Excellent Anti-lung Cancer Activity of Populus nigra and Phylogenetic Analysis

activity Populus Abstract: Lung cancer has the highest incidence rate among malignant tumors all over the world, and it is also the leading cause of death. In this present research, we aimed to evaluate the anti-cancer activity of the Populus nigra extract against the lung cancer and study the genome evolution of the Populus nigra . Firstly, the inhibitory activity of the Populus nigra extract on the NCI-H292 lung cancer cell viability was determined with Cell Counting Kit-8 (CCK-8) assay. The trans-well assay was conducted and the influence of the Populus nigra extract on the NCI-H292 lung cancer cell migration and invasion ability was determined. In addition to this, the chloroplast (cp) genome of Populus nigra was sequenced with high-throughput Illumina pair-end sequencing, which was a classical useful model for genome evolution assessment. The CCK-8 and trans-well assay indicated the Populus nigra extract exhibited excellent inhibitory activity on the NCI-H292 lung cancer cell viability, migration and invasion ability. The circular cp genome of the Populus nigra was 156,354 bp in size, including a large single-copy (LSC) region of 84,528 bp and a small single-copy (SC) region of 16,564 bp, which were separated by two inverted repeat (IR) regions (38,612 bp each). A total of 132 genes were predicted, including 8 ribosomal RNAs (rRNAs), 37 transfer RNAs (tRNAs), and 90 protein-coding genes (PCGs). Furthermore, phylogenetic analysis revealed that Populus nigra has the closest relationship with Populus alba var. pyramidalis . In addition to Populus alba var. pyramidalis , Populus adenopoda and Populus tomentosa are also has closely relationship with Populus nigra .

nigra is still need to be explored. According to the intraspecific genetic diversity, genome evolution assessment was carried out to reveal the evolutionary relationship of the Populus nigra with other species 8,9 . In this present research, the whole chloroplast genome of Populus nigra was constructed for population genetics studies. Above all, the Populus nigra extract provide a new choice for the lung cancer treatment by inhibiting the cancer cell viability, migration and invasion.

Extraction
In this study, soxhlet apparatus was used for the extraction of Populus nigra 10 . Typically, fresh leaves of Populus nigra 100 mg was dissolved in EtOH 100 mL in a 50 mL flask, then the soxhlet extractor was placed onto the flask containing the extraction solvent. After heating the flask for 4-5 h, the desired extraction compound was obtained via evaporation of the solvents by the rotatory evaporator as white color powder.

CCK-8 assay
The Cell Counting Kit-8 was conducted firstly in this present research to evaluate the inhibitory activity of the Populus nigra extract on the NCI-H292 lung cancer cell viability. This preformation was finished totally under the guidance of the instructions with some modifications 11, 12 . In brief, the NCI-H292 lung cancer cells in the logical growth phage were collected and then seeded into the 96 well plates at the final destiny of 10 5 cell per well. After 12 h incubation in an incubator at the condition of 37 , 5 CO 2 , the Populus nigra extract was added into the wells for another 12 h incubation. Then, the culture medium was discarded and the fresh medium containing the CCK-8 reagent was added. Finally, the absorbance of each well was measured with microplate reader at 450 mm. This preformation was conducted at least three times and the results were presented as mean SD.

Trans-well assay
The trans-well assay was conducted in this present research to evaluate the influence of the Populus nigra extract on the migration and invasion ability of the NCI-H292 lung cancer cells. This conduction was finished strictly in accordance with the protocols with only little change 13 . Briefly, the 24-well trans-well chambers 8μm pore size, 6.5 mm diameter; Corning, NY, USA were precoated with or without of Matrigel matrix 50 μL/well, 200 mg/mL; BD Biosciences, Franklin Lakes, NJ, USA , respectively for the migration and invasion assays. Then, the chambers were placed into the 24-well plates, and the cancer cells in the logical growth phage were collected and seeded into the upper chamber at the destiny of 5 10 4 cells per well, cultured with free-FBS medium. After 24 h incubation in an incubator at the 37 , 5 CO 2 condition, the cells remaining on the upper sides of the membranes were carefully wiped off, and the cells on the lower side of the membranes were labeled with 0.5 crystal violet. The cell numbers on the lower surface of the membrane were quantified in six randomly independent fields under a microscope.

Plant materials and DNA isolation
Fresh leaves of Populus nigra were collected from Xinjiang, China 86 06 E, 41 68 N , and further analyses was conducted in the Kunming Institute of Botany, Chinese Academy of Sciences. The duplicate specimens were saved in the herbarium of Kunming Institute of Botany KIB at 80 condition. The chloroplast genomic DNA was extracted from 25 mg silica-gel-dried leaves with Ezup Plant Genomic DNA Prep Kit Sangon Biotech, Shanghai, China 14,15 . The quality and quantity of the chloroplast genomic DNA was measured with Agarose gel electrophoresis.

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 16 18 . 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 nigra. Complete cp genome includes protein coding genes, tRNAs and rRNAs, the an-

Comparative and phylogenetic analysis
The phylogenetic analysis was conducted based on the General Time Reversible model with Maximum Likelihood ML method 19 . The tree with the highest log likelihood 297751.66 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 automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood MCL approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 35 nucleotide sequences. There was a total of 169964 positions in the final dataset. The ML phylogenetic analysis were conducted with MEGA v7.0.26 generating 1000 bootstrap replicates to determine measures of nodal support with each run initiating from a random starting tree 20 .

