| Objectives: To observe the expression of calcium channel TRPC1 in laryngeal squamous cell carcinoma and hypopharyngeal squamous cell carcinoma.To investigate the effects of TRPC1 on the cytological functions of proliferation,invasion,migration and apoptosis of human pharyngeal squamous cell carcinoma pleural fluid metastasis cell D562 and laryngeal carcinoma cell TU212.To explore the mechanism of TRPC1 on PI3K/AKT pathway.Methods: Twenty patients diagnosed as cancer by surgical examination of laryngeal squamous cell carcinoma and hypopharyngeal squamous cell carcinoma in hospital from January 2022 to January 2023 were collected according to the inclusion and exclusion criteria.Tissue samples of laryngeal squamous cell carcinoma and hypopharyngeal squamous cell carcinoma and 20 pairs of corresponding paracancer tissue samples were collected.The expression of Transient Receptor Potential Channel 1(TRPC1)in cancer tissues and adjacent tissues was detected by real-time quantitative polymerase chain reaction(qPCR),and the data were analyzed statistically.A plasmid knocking down TRPC1 gene expression and an empty vector plasmid were constructed and transfected into human laryngeal cancer cell TU212 and human pharyngeal squamous cell carcinoma pleural effusion metastatic cell D562,and the experiment was divided into three groups,namely: Blank control group(Blank),Negative control group(sh-NC),Experimental group(sh-TRPC1 group).qPCR was used to detect the expression level of TRPC1 in the cells of each group to verify whether the transfection efficiency was successful.If the expression level of TRPC1 decreased by more than 50%,the transfection was successful.Next,MTT colorimetry and cell plate cloning were used to detect the growth ability and proliferation ability of the three groups of cells;the cell scratch test was used to detect the migration ability of the three groups of cells;The transwell chamber invasion test was used to detect the invasion ability of the three groups of cells;flow cytometry The apoptosis ability of D562 cells and TU212 cells after knocking down TRPC1 was detected by experiment.Statistical analysis: The statistical software SPSS 26.0 was used for data processing and analysis.The experimental data was expressed by mean± standard deviation(x ± s).The difference between the two groups was compared by independent sample t test,and the difference between multiple groups was compared by single Factor analysis of variance.Image J software was used to analyze the gray value of protein bands,clones,and invasion calculations,and Graph Prism9.3.1software was used to make statistical maps.Results:1.qPCR was used to measure the expression of TRPC1 in various head and neck squamous cell lines,and human pharyngeal squamous cell carcinoma pleural effusion metastatic cell D562 and human laryngeal carcinoma cell TU212 were selected as the follow-up experimental research objects(M=5.0864 and M=4.1894).2.qPCR,the experimental results showed that the ΔCT value of TRPC1 in cancer tissue was higher than that in adjacent normal tissue,and the relative expression of TRPC1 in cancer tissue was significantly up-regulated,[(156.5978 ±270.8670)VS(1.3811 ± 1.3710),p<0.01],the difference was statistically significant.3.A plasmid knocking down TRPC1 gene expression was constructed and transfected into two cell lines D562 and TU212,and the expression level of TRPC1 was detected by qPCR.The ΔCT value was significantly lower,respectively [(0.2298± 0.0864)VS(1.2402 ± 0.0453),p<0.001],the difference was statistically significant;[(0.3838 ± 0.0320)VS(0.8163 ± 0.1228),p<0.01],The difference was statistically significant,indicating that the TRPC1 low expression plasmid was successfully transfected.4.Western blot experiments showed that,based on the gray value of the Blank group,compared with the sh-TRPC1 group,D562 cells and TU212 cells were[(1.0037 ± 0.1546)VS(0.3413 ± 0.2453),p<0.05];[(0.7420 ± 0.1363)VS(0.0960 ±0.0361),p<0.01],the difference was statistically significant,suggesting that the lentivirus transfection directly interfered with the protein translation of TRPC1 and significantly reduced the expression.5.The cell growth curve experiment showed that compared with the Blank group,the cell growth ability of the sh-NC group had no significant decrease,p>0.05,and the difference was not statistically significant;however,in the sh-TRPC1 group,the growth ability of D562 cells and TU212 cells was significantly reduced,p<0.05,the difference was statistically