Urine miRNA signature as a potential non-invasive diagnostic and prognostic biomarker in cervical cancer

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Prevalence of HPV infection in urine compared with paired cervical scrapes and biopsies in cervical pre-cancer, cancer and control

A noninvasive urine sampling has been utilized to establish if urine can serve as an alternative clinical material for reliable detection of HPV and other sensitive biomarkers such as miRNA expression for early detection of cervical cancer. Therefore, in the present study, paired urine samples, cervical scrapes and tissue biopsies from 50 subjects each of pre-cancer, cancer along with adjacent normal tissues and normal subjects were collected and subjected to detection and genotyping of HPV types 16 and 18 which are the most common high-risk oncogenic HPV types worldwide and are present in over 90% of carcinomas of Indian women. The analysis of HPV infection and its genotyping was conducted by PCR using consensus and HPV type-specific primers.

PCR-based detection revealed that 30 (60%) out of a total of 50 urine samples of pre-cancer lesions were HPV positive. Out of these 50 precancer urine samples, 17 LSIL and 33 HSIL had 4 (8%) and 26 (52%) HPV positives, respectively. HPV infection was detected in the cervical scrape of 35/50 (70%) of precancer lesions with 14% of LSIL and 56% of HSIL found to be HPV positive. However, in control samples (n = 50), only 3 (6%) urine samples and 4 (8%) normal cervical samples were found to be HPV positive (Table 1). Subsequently, HPV type specific PCRs performed in these cases to determine the prevalence of two most prevalent HR-HPV types 16 and 18 in urine samples revealed the presence of HPV16 DNA sequence in 28 out of 50 (56%) cervical pre-cancer cases [4(8%) of LSIL and 24 (48%) of HSIL] whereas in cervical scrapes it was 32 out of 50 (64%) with 4 (8%), LSIL and 28 (56%), HSIL and 2 (4%) were positive both in urine and cervical scrapes controls. No sample was found positive for HPV type 18.

PCR-based detection revealed that 30 (60%) out of a total of 50 urine samples of pre-cancer lesions were HPV positive. Out of these 50 precancer urine samples, 17 LSIL and 33 HSIL had 4 (8%) and 26 (52%) HPV positives, respectively. HPV infection was detected in the cervical scrape of 35/50 (70%) of precancer lesions with 14% of LSIL and 56% of HSIL found to be HPV positive. However, in control samples (n=50), only 3 (6%) urine samples and 4 (8%) normal cervical samples were found to be HPV positive (Table 1). Subsequently, HPV type specific PCRs performed in these cases to determine the prevalence of two most prevalent HR-HPV types 16 and 18 in urine samples revealed the presence of HPV16 DNA sequence in 28 out of 50 (56%) cervical pre-cancer cases [4(8%) of LSIL and 24 (48%) of HSIL] whereas in cervical scrapes it was 32 out of 50 (64%) with 4 (8%), LSIL and 28 (56%), HSIL and 2 (4%) were positive both in urine and cervical scrapes controls. No sample was found positive for HPV type 18.

Table 1 Clinicopathological characteristics and HPV status of cervical pre-cancer, cancer patients and controls.

In cancer cases (n = 50), HPV positivity was detected in 40 out of 50 (80%) urine samples and 36 out of 40 (90%) tumor tissue biopsies. HR-HPV type 16 was the most frequently detected HPV type in the urine (74%) and tumor biopsies (85%) of cervical cancer patients. Interestingly, HPV18 infection was found only in 2 (4%) each in urine and tumor biopsy of cancer patients. We also collected paired adjacent normal tissues as controls from 30 cervical cancer cases which revealed the presence of HPV16 infection in only 4 (8%) cases. Interestingly, none of the paired adjacent normal tissues were positive for HPV 18 infection (Table 1).

When we examined the distribution of HPV prevalence with respect to differentiation and histopathological grades, we obtained HPV positivity in 23 (46%), 10 (20%), 7 (14%) in urine, while 16 (40%), 10 (25%), 10 (25%) in tumor tissue of WDSCC, MDSCC and PDSCC, respectively (Table 1). The HPV status along with histopathological grades and differentiation status of the tumors are presented in Table 1. HPV positivity in urine, cervical scrapes, and tissue biopsies was also correlated with clinical stage of the tumors (Table 1). HPV was found present in urine of 10 (20%) cases of stage I, 17 (34%) of stage II, 15 (30%) of stage III and 3 (6%) in stage IV. Majority of HPV positive tumors had HPV16 genotype regardless of tumor grade and sample types (Table 1). The urine samples of all stage I and II cervical cancer patients were HPV positive, while in paired tissue biopsies it was found that 8 out of 10 cases of stage I and 15 out of 17 of stage II patients were HPV positive. Further, in the urine samples, 15 out of 18 of stage III and 3 out of 5 of stage IV cervical cancer patients were found to be HPV positive, while in paired tumor tissue biopsies, all stage III and stage IV patients were HPV positive (Table 1).

