1. Regulatory effect of designed saRNAs on the expression of KLK1 in different prostate cell lines
To activate KLK1 expression through RNAa in human prostate cells, we have designed 6 saRNAs and each duplex was transfected into RWPE-1, WPMY-1, BPH-1 and DU-145 cells. KLK1 gene expression was evaluated via qRT-PCR and western blot 72 hours later. As shown in Fig. 1, we found that in the normal prostate cell lines RWPE-1 and WPMY-1, all saRNAs were difficult to up-regulate the expression of KLK1, while in the prostate tumor cell lines BPH-1 and DU-145, saKLK1-374 and saKLK1-463 had an up-regulation effect on the expression of KLK1. We chose saKLK1-374 with a higher up-regulation amplitude in BPH-1 and DU-145, for subsequent experiments.
It should be noted that BPH-1 was only used in the preliminary screening of effective saRNAs. After we discovered the unexpected results described above, the main purpose was to learn the difference between normal cells and tumor cells in RNAa. However, to reduce the difficulty of research, currently we only used malignant tumor cell lines as the representative of tumor cell lines rather than including the benign tumor cell line BPH-1 into the research. In subsequent experiments, DU-145 and 22RV-1 were used as representatives of prostate malignant tumor cell lines.
2. The chemical modification also failed to enable saKLK1-374 to activate KLK1 expression in normal prostate cell lines
To improve the stability of saKLK1-374 and rule out saKLK1-374 lost its function due to enzyme degradation, we created a localized modified saKLK1-374 (saKLK1-374-2'F) in which all cytidine and uridine nucleosides within the guide strand contained a 2'-fluoro-modified ribose sugar. The saKLK1-374 and saKLK1-374-2'F were transfected into cells by the same method as above. As shown in Fig. 2, saKLK1-374-2'F also could not up-regulate the expression of KLK1 in normal prostate cell lines RWPE-1 and WPMY-1. In prostate tumor cell lines, saKLK1-374-2'F had the same effect of up-regulating KLK1 expression as saKLK1-374. And it was worth noting that in this experiment, fluorine modification could not reverse the ineffectiveness of saRNA, nor could it significantly change the up-regulation amplitude of genes by saRNA. This meant that in this experiment, there was no failure of gene up-regulation due to the loss of activity of saRNAs.
3. In normal prostate cells, saP21-322 and saP21-322-2'F could not activate p21WAF1/CIP1
Using another gene, p21WAF1/CIP1, as the target of exogenous saRNA could help us eliminate many problems (but not absolutely). Under the same experimental operation, if the RNAa of other target genes was successful, then our conjecture was certainly wrong. But if RNAa for p21WAF1/CIP1 also failed in normal prostate cell lines, then the answer becomes unpredictable. As shown in Fig. 3, the saP21-322 and saP21-322-2'F still could not up-regulate the expression of p21WAF1/CIP1 in normal prostate cell lines RWPE-1 and WPMY-1, but they could up-regulate the expression of p21WAF1/CIP1 in prostate tumor cell lines DU-145 and 22RV-1. The RNAa experiments with the two target genes of p21WAF1/CIP1 and KLK1 yielded similar results, making us suspect that the key lay in the cell rather than the target gene.
The last step of RNAa is to bind the exogenous saRNA to the promoter of the target gene to promote the transcription of the target gene. The epigenetic characteristics of target gene promoters are uncertain, and their epigenetic characteristics may be different in different types of cells from the same tissue. The saP21-322 is a saRNA that has been successfully reported to activate the expression of p21WAF1/CIP1 in prostate cancer cell lines. However, it is not certain that saP21-322 would bind to p21WAF1/CIP1 promoter in normal prostate cells, because we did not detect the difference in the degree of methylation of the p21WAF1/CIP1 promoter between normal prostate cells and prostate cancer cells. If you need to detect the methylation degree of the target gene promoter every time you try RNAa, it will undoubtedly significantly increase the cost of RNAa experiments. To answer the question " Are genes more easily up-regulated by exogenous saRNA in tumor cells? ", the core still needs to return to normal cells and tumor cells themselves. Therefore, in this article, we did not study the difference in the degree of methylation of KLK1 and p21WAF1/CIP1 promoters in normal prostate cells and prostate cancer cells, or test any other genes via RNAa on these cells. Rather, it was assumed that the failure of RNAa occurs in the process before the exogenous saRNA binds to the promoter of the target gene.
