||Dysregulated microRNAs Enhance ZNF322A Expression and Transcriptional Activity in Lung Cancer
||Department of Pharmacology
研究背景: ZNF322A (zinc finger protein 322A) 是一個致癌性鋅指蛋白轉錄因子，它會使其下游基因例如Cyclin D1 (CCND1)、P53、Alpha-adducin (ADD1) 的表現失調進而促進癌細胞生長及移動能力。先前實驗室的研究指出約有70%的東西方族群肺癌病患其ZNF322A的mRNA及蛋白都有過度表達的情形。除此之外，實驗室也發現ZNF322A蛋白會受到CK1δ/GSK3/FBXW7 路徑調控而降解；然而我們對於ZNF322A mRNA的表現是透過什麼機制達成動態平衡仍是未知的。
研究目的: 本研究旨在以肺癌細胞及動物模式去探討微小核醣核酸 (microRNAs) 對於ZNF322A的轉錄調控；並以肺癌病人檢體闡明ZNF322A與其上游微小核醣核酸調控子的關聯性。
研究結果: 藉由預測軟體分析，在ZNF322A基因的3′端非轉譯區 (3′UTR) 上可能擁有miR-98-5p、miR-135a-5p、miR-326以及miR-484的結合位，為了驗證這些候選的微小核醣核酸是否會調控ZNF322A的mRNA表現，我們將這些候選的微小核醣核酸類似物 (mimics) 轉染 (transfect) 至多種肺癌細胞株中並進行後續的實驗分析。首先，我們針對這四種微小核醣核酸建構ZNF322A 3′端非轉譯區的冷光表現載體 (3′UTR luciferase reporter) 並去檢測候選的微小核醣核酸是否會抑制冷光表現。在這四個候選微小核醣核酸中，只有miR-326以及miR-484可以抑制野生型 (wild-type) 的3′端非轉譯區的冷光活性 (luciferase activity)，但是將miR-326與miR-484的預測結合位做突變 (mutation) 則冷光活性就不會受到miR-326與miR-484所抑制。再者，miR-326 mimics的大量表現會抑制ZNF322A的mRNA與蛋白表現；但是miR-484 mimics僅在mRNA層級去抑制ZNF322A表現而非蛋白層級。重要地，miR-326會透過減少ZNF322A下游標的CCND1及ADD1表現來抑制肺癌細胞的生長及爬行能力。在重建實驗 (reconstitution) 我們發現miR-326/ZNF322A/CCND1路徑對於細胞生長調控非常重要，而miR-326/ZNF322A/ADD1路徑則調控細胞移動能力。臨床檢體的檢測上，我們發現ZNF322A mRNA與miR-326的表現在肺癌病人上有著負相關的趨勢，此外，我們的分析結果也指出低表達ZNF322A/高表達miR-326 (ZNF322A-L/miR326-H) 的病人族群擁有較好的整體存活期(overall survival)。
結論： 我們的結果顯示了miR-326/ZNF322A路徑藉由減少CCND1與ADD1來抑制腫瘤進程 (tumor progression)。除此之外，腫瘤組織中低ZNF322A/高miR-326 (ZNF322A-L/miR326-H) 的表達形式 (profile) 在未來臨床應用上可能可以作為一個有潛力的生物標記(biomarker)。
Background: ZNF322A is an oncogenic zinc-finger transcription factor, which dysregulates genes in control of cell growth and cell motility such as cyclin D1 (CCND1), p53 and alpha-adducin (ADD1). Our published reports show that overexpression of ZNF322A mRNA and protein is found in about 70% of Asian and Caucasian lung cancer patients. In addition, ZNF322A protein degradation is regulated in part by the CK1δ/GSK3/FBXW7 axis. However, the mechanism involved in homeostasis of ZNF322A mRNA remains unclear.
Purpose: This study aims to investigate the microRNAs regulation on ZNF322A transcription in lung cancer cell and xenograft models. In addition, we explore the relationship between ZNF322A and its upstream miRNA regulators using clinical studies.
