| Objective:To explore the possible mechanism of Hypoxia Inducible Factor-1α in the proliferation of lung cancer cells induced by human umbilical cord-derived mesenchymal stem cells under hypoxia,and to explore its role in the development of lung cancer.Methods:(1)Isolation,culture and expansion of primary hucMSCs: fresh umbilical cords of newborns were taken,and mesenchymal stem cells of umbilical cord tissue were isolated by tissue adherence method,and immunophenotypic analysis was performed by flow cytometry.(2)According to the effect on the proliferation of lung adenocarcinoma A549,it was divided into the following 6 groups:(1)normoxia A549 cell group,(2)normoxic A549 cells and hMSCs co-culture group,(3)hypoxic A549 cell group,(4)hypoxic A549 cells and hMSCs co-culture group,(5)hypoxic interferes with HIF-1α gene A549 cell group,(6)hypoxic interferes with HIF-1α gene A549 cells and hMSCs co-culture group.(3)Real-time PCR and western blot were used to detect the expression level of gene and protein in A549 cells,A549 cells co-cultured with hMSCs,A549 cells after interfering with HIF-1α gene,A549 cells co-cultured with hMSCs after interfering with HIF-1α gene under normoxia and hypoxia.MTT assay was used to detect the effect of h MSCS on the proliferation of lung cancer A549 cells under normoxia and hypoxia.The cell scratch test was used to detect the different proliferative capacity of each group of cells.Resluts:(1)Mesenchymal stem cells were isolated from the umbilical cord by tissue block adherence.About 2 weeks after the growth,the spindle cells began to climb out from around the umbilical cord.About 3 weeks later,the cells gradually merged and spread the T-25 cm2 culture flask.It can be observed that cells form colonies,spiral growth,and the morphology is consistent after passaging cells to 3rd generation.Flow cytometry showed that HucMSCs expressed stromal cell-associated surface markers CD73,CD90,CD105;did not express the markers CD14,CD19,CD34 on the surface of monocytes,lymphocytes and hematopoietic stem cells.The test results are consistent with the phenotypic characteristics of mesenchymal stem cells.(2)MTT assay showed that A549 cells co-cultured with hMSCs under hypoxia showed the strongest proliferative power;followed by A549 cells under hypoxia.The least proliferative was A549 cells that interfered with the HIF-1α gene co-cultured with hMSCs.Compared with normal oxygen state,the proliferation of A549 cells was increased in the hypoxic state(p<0.05).Under hypoxia,hMSCs could further increase the proliferation of A549 cells(p<0.05).Whether it is hypoxic conditions or hMSCs co-culture conditions,interferencing with HIF-1αcan inhibit proliferation.(3)The cell scratch test showed that the proliferative ability of the co-cultured group with hMSCs decreased under normoxia;the proliferative ability increased under hypoxic conditions;the proliferation ability of hMSCs co-culture was further enhanced under hypoxia;the interference of HIF-1α gene proliferation under hypoxia Decrease in ability.The proliferation ability after co-culture with hMSCs was not significantly enhanced and attenuated.(4)The expression of survivin was higher in A549 cells under hypoxia than in normoxic A549 cells(2.0798±0.1498 vs 1±0.1441),the difference was significant on statistically(p<0.01);A549 and MSC under hypoxia The survivin gene in the co-culture group was higher than the A549 cell group that interfered with HIF-1αgene and the A549 cell and MSC co-culture group that interfered with HIF-1α gene(2.4835±0.2021 vs 0.6321±0.1502;2.4835±0.2021 vs 0.8882± 0.1134),and the difference was significant on statistically(p < 0.01).There was no statistically significant difference in the expression level of co-cultured genes between A549 cells andMSCs that interfered with HIF-1α gene under hypoxia(0.6321±0.1502 vs0.8882±0.1134,p>0.05).The protein expression of HIF-1α in the A549 cell group under hypoxia was higher than that in the normoxic A549 cell group(1.2462±0.0127 vs 1.0856±0.1757),and the difference was significant on statistically(p<0.01);The protein expression of HIF-1α in A549 and MSC co-culture group was higher than that in A549 cells under hypoxia(1.8983±0.0394 vs 1.2462±0.0127),and the difference was significant on statistically(p<0.01);A549 and The MSC co-culture group was higher than the A549 cell group that interfered with HIF-1α gene and the A549 cell and MSC co-culture group that interfered with HIF-1α gene(1.8983±0.0394 vs 0.9669±0.0331;1.8983±0.0394 vs 1.1037±0.0363).And the difference was significant on statistically(p <0.01).There was no statistically significant difference in the expression level of co-cultured genes between A549 cells andMSCs that interfered with HIF-1α gene under hypoxia(0.9669±0.0331 vs 1.1037±0.0363,p>0.05).The expression of survivin protein in A549 cells was higher than that in normoxia A549 cells(0.9854±0.0213 vs 0.8284±0.0200),and the difference was significant on statistically(p<0.01).A549 and MSC co-culture The expression of A549 cells was higher in the hypoxia group(1.0956±0.0278 vs 0.9854±0.0213),and the difference was significant on statistically(p<0.01).The protein expression of survivin in A549 and MSC co-cultured groups interfered with HIF.The A549 cell group of-1α gene and A549 cells interfering with HIF-1α gene were higher in the co-culture group than MSC(1.0956±0.0278 vs 0.5276±0.0069;1.0956±0.0278 vs0.7554±0.0328),the difference was significant on statistically(p<0.01).There was no statistically significant difference in the expression level of co-cultured genes between A549 cells andMSCs that interfered with HIF-1α gene under hypoxia(0.5276±0.0069 vs 0.7554±0.0328,p>0.05).Conclusion:Under hypoxic conditions,hMSCs can cause over-expression of HIF-1α and survivin in A549 cells,resulting in continuous proliferation of lung cancer cells. |
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