When the results of three paired clinical/biological samples viz. urine, cervical scrapes and tumor biopsies were compared for the presence of HPV infection, there was no significant difference, rather very good correlation of results.

Expression profiling of selected six miRNAs in paired urine, serum, cervical scrape, and tissue biopsy in control, cervical pre-cancer and cancer

We analyzed the expression of six miRNAs (miR-21-5p, miR-199a-5p, miR-155-5p, miR-145-5p, miR-34a-5p and miR-218-5p) in aforementioned samples using qRT-PCR (Table 2). A total of 460 samples were analyzed for miRNA expression (in 150 urine and serum samples collected from 50 subjects each of normal, cervical pre-cancer, and cancer, 50 cervical scrapes from pre-cancer patient, 40 cervical scrapes from normal controls, 40 tumor tissue biopsies and 30 paired adjacent normal tissues from cervical cancer patients). We found a significant upregulation of miR-21-5p, miR-199a-5p, and miR-155-5p and downregulation of miR-145-5p, miR-34a-5p, and miR-218-5p in urine, paired serum, tumor biopsies and cervical scrape (Table 2, Fig. 1 and Figure S1a, S1b, S1c and S1d) as well as in cervical cancer cell lines (Table S1). Compared to paired adjacent normal tissues, the expression of miR-21-5p (FC = 3.4), miR-199a-5p (FC = 2.2), and miR-155-5p (FC = 2.6) was upregulated while that of miR-145-5p (FC = 0.12), miR-34a-5p (FC = 0.14), and miR-218-5p (FC = 0.13) was downregulated in tissue biopsies of cervical cancer patients (Table 2; Fig. 1a,b; Figure S1c). Similar results were also obtained for cervical scrapes from cervical pre-cancer cases when compared with cytological normal samples (Table 2; Fig. 1c,d, and Figure S1d).

Table 2 Relative expression level of miRNAs in paired urine, serum, cervical scrapes, and tissue biopsies.
Figure 1
figure1

Comparative differential expression profile of six miRNAs in urine, serum, tissue biopsies and cervical scrape derived from healthy controls, cancer and pre-cancer. (a & c) The miRNA expression level of upregulated miRNAs (miR-21-5p, miR-155-5p, miR-199a-5p) and (b & d) down regulated miRNAs (miR-145-5p, miR218-5p, and miR-34a-5p). Urine and serum samples were taken from pre-cancer and cervical cancer patients and compared to samples from healthy controls. In case of tissue biopsies, the samples are derived from cancer patients compared with samples from adjacent non-malignant normal tissues. **P ≤ 0.01, ns (nonsignificant), while in case of cervical scrape, the samples are derived from pre-cancer patients and compared with samples from healthy volunteers.

To determine whether there is a similar trend of miRNAs expression in urine as in tumor biopsies, the correlation of all six miRNAs expression in urine and paired tumor biopsies was calculated using Pearson’s correlation coefficient. Interestingly, the expression of all six miRNAs significantly and positively correlated in urine and paired tumor biopsies of cervical cancer cases (Figure S2) suggesting that miRNA expression profile of urine might accurately reflects to that in tumor cells.

Correlation of miRNA expression with HPV infection status in HPV + ve, HPV -ve cervical cancer cell lines and samples.

We checked the expression of all six miRNAs in HPV16 positive cervical cancer cell line SiHa, HPV18 positive cervical cancer cell line HeLa, and HPV negative cervical carcinoma cell line C-33A. Similar to the results in paired urine, serum, cervical scrape and tissue biopsies, qRT-PCR assay showed a marked upregulation in the expression of miR-21-5p, miR-155-5p, and miR-199a-5p and downregulation in the expression of miR-34a-5p, miR-218-5p, and miR-145-5p in HPV positive and negative cell lines (Table S1). The results of expression of upregulated miRNA in paired samples of urine, serum, cervical scrapes and tumor biopsies of HPV positive and negative infection showed no significant difference rather perfect correlation of results with cell lines (Fig. 2).

Figure 2
figure2

Expression levels of miR-21-5p, miR-155-5p, miR-199a-5p, miR-145-5p, miR-218-5p, and miR-34a-5p in HPV16-positive (a & c) and HPV16-negative (b & d) urine, serum, and cervical scrape samples of cervical pre-cancer and in HPV16-positive (e & g) and HPV16-negative (f & h) urine, serum, and tissue biopsies of cervical cancer patients.

