| 【BACKGROUND AND OBJECTIVE】:Oleanolic acid(OA) is a natural compound from plants with anti-tumor activities. However, the mechanism of the inhibitory effect of OA on cell cycle progression has not been completely explored. We employed several lung carcinoma cell lines to investigate the cell cycle-related molecular pathway affected by OA.The data revealed that OA suppressed the proliferation of lung cancer cells in both dose- and time-dependent manners, along with an increase in mi R-122 abundance. The suppression of mi R-122 abolished the effect of OA on lung cancer cells. CCNG1 and MEF2 D, two putative mi R-122 targets, were found to be downregulated by OA treatment. Restoring their expression counteracted the effect of OA on lung carcinoma cells. OA was further shown to induce the expression of mi R-122-regulating transcriptional factors in lung cancer cells. Collectively, OA induced cell cycle arrest in lung cancer cells through mi R-122/Cyclin G1/ MEF2 D pathway. This finding may contribute to the understanding of the molecular mechanism of OA’s antitumor activity.【METHODS】:1. The proliferation rates of A549、NCI-H460、NCI-H1299、NCI-H1299、Primary LC1、Primary LC1 and MRC-5 which were treated by in time- and dose-dependent fashions were tested by MTT assays.2. The level of mi RNA 122 of A549、NCI-H460、NCI-H1299、NCI-H1299、Primary LC1、Primary LC1 and MRC-5 which were treated by in time- and dose-dependent fashions were tested by Quantitative-PCR.3. The decline in mi R-122 level by the inhibitor of mi RNA122, the effect of OA on lung cancer cells was observed.4. We wanted to discuss the effect of OA inhibited the proliferation of tumor by mi R-122, the level of CCNG1 and MEF2D was tested by Western Blot.5. We transfered pc DNA3-CCNG1-pc DNAMEF2 D to the cell line of A549 and Primary LC1,the level of CCNG1 and MEF2D was tested by Western Blot.6. We wanted to discuss the effect of OA increased the level of mi R-122, the cell line of A549 was treated by OA in time- and dose--dependent fashions, the protein level of HNF1α、H NF3β、H NF4αand HNF6 was was tested by Western Blot at last.7. We built the mouse mode of lung cancer, and treated the mouse with different dose of OA, to observe the volume of the tumor, tested the level of mi R-122 by Quantitative-PCR and the protein level of CCNG1 and MEF2D by western bolt in the tumor.【RESULTS】:1. The cell line of A549、NCI-H460、NCI-H1299、NCI-H1299、Pri mary LC1、Pri mary LC1 and MRC-5 was treated by OA in the dose of 0ug/ ml、15ug/ ml、30ug/ ml、60ug/ ml,cultured after 48 h, we found that OA significantly reduced the proliferation of the tested cells by different extents(A549, 10.7–39 %; NCIH460, 1.3–41.8 %; NCI-H1299, 2–50.4 %; primary lungcancer 1, 4.5–64.3 %; primary lung cancer 2, 1.8–46.9 %) in a dose-dependent manner, but there was no change in the cell line of MRC-5. We also found than OA suppressed their proliferation in a time-dependent manner at the concentration of 30 lg ml-1. In addition, normal lung fibroblast cell lines, MRC-5, had no significant reduction in its proliferation rate when OA was added to the media.2. The cell line of A549、NCI-H460、NCI-H1299、NCI-H1299、Pri mary LC1、Pri mary LC1 and MRC-5 was treated by OA in the dose of 0ug/ ml、15ug/ ml、30ug/ ml、60ug/ ml,cultured after 48 h. Compared with the group of 0ug/ ml, the level of mi RNA 122 was no different change in the group of 15ug/ ml, but increased in the group of 30ug/ ml(P<0.05) and obviously increased in the group of 60ug/ ml(P<0.01). By the same time,we found than those cell lines wa s treated by30ug/ ml in different times, the results indicate than compared with the group of 0ug/ ml, the level of mi RNA 122 was no different change in the group 24 h, but increased in the group of 