| Objective:1.To investigate the feasibility of cisplatin combine serum-free suspension culture method in screening and enriching of cancer stem cells in lung adenocarcinoma;2.To determine the expression difference of mi R-192 between lung adenocarcinoma cells and CSCs;3.To detect the function of mi R- 192 in regulation of stem cells in lung adenocarcinoma cancer. Methods:1.Screen PC9 lung adenocarcinoma cancer cells with cisplatin,and culture in serum-free DMEM/F12 medium for 3 weeks.2.Draw growth curve of CSCs and PC9 cells by CCK-8,and compare the difference;Compare the clone-ability difference between CSCs and PC9 cells by Colony-forming assay;Flow cytometry is used to test the difference of SP proportion between CSCs and PC9 cells; Detect the expression difference of stem markers between CSCs and PC9 cells by RT-PCR and Western-blot technology.3.Detect the expression differences of mi R- 192 between CSCs and PC9 cells by RT-PCR technique.4.Detect the difference of shpere-forming ability of PC9 cells and CSCs by suspension culture method after mi R-192 mimic or mi R-192 inhibitor transfection;CCK-8 method is used to draw the growth curve of PC9 cells and CSCs after mi R-192 mimic or mi R-192 inhibitor transfection;Flow cytometry technology is used to detect the difference of SP proportion in CSCs after mi R-192 or mi R-192 inhibitor transfection; Flow cytometry technology is used to detect the difference of cell cycle proportion in PC9 cells after mi R-192 mimic transfection;Detect the expression difference of stem markers in PC9 cells and CSCs by the RT-PCR and Western blot technology after mi R-192 mimic or mi R-192 inhibitor transfection. Results:1.After cisplatin screening combine serum-free culturing for 3 weeks, CSCs show obvious morphology difference from parental PC9 cells.2.The cell growth curve and Colony-forming assay demonstrate that CSCs have greater ability of proliferation than parental PC9 cells(P<0.000),(P=0.003).3.SP proportion in PC9-CSCs is significantly increased compared with parental PC9 cells(13.16±1.12% vs.1.21±0.64%)(P<0.000).4.The m RNA expressions of ABCG2, CD133, Oct4, Nanog are significantly increased in PC9-CSCs compared to these of parental PC9 cells,increased to 3.44±0.12,2.06±0.11,5.51±0.20,6.04±0.22 times respectively(P<0.000~0.000).5.The protein expressions of abcg2, cd133, oct4 and nanog are significantly increased in PC9-CSC compared to these of parental PC9 cells. The grey values of PC9-CSC increased to 1.54 ± 0.13,1.19 ± 0.08,1.372 ± 0.11,1.31 ± 0.12 times respectively compared to these of parental PC9 cells(P=0.002~0.012);The grey value of internal reference protein beta-actin has same expression level(P=0.196).6.The expression of mi R-192 in CSCs is significantly higher than that of PC9 cells,and is 4.21±0.46 times compared to that of PC9 cells(P<0.000).7.The sphere-forming ability of PC9 is increased after mi R-192 mimic transfection compared to the control group(17.00±3.00 vs.54.22±13.53)(P=0.009).The sphere-forming ability of PC9-CSCs is increased after mi R-192 mimic transfection compared to the control group(153.667±14.744 vs.107.00±11.00)(P=0.008).The sphere-forming ability of PC9-CSCs is not significantly decreased after mi R-192 inhibitor transfection compared to the control group(92.00±7.00 vs.112.333±12.51)(P=0.406).8.Growth curve shows that the proliferation ability of PC9 is enhanced after mi R-192 mimic transfection compared to the control group(P<0.000);Growth curve shows that the proliferation ability of PC9-CSC is enhanced after mi R-192 mimic transfection compared to the control group(P<0.000);Growth curve shows that the proliferation ability of PC9-CSCs is weakened after mi R-192 inhibitor transfection compared to the control group(P<0.000).9.SP proportion in PC9-CSC is increased after mi R-192 mimic transfection compared to the control group(16.32±0.87%vs.13.26±0.74%)(P=0.018). SP proportion in PC9-CSCs is decreased after mi R-192 inhibitor transfection compared to the control group(9.43±0.71%vs.12.76±0.54%)(P=0.002).10.The the result of cell cycle shows that G2/M phase ratio of PC9 is obviously increased after mi R-192 mimic transfection compared to the control group(28.1%vs13.4%)(P<0.000).11.The m RNA expressions of ABCG2, CD133, Oct4, Nanog are significantly increased in PC9 after mi R-192 mimic transfection,increased to 2.19±0.12,2.06±0.11,3.21 ± 0.21,2.89 ± 0.27 times respectively( P<0.000~0.001);The m RNA expressions of ABCG2, CD133, Oct4, Nanog are significantly increased in PC9-CSCs after mi R-192 mimic transfection,increased to 1.61±0.14,1.42±0.13, 1.49±0.17,1.34±0.12 times respectively(P=0.004~0.033); The m RNA expressions of ABCG2, CD133 are decreased in PC9-CSC after mi R-192 inhibitor transfection,decreased to 0.73±0.09,0.62±0.14 respectively as many these as the control group(P=0.020~0.041).The reduction of Oct 4, Nanog are not obvious(P>0.05).12.According to the results of Western blot,the protein expression of cd133 is increased remarkably in PC9 after mi R-192 mimic transfection.The grey values of it is increased 1.18±0.04 times compared to that of control group(P<0.002).The grey values of abcg2, oct4, nanog have no significant difference compared to these of control group(P>0.05);The protein expression of oct4, nanog are increased remarkably in PC9-CSCs after mi R-192 mimic transfection.The grey values of them are increased 1.12±0.04,1.13±0.03 times compared to these of control group(P=0.003~0.014).The grey values of abcg2, cd133 have no significant difference compared to these of control group(P>0.05);The protein expression of abcg2, cd133, oct4, nanog are decreased remarkably in PC9-CSCs after mi R-192 inhibitor transfection.The grey values of them are decreased to 0.88±0.04,0.90±0.04, 0.87±0.02,0.90±0.02 respectively as many these as the control group(P=0.001~0.026);The grey values of internal reference protein beta-actin has same expression level(P > 0.05). Conclusion:1.Cisplatin combine serum-free suspension culture method can screen and enrich cancer stem cells in PC9 lung adenocarcinoma;2.CSCs have greater ability of proliferation, higher proportion of SP cells, higher expression of the stem markers;3.The expression of mi R-192 increased obviously in the CSCs, and might play a regulator role in lung adenocarcinoma cancer stem cells. |
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