Engineering Success
1. The LncRNA MALAT1 can act as “sensor ” to detect cervical cancer.
Our project aim to effectively diagnose the cervical cancer patients. LncRNAs are generally more than 200 nucleotides long but they are rarely able to translated into protein. In cervical cancer research, lncRNA MALAT1 are identified as tumor driving oncogenic lncRNA [1]. They are involved in cell growth, apoptosis, cell migration and invasiveness as well as radioresistense. microRNAs (miRNAs) are short (∼23nt) single-stranded non-coding RNAs that act as potent post-transcriptional gene expression regulators[2]. Previous studies indicated miRNAs also play an important role in the pathogenesis of cervical cancer. For example, miR-145 was significantly downregulated in the cervical cancer patients and could be potential biomarker of cervical cancer[3]. lncRNAs could bind to specific miRNA and act as sponges to compete miRNAs[4]. This mechanism gives rise to our idea about fusing a sponge RNA based on the sequences of lncRNA MALAT1 with binding sites complementary to the sequence of miRNA to a plasmid that has reporter gene, pepper for instance, which will monitor the expression of miRNA in the cells.
We use the “Pepper” plasmid containing the part sequence of LncRNA MALAT1 in order to reflect miRNA expression in vivo. lncRNAs can interact with miRNAs as “sponges”. And the fluorescence reflect the miRNA expression in the cervical cancer in turn. We tested the sensitivity and specificity of the vectors in Hela cells. In the future, the miRNA-LncRNA MALAT1 complex could be used to screen and detect the cervical cancer. The patients could benefit from our work.
2. miR-145/ miR-22 could bind with LncRNA MALAT1
According to a database mircode (www.mircode.org), we found miR-145 and miR-22 could bind to the sequence of MALAT1. Moreover, a growing number of studies highlights the role of miR-145/miR-22 in cervical cancer development. Given the fact that lncRNA could act as miRNA sponge, we designed the sequence that could bind to miR-145/miR-22 based on the MALAT1 sequence (Fig 1).
Fig 1. The diagram of miR-145/miR-22 sensor
3. Construction of the miR-145-sponge-pepper/ miR-22-sponge-pepper plasmids
We constructed four plasmids miR-145-sponge-pepper, pre-miR-145, miR-22-sponge-pepper,and pre-miR-22. The plasmids of miR-145-sponge-pepper and miR-22-sponge-pepper were used to monitor the expression of miR-145/miR-22 in cells. Pre-miR-145 andpre-miR-22 were applied to overexpress miR-145/miR-22 in cells. These plasmids were synthesized by Nanjing Genscript Biotechnology corporation.
The map of the original pepper plasmid was listed below (Fig 2). The plasmid is cut by specific restriction endonuclease enzyme. Part sequence of MALAT1 (contain the miR-145/miR-22 binding site) was amplified by Polymerase Chain Reaction (PCR) technique. After the amplification, linear pCDNA3-MCS-nPepper and the part sequence of MALAT1 were ligated by T4 DNA ligase. The recombinant plasmids were then transformed into competent E. coli and screened the positive clones on LB solid plates containing ampicillin (Fig 3). To test whether the target fragment was inserted in the plasmid, miR-145-sponge-pepper and miR-22-sponge-pepper was analyzed on 1% Agarose Gel (Fig 4.). Moreover, the recombinant plasmids were also confirmed by using Sanger sequencing (Fig 5) .
Fig 2.Map of pCDNA3-MCS-nPepper plasmids
Fig 3.Solid plates of miR-145-sponge-pepper and miR-22-sponge-pepper
Fig4.Electrophoresis of miR-145-sponge-pepper (miR-145 sensor, left), miR-22-sponge-pepper (miR-22 sensor, middle) and original pepper plasmid (right)
Fig 5. The miR-145-sponge-pepper (up) and miR-22-sponge-pepper (down) sensor validated by Sanger sequencing
4.1.1 Function verification of miR-145 sensor
In order to test the ability of miR145 sensor, we transfected miR-145-sponge-pepper and pre-miR-145 (overexpress miR-145 in cells) into Hela cells. The control group only transfected with miR-145-sponge-pepper (2 ug), the experimental group transfected with both miR-145-sponge-pepper (2 ug) and pre-miR-145 (1 ug). 48 hours later, we add 2 μm HBC fluorescent dye into per well. After incubation for 2 hours, cells were harvested and the green fluorescence was measured by plate reader (SpectraMax i3). The result showed that miR-145 could inhibit the fluorescence of Pepper in cells transfected with miR-145-sponge-pepper (Fig 6). The result suggested that miR-145 sensor can detect the alteration of miR-145 expression in cells.