4. RNAa experimental results of KLK1 and p21WAF1/CIP1 in bladder cell lines
It was necessary to conduct RNAa experiments in cells derived from tissues other than the prostate to verify our conjecture. We used the bladder-derived cell lines available in our laboratory, which were the normal urothelial cell line SV-HUC-1, and the bladder cancer cell lines T24 and 5637. RNAa experiments were performed for KLK1 and p21WAF1/CIP1 in these cells. As shown in Fig. 5A, in all bladder cell lines, neither saKLK1-374 nor saKLK1-374-2'F up-regulated KLK1 expression. We speculated that this result was due to the extremely low expression of KLK1 in bladder cells. We detected the basic expression of KLK1 mRNA in prostate cells and bladder cells by qRT-PCR, and found that the expression of KLK1 in bladder cells was significantly lower than that in prostate cells (Fig. 5B, left). Therefore, only qRT-PCR rather than western blot was performed to detect changes in KLK1 expression. The extremely low expression in the basal state means that the KLK1 gene is inhibited by a powerful silencing mechanism in bladder cells. While RNAa is epigenetic regulation of a targeted promoter, and it cannot counteract powerful silencing mechanisms. Therefore, it was foreseeable that saKLK1-374 could not activate KLK1 expression in all bladder cell lines.
Different from KLK1, p21WAF1/CIP1 was significantly up-regulated by saP21-322 and saP21-322-2'F in bladder cancer cell lines T24 and 5637 (about 3–4 times as much as mock; Fig. 5D&E), while the up-regulation rate is lower in normal urothelial cell line SV-HUC-1 (about twice as much as mock; Fig. 5C). Unlike KLK1, the expression of p21WAF1/CIP1 in prostate and bladder cell lines is relatively normal. We detected the basic expression of p21WAF1/CIP1 mRNA in prostate cells and bladder cells by qRT-PCR (Fig. 5B, right). Therefore, the activation effect of the p21WAF1/CIP1 would not be affected by the gene itself. In the bladder, tumor cells had a higher magnitude of RNAa effect than normal cells, and it seemed that this phenomenon should also be explained in the process before the saRNA bonded to the target gene.
5. Prolonging the time that the exogenous saRNA acted on the cell did not change the effect of the saRNAs
RNAa usually takes effect more slowly than RNAi. RNAi usually has obvious gene down-regulation 24 hours after transfection, while the obvious gene up-regulation of RNAa appears 72 hours after transfection. The poor RNAa performance in normal cell lines might be caused by the insufficient duration of saRNA action. Therefore, we extended the time to 7 days and and detected the expression level of the target gene every 24 hours. To obtain more credible results and appropriately simplify the experiment, we only performed the RNAa experiments for KLK1 in prostate cells, and only performed the RNAa experiments for p21WAF1/CIP1 in the bladder cell line. As shown in Fig. 6A (upper), saKLK1-374 could not up-regulate the expression of KLK1 in normal prostate cell lines RWPE-1 and WPMY-1 for 7 days after transfection. However, in the prostate cancer cell lines DU-145 and 22RV-1, the expression of KLK1 reached a peak on the 3rd day after saKLK1-374 transfection, and fluctuated around this peak in the following 4 days (Fig. 6A, lower). In the 3 bladder cell lines, the expression of p21WAF1/CIP1 peaked on the 3rd day after saP21-322 transfection, and fluctuated in the vicinity of the peak 4 days later (Fig. 6B). This meant that extending the time could not change the phenomenon that "normal cells were more difficult to achieve effective RNAa". And we could speculate that normal cells might lack certain components necessary for RNAa.
6. The difference in transfection efficiency of exogenous saRNA between normal cells and tumor cells
The process of RNA activation is that the exogenous saRNA crosses the cell membrane into the cytoplasm with the assistance of the carrier, and then enters the nucleus with the help of some key proteins such as Ago2 and targets the promoter to work. After excluding the target gene and saRNA, the cause of RNAa failure should be found in the process of exogenous saRNA entering the nucleus from the culture medium.
RNAiMAX has been the best cationic-lipid transfection reagent currently available for dsRNA. First, exogenous saRNA was combined with the cationic-lipid transfection reagent and added to the cell culture medium. Because of the cell membrane, only part of the saRNA-lipid complexes could enter the cytoplasm. The amount of saRNA-lipid complexes entering the cytoplasm could be assessed by fluorescently labeled dsRNA. As shown in Fig. 7, we detected the content of fluorescently labeled dsControl-5cy3 (red) entering the cytoplasm in four prostate cell lines and 3 bladder cell lines. After transfection with the same concentration of dsControl-5cy3, prostate cancer cell lines DU-145 and 22RV-1 took in more dsRNA-5cy3 than normal prostate cell lines RWPE-1 and WPMY-1 (Fig. 7A). After zooming in on the photo, it could be found that the nuclei of prostate cancer cell lines DU-145 and 22RV-1 have also taken in more dsRNA-5cy3 than normal prostate cell lines RWPE-1 and WPMY-1 (Fig. 7B). But the situation was different in the bladder cell lines. The intake of dsControl-5cy3 in bladder cancer cell lines T24 and 5637, and normal urothelial cell line SV-HUC-1 were similar (Fig. 7C). The intake of dsControl-5cy3 by the nucleus in the bladder cell lines was also similar (Fig. 7D).
