Role And Mechanism Of MicroRNA-301b-3p In Colorectal Cancer

Colorectal cancer is one of the most common digestive tract cancers in China.It has high incidence rate and high mortality,seriously threaten people’s health,Despite that the treatment for colorectal cancer greatly improve in recent these years,the five-survive rate of colorectal cancer is not satisfactory.Therefore,it is important to study the pathogenesis of colorectal cancer and explore new therapeutic targets for colorectal cancer.Previous studies revealed that miR-301b-3p is highly expressed in a variety of tumor tissues.MiR-301b-3p can promote tumor proliferation and migration,which is associated with the development and progression of several cancers,but the potential functions of miR-301b-3p in colorectal cancer remained largely unclear.The current study is aimed at exploring the potential roles and possible mechanism of miR-301b-3p in colorectal cancer,which may contribute to providing novel biomarkers and therapeutic targets for colorectal cancer treatment.Part1: The role of miR-301b-3p in the development of colorectal cancer Purposes:To analyze the expression of miR-301b-3p in colorectalr cancer tissues and cell lines,and to explore the role of miR-301b-3p in the malignant biology of colorectal cancer.Materials and Methods:RT-qPCR was used to examine the expression level of miR-301b-3p in colorectal cancer tissues and adjacent normal tissues,as well as in colorectal cancer cell lines Caco-2(MSI-H),SW480((MSS),HCT-116(MSI-H),HT-29(MSS),DLD-1(MSI-H)and normal colon epithelial cell NCM460.DLD1 and HT29 cells were transfected with miR-301b-3p mimics,cell proliferation and migration were detected using cell counting kit-8(CCK-8),colony formation,wound healing and transwell assays.Results:RT-q PCR showed that miR-301b-3p expression was higher in colorectal cancer tissues than that in adjacent normal tissues.Also,miR-301b-3p was associated with malignant prognosis in colorectal cancer patients.miR-301b-3p was obviously upregulated in colorectal cancer cell compared to normal colon epithelial cells(DLD-1 and HT-29 ranked as the lowest expression level among all the colorectal cancer cell and they were represented MSI-H(DLD-1)and MSS(HT-29)two different types of colorectal cancer,so we chose them to perform subsequent functional experiment).RT-q PCR suggested that miR-301b-3p was significantly up-regulated in DLD-1 and HT-29 celllines after transfected with its mimics.The results of CCK-8,colony formation,wound healing and transwell assays found that overexpression of miR-301b-3p promoted colorectal cancer cell proliferation and migration.Conclusions:Expression of miR-301b-3p is upregulated in colorectal cancer.Overexpression of miR-301b-3p can significantly promote the cell proliferation and migration in colorectal cancer.MiR-301b-3p may serve as a tumor promoter in colorectal cancer,it mediate the malignant biological behavior of colorectal cancer.Part2: The mechanism of miR-301b-3p in the progression of colorectal cancer Purposes:To explore the molecular mechanism of mir-301b-3p in regulating the occurrence and development of colorectal cancer.Materials and Methods:Target Scan database predicted that the 3’UTR of the HOXB1 m RNA was complementary to miR-301b-3p,which suggested that HOXB1 could be a putative downstream target of miR-301b-3p.In order to verify the binding site between miR-301b-3p and HOXB1,Dual-luciferase report experiment was used to detect the change of luciferase activity.HT-29 and DLD-1 cells were transfected with designed HOXB1 si RNAs,then knock out HOXB1,and the transfection efficiency was determined by RT-q PCR and Western blot.CCK-8,colony formation,wound healing and transwell assays were used to detect the effect of HOXB1 knockdown on proliferation and invasion of colorectal cancer cells.To ascertain whether miR-301b-3p accelerated the proliferation and migration of HT-29 and DLD-1 cells through HOXB1,we treated them with mimics of miR-301b-3p or NC(mimics)and(or)pc DNA-HOXB1 plasmid.RT-q PCR,Western blot,CCK-8,colony formation,wound healing and transwell assays were used to verify the relationship between miR-301b-3p and HOXB1.Results:Bioinformatics and Dual-luciferase reporter assays verified that HOXB1 acted as the downstream targeted m RNA.HOXB1 inhibition accelerated proliferation and migration of colorectal cancer cells.MiR-301b-3p significantly reduced the abundance of HOXB1,which were partly reversed by HOXB1 overexpression.Conclusions:MiR-301b-3p facilitated proliferation and invasion of colorectal cancer cells via targeting HOXB1.The signal axis of miR-301b-3p/HOXB1 plays an critical character in the pathogenesis of colorectal cancer and may be an important pathway for the occurrence and development of colorectal cancer.