Results: The 3′UTR of ZNF322A contained target sites for microRNAs miR-98-5p, miR-135a-5p, miR-326 and miR-484 according to three prediction software or database. To validate the reciprocal transcription regulation of microRNA candidates on ZNF322A mRNA expression, multiple lung cancer cells were transfected with these indicated miRNAs mimics. We constructed 3′UTR luciferase reporter assay for all four miRNA candidates. Among them, miR-326 and miR-484 inhibited the ZNF322A wild-type 3′UTR luciferase activity, but the 3′UTR luciferase activity with the mutated seed regions of the corresponding microRNAs had no response to miR-326 or miR-484. Furthermore, expression of both ZNF322A mRNA and protein was attenuated by overexpressing of miR-326 mimics, but miR-484 inhibited ZNF322A expression only at the mRNA level, not protein level. Importantly, miR-326 suppressed proliferation and migration ability through inhibition of expression of ZNF322A and its downstream target genes CCND1 and ADD1 in lung cancer cells. Reconstitution experiments indicated that miR-326/ZNF322A/CCND1 was important for cell growth regulation while miR-326/ZNF322A/ADD1 was important for cell motility. Clinically, a trend of inverse correlation between miR-326 and ZNF322A mRNA expression was observed in lung cancer patients. Moreover, low ZNF322A/ high miR-326 (ZNF322A-L/miR326-H) profile showed better overall survival.
Conclusions: Our results reveal that miR-326/ZNF322A axis inhibits lung cancer progression by reducing CCND1 and ADD1 expression. Furthermore, our clinical studies suggest that low ZNF322A/ high miR-326 (ZNF322A-L/miR326-H) profile may be a potential prognosis biomarker for clinical application in the future.
I. Lung cancer
(A). Epidemiology of lung cancer 1
(B). Genetic alteration in lung cancer 1
II. MicroRNAs (miRNAs) in cancer
(A). Overview of miRNAs 2
(B). miRNAs in lung cancer 2
(C). Biomarkers and therapeutic strategies of miRNAs in cancer 3
(D). miR-326 in cancer 4
(E). miR-484 in cancer 4
III. Transcription regulation of ZNF322A in lung cancer cells
(A). Structure and function of ZNF322A 5
(B). ZNF322A in lung cancer progression 5
IV. The roles of centrosomal protein 170 (CEP170) and protein O-mannose kinase (SGK196)
(A). The function of CEP170 6
(B). The function of SGK196 7
Study basis and specific aims 8
Materials and methods 10
I. Cell lines and culture conditions 10
II. miRNA target prediction software programs 10
III. Transfection of plasmids and miRNA mimics 10
IV. 3′UTR reporter construct and site-directed mutagenesis 10
V. 3′UTR reporter assay 11
VI. Dual luciferase promoter activity assay 11
VII. RNA extraction and quantitative reverse transcriptase-polymerase
chain reaction (RT-qPCR) assay 12
VIII. TaqMan miRNA RT-qPCR assay 12
IX. Protein extraction 12
X. Western blot 13
XI. Cell proliferation assay 13
XII. Foci formation assay 14
XIII. Transwell migration assay 14
XIV. Tail vein injection metastasis assay in vivo 14
XV. Clinical samples of lung cancer patients 15
XVI. Statistical analysis 15
I. Identification of candidate miRNAs that may target on ZNF322A 3′UTR
(A). The schematic diagram of candidate miRNAs selected from
miRNA target prediction software 16
(B). miR-326 and miR-484 inhibited the ZNF322A wild-type 3′UTR luciferase activity, but the 3′UTR with the mutated seed regions was not responding to the corresponding miRNAs 16
II. miR-326 inhibited ZNF322A expression and cancer cell proliferation and migration ability in vitro
(A). miR-326 downregulated expression of ZNF322A mRNA and protein 18
(B). miR-326 attenuated lung cancer cell proliferation and
migration ability in vitro 18
III. miR-326/ZNF322A/cyclin D1 axis regulated lung cancer cell growth in vitro
(A). miR-326 attenuated cyclin D1 (CCND1) mRNA and protein expression 19
(B). miR-326 decreased CCND1 expression through ZNF322A
transcriptional regulation in vitro 19
(C). miR-326 suppressed cancer cell proliferation through miR-326/ZNF322A/CCND1 axis in vitro 20