48h(P<0.05) and obviously increased in the group of 72h(P<0.01).3. The decline in mi R-122 level by the inhibitor of mi RNA122, the effect of OA on lung cancer cells. The results indicated than compared with the group of Antagomir/OA(-/-), the level of mi RNA 122 was obviously increased in the group of(-/+)(P<0.01), the rate of proliferation was decreased(P<0.05).but there were no changes in the group of(+/-)and(+/+).4. We further wanted to discuss the effect of OA inhibited the proliferation of tumor by mi R-122, the cell line of A549 and Primary LC1 was treated by OA in the dose of0ug/ ml、15ug/ ml、30ug/ ml、60ug/ ml after 48 h. The results indicated than compared with the group of 0ug/ ml, the protein level of CCNG1 was decreased in the group of 15ug/ ml(P<0.05), obviously decreased in the group of 30ug/ ml and 60ug/ ml(P<0.01).Compared with the group of 0ug/ ml, the protein level of MEF2 D was obviously changes in the group of 15ug/ ml,30ug/ ml and 60ug/ ml( P<0.01). The cell line of A549 and Primary LC1 was treated by30ug/ ml OA in the time-dependent fashions, The results indicated than compared with the group of 0h, the protein level of CCNG1 and MEF2 D was decreased in the group of 24h(P<0.05), obviously decreased in the group of 48 h and 72h(P<0.01).5. We transfered pc DNA3-CCNG1-pc DNAMEF2 D to the cell line of A549 and Primary LC1,the cell line of A549 and Primary LC1 was treated by OA in the dose of 30ug/ ml,cultured 48 h. The results indicated than compared with the group of Antagomir/OA(-/-), the protein level of CCNG1 and MEF2D was obviously increased in the group of(-/+)and(+/+)(P<0.01), but the group of(+/-) obviously decreased(P<0.01). The rate of proliferation indicated than compared with the group of Antagomir/OA(-/-), the group of(-/+) decreased(P<0.05), and there was no different in the group of(+/-) and(+/+).6. The cell line of A549 was treated by OA in the dose of 0ug/ ml 、15ug/ ml、30ug/ ml、60ug/ ml,cultured 48 h. Western bolts results indicated than compared with the group of 0ug/ ml, the protein level of HNF1α、H NF3β、H NF4αwas no changes in the group of 15ug/ ml, but the protein of HNF6 decreased(P<0.05),those protein obviously decreased in the group of 30ug/ ml and 60ug/ ml(P<0.01).The cell line of A549 was treated by 30ug/ ml OA in the time-dependent fashions, The results indicated than compared with the group of 0h,the protein level of HNF1α、H NF3β、H NF4αwas no changes in the group of 24 h,but the protein of HNF6 dec reased(P<0.05)the protein of HNF1α、H NF3β、H NF4α、H NF6 obviously decreased in the group of 48 h and72h(P<0.01).7. We built the mouse mode of lung cancer, and treated the mouse with OA in the low dose( 40ug/ ml) and high dose( 120ug/ ml), fed 35 days. The results indicated than compare with the CON group, the volume of tumor in the group of low dose( 40ug/ ml) was decreased, and obviously decreased in the group of high dose( 120ug/ ml)(P<0.05); Compare with the Con group, the level of mi RNA 122 in the group of low dose( 40ug/ ml) was no changes, but he group of high dose( 120ug/ ml)obviously decreased(P<0.01); Compare with the Con group, the protein level of CCNG1 in the group of low dose( 40ug/ ml) was decreased( P<0.05), obviously decreased in the group of high dose( 120ug/ ml)(P<0.05); by the same time, the protein level of MEF2 D in the group of low dose( 40ug/ ml) was no different, but the high group obviously decreased(P<0.01);【CONCLUSION】: 1. Oleanolic acid can decreased the rate of lung carcinoma cell proliferation, increased the level of mi R-122.2. Oleanolic acid can decreased the protein level of CCNG1 and MEF2 D.3. Oleanolic acid suppresses the lung tumor by mi R-122/CCNG1/ MEF2 D axis. |
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