When we compared the miRNA expression in HPV16 positive and negative samples of cervical pre-cancer and cervical cancer patients, we found that the expression of miR-145-5p was significantly lower in HPV16 positive urine samples of cervical pre-cancer (Figure S3a). Further, the expression of miR-21-5p was significantly higher in the serum of HPV16 positive than HPV negative cervical pre-cancer patients (Figure S3b). Moreover, the expression of miR-21-5p, miR-155-5p, and miR-199a-5p was significantly higher in HPV16 positive than HPV16 negative cervical scrapes of cervical pre-cancer patients (Table S2; Figure S3c ) and the expression of miR-145-5p was significantly lower in HPV16 positive in urine and serum samples of cervical cancer patients (Figure S3d and S3e) and the expression of miR-21-5p, miR-155-5p, and miR-199a-5p was significantly higher in HPV16 positive than HPV16 negative tissue biopsies of cervical cancer patients (Figure S3f.).

Correlations of miRNA expression level with clinicopathological characteristics of cervical pre-cancer and cancer.

The expression of all six miRNAs in urine, serum, cervical scrape, and tissue biopsies was correlated with various clinicopathological characteristics and known risk factors for cervical cancer. Except miR-34a-5p in the urine and miR-34a-5p and miR-199a-5p in the serum, the expression of all other miRNAs was significantly correlated with the age of the cervical pre-cancer patients (Table S3). The expression of miR-21-5p (in urine and serum), miR-145-5p (in urine), miR-155-5p (in serum), and miR-199a-5p (in urine and serum) correlated with the age of marriage of cervical pre-cancer patients (Table S3). Further, the expression of miR-21-5p (in urine and serum), miR-145-5p (in serum), and miR-199a-5p (in urine and serum) correlated with the parity of cervical pre-cancer patients (Table S3).

In cervical cancer patients, the expression of miR-21-5p (in serum), miR-218-5p (in urine and serum), miR-155-5p (in urine and serum), and miR-199a-5p (in serum) were correlated with the age (Table S3). Further, the expression of miR-21-5p, miR-155-5p, and miR-199a-5p in urine and serum and the expression of miR-145-5p and miR-218-5p in serum showed correlation with the age of marriage of cervical cancer patients (Table S3). The expression of all 6 miRNAs in urine and serum significantly correlated with the stage of cervical cancer (Table S3).

In cervical scrapes of pre-cancer patients, we found a significant correlation between the expression of miR-145-5p, miR-218-5p, miR-34a-5p, and miR-199a-5p with age (Table S3). Further, the expression of miR-21-5p and miR-155-5p correlated with the age of marriage while that of miR-21-5p, miR-155-5p, and miR-199a-5p significantly correlated with parity (Table S3). In tissue biopsies of cervical cancer patients, except miR-145-5p and miR-34a-5p, the expression of all other miRNAs significantly correlated with the age (Table S4). Additionally, the expression of all 6 miRNAs in tumor biopsies significantly correlated with the stage of cervical cancer patients (Table S4).

Diagnostic performance of miRNAs for cervical cancer as revealed by ROC analysis

To evaluate the diagnostic utility of urinary and tissue miRNAs for cervical cancer, ROC analysis was performed (Table 3; Fig. 3 and S4). ROC analysis demonstrated that urinary miRNAs may serve as useful non-invasive biomarkers for discriminating cervical pre-cancer and cancer from healthy controls. miR-21-5p exhibited the best AUC (0.971) with a sensitivity and specificity of 88% and 98%, respectively (Table 3). In an attempt to improve the diagnostic performance of miRNAs, we constructed a binary logistic regression model to evaluate the performance of the combined use of 3 or all 6 urinary miRNAs together. Interestingly, a combination of miR-145-5p, miR-218-5p and miR-34a-5p yielded a 100% sensitivity and 92.8% specificity for distinguishing cervical cancer patients from healthy controls (Table 3). As evident from Table 3, the diagnostic performance of miRNA expression evaluated in tumor biopsies was better than urinary miRNAs. A combination of 3 miRNAs (miR-21-5p, miR-155-5p, and miR-199-5p) as well as all 6 miRNAs yielded a 100% sensitivity and specificity for distinguishing cervical cancer patients from healthy controls (Table 3). The absolute sensitivities and specificities achieved after combining multiple miRNAs indicate that detection of a combination of miRNAs in liquid biopsy sample like urine can prove to be a useful biomarker for the early diagnosis of cervical pre-malignant lesions and for reducing the incidence of cervical cancer and better management.