significant.6.The cell cloning experiments showed that the cloning abilities of D562 cells and TU212 cells were not significantly different between the Blank group and the sh-NC group after two weeks,which were[(50.0000 ± 6.5574)VS(49.0000 ±5.0000)],[(59.3333 ± 8.3267)VS(53.3333 ± 2.0817)],the difference was not statistically significant,however,in the sh-TRPC1 group,a significant reduction in the number of cell clones could be seen,D562 cells and TU212 cells were[(50.0000 ±6.5574)VS(36.0000 ± 2.6458),p<0.05];[(59.3333 ± 8.3267)VS(29.6667 ± 2.8868),p<0.01],the difference was statistically significant,indicating that the clone ability of D562 cells and TU212 cells after knocking down TRPC1 was significantly weakened.7.The cell scratch test showed that after 48 h,the blank area showed an increasing trend compared with the Blank group and the sh-NC group,and the D562 cells and TU212 cells were [(0.6851 ± 0.1586)VS(0.6308 ± 0.0243),p>0.05];[(0.5283 ± 0.0444)VS(0.5313 ± 0.0082),p>0.05],the difference was not statistically significant.However,in the sh-TRPC1 group,the blank area was significantly increased.Compared with the Blank group,D562 cells and TU212 cells were[(0.3284 ± 0.0581)VS(0.6851 ± 0.1586),p<0.05];[(0.3853 ± 0.0082)VS(0.5283 ±0.0444),p<0.05],the difference was statistically significant,indicating that the migration ability of D562 cells and TU212 cells was significantly reduced after knocking down TRPC1.8.Transwell chamber invasion experiments showed that compared with the Blank group and the sh-NC group,the cell invasion ability showed a downward trend,and the D562 cells and TU212 cells were [(53.6667 ± 1.5275)VS(50.3333 ± 1.5275),p>0.05];[(39.6667 ± 5.0332)VS(40.0000 ± 5.2915),p>0.05],the difference was not statistically significant.However,compared with the Blank group,the sh-TRPC1 group showed a significant decrease in cell invasion ability,D562 cells and TU212 cells were [(18.3333 ± 1.5470)VS(53.6667 ± 1.5275),p<0.0001];[(26.6667 ±1.5275)VS(39.6667 ± 5.0332),p<0.05],the difference was statistically significant,indicating that the invasion ability of D562 cells and TU212 cells was significantly inhibited after knocking down TRPC1.9.Flow cytometry assay for cell apoptosis showed that compared with Blank group and sh-NC group,the apoptosis rate of D562 cells and TU212 cells showed a certain increasing trend,but the difference was not statistically significant.However,compared with the Blank group,the apoptosis rate in the sh-TRPC1 group was significantly increased,and the rates of D562 cells and TU212 cells were [(0.0700 ±0.0200)VS(54.1633 ± 0.8920),p<0.0001],[(0.0600 ± 0.0000)VS(53.3800 ±0.4417),p<0.0001],the difference was statistically significant,indicating that D562 cells and TU212 cells were more prone to apoptosis after knocking down TRPC1.10.Western blot experiments showed that there was no significant difference in the protein expression levels of PI3 K and AKT in each group,but there was a significant difference between P-PI3 K and P-AKT,and the comparison was made by the multiples of the gray value of the proteins in each group.The blank group,sh-NC group,sh-TRPC1 group,and Blank+2-APB group of D562 cells and TU212 cells of P-PI3 K are[(1.0238 ± 0.0958)VS(0.9824 ± 0.0905)VS(0.4065 ± 0.1004)VS(0.5710 ± 0.1747),p<0.05][(1.1505 ± 0.1371)VS(1.1552 ± 0.0825)VS(0.7202 ±0.0924)VS(0.2644 ± 0.0267),p<0.05],the difference was statistically significant,compared with sh-NC group and Blank group,the expression of P-PI3 K in sh-TRPC1 group and Blank+2-APB group decreased significantly.The blank group,sh-NC group,sh-TRPC1 group and Blank+2-APB group of D562 cells and TU212 cells of P-AKT protein are [(1.0238 ± 0.0958)VS(0.9824 ± 0.0905)VS(0.4065 ± 0.1004)VS(0.5710 ± 0.1747),p<0.05];[(1.1505 ± 0.1371)VS(1.1552 ± 0.0825)VS(0.7202± 0.0924)VS(0.2644 ± 0.0267),p<0.05],the difference was statistically significant,comparable Compared with sh-NC group and Blank group,the expression of P-AKT in sh-TRPC1 group and Blank+2-APB group was significantly decreased.Conclusions:1.TRPC1 is highly expressed in laryngeal squamous cell carcinoma and hypopharyngeal squamous cell carcinoma.2.TRPC1 was highly expressed in D562 cells and TU212 cells,and TRPC1 knockdown significantly inhibited the proliferation,migration and invasion of D562 cells and TU212 cells,and significantly promoted the apoptosis of D562 cells and TU212 cells.3.TRPC1 knockdown can inhibit the activation of PI3K/AKT pathway. 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