Through semi-quantitative analysis, it could be found that the average single-cell intake of dsControl-5cy3 in prostate cancer cell lines was higher than that in normal prostate cell lines, while the average single-cell intake of dsControl-5cy3 in the 3 bladder cell lines was similar and also higher than that of normal prostate cell lines (Fig. 7E). It was worth mentioning that in this experiment, although the number of cells was the same in the initial procedure of seeding plate, the growth rate of different cell lines was still different, so the number of cells in each subsequent test was not equal. However, in our experiments, the growth rate of prostate cancer cell lines DU-145 and 22RV-1 was significantly faster than that of normal prostate epithelial cells RWPE-1, and was similar to the normal prostate stromal cell line WPMY-1. This meant that prostate cancer cells would have a larger number of cells in subsequent detections. In the presence of the same amount of dsControl-5cy3, assuming that the transfection efficiency of all cells was equal, the average single-cell intake of dsControl-5cy3 of prostate cancer cells could only be lower. However, the experimental results showed that the single-cell intake of dsControl-5cy3 of prostate cancer cells was higher than that of normal prostate cell lines, which means that the transfection efficiency of prostate cancer cell lines must be higher than that of normal prostate cell lines. It should be noted that the average single-cell dsControl-5cy3 intake obtained by analyzing the picture referred to the average fluorescence intensity of a single cell. However, limited by analytical methods, we could not calculate the fluorescence intensity in a single cell nucleus.
The above experimental results indicated that effective RNAa could not be achieved in the normal prostate cell lines RWPE-1 and WPMY-1 probably because they did not take in enough exogenous saRNA. However, the average single-cell intake of dsControl-5cy3 of the 3 bladder cell lines was similar, which meant that the up-regulation of p21WAF1/CIP1 gene in normal urothelial cell lines was lower than that of bladder cancer cell lines T24 and 5637 was not due to the insufficient intake of exogenous saRNA.
7. The difference of RNAa indispensable accessory protein between normal cell line and tumor cell line
Currently, Ago2 and importin 8 (IPO8) have been found to play an indispensable role in the transport of dsRNA from the cytoplasm to the nucleus. To regulate gene expression, the exogenous saRNA absorbed into the cytoplasm needs to enter the nucleus with the help of Ago2 and IPO8. Similar to the role of Ago2 in RNAi, in RNAa, Ago2 serves the role of a navigator and a recruiting platform on which an RNAa effector complex is assembled. IPO8, a member of the karyopherin family, has been identified to interact with Ago2 and localize to cytoplasmic processing body which is a structure involved in RNA metabolism, and IPO8 has been demonstrated to play a critical role in mediating the cytoplasm-to-nucleus transport of mature micro RNAs16,17.
As shown in Fig. 8, we detected the expression of Ago2 and IPO8 in all bladder and prostate cell lines by qRT-PCR and western blot. The qRT-PCR results showed that the expression of AGO2 and IPO8 was not different between untreated cells and cells transfected with dsControl, indicating that transfection of exogenous saRNA did not affect the expression of these two proteins. Among all cell lines, the expression of Ago2 and IPO8 of RWPE-1 was the lowest, which might explain the complete loss of function of RNAa in RWPE-1. In the bladder cell lines, the expression of Ago2 and IPO8 in the normal urothelial cell line SV-HUC-1 was lower than that of the two bladder cancer cell lines, was still also significantly higher than that of RWPE-1. Considering that the previous experimental results showed that the exogenous dsRNA in the cytoplasm of SV-HUC-1 was similar to the two bladder cancer cell lines, this could explain that the exogenous saRNA in the SV-HUC-1 could up-regulate the expression of p21WAF1/CIP1 at lower amplitude, instead of completely ineffectiveness. In summary, to successfully use exogenous saRNA to up-regulate target gene expression, the transfected cells must absorb enough exogenous saRNA and express enough Ago2 and IPO8. However, due to lower transfection efficiency or lower expression of Ago2 and IPO8, or both, the normal cells may not be able to effectively activate target gene expression through exogenous saRNA or had a low amplitude of up-regulation. In contrast, tumor cells generally could absorb more exogenous saRNA and had higher expression of Ago2 and IPO8. It needs to be emphasized again that our experimental results and inferences were based on in vitro experiments using cationic-lipid transfection reagents to carry exogenous saRNA.
