Anti-oral Cancer Biological Activity Evaluation and Chloroplast Genome Analyses of Populus euphratica

: In this research, the anti-cancer activity of the Populus euphratica extract was evaluated with Cell Counting Kit-8 (CCK-8) assay. The inhibitory activity of the Populus euphratica extract on the activation levels of VEGF signaling pathway in the cancer cells was measured with real time RT-PCR. Next, the high-throughput Illumina pair-end sequencing was performed to detect the chloroplast (cp) genome of Populus euphratica for genome evolution assessment. The CCK-8 results indicated that the extract of Populus euphratica exhibited the significantly suppression effect on the viability of the cancer cells, and the data of the real time RT-PCR showed the activation levels of VEGF signaling pathway in the cancer cells was also reduced obviously by the Populus euphratica extract. The circular cp genome of the Populus euphratica is 157,806 bp, encoding 131 genes, containing 8 Ribosomal RNA genes (rRNAs), 37 Transfer RNA genes (tRNAs) and 86 Protein coding genes (PCGs). And the results of the phylogenetic analysis indicated that the Populus euphratica . Furthermore, phylogenetic analysis revealed that Populus euphratica has the closest relationship with Populus pruinosa . In addition to Populus pruinosa , Populus ilicifolia also has closely relationship with Populus euphratica . These three species could be clustered on the same clade.

used for hypertension treatment. The inflorescence of the Populus euphratica was used for hemostasis 7 10 . While, the anti-cancer effect of the Populus euphratica has not be evaluated up to now. And the phylogenetic relationship between Populus euphratica and other Populus species remains unclear. Thus, in this present research, the inhibitory activity of the Populus euphratica extraction against the oral cancer was evaluated and the phylogenetic analysis was conducted for evolution revealing of the Populus species. We aimed to develop new candidates for the tumor treatment. In this present research, the biological activity evaluation revealed that the of the Populus euphratica was excellent on the oral cancer treatment by inhibiting the cancer cell viability, migration and invasion, and the intraspecific genetic diversity, genome evolution assessment was carried out for the reveal of relationship between Populus euphratica and other species by measuring the whole chloroplast genome of Populus euphratica.

Extraction
In this present research, for the extraction of mainly material in Populus euphratica, the soxhlet apparatus was applicated. Briefly, 100 mg Populus euphratica fresh leaves were dissolved with 100 mL EtOH in a 50 mL flask. Next, the then the soxhlet extractor was placed onto the flask containing the extraction solvent. The flask was heated for 4-5 h, the solvent was evaporated and the desired extraction compound white powder was obtained 11 .

CCK-8 assay
In this present research, the inhibitory activity of the Populus euphratica extract on the oral cancer treatment was evaluated with Cell Counting Kit-8 assay. This preformation was conducted strictly under the guidance of the instructions with some modifications for the viability evaluation of the oral cancer cells after treated with the Populus euphratica extract 12 . In brief, the oral cancer cells in the logical growth phage were collected and seeded into the 96 well plates at the final destiny of 10 4 cells per well. Then, the cells were placed in the incubator at the condition of 37 and 5 CO 2 for 12 h. Next, the Populus euphratica extract was added into the wells for treatment for 48 h with serial different dilutions 1, 2, 4, 8, 10, 20, 40 and 80 μM . Subsequently, the culture medium was discarded and the fresh medium containing 10 μL CCK-8 reagent was added. Finally, the absorbance of each well was measured with microplate reader at 450 mm. This preformation was conducted triplicate and the results were presented as mean SD.

Real time RT-PCR
After treated with Populus euphratica extract, the changes of the VEGF signaling pathway activation levels in the oral cancer cells was determined with real time RT-PCR. This experiment was performed totally in accordance with the instructions with only a little change 13 . Shortly, the oral cancer cells in the logical growth phage were collected and seeded into the 6-well plates at the destiny of 5 10 5 cells/well in DMEM-FBS medium. After 24 h incubation in an incubator at the 37 , 5 CO 2 condition, the cells were collected and the total RNA in the cells were extracted with TRIZOL reagent. The quality of the total RNA was evaluated using the OD260/OD280 ratio, and the cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit Applied Biosystems, Cambridge, MA, USA . The PCRs were conducted using the qRT-PCR miRNA Detection Kit Invitrogen : 95 for 15 min, followed by 40 cycles of denaturation at 95 for 5 s, annealing at 55 for 30 s and extension at 72 for 30 s. Each experiment was performed in triplicate and the relative quantification was analyzed by the 2 ΔΔCt method. All the results were presented as mean SD.

Populus euphratica materials and DNA isolation
Fresh leaves of Populus euphratica were collected from Xinjiang, China 86 06 E, 41 68 N , and Kunming Institute of Botany, Chinese Academy of Sciences was cooperated with our lab for further analyses 14 . The duplicate specimens were also saved in the herbarium of Kunming Institute of Botany KIB at the condition of 80 . Ezup Plant Genomic DNA Prep Kit Sangon Biotech, Shanghai, China 13,14 . Ezup Plant Genomic DNA Prep Kit Sangon Biotech, Shanghai, China was used to extracted with the chloroplast genomic DNA, and the chloroplast genomic DNA was used to measure the quality and quantity of the chloroplast genomic DNA.

