Preliminary Study On The Effect Of Moderate Heating Temperature(39℃)on The Viability Of Cultured Pancreatic Cancer Cells And Its Mechanism

Objective:（1）To investigate the effects of moderate heating temperature（39℃）on the proliferation,migration and chemotherapy of pancreatic cancer cells in vitro;（2）To explore the changes of transcripts and alternative splicing of G1 phase related genes when the ambient temperature increased to 39℃;（3）Verifying whether the heat shock protein（HSPS）gene（a marker of heat stress response gene）is also expressed and altered by alternative splicing when the ambient temperature rises to 39℃.Methods:（1）Culturing human and murine pancreatic cancer cell lines.In high density（10,000/35 mm）or（20,000/35 mm）and low density（1,000/35 mm）or（1,500/35 mm）grown in 6 orifice,respectively in different temperature environment of incubator,normal temperature（37℃）and moderate fever temperature（39℃）,high density in 3,5,7 days,low density in 9,12,15 days after the fixed liquid crystal violet dye to check cell clone forming ability.（2）Pancreatic cancer cell lines Panc02 and Panc28 at the growth logarithmic stage at 37℃were planted in 6-well plates,respectively,and the cell state was maintained by serum-free medium overnight scratch.Photographs were taken and analyzed.（3）take 37℃logarithmic growth phase of anthropogenic sex（PANC1,ASPC-1）and the source of the rat pancreatic cancer cell line（PANC02）,respectively,to plant 96 hole board in 39℃,the temperature and humidity and 37℃and 5%CO₂ incubator,first-line drugs gemcitabine in pancreatic cancer chemotherapy（gemcitabine,GEM）with 72 hours（hour,h）,respectively CCK8 method to detect 39℃to 37℃and cell under the environment of absorbance values,analysis the GEM in 39℃ambient temperature effect on pancreatic cancer cell proliferation ability and the effect of chemotherapy.（4）Pancreatic cancer cell lines（PANC1,ASPC-1,PANC02）were cultured at 37℃and 39℃respectively,and GEM-treated cells were added.Flow cytometry with Propidium iodide（PI）staining was used to detect the cycle distribution of pancreatic cancer cells at different temperatures and the cycle distribution of cells treated with GEM-treated cells.FITC-Annexin V/PI double staining was used to detect the apoptotic ability of pancreatic cancer cells under different temperature conditions and the effect of adding GEM on the apoptotic ability of pancreatic cancer cells.（5）Cervical cancer,colorectal cancer,pancreatic cancer and human embryonic kidney cell lines were cultured and placed at different ambient temperatures of 37℃and 39℃,respectively.After 3 days,the cells were collected to extract ribonucleic acid（RNA）and then reverse transcribed into complementary deoxyribonucleic acid（c DNA）to design specific primer sequences.The CDK2,HSPS and HSF1 genes were amplified by Polymerase Chain Reaction（PCR）.Interests of HSPB1 gene were detected,amplified by 5’RACE and sequenced.Nucleotide sequences were analyzed using BLAST and other tools.Results:（1）The results of the clone formation experiment showed that compared with the normal temperature（37℃）,the moderate temperature（39℃）had a certain effect on the proliferation of pancreatic cancer cells.There was no significant difference in promoting the formation of PANC02 cells,inhibiting the formation of PANC1 cells and inhibiting the formation of ASPC-1 cells,respectively.（2）The scratch test results showed that cell lines with rapid proliferation and clone formation（PANC28,PANC02）had faster migration at 39℃.（3）CCK8 proliferation assay showed that 39℃promoted the inhibitory effect of GEM on pancreatic cancer cell Panc1 and Panc02 compared with 37℃;The inhibitory effect of GEM on ASPC-1 was not significantly different between 37℃and 39℃.（4）Flow cytometry PI staining cycle detection results showed that compared with the corresponding group at 37℃,39℃promoted the distribution of PANC02 cells in the G1 phase,and GEMs inhibited the distribution of cells in the G1 phase of the growth cycle.Compared with the corresponding group at 37℃,the number of cells distributed in S phase of growth cycle of Panc1 and ASPC-1 increased slightly at 39℃,and cells were inhibited in S phase by the addition of GEM.5)Flow cytometry results of FITC-Annexin V/PI double staining showed that the apoptosis rate of pancreatic cancer cell lines（PANC1,ASPC-1,PANC02）treated with GEM at 39℃was higher than that of the group treated with GEM at 37℃.（6）RT-PCR sequencing results showed that there was alternative splicing of CDK2 in G1 phase under our culture conditions（37℃and 39℃）.Alterable splicing of RNA was observed in HSPs and its regulatory gene HSF1 at 39℃,and four new variants of HSPB1 were found by5’RACE amplification.Conclusions:（1）Moderate heating temperature（39℃）had different effects on the proliferation of different pancreatic cancer cell lines,which could either promote or inhibit their growth.39℃promoted the inhibitory effect of GEM on most pancreatic cancer cell lines,but not on others.（2）Compared with 37℃,39℃affected the cycle distribution of pancreatic cancer cell lines.Compared with 37℃and GEM treatment group,the distribution of cell cycle was affected by the treatment of GEM at 39℃.（3）Alternative splicing of CDK2 RNA was present in our culture conditions.Under heat stress（39℃）,HSPs and HSF1 RNA were alternatively spliced in the organism.