Table 3 Diagnostic performance of miRNAs in cervical cancer patients.
Figure 3
figure3

Receiver operating characteristics (ROC) plots of miRNA expression in urine sample of cervical cancer patients. ROC plots evaluating diagnostic potential of (a) miR-21-5p, (b) miR-155-5p, (c) miR-199a-5p, (d) combined ROC plots of three upregulated miRNAs (miR-21-5p, miR-155-5p, and miR-199a-5p), (e) miR-218-5p, (f) miR-145-5p, (g) miR-34a-5p, (h) combined ROC plots of three downregulated miRNAs (miR-218-5p, miR34a-5p, and miR-145-5p), and (l) combined ROC plots of all six miRNAs.

Prognostic value of miRNA expression levels in cervical cancer

We constructed a prognostic signature by integrating the expression profiles of six miRNAs after a median follow up of 12–15 months and calculated corresponding estimated regression coefficient. To evaluate the ability of these miRNAs as a signature to predict prognosis, we first carried out a univariate Cox proportional hazards regression analysis to evaluate the correlation between each of the six miRNAs and the survival outcome. The analysis revealed a significant correlation of miR-155-5p, miR-21-5p, miR-199-5p, miR-218-5p, and miR-34a-5p expression with the overall survival (OS) (Table 4). We also performed multivariate Cox regression analysis to find independent prognostic power of aforementioned miRNAs within the context of several common clinical parameters, including age at diagnosis, age of marriage, parity, HPV infection, clinical stage and histopathological grade. The multivariate analysis revealed that miR-34a-5p (HR = 5.721; P = 0.003), miR-218-5p (HR = 0.218; P = 0.011), and HPV 16 (HR = 0.352; P = 0.08) were independent prognostic factors of OS (Table 4). Further, a combination of 3 upregulated miRNA signature (HR = 3.584; P = 0.01) and 3 downregulated miRNA signatures (HR = 0.239; P = 0.01) also retained their significance as independent prognostic factors of OS while performing univariate analysis, however, it was not significant in multivariate analysis (Table 4).

Table 4 Univariate and multivariate Cox regression analysis to evaluate the prognostic value of individual miRNAs.

Further, we have done the Kaplan Meier survival analysis to examine the potential prognostic power of the six miRNAs (Fig. 4). Notably, different expression levels of miR-155-5p, miR-21-5p, miR-199a-5p, miR-218-5p, miR-34a-5p have led to statistically significant different survival rates (log-rank test P-value < 0.0001, P < 0.001, P < 0.0001, P = 0.007 and P = 0.006, respectively), while no survival differences were observed between the high and low expression set of miR-145-5p (P-value = 0.09). We divided these six miRNAs among all samples into a high expression group and a low expression group to the cut off expression level.

Figure 4
figure4

The correlation of miRNA expression with overall survival of cervical cancer patients calculated using Kaplan Meier curve and Log-rank test. The patients were stratified into high expression and low expression group according to median of each miRNA. (a) miR-21-5p, (b) miR-199a-5p, (c) miR-155-5p, (d) miR-218-5p, (e) miR-145-5p, (f) miR-34a-5p, (g) three upregulated miRNA signatures, and (h) three downregulated miRNA signatures. The patients were stratified into high-risk group and low risk group based on median.

Target gene prediction and functional analysis of miRNAs

To further understand the functions of all 6 miRNAs, the online target gene prediction was performed using three prediction algorithms: miRDB, Target Scan, and microT-CDS. As evident from Figure S5, all 6 miRNAs can potentially regulate the expression of a large number of genes. The number of overlapping targets for miR-21-5p, miR-155-5p, miR-199a-5p, miR-34a-5p, miR-145-5p, and miR-218-5p were 274, 208, 234, 568, 1708, and 508, respectively (Figure S5). Subsequently, pathway enrichment analysis was performed on all overlapping potential target genes of six miRNAs to investigate their potential biological functions (Figure S6A). We observed that these genes were involved in PI3K-AKT pathway, mTOR signaling pathways, microRNA in cancer pathway along with other pathways involved in cancer (Figure S6B) as per the analysis performed using DAVID25,56. The pathway analysis revealed the enrichment of genes involved in signaling transduction, RNA polymerase II transcription pathway, and immune system. Since most of these pathways are well known for their role in tumor cell proliferation, angiogenesis, apoptosis, tumor cell migration and invasion, we can emphasize that these 6 miRNAs can be involved in the development, progression and prognosis of cervical cancer.

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