Objective: To investigate the effect of P62 on the migration and motility of human epidermal cell line HaCaT in high glucose microenvironment and its possible molecular mechanism, so as to explore the mechanism of refractory diabetic foot wound healing. Methods: The method of experimental research was used. HaCaT cells in logarithmic growth phase was taken for experiment. The cells were collected and divided into normal control group (culture solution containing glucose with final molarity of 5.5 mmol/L) and high glucose (culture solution containing glucose with final molarity of 30.0 mmol/L) 24 h group, high glucose 48 h group, and high glucose 72 h group according to the random number table (the same grouping method below). The cells in normal control group were routinely cultured for 72 h, cells in high glucose 72 h group were cultured with high glucose for 72 h, cells in high glucose 48 h group were routinely cultured for 24 h then cultured with high glucose for 48 h, cells in high glucose 24 h group were routinely cultured for 48 h then cultured with high glucose for 24 h. Then the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal control group and high glucose group. After being correspondingly cultured for 48 h as before, the protein expression of P62 was detected by immunofluorescence method (indicated as green fluorescence). The cells were collected and divided into negative control small interfering RNA (siRNA) group, P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group, and transfected with the corresponding reagents. At post transfection hour (PTH) 72, the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group. After the corresponding treatment, the protein expression of P62 was detected by Western blotting at PTH 72 h, the cell migration rate was detected and calculated at 24 h after scratching by scratch test, with the number of samples being 9; and the range of cell movement was observed and the trajectory velocity was calculated within 3 h under the living cell workstation, with the number of samples being 76, 75, 80, and 79 in normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group, respectively. The cells were collected and divided into normal glucose+phosphate buffered solution (PBS) group, high glucose+PBS group, and high glucose+N-acetylcysteine (NAC) group. After the corresponding treatment, the protein expression of P62 at 48 h of culture was detected by Western blotting and immunofluorescence method, respectively. Except for scratch test and cell motility experiment, the number of samples was all 3 in the rest experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: Compared with the protein expression in normal control group, the protein expressions of P62 of cells in high glucose 24 h group, high glucose 48 h group, and high glucose 72 h group were significantly increased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose group was stronger than that in normal control group. At PTH 72, compared with the protein expression in negative control siRNA group, the protein expressions of P62 of cells in P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group were significantly decreased (P<0.01). At PTH 72, compared with the protein expression in normal glucose+negative control siRNA group, the protein expression of P62 of cells in normal glucose+P62-siRNA group was significantly decreased (P<0.01), while the protein expression of P62 of cells in high glucose+negative control siRNA group was significantly increased (P<0.01); compared with the protein expression in high glucose+negative control siRNA group, the protein expression of P62 of cells in high glucose+P62-siRNA group was significantly decreased (P<0.01). At 24 h after scratching, compared with (55±7)% in normal glucose+negative control siRNA group, the cell migration rate in normal glucose+P62-siRNA group was significantly increased ((72±14)%, P<0.01), while the cell migration rate in high glucose+negative control siRNA group was significantly decreased ((37±7)%, P<0.01); compared with that in high glucose+negative control siRNA group, the cell migration rate in high glucose+P62-siRNA group was significantly increased ((54±10)%, P<0.01). Within 3 h of observation, the cell movement range in high glucose+negative control siRNA group was smaller than that in normal glucose+negative control siRNA group, while the cell movement range in normal glucose+P62-siRNA group was larger than that in normal glucose+negative control siRNA group, and the cell movement range in high glucose+P62-siRNA group was larger than that in high glucose+negative control siRNA group. Compared with that in normal glucose+negative control siRNA group, the cell trajectory speed in normal glucose+P62-siRNA group was significantly increased (P<0.01), while the cell trajectory speed in high glucose+negative control siRNA group was significantly decreased (P<0.01); compared with that in high glucose+negative control siRNA group, the cell trajectory speed in high glucose+P62-siRNA group was significantly increased (P<0.01). At 48 h of culture, compared with that in normal glucose+PBS group, the protein expression of P62 of cells in high glucose+PBS group was significantly increased (P<0.01); compared with that in high glucose+PBS group, the protein expression of P62 of cells in high glucose+NAC group was significantly decreased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose+PBS group was stronger than that in normal glucose+PBS group, while the green fluorescence of P62 of cells in high glucose+NAC group was weaker than that in high glucose+PBS group. Conclusions: In HaCaT cells, high glucose microenvironment can promote the protein expression of P62; knockdown of P62 protein can promote the migration and increase the mobility of HaCaT cells; and the increase of reactive oxygen species in high glucose microenvironment may be the underlying mechanism for the increase of P62 expression.
Humans ; RNA, Small Interfering/genetics* ; Cell Line ; Epidermis ; Glucose/pharmacology* ; Epidermal Cells