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Functional analysis of let-7b in Basigin expression and cellular physiology of melanoma cells and endometrial cells in mice
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|摘要:||MicroRNAs（miRNAs）為內源性非蛋白質編碼（endogenous nonprotein-coding）之小分子RNAs，其長度約為18-25個核苷酸（nucleotides），已被證實可藉由鹼基配對方式（base-pairing）對基因進行轉錄後調控（post-transcriptional regulation），並參與多種不同的生理過程。Basigin（Bsg）表現於細胞膜上，為一細胞黏附因子，又稱作extracellular matrix metalloproteinase inducer（EMMPRIN）。研究發現，Bsg可表現於癌細胞，當發生腫瘤轉移（metastasis）時，Bsg促使周圍纖維母細胞或腫瘤細胞產生基質金屬蛋白酵素（matix metalloproteinases, MMPs）。依miRGen資料庫分析比對可知，let-7家族均為調控bsg之候選miRNA，其中尤以let-7b互補之程度最高。藉由構築含有bsg 3’UTR之luciferase報導基因載體，將其轉染至具有內源性表現let-7b之小鼠黑色素瘤細胞（B16-F10 cells）中，發現let-7b可直接與bsg 3’UTR結合而達到抑制luciferase表現之效果。以西方吸漬法分析經轉染let-7b之B16-F10 cells，發現可顯著抑制Bsg與MMP-9之表現，同時亦具有抑制腫瘤細胞轉移與生長之功能。此外，亦探討於小鼠懷孕過程中，let-7b於子宮內膜中可能所扮演之角色。let-7b於著床前之內膜上皮細胞中之表現顯著上升，然基質細胞則於懷孕第1日最高，而後顯著下降，於第4日再顯著上升。以寡核苷酸序列LNATM（Locked Nucleic Acid）作為probe進行偵測，可知let-7b表現於懷孕小鼠子宮（第6日至第8日）之著床點。小鼠懷孕期間血液中具高濃度的助孕素（progesterone, P4）與低濃度的雌激素（17β-estradiol, E2），以雌激素、助孕素培養未發身（prepubertal）小鼠之內膜上皮與基質細胞，發現上皮細胞於培養72 h後，P4與E2處理組之let-7b，其表現顯著上升；基質細胞於培養96 h後，以P4處理組表現最高，且可抑制其細胞增生。再者，轉染let-7b於懷孕第7日之內膜基質細胞可抑制其生長，且顯著抑制Bsg與MMP-9之表現。綜合上述，可知let-7b為一腫瘤抑制因子，可抑制細胞增生與Bsg之表現，且於小鼠著床過程可能扮演重要角色。|
The endogenous nonprotein coding microRNAs (miRNAs) of 18-25 nucleotides (nt) have been shown to involve in a wide variety of cellular processes as the posttranscriptional regulators by base-pairing to their target mRNAs. Basigin (Bsg), is highly expressed on the cell surface as an adhesion molecule, which is found in the tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (MMPs). Therefore, Bsg is also called extracellular matrix metalloproteinase inducer (EMMPRIN). In this study, we postulated that Bsg was a potential target of let-7b according to the computer analysis in miRGen database. Based on the result of luciferase assay, we proved that let-7b could interact with the 3'UTR of Bsg directly; The immunoblotting analysis revealed that the protein level of Bsg in mouse melanoma cell line B16-F10 was also down-regulated by let-7b. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. According to real-time RT-PCR analysis, the expression of let-7b in the epithelial cells increased gradually from day 1 to day 4 of pre-implantation stages and reached the highest level on day 4. On the other hand, the highest level of let-7b in the stromal cells was observed on day 1, although the expression was decreased on day 2 and increased significantly on day 4. By in situ hybridization, let-7b was also found to express in the uteri during days 6-8 of pregnancy. The endometrial cells isolated from prepubertal mice were treated with steroid hormones, progesterone (P4), estradiol (E2) and P4 plus E2. After 96 h of culture in the presence of steroid hormones, the expression levels of let-7b were increased in the endometrial cells, although significant differences were only observed after P4 treatment in the stromal cells and after individual E2 and P4 treatments in the epithelial cells. In association with the increased let-7b expression, the cell proliferation slope, measured by MTT assay, significantly decreased in the presence of P4 and P4 plus E2 than that in the groups of non-hormone and E2 treatments during 72-108 h of culture. Results from the transfection of let-7b into the stromal cells isolated from day 4 pregnant mice or prepubertal mice demonstrated that let-7b attenuated the proliferation during the periods of time examined. After transfection of let-7b into mouse stromal cells isolated from day 7 of pregnancy, the expression of Bsg was suppressed, as well as MMP-9. In conclusion, this study clarifies the expression pattern of let-7b in the uterine epithelial and stromal cells during pre-implantation stages in mice, as well as the inhibitory effect of let-7b associated with steroid hormones on stromal cell proliferation and on the expression.
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