IV. miR-326/ZNF322A/alpha-adducin axis regulated tumor metastasis of lung cancer
(A). miR-326 decreased alpha-adducin (ADD1) expression through ZNF322A transcriptional regulation in vitro 20
(B). miR-326 attenuated lung cancer cell migration ability through miR-326/ZNF322A/ADD1 axis in vitro 21
(C). miR-326 inhibited lung tumor metastasis through miR-326/ZNF322A/ADD1 axis in vivo 21
V. miR-326 expression negative correlated with ZNF322A mRNA level in lung cancer patients
(A). miR-326 expression negative correlated with ZNF322A mRNA level in lung cancer patients 22
(B). The patients with low ZNF322A/ high miR-326 (ZNF322A-L/miR-326-H) expression profile showed the better overall survival than other patients 23
VI. miR-484 negatively regulated ZNF322A mRNA expression
(A). miR-484 downregulated ZNF322A mRNA without changing the protein expression of ZNF322A 24
(B). miR-484 did not inhibit mRNA expression of FBXW7α, the E3-ligase of ZNF322A 25
VII. ZNF322A activated promoter activity of CEP170 and SGK196 genes
(A). ZNF322A activated promoter activity of CEP170 and SGK196 25
(B). ZNF322A transcriptionally activated expression of CEP170 gene, but not SGK196 gene 26
I. Dysregulated miR-326/ZNF322A/CCND1 axis and miR-326/ZNF322A/ADD1 axis contribute to lung tumorigenesis 27
II. Low ZNF322A/ high miR-326 (ZNF322A-L/miR326-H) profile may be selected as a potential prognosis biomarker 28
III. miR-484 may inhibit other ZNF322A protein ubiquitination pathways in lung cancer 29
IV. ZNF322A promoted CEP170 promoter activity and mRNA expression, while ZNF322A promoted SGK196 promoter activity but not the SGK196 mRNA expression 30
V. Conclusion and perspectives 31
Appendix Figures 68
Appendix Tables 71
Table 1. The plasmids and their characteristics used in the current study 40
Table 2. The primers used in the current study 42
Table 3. Antibodies and their reaction conditions used in the current study 44
Table 4. Alteration of ZNF322A mRNA and miR-326 expression in relation to clinicopathological parameters in 120 lung cancer patients’ samples 45
Table 5. Cox regression analysis of risk factors for cancer-related death in lung cancer patients 46
Figure 1. The schematic diagram of candidate miRNAs selected from miRNA target prediction software 49
Figure 2. miR-326 and miR-484 inhibited the ZNF322A wild-type 3′UTR luciferase activity, but the 3′UTR with the mutated seed regions was not responding to the corresponding miRNAs 51
Figure 3. miR-326 downregulated expression of ZNF322A mRNA and protein 53
Figure 4. miR-326 attenuated lung cancer cell proliferation and migration ability in vitro 54
Figure 5. miR-326 attenuated CCND1 mRNA and protein expression 56
Figure 6. miR-326 decreased CCND1 expression through ZNF322A transcriptional regulation in vitro 57
Figure 7. miR-326 suppressed cancer cell proliferation through miR-326/ZNF322A/CCND1 axis in vitro 58
Figure 8. miR-326 decreased ADD1 expression through ZNF322A transcriptional regulation in vitro 59
Figure 9. miR-326 attenuated lung cancer cell migration ability through miR-326/ZNF322A/ADD1 axis in vitro 60
Figure 10. miR-326 inhibited lung tumor metastasis through miR-326/ZNF322A/ADD1 axis in vivo 61
Figure 11. miR-326 expression negative correlated with ZNF322A mRNA level in lung cancer patients 62
Figure 12. miR-484 downregulated ZNF322A mRNA without changing the protein expression of ZNF322A 64
Figure 13. miR-484 did not inhibit mRNA expression of FBXW7α, the E3-ligase of ZNF322A 65
Figure 14. ZNF322A enhanced promoter activity of CEP170 gene and SGK196 gene 66
Figure 15. The schematic model of miR-326/ZNF322A/CCND1 axis and miR-326/ZNF322A/ADD1 axis in regulation of lung tumor growth and metastasis 67
Appendix Figure 1. The biogenesis and function of miRNA 69
Appendix Figure 2. The experimental procedure of 3′UTR reporter assay used in this study 70
Appendix Table 1. The well-known suppressor-miRNAs and onco-miRNAs in lung cancer 72
Appendix Table 2. The downstream targets of miR-326 or miR-484 in lung cancer 74
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