Aptamers Evoloved From Human Gastric Tumor Cells And Human Liver Cancer Cells And Its Clinical Applications

Human gastric cancer and human liver cancer has been top to the "first health killer". Recently, cancers been treated were come to the later stage, the existed methods had little effect on treatment, so the early diagnosis of cancer may has a good curative effect. In the present study, we use human gastric cancer cells and human liver cancer cells as the targets, the aptamers were screened via cell-SELEX, and their values in the diagnosis of human gastric cancer and human liver cancer were preliminarily evaluated, in which to lay a foundation for establishing a new method for human gastric cancer and human liver cancer diagnosis and treatment.Aptamers selected against human gastric cancer cells:In this part, we chosen SGC7901human gastric cancer cells as the target cells, GES-1were used as negative cells. Using cell-SELEX technology, under the optimized SELEX conditions, we performed10rounds of selection, cloned the8th with highest binding affinity with SGC7901human gastric cancer cells, sequenced and finally obtained7aptmers.DNAMAN software was employed to imitate the secondary structures of the7sequences. All the sequences were truncated based on its secondary structure then we obtained16new aptamers, these aptamers possessed different secondary structures, but most of them contained loop-stem structure.Then we used flow cytometry to analysis the binding affinity of aptamers against human gastric tumor cells, all the aptamers showed nanomole binding affinity to SGC7901cells. Almost all the truncatured aptamers have better binding affinity than the original length (78nt). The binding affinity of aptamer S1a (Kd=141.6nM)was4fold higher than aptamer S1(Kd=441.7nM); the binding affinity of aptamer S2a(Kd=261.2nM)was two times than aptamer S2(Kd=566.9nM); aptamer S4a showed the highest binding affinity to SGC7901gastric cancer cells, the Kd was about59.71nM, the binding affinity of some other aptamers were not so well.Confocal image indicated that aptamer S1a、S1b、S2a and S4a bound well with SGC7901gastric cancer cells, emitting very bright fluorescence, while these aptamers would not bind with GES-1gastric mucosal cells. More importantly, these aptamers did not bind with other non-target cancer cells, such as:97L human liver cancer cells、 HT29Colon adenocarcinoma cells-. A498human renal cell carcinoma and MDA-MB-231huamn breast cancer cells.Then we invested the binding target of aptamers, flow cytometry indicated that aptamers did not bind with SGC7901gastric cancer cells when cells were treated with trypsin or protein K, this indirectly illustrated that the aptamers might bind with cell membrane protein.Finally we invested the clinical application of aptamers:①Stained of tissues:aptamers of S1a、S1b、S2a and S4a could bind with human gastric tumor tissues well,so stained tumor tissues emitted bright fluorescence, in comparision, the normal tissues could not stained by aptamers.②Capture and release cancerous cells: aptamer fouctionalized magnetic nanospheres captured SGC7901gastric cancer cells greatly, resulting in larget amout of nanosphers clustered aroud gastric cancer cells,when couted cell numbers, the capture efficiency was abou93%; however, the capture efficiency for negative cells (GES-lcells)was less than7%. When the complex of aptamer founctionalized magnetic nanosphers-cells was treated with benzonase nuclease, nearly all the captured cells were detached from nanospheres, and the release efficiency was related with incubation time of benzonase nuclease, when treated with benzonase nuclease for40min, the release efficiency could reach to80%.③Aptamer fluorescence probes detection cancer cells in vitro:in the absence of target cancer cell (prescence of GES-1cells), the fluorescence of the aptamer probes was quenched, while the fluorescence was activated by target SGC7901gastric cancer cells. And the aptamer fluorescence probes could detect SGC7901gastric cancer cells range from30000to300, the aptamer fluorescence probe could response to102Order of magnitude of SGC7901gastric cancer cells.④Aptamer fluorescence probes detection cancer cells in vivo:aptamer fluorescence circulated into the mice transplanted with SGC7901gastric cancer cells within15min, but the probe targeted tumor site and enriched, emitting very bright fluorescence. This results indicated that the aptamer had great potential to be applied for cancer diagnosis.Aptamer selected human liver cancer cells:In this part, we used Huh7.5.1human liver cancer cells as the target cells and HeLa cervical cancer cells were used as the negative cells, we performed13rounds of selection, flow cytometry assay indicated that the pool was enriched greatly.Then PCR amplified the13th pool with highest binding rate against Huh7.5.1human liver cancer cells, cloned and sequenced, we obtained7sequences.DNAMAN software was employed to imitate the secondary structures of the7sequences. All the sequences were truncated based on its secondary structure then we obtained16new aptamers, these possessed different secondary structures, but most of them contained loop-stem structure.We further analyzed the binding affinity of aptamers against human liver tumor cells:the aptamers were truncatured based on its secondary structures, then we obtained19modified aptamers.Aptamer H4a and H4b has better bindind affinity (H4a, Kd=22.92nM; H4b, Kd=8.496nM), aptamer H7a showed not good bindind affinity agsinst human liver tumor cells with Kd constants of116.1nM, the binding affinity of other aptamers was between88nM and30nM.We can obvserved that the aptamer H2a、H2b、H2、B3a、H3b and H4a bound well with Huh7.5.1human liver cancer cells, it showed very bright fluorescence from the confocal image, while not bind with HeLa cancer cells. More importantly, these aptamers showed similar binding rate to HepG2human liver cancer cells, but little binding rate to other human liver cancer cells and non-target cancer cells, such as 97L human liver cencer cells、LM9human liver cancer cells and HT29human colon adenocarcinoma cells. Aptamers showed bindin rate about30%to GES-lgastric mucosal cells. Interestingly, most of the aptamers bound well with Hs578Bst human mammary epithelial cells, only aptamer H1c and H1d had high binding rate and specificity to Huh7.5.1and HepG2human liver cancer cells, they showed about15%binding rate to all of the non-target cells including L-02human normal liver cells.We further studied the aptamers binding with human liver cancer tissues and normal liver tissues. All the aptamers could clearly distinguish human liver tumor tissues from the normal liver tissues, this implied that the aptamer may have a great potential for diagnosisi of human liver cancer.Last, in order to increase the binding rate of aptamers to target liver cancer cells, we modified the base of aptamers. Here the base T of aptamer Hla was modified by D-/L-IsoT, we obtained10modified sequences, then determined its binding rate with Huh7.5.1huamn liver cancer cells and L-02normal human liver cells via UV-Vis, the results showed that:sequence DT-2-22TD-. DT-2-36TL、DT-2-32TL all showed better binding rate and specificity than the original aptamers.