Results
3.1 Populus nigra extract signi cantly reduce the viability of the cancer cells As the excellent pharmacological activities of the Populus nigra extract, such as anti-inflammatory and anticoagulant activity, which application values on the lung cancer treatment was firstly explored. Thus, the CCK-8 assay was conducted in this research to assess the inhibitory activity of the Populus nigra extract on the NCI-H292 lung cancer cell viability. As the results showed in Fig. 1, we can see that the compared with the control group, the Populus nigra extract could significantly reduce the viability of the NCI-H292 lung cancer cells with p 0.005, which is even better than the positive anti-cancer drug 5-Fu. This result suggested the Populus nigra extract has the excellent clinical application values for the cancer therapy.
3.2 Populus nigra extract obviously inhibit the migration and invasion ability of the cancer cells In the above experiment, we have proved that the Populus nigra extract could significantly reduce the NCI-H292 lung cancer cell viability. However, whether the Populus nigra extract could also inhibit the NCI-H292 lung cancer cell migration and invasion ability was still need to be explored. So, the trans-well assay was conducted for this assessment. The results in Fig. 2 showed that the migration and invasion ability of the NCI-H292 lung cancer cell was much stronger than that of the normal human cells, with p 0.005, while after the Populus nigra extract exposure, the NCI-H292 lung cancer cell migration  and invasion ability was obviously reduced.

Chloroplast genome features
According to the annotation analysis of the chloroplast genome of Populus nigra, there are four categories of 132 genes in the chloroplast genome, which can be divided into self-replicating genes, photosynthesis genes, other functional genes and unknown function genes. The Self-repli-cating genes consisted of 4 duplicated Ribosomal RNA genes rrn4.5, rrn5, rrn16, and rrn23 , 37 transfer RNA genes, 14 Small subunit of ribosome genes, 10 Large subunit of ribosome genes, 4 RNA polymerase subunits genes. The Photosynthesis genes comprised of 21 genes in Subunits of photosystem I and Subunits of photosystem II, 6 genes in Subunits of cytochrome, 1 gene in Rubisco large subunit, 12 genes coding Subunits of NADH Dehydroge- Table 1 Genes contained in Populus nigra chloroplast genome 133 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 two-intron containing genes, respectively.
nase. There are 6 genes infA, matK, cemA, accD, ccsA, clpP belong to the Other functional genes and 7 genes ycf4, ycf2 a , ycf15 a , ycf1 a belonging Unknown function genes. The repetitive genes appearing in the inverted repeat region have been marked with "a" in the upper corner of Table 1.

Gene map of chloroplast genome
The annotated chloroplast genome was saved as .gb format file, which was then uploaded onto OGDraw https:// chlorobox.mpimpgolm.mpg.de/OGDraw.html , an online analysis tool for the chloroplast genome physical map preparation. The chloroplast genome of Populus nigra has a typical tetrad structure Fig. 3 . The circular cp genome of the Populus nigra was 156,354 bp in size, including a large single-copy LSC region of 84,528 bp and a small single-copy SC region of 16,564 bp, which were separated by two inverted repeat IR regions 38,612 bp each . A total of 132 genes were predicted, including 8 ribosomal RNAs rRNAs , 37 transfer RNAs tRNAs , and 90 proteincoding genes PCGs . The LSC consisted of 81 genes and the SSC comprised of 13 genes. The IRA and IRB regions have the same 20 genes with opposite directions, such as rps19, rpl2, rpl23, trI-CAU, ycf2, ycf15, trnL-CAA, ndhB, rps7, rps12, trnV-GAC, rrn16S, trnI-GAU, trnA-UGC, rrn23S, rrn4.5S, rrn5S, trnR-ACG, trnN-GUU and ycf1.

Phylogenetic analysis
Phylogenetic analysis was performed according to the whole chloroplast complete sequences of 35 species, including 31 Populus and 4 Salix species Salix matsudana f. tortuosa, Salix tetrasperma, Salix oreinoma and Salix taoensis as outgroup species Fig. 4 . The reconstructed phylogenetic tree clustered all the species into two groups. The four Salix species were clustered into the same evolutionary group, while all other Populus species were clustered into another major evolution group. The Populus nigra has the closest relationship with Populus alba var. pyramidalis. In addition to Populus alba var. pyramidalis, Populus adenopoda and Populus tomentosa are also has closely relationship with Populus nigra.

Conclusion
In this present research, the anti-cancer activity of the Populus nigra extract was evaluated and its phylogenetic analysis was conducted for evolution revealing as well. The results of the CCK-8 showed that the Populus nigra extract could significantly reduce the viability, migration and invasion ability of the NCI-H292 lung cancer cells, suggesting the excellent application prospect the Populus nigra. The length of Populus nigra cp genome reported in this present research is 156,354 bp, which is 392 bp similar than that of closely related species Populus adenopoda 156,746 bp 21 . The Populus species also have and similar gene orders, gene numbers and GC contents. The Populus nigra cp genome is AT-rich 63.23 , which is consistent with other species from Populus family, for example, Populus adenopoda 63. 21 , Populus alba 63.26 22 , Populus alba var. pyramidalis 63.21 and Populus tremula var. davidiana 63.21 21 . This evolutionary relationship suggesting the other related species may also has the anti-lung cancer activity as above described. Through this research, we proved that the Populus nigra extract provide a new choice for the lung cancer treatment by inhibiting the cancer cell viability, migration and invasion. And the closely related species also have the same anti-cancer activity, which also need to be explored.

Author Contributions
Jun Ma, Yang Gao, Tao Jiang and Feng Tian contributed equally to this research. Tao Jiang and Feng Tian designed this research together; Jun Ma finished the biological evaluation of the Populus nigra extract; Yang Gao finished the circular cp genome sequence of the Populus nigra and related evolutionary relationship analysis.