)%and(0.18±0.06)%).These cells were identified as ESCs.4.The results of MTT assay revealed that:i)OD values of the cells in pc-HOTAIR group at 24 h,48 h,72 h and 96 h after culture were significantly higher than that of the cells in pc-HOTAIR group and con-ESCs(P<0.05).ii)OD values of the cells in sh-HOTAIR group at 24 h,48 h,72 h and 96 h after culture were significantly lower compared with that of the cells in sh-NC group and con-ESCs(P<0.05).iii)OD values of the cells in pc-vectorgroup,sh-NC group and the con-ESCs at 24 h,48 h,72 h and 96 h after culture were not significantly different(P>0.05).5.The results of BrdU assay revealed that:i)the percentage of BrdU-positive cells in pc-HOTAIR group at 10 d after culture was(64.18±1.42)%,which was significantly higher than that in pc-HOTAIR group and con-ESCs((45.82±2.74)%,(46.60±2.46)%)(P<0.05).ii)the percentage of BrdU-positive cells in sh-HOTAIR group at 10 d after culture were significantly lower compared with that in sh-NC group and con-ESCs((44.13±4.09)%,(46.60±2.46)%))(P<0.05).6.The results of qRT-PCR revealed that:i)There was no significant difference of CK10 m RNA expression among the groups at 24 h after culture.ii)At 48 h,72 h and 96 h after culture,CK10 m RNA expression of the cells in pc-HOTAIR group were significantly lower than that of pc-HOTAIR group and con-ESCs(P<0.05),while that of the cells in sh-HOTAIR group were significantly higher compared with that of cells in sh-NC group and con-ESCs(P<0.05).7.The results of qRT-PCR revealed that:The expression of NANOG in pc-HOTAIR group at 10 d after culture was(3.53±0.07),which was significantly higher than that of pc-HOTAIR group and con-ESCs((1.22±0.05),(1.41±0.03))(P<0.05).The expression of NANOG in sh-HOTAIR group at 10 d after culture were significantly lower compared with that in sh-NC group and con-ESCs((1.25±0.08),(1.41±0.03))(P<0.05).8.Expression of HOTAIR is elevated during wound healing.The expression level of HOTAIR at day 0 post-injury was low.Along with wound healing,the expression of HOTAIR gradually increased and peaked at day 7 post-injury and remained at a significantly higher level until day 14 post-injury compared with the baseline level at day 0 post-injury.(both P<0.05).9.On PID 0,the wounds of rats in four groups were approximately similar,which were round-like wound.On PID 3,normal skin tissues of wound margins slightly contracted in pc-HOTAIR group,which were earlier than those in the other three groups.On PID 7,normal skin tissues of wound margins significantly contracted and the new generated epidermis partly covered wounds in pc-HOTAIR group,normal tissues around wound slightly contracted and few epithelization were detected in wound margin in control group.The wound state of pc-vector group and con-ESCs group were between pc-HOTAIR group and control group.On PID 14,the new generated epidermis almose covered all wounds in pc-HOTAIR group,while normal skin tissues of wound margins significantly contracted and the new generated epidermis covered most of wounds in pc-vector group and con-ESCs group and a few of wounds were unhealed in control group.On PIN 3,the wound healing rates of four groups were close(P>0.05).On PIN 7 and 14,the wound healing rates of pc-HOTAIR group were respectively(36.13±1.65)%and(86.77±3.16)%,which were significantly higher than that of the other three groups(P<0.05).On PIN 7and 14,the wound healing rates of pc-vector group and con-ESCs group were respectively((19.90±2.99)%,(54.28±5.12)%)and((21.89±1.50)%,(51.98±4.32)%),which were significantly higher than that of control group((10.90±1.15)%,(29.87±2.73)%)(P<0.05).10.On PID 0,the burn lesions of wounds of rats in four groups were confined to the superficial dermis of the skin.On PID 3,formation of granulation was observed in pc-HOTAIR group,which was earlier than those in the other two groups.On PID 7,a large number of epidermal cells and the new generated epidermis and hair follicles were detected in pc-HOTAIR group,while few of the new generated epidermis was detected in pc-vector group and con-ESCs group.On PID 14,the newly generated epidermal cells covered all wounds in pc-HOTAIR group,while the newly generated epidermal cells covered part of the wounds in control group and the wound state of pc-vector group and con-ESCs group were between pc-HOTAIR group and control group.On PIN 3,the histological scores of four groups were close(P>0.05).On PIN7 and 14,the histological scores of pc-HOTAIR group were respectively(8.17±0.31)%and(9.67±0.21)%,which were significantly higher than that of the other three groups(P<0.05).On PIN 7 and 14,the histological scores of pc-vector group and con-ESCs group were respectively((6.00±0.37)%,(7±0.36)%)and((5.83±0.40)%?(7.33±0.42)%),which were significantly higher than that of control group((3.50±0.22)%?(5.0±0.26)%)(P<0.05).Conclusions:1.The expression of HOTAIR was significantly different between human burned tissue and normal skin tissue.During wound healing,HOTAIR expression showed obvious temporal expression characteristics.This suggests that HOTAIR may be involved in wound healing.2.The upregulation of HOTAIR expression can maintain the high proliferation ability and multidirectional differentiation potential of epidermal stem cells in vitro.This suggests that HOTAIR may be involved in wound healing.This indicates that HOTAIR has a regulatory effect on epidermal stem cells.3.Epidermal stem cells targeting the overexpression of HOTAIR were proved to promote the re-epithelialization of the wound and shorten the repair time.