CMB Carrying PTX And CRISPR/Cas9 Targeting C-erbB-2 Plasmid Interferes With The Study Of Endometrial Cancer Cells

Objectives:Endometrial cancer is one of the gynecological malignant diseases that threaten women’s life and health.In recent years,data has shown that the incidence of endometrial cancer has gradually increased and is showing a younger trend.Paclitaxel,as a common clinical first-line chemotherapy drug,has poor side effects and curative effects.Well balanced,the C-erb B-2 gene is a member of the RAS/RAF/MEK/ERK(MERK)signaling cascade and PI3K/PTEN/AKT/m TOR cell proliferation pathway.It is in the process of cell growth and proliferation.Both play an important role.This paper uses cationic microbubbles loaded with paclitaxel combined with Ultrasound-targeted microbubble destruction(UTMD)to explore whether this method can effectively improve the local efficacy of the drug and reduce systemic side effects.Simultaneously study cationic microbubbles to deliver CRISPR/Cas9 plasmids for C-erb B-2gene knockout,evaluate the impact on the biological functions of endometrial cancer cell lines,and conduct preliminary explorations of its specific regulatory mechanisms,and discuss cationic microbubbles.The chemotherapeutic-gene combination therapy mode of carrying paclitaxel chemotherapeutic drugs and simultaneously carrying the plasmid for C-erb B-2 knockout has the interference effect on endometrial cancer cells.Methods:Construct cationic microbubbles,cationic microbubbles carrying paclitaxel,and knock out the plasmid of the C-erb B-2 gene to identify the binding;endometrial cancer cell line HEC-1A is well cultured and divided into five groups:CMB(cationic microbubbles)Vesicle group),PTX(paclitaxel group),PTX-CMB(cationic microbubble carrying paclitaxel group),CRISPR/Cas9-CMB(cationic microbubble carrying plasmid group),CRISPR/Cas9-PTX-CMB(cationic microbubble carrying paclitaxel and Carrier plasmid group),combined with UTMD to carry out the blasting of cationic microbubbles to release the carrier.The ultrasonic irradiation blasting conditions are 1MHz,0.75W/cm~2,the 30s,and the final concentration of PTX are 1μM/L.After the plasmid transfection group,Real Time-PCR and WB were performed to verify the effective knockout of the C-erb B-2 gene for subsequent cell function experiments.The cell invasion experiment was performed using Transwell 24-well plates to evaluate the invasion ability of cells in each group.The cell scratch experiment was carried out to evaluate the planar migration and healing ability of each group of cell lines;the cell plate clone formation experiment was used to analyze the proliferation ability of each group of cells;the CCK-8 experiment was used to analyze the growth and proliferation of each group of cells.Real-Time PCR analyzes the m RNA levels of PI3K/AKT/m TOR cell proliferation pathway-related genes P21,P27,Bad,and m TOR in each group of cells again.Results:RT-PCR and WB results showed that the C-erb B-2 gene was effectively knocked out(P<0.05);in the invasion experiment,compared with the CMB group,PTX,PTX-CMB,CRISPR/Cas9-CMB,CRISPR/Cas9-The number of cell invasions in the PTX-CMB group was significantly reduced,and there was a statistical difference(P<0.05).The number of invasions in the PTX-CMB group was less than that of the PTX,and the CRISPR/Cas9-PTX-CMB group was compared to CRISPR/Cas9-CMB is less,and there are statistical differences(P<0.05);in the scratch experiment,compared with the CMB group,the migration of cells in the PTX,PTX-CMB,CRISPR/Cas9-CMB,and CRISPR/Cas9-PTX-CMB groups The ability was significantly weakened(P<0.05).Among them,the PTX-CMB group had a more pronounced weakening effect than the PTX group,and the CRISPR/Cas9-PTX-CMB group had a more pronounced weakening effect than the CRISPR/Cas9-CMB group.In the plate clone formation experiment,PTX,The number of clones of cells in the PTX-CMB,CRISPR/Cas9-CMB,and CRISPR/Cas9-PTX-CMB groups was significantly less than that of the CMB group(P<0.05).The PTX-CMB group had fewer clones than the PTX group and had statistical differences(P<0.05),there was no statistical difference between CRISPR/Cas9-CMB and CRISPR/Cas9-PTX-CMB between the two groups;in the CCK-8experiment,PTX,PTX-CMB,CRISPR/Cas9-CMB,CRISPR/Cas9-PTX-The proliferation rate of the four groups of CMB slowed down(P<0.05),among which the proliferation rate of PTX-CMB slowed down more significantly than that of PTX(P<0.05),and the proliferation rate of CRISPR/Cas9-PTX-CMB slowed down compared with CRISPR/Cas9-CMB group More significant(P<0.05);RT-PCR detection of P21,P27,m TOR,and Bad gene expression in each group of cells showed that the two groups with C-erb B-2 gene knockout were compared with CMB,PTX,PTX-In the three CMB groups,the expression of P21,P27,and Bad genes increased significantly(P<0.05),while m TOR decreased(P<0.05),while the C-erb B-2 gene knockout group had no gene expression Statistical difference(P>0.05).Conclusion:1.CMB can effectively carry drugs and genes for delivery at the same time.2.PTX-CMB is more effective than EC with pure PTX.3.CRISPR/Cas9-PTX-CMB this chemotherapy-gene combination therapy is effective in inhibiting the endometrial cancer cell line HEC-1A.4.The C-erb B-2 gene may promote the growth and proliferation of EC tumor cells by up-regulating m TOR,down-regulating P21,P27,and Bad genes.