Genome assembly and annotation
The TruSeq DNA sample preparation kits Illumina, San Diego, CA, USA was used to construct a paired-end library firstly. This preformation was carried out totally under the guidance of the instructions with some modifications. The parameter was set as follows: Standard Mode, "Sequence source" was set as "Plastid". Then, selecting display photosystem I, photosystem II, cytochrome b/f complex, ATP synthase, NADH dehydrogenase, RubisCO large subunit, RNA polymerase, Ribosomal proteins SSU , ribosomal proteins LSU , clpP, matK, other genes, hypothetical chloroplast reading frames ycf , ORFs, transfer RNAs, ribosomal RNAs, origin of replication and polycistronic transcripts and other gene and characteristic information. Selecting "Draw GC content graph" and "Label intron-containing genes with *". The export file format was set to "PDF". Finally, submit the data and parameter settings for calculation, and finally generate a gene map of the chloroplast genome of the Populus euphratica. Complete cp genome includes protein coding genes, tRNAs and rRNAs, the annotations of the cp genome was carried out using Dual Organellar GenoMe Annotator DOGMA according to

Molecular phylogenetic analysis
The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible GTR model 16,17 . The tree with the highest log likelihood -286231.95 is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree s for the heuristic search were obtained by applying the BioNJ method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood MCL approach. The rate variation model allowed for some sites to be evolutionarily invariable I , 0.00 sites . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 30 nucleotide sequences. There was a total of 169844 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 18 .

Excellent inhibitory activity of the Populus euphratica
extract on oral cancer cells The widely biological activity of the Populus euphratica extract, such as anti-inflammatory and anticoagulant activity has drawn the attention of most researchers. However, the application values of the Populus euphratica extract on cancer treatment was still need to be explored. Thus, in this present research, the CCK-8 assay was firstly performed to evaluate the anti-viability of the Populus eu-phratica extract on the oral cancer cells. The results in Figure 1 showed that the viability of the oral cancer cells after Populus euphratica extract treatment was signifi-  cantly reduced, which is significantly different from the control group, with p 0.005. The inhibition of the Populus euphratica extract was even much better than the positive anti-cancer drug 5-Fu. This experiment revealed that the Populus euphratica extract exhibited pro-tentional application values against the oral cancer.

Strong inhibition effect of the
Populus euphratica extract on VEGF signaling pathway in the cancer cells As we have showed in Fig. 1, the Populus euphratica Table 1 Genes contained in Populus euphratica chloroplast genome 131 genes .

Category
Group of gene Name of gene Note: a Two gene copies in IRs; b gene divided into two independent transcription units; one and two asterisks indicate one-and twointron containing genes, respectively.
extract has excellent inhibitory activity on the viability of the oral cancer cells. In addition to the character of abnormal proliferation, the activation levels of VEGF signaling pathway in the cancer cells was also need to be paid attention, which is important for the cancer cell proliferation. As the results showed in Fig. 2, we can see that there was a higher activation level of the oral cancer cells compared with the normal cells. However, after the treatment of the Populus euphratica extract, the activation levels of VEGF signaling pathway in the cancer cells was reduced obviously.

Chloroplast genome features
After the chloroplast genome sequence of Populus euphratica and the annotation analysis, we can see in Table  1, there were four categories and totally 131 genes in the chloroplast genome Self-replicating genes, Photosynthesis genes, Other functional genes and Unknown function genes . In the Self-replicating genes, there were total 8 Ribosomal RNA genes duplicated rrn4.5, rrn5, rrn16, and rrn23 , 37 transfer RNA genes tRNAs , 14 Smalls subunit of ribosome genes, 10 Smalls subunit of ribosome genes, 4 RNA polymerase subunits. In the Photosynthesis genes, there are 21 Subunits of photosystem I and Subunits of photosystem II genes, 6 Subunits of ATP synthase genes, 1 Large subunit of Rubisco gene, 12 Subunits of NADH Dehydrogenase. In addition to this, the matK, cemA, accD, ccsA and clpP belong to the Other genes. And unknown function genes include of 7 genes duplicated ycf 2, ycf 15, ycf 1 and a ycf 4 . The repetitive genes appearing in the inverted repeat region have been marked with "a" in the upper corner of Table 1.

Chloroplast genome gene map
The annotated chloroplast genome of Populus euphratica was saved as .gb format file, and then uploaded onto OGDraw https://chlorobox.mpimpgolm.mpg.de/OGDraw. html , an online analysis tool for the chloroplast genome physical map preparation. As the results showed in Fig. 3

Phylogenetic analysis
Phylogenetic analysis of Populus species was performed based on the whole chloroplast complete sequences of 30 species, including 29 Populus species and 1 Salix specie Salix taoensis as the outgroup specie Fig. 4 . As the results showed in the reconstructed phylogenetic tree, all species could be clustered into two groups, the Populus evolution group and the Salix evolutionary group. according to the evolutionary relationship, Populus euphratica has the closest relationship with Populus pruinose, with the evolutionary similarity of 100.

Conclusion
In this present research, for the treatment of oral cancer, the biological activity of the Populus euphratica extract was evaluated, and the phylogenetic analysis of the Populus euphratica with other species was analyzed as well. Through the CCK-8 assay, we proved that the extract of Populus euphratica could significantly reduce the viability of the oral cancer cells. Besides, the activation levels of VEGF signaling pathway in the oral cancer cells was also reduced obviously by the Populus euphratica extract, suggesting the excellent application values of the Populus euphratica extract on oral cancer. Additionally, the Populus euphratica has a 157,806 bp length cp genome. Furthermore, phylogenetic analysis revealed that Populus euphratica has the closest relationship with Populus pruinosa. In addition to Populus pruinosa, Populus ilicifolia also has closely relationship with Populus euphratica. The above results suggested that these three species may also has similar anti-cancer application values, which need to be further explored.