Reducing ROCK2 Enhances Chemosensitivity Of Pancreatic Cancer Cells By Inhibiting Gemitabine-induced Protective Autophagy

Background:Resistance of pancreatic cancer cells to chemotherapy has been an important reason for the poor effect of chemotherapy in pancreatic cancer.Gemcitabine,as a first-line drug,still has an unsatisfactory therapeutic effect.Therefore,it is urgent to explore ways to increase the efficacy of gemcitabine.Autophagy is a conserved biological process that can degrade damaged organelles and proteins in cells and recycle them.It has been shown that gemcitabine induces an increase in autophagy levels in pancreatic cancer cells and protects cells from gemcitabine-induced apoptosis,thereby producing chemoresistance.ROCK2 is a protein kinase that phosphorylates its substrates,and ROCK2 can affect biological processes such as tumor invasion and metastasis,and tumor epithelial-to-epithelial transformation through a variety of mechanisms.However,the process of autophagy is also accompanied by the phosphorylation of many proteins,and whether ROCK2 as a protein kinase can affect autophagy in pancreatic cancer cells is unknown.Therefore,the aim of this study was to investigate the relationship between ROCK2 and autophagy and the effect on the chemosensitivity of gemcitabine.Methods:1,The microarray data on pancreatic cancer in the GEO database and GEPIA website were searched to analyze the relative expression of ROCK2 m RNA in pancreatic cancer tissues and tissues and the effect of ROCK2 expression on patient survival.Then tissue samples were collected from patients with pancreatic cancer,and the m RNA and protein expression of ROCK2 in pancreatic cancer and corresponding tissues were verified by real-time PCR(q RT-PCR),immunohistochemistry(IHC)and Western blot.2,By changing the expression of ROCK2 in different pancreatic cancer cell lines,the effect of changing ROCK2 expression on autophagy levels in pancreatic cancer cells was detected by WB,electron microscopy,and cell immunofluorescence.3,Explore the specific biological mechanism by which ROCK2 affects autophagy in pancreatic cancer cells.4,Verify whether ROCK2 affected the chemosensitivity of pancreatic cancer cells through autophagy in pancreatic cancer cells.5,The correlation between ROCK2 and its downstream autophagy regulation-related genes was validated by immunohistochemistry,WB,and regression analysis in tissues from pancreatic cancer patients.Results:1,Data analysis of the GEO database and GEPIA website showed that the m RNA expression of ROCK2 was relatively high in pancreatic cancer tissues,and in addition,survival analysis revealed that pancreatic cancer patients with high ROCK2 expression had shorter survival.QRT-PCR,IHC and westernblotting were performed on the cancer and tissues of 30 patients with pancreatic cancer,and the results revealed that the m RNA and protein expression of ROCK2 was increased in pancreatic cancer tissues.2,The expression of ROCK2 was interfered in pancreatic cancer cells PANC-1,and ROCK2 was overexpressed in Bx PC-3 cells,and autophagy-related parameters were detected by westernblotting,electron microscopy,and cell immunofluorescence revealed that reducing the expression of ROCK2 could inhibit the autophagy level of pancreatic cancer cells,and epi-ROCK2 could increase the autophagy level of pancreatic cancer cells.3,Mechanistically,we found that ROCK2 did not affect the protein expression of the autophagy-initiating complex Bcl2-Beclin1,but,would affect the binding of the Bcl2-Beclin1 complex.Interference with ROCK2 increased the binding of the Bcl2-Beclin1 complex,and overexpression of ROCK2 decreased the binding of the Bcl2-Beclin1 complex.Further experiments revealed that ROCK2 could affect the dissociation of Bcl2-Beclin1 complex by regulating the phosphorylation of Bcl2(S70).We show that ROCK2 does not bind directly to Bcl2,but rather by binding to ERK1/2 and affecting the phosphorylation of Bcl2(S70).As described in the review,we found that ROCK2 can promote the phosphorylation of ERK1/2,leading to increased phosphorylation of Bcl2(S70),which dissociates the Bcl2-Beclin1 complex and increases autophagy levels.4,Through further in vivo and in vitro experiments,we found that gemcitabine could induce an increase in autophagy levels in pancreatic cancer cells,and interference with ROCK2 could restore the gemcitabine-induced increase in autophagy.Apoptosis-related experiments showed that gemcitabine could lead to increased apoptosis,and interference with ROCK2 could increase gemcitabine-induced apoptosis.Interference with ROCK2 in combination with the autophagy agonist rapamycin can revert to the increased efficacy of gemcitabine resulting from interference with ROCK2.These results indicate that reducing ROCK2 expression can increase the sensitivity of pancreatic cancer cells to gemcitabine by inhibiting gemcitabine-induced autophagy.5,The protein expression of ROCK2,ERK1/2,and p-Bcl2 was found to be positively correlated by IHC and westernblotting of pancreatic cancer tissues,as well as correlation analysis.Conclusion:ROCK2 can regulate the level of autophagy in pancreatic cancer cells through the ERK1/2-Bcl2 axis,and reducing the expression of ROCK2 can increase the sensitivity of pancreatic cancer cells to gemcitabine by inhibiting gemcitabine-induced autophagy,which provides new insights into the mechanism of chemoresistance in pancreatic cancer cells and provides a new theoretical basis for chemotherapy regimens for pancreatic cancer.