8. saKLK1-374 caused the death of prostate cancer cells instead of normal prostate cells
While conducting the above-mentioned RNAa experiments, we accidentally discovered that in prostate cancer cell lines DU-145 and 22RV-1, a large number of floating cells appeared in the culture medium after transfection with saKLK1-374 (Fig. 9C&D, left). However, there was no significant increase in floaters in normal prostate epithelial cell line RWPE-1 and normal prostate stromal cell line WPMY-1 (Fig. 9A&B, left). This seemed to indicate that saKLK1-374 had a targeted killing ability on prostate cancer cells. To more accurately determine the inhibitory effect of saKLK1-374 on prostate cancer cell lines, we conducted a cytotoxicity test. As shown in Fig. 9 (right), saKLK1-374 and saKLK1-374-2'F did not inhibit the growth of RWPE-1 and WPMY-1 within five days after transfection, while in DU-145 and 22RV-1, cell viability started to decrease on the first day after transfection.
9. saKLK1-374 increases intracellular ROS and promotes prostate cancer cell apoptosis
We started with common cell death mechanisms and briefly studied the causes of the death of prostate cancer cells caused by saKLK1-374. As shown in Fig. 10A&B, the intracellular ROS of prostate cancer cell lines DU-145 and 22RV-1 were significantly up-regulated after saKLK1-374 transfection. The qRT-PCR results showed that the ratio of BAX/Bcl-2 in DU-145 and 22RV-1 increased after saKLK1-374 transfection (Fig. 10C&D, left). The results of qRT-PCR and western blot showed that saKLK1-374 also up-regulated Caspase3 in DU-145 and 22RV-1 (Fig. 10C&D).
Up to now, our experimental results seemed to show that saKLK1-374 up-regulated the expression of KLK1 in prostate cancer cell lines DU-145 and 22RV-1 to cause cell oxidative stress and apoptosis. However, according to many previous reports, KLK1 often played an anti-oxidative stress role and its downstream bradykinin promoted the proliferation of prostate cancer cells.
10. KLK1 could not be detected outside the cell and recombinant KLK1 did not change ROS and cell viability.
We suspected that the cause of cell death by saKLK1-374 was not the increase in KLK1 gene expression. Considering that KLK1 usually played a role after being activated by other enzymes outside the cell, we first detected the content of KLK1 protein in the prostate cancer cell culture medium after transfection with saKLK1-374. But surprisingly, the KLK1 in the medium of all samples at 72 hours, 96 hours and 120 hours after transfection of saKLK1-374 were below the minimum detection limit (The KLK1 standard could be detected normally; The annotated detection range of the Human KLK1 ELISA Kit used in this experiment is “156pg/ml − 10000pg/ml”). We also used the recombinant KLK1 protein (concentration range: 10ng/ml to 10µg/ml) to directly interfere with prostate cancer cells, but no obvious oxidative stress or cell death was found. As shown in Fig. 11B&C, the recombinant KLK1 protein had no significant effect on the viability of prostate cancer cell lines DU-145 and 22RV-1. As shown in Fig. 11A, the recombinant KLK1 protein did not upregulate ROS in DU-145 and 22RV-1.
Since KLK1 protein did not be detected outside the cells transfected with saKLK1-374, and the recombinant KLK1 protein did not have the same effect as saKLK1-374, it was almost certain that saKLK1-374 did not increase the expression of KLK1 to cause the death of prostate cancer cells.
11. Interference with KLK1 mRNA expression could not completely reverse the inhibition of saKLK1-374 on the growth of prostate cancer cells
Through previous experiments, we ruled out the possibility of extracellular KLK1 inhibiting growth of prostate cancer cells. Next, we used RNAi to investigate whether intracellular KLK1 was the cause of prostate cancer cell death. As shown in Fig. 12A&B, we tested the inhibitory effect of the 3 purchased siRNAs on KLK1 expression, and found that siKLK1-1 has the strongest inhibitory effect on both DU-145 and 22RV-1, so we chose siKLK1-1 for subsequent experiments. When saKLK1-374 activated the expression of KLK1, siKLK1-1 could also suppress the expression of KLK1 below the baseline level (compared to the mock samples; Fig. 12C&D). In the cytotoxicity test, siKLK1-1 reduced the inhibition of saKLK1-374 on prostate cancer cell lines DU-145 and 22RV-1, but the inhibition could not be completely reversed (Fig. 12E)
Considering when both saKLK1-374 and siKLK1-1 were transfected into prostate cancer cells, KLK1 mRNA was lower than the baseline level, so it is difficult to entirely attribute the cell growth inhibition still existed in this case to the increase of intracellular KLK1. Therefore, there must be other reasons for cell death besides RNAa of the KLK1 gene.