Fig 6. The images of Hela cells transfected with different plasmids. (A). miR-145-sponge-pepper were transfected into Hela cells. (B) miR-145-sponge-pepper and pre-miR-145 were transfected into Hela cells
4.1.2 The sensitivity of miR-145 sensor
To further test the sensitivity of miR-145-sponge-pepper (miR-145 sensor) as a monitor to detect the expression of miR-145, Hela cells were transfected with the same amount of miR-145-sponge-pepper and different amount of pre-miR-145 (0 ug, 0.5 ug, 1ug, 2ug) (Fig 7). Down-regulation of green fluorescence value was observed in cervical cancer cells transfected with different concentration of pre-miR-145 compared with control cells (Table1). Moreover, the fluorescence was significantly decreased in a dose dependent manner (p<0.05, Fig 8). Based on the values in cell treated with different concentration of miRNA-145, the standard curve of the relationship between fluorescence and pre-miR-145 amount were made by EXCEL. The correlation coefficient (R2 value) of miRNA-145 was 0.9495. The linear fitting graph equation is y=-19962x+228042(Fig 9).
Fig 7. Hela cells were transfected with different miR-145 in 24-well plates.
1 The value of fluorescence of miR-145 sensor
Fig 8. The value of green fluorescence in cells. Hela cells were co-transfected with miR-145-sponge-pepper and different amount of miR-145 for 48 h (* mean p<0.05 ).
Fig 9. The standard curve of miR-145-sponge-pepper in Hela cells
4.2.1 Function verification of miR-22 sensor
In order to test the ability of miR22 sensor, we transfected miR-22-sponge-pepper and pre-miR-22 (overexpress miR-22 in cells) into Hela cells. The control group only transfected with miR-22-sponge-pepper (2 ug), the experimental group transfected with both miR-22-sponge-pepper (2 ug) and pre-miR-22 (1 ug). 48 hours later, we add 2 μm HBC fluorescent dye into per well. After incubation for 2 hours, cells were harvested and the green fluorescence was measured by plate reader (SpectraMax i3). The result showed that miR-22 could inhibit the fluorescence of Pepper in cells transfected with miR-22-sponge-pepper (Fig 10). The result suggested that miR-22 sensor can detect the alteration of miR-22 expression in cells.
Fig 10. The images of Hela cells transfected with different plasmids.
(A). miR-22-sponge-pepper were transfected into Hela cells. (B) miR-22-sponge-pepper and pre-miR-22 were transfected into Hela cells
4.2.2 The sensitivity of miR-22 sensor
To further test the sensitivity of miR-22-sponge-pepper (miR-22 sensor) as a monitor to detect the expression of miR-22, Hela cells were transfected with the same amount of miR-22-sponge-pepper and different amount of pre-miR-22 (0 ug, 0.5 ug, 1ug, 2ug) (Fig 11). Down-regulation of green fluorescence value was observed in cervical cancer cells transfected with different concentration of pre-miR-22 compared with control cells (Table 2). Moreover, the fluorescence was significantly decreased in a dose dependent manner (p<0.05, Fig 12). Based on the values in cell treated with different concentration of miRNA-22, the standard curve of the relationship between fluorescence and pre-miR-22 amount were made by EXCEL. The correlation coefficient (R2 value) of miRNA-22 was 0.9958. The linear fitting graph equation is y=-86524x+ 2*10^6 (Fig 13).
Taken together, both miR-145 sensor and miR-22 sensor can act as “sensor” to monitor the cervical cancer progression. When it comes to the sensitivity and fluorescence intensity, miR-22 sensor is better than miR-145 sensor.
Fig 11.Hela cells were transfected with different miR-22 in 24-well plates.
2 The value of fluorescence of miR-22 sensor
Fig 12. The value of green fluorescence in cells. Hela cells were co-transfected with miR-145-sponge-pepper and different amount of miR-22 for 48 h (* mean p<0.05 ).
Fig 13. The standard curve of pcDNA3-miR-22-sponge-npepper in Hela cells
References
[1] Hao Chenjun,Lin Shaodan,Liu Ping et al. Potential serum metabolites and long-chain noncoding RNA biomarkers for endometrial cancer tissue.[J] .J Obstet Gynaecol Res, 2023, 49: 725-743.
[2] Tornesello Maria Lina,Faraonio Raffaella,Buonaguro Luigi et al. The Role of microRNAs, Long Non-coding RNAs, and Circular RNAs in Cervical Cancer.[J] .Front Oncol, 2020, 10: 150.
[3] Zhang Xian-Yu,Ma Huan,Li Jing et al. Functional implications of miR-145/RCAN3 axis in the progression of cervical cancer.[J] .Reprod Biol, 2020, 20: 140-146.
[4] Tornesello Maria Lina,Faraonio Raffaella,Buonaguro Luigi et al. The Role of microRNAs, Long Non-coding RNAs, and Circular RNAs in Cervical Cancer.[J] .Front Oncol, 